That is so sad. Poor guy.

I am a little disappointed in the lack of resources on executive dysfunction/the urge to procrastinate. Oftentimes I feel as if I literally cannot start working on a project, essay, or other assignment until the last possible moment. It makes research projects feel cramped and rushed, but most advice about starting slow or starting small doesn't get through. Unfortunately I don't have advice for anyone else who struggles with this, except attempting to trick your brain into thinking there's a sooner deadline. But if you also suffer from executive dysfunction, know you're not alone!

Poverty in Rural America, 1965. http://archive.org/details/0223PovertyInRuralAmerica.

See this with respect to White Trash (Isenberg, 2016) and the quote about Charles Murray's idea that 1963 was peak America.

judgments that favour the essential rights of an individual person

Intuitive heuristics are like mental shortcuts or rules of thumb that help us make decisions quickly and efficiently, based on our intuition, rather than extensive reasoning or analysis. Instead of carefully weighing all the options and considering all the information available, we often rely on these intuitive heuristics to make judgments and choices.

interaction

the way in which two or more things have an effect on each other

relating to the process of acquiring knowledge and understanding through thought, experience, and the senses

prohibition: the action of forbidding something, especially by law

here: tendency to make a specific choice based on cultural socialization

resonance: a feeling, thought, memory, etc. that a piece of writing or music makes you have, or the quality in a piece of writing, etc. that makes this happen

here: emotional resonance of a foreign language

the ease with which people understand and process information

considerations regarding the relation between the cost of an undertaking and the value of the resulting benefits

post hoc (latin) means "after this"

statistical analysis after a study has been concluded and the data collected

deny the truth of (a statement) by asserting the opposite

A ceiling effect occurs when the highest possible score or outcome on a measurement instrument is too low. Consequently, a significant number of respondents or subjects score at or near that highest score. As a result, the test or instrument does not differentiate adequately among participants at the higher end of the performance spectrum. To summarise, they “hit the ceiling.”

source: https://www.researchprospect.com/what-is-a-ceiling-effect/

relating to the practice or principle of giving a group priority over each individual in it

this term relates to a cognitive bias which describes why, for individuals, the pain of losing is psychologically twice as powerful as the pleasure of gaining

example: losing $10 can feel worse than gaining the same amount of money

source: https://thedecisionlab.com/biases/loss-aversion

principles concerning the distinction between right or wrong or good and bad behaviour

judgments favouring the greater good

here: the action of solving a problem

the doctrine that actions are right if they are useful or for the benefit of a majority

e.g. Deciding to donate money to a charity that helps a lot of people, even if it means sacrificing something you want for yourself.

eLife assessment

This study address a fundamental question: Do lipid rafts play a role in trafficking in the secretory pathway? By performing carefully controlled experiments with synthetic membrane proteins derived from the transmembrane region of LAT, the authors describe, model and quantify the importance of transmembrane domains in the kinetics of trafficking of a protein through the cell, from the ER to the cell surface via the Golgi. While their findings are solid, further experiments that relate to the existence and nature of domains at the TGN are necessary to provide a direct connection between the phase partitioning capability of the transmembrane regions of membrane proteins and the sorting potential of rafts.

Reframing what we think "citizenship" means

(#34)

*N2 (34) (Rebecca): I’m not sure if I understand deviance (from the lecture notes ) – is it something comparable to R2 in an OLS model? To what extent does it really “matter” in interpreting results from maximum likelihood estimation?

Response: First, note that every model can be fit by maximum likelihood maximization. After OLS, R2 doesn’t work anymore. The deviance is related to the log likelihood by the equation in 3.7 of the notes.

(#17)

*N7 (17) (Rita): From the lecture note, when calculating the likelihood function(9:13), what is the difference between the P and phat for logit equation? Could you further explain the likelihood table too?

Response: P would refer to the actual P for that case, and Phat is our prediction of it. Of course, we don’t observe P only Y=1 or 0.

The reverse problem

• N5 (#14, 26) (Syl): Can you explain to me like I’m 5 what you mean by “we’re working backwards” on slide 4? I’ve never thought about logit models like that and for some reason it’s not clicking and maybe even confusing me a little.

Response: This gets at Q2:

(#14)

*N1 (14) (Lily): There are many different phrases discussing “likelihood.” Can we go over the difference between them (i.e., log likelihood versus maximum likelihood)? (Osamudia)

Response: Sure. Likelihood (L) = the probability that a specific event (or combination of events will occur), given the set of coefficients. Log likelihood is just the log of L. Maximum likelihood is the process of solving a likelihood equation.

*N3 (#14)(Savannah): Does the MLE get reported in a typical logit regression table in R, or do we have to do an extra command to see it? Additionally, can we compare MLEs across different model specifications to see which model has the best fit (like we do with R^2 in OLS)?

Response: The MLE would mean the maximum likelihood estimates--those are the coefficients. The log likelihood gets reported--or the deviance--which is just another way of reporting it.

*N4 (#14) (Osamudia)--could we contextualize this against probit and logit models? I’m having trouble understanding the scenarios for which this sort of analyses would be useful.

Response: Maximum likelihood estimation is the process by which all models get estimated (aside from OLS). You can’t avoid it. The results (i.e. the coefficients) are what we interpret.

• N5 (#14) (Syl): Can you explain to me like I’m 5 what you mean by “we’re working backwards” on slide 4? I’ve never thought about logit models like that and for some reason it’s not clicking and maybe even confusing me a little.

Response: This gets at Q2:

*N6 (#14) (Mia): I’m trying to wrap my head around calculating the best probability to give us similar data observations. Are there no implications to research when our predicted probability is a better fit for some demographic groups then others? How important is sample size in accurately getting to maximized likelihoodness?

Response: Note that this is what the program does given the variables (and the paramaterization--i.e., do you want interaction terms? Do you want to stratify by race? Do you want to include age squared etc.--> those are the choice you make. Then, given those choices, the algorithm finds the best fitting coefficients.

As NPR puts it, "Texas won its independence from Mexico in 1836 and eventually joined the U.S. as a slave state."

Looking #6 and #8... So San Antonio is between Philadelphia and San Diego.

Indication for EMG

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

Learn more at Review Commons

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Reply to the reviewers

1. General Statements__: The manuscript entitled "__Dual antiviral mechanisms of Herbacetin and Caffeic acid phenethyl ester against Chikungunya and Dengue viruses with insights into Dengue methyltransferase-CAPE crystal structure" is the first report of broad spectrum alphavirus and flavivirus inhibitors with dual roles that efficiently inhibit virus replication by diminishing the levels of polyamines in the host cells as well as inhibit the enzymatic activity of the virus-specific methyltransferase (MTases). Chikungunya virus (CHIKV) and Dengue virus (DENV) are re-emerging alpha- and flaviviruses respectively. Until now, no antivirals are commercially available to combat these two viral infections. This study delves into the antiviral mechanisms of Herbacetin (HC) and Caffeic acid phenethyl ester (CAPE) against DENV and CHIKV. Treatment of Vero cells with these compounds resulted in polyamine depletion. However, adding exogenous polyamines did not completely rescue the virus, suggesting alternative antiviral mechanisms. Interestingly, these compounds exhibited anti-MTase activity against purified viral MTases of CHIKV and DENV. The crystal structure of the DENV 3 MTase in complex with CAPE revealed its binding site within the GTP-binding region of DENV MTase. This study presents the novel dual inhibition mechanism of HC and CAPE, offering promising prospects for developing broad-spectrum antivirals.

2. Point-by-point description of the revisions

We express our gratitude to the reviewers for their time and insightful comments, which have significantly contributed to the improvement of the manuscript. We believe that the thoughtful critiques and suggestions have significantly enhanced the overall quality of our work. Below, we provide a point-by-point response to each comment, addressing the concerns raised by the reviewers.

Reviewer 1: -

Comment 1: My main concern is that the depletion of polyamines is likely to have broad implications for host cell metabolism. Polyamines are critical for genome folding and stability. Hence, polyamine depletion will likely compromise cellular metabolic homeostasis. My suggestion is to perform a literature survey on this topic, identify appropriate assays of cellular homeostasis, and add at least one such assay in the relevant HC and CAPE concentration range to address my question..

I also suggest adding the potential negative effects of polyamine depletion on host cell metabolism in the discussion section

• Response: We appreciate the reviewer's constructive feedback for their insightful remarks on the potential extensive influence of polyamine depletion on host cell metabolism. We acknowledge the critical role polyamines play in genome folding and stability, and their depletion could indeed disrupt cellular homeostasis. In response to this valuable feedback, we conducted a comprehensive literature review. This literature review uncovered studies investigating the targeting of the polyamine biosynthetic pathway as a potential therapeutic strategy for combating various infections and diseases. Additionally, DFMO , a drug that targets polyamine biosynthetic pathway enzyme is an FDA-approved drug for African sleeping sickness and high-risk neuroblastoma (Bouteille & Dumas, 2003; Nazir et al., 2024) indicating that despite the critical role of polyamines in cellular metabolic homeostasis, the host polyamine pathway can also be successfully targeted for antiviral drug discovery. As recommended, we have added this information in the revised manuscript. * Additionally, ribavirin, an FDA-approved antiviral agent, employs various mechanisms to inhibit viral replication, including the reduction of polyamine levels (Tate et al., 2019). Furthermore, we have also examined the protocols available in the literature for CAPE, HC, and DFMO treatment. Most of these studies have employed MTT assay, as illustrated in the research conducted by Arisan et al. 2012 and Shen et al. 2013 (Arisan et al., 2012; Shen et al., 2013). Notably, Aljabr et al.,2016 also employed the MTT assay for viability testing, underscoring its relevance (Aljabr et al., 2016). Similarly, our manuscript employed the MTT assay at various compound concentrations to ensure the utilization of non-cytotoxic concentrations for antiviral activity testing. *

As per reviewer's recommendation, we have discussed the potential adverse effects of polyamine depletion on cellular processes in the revised manuscript's discussion section.

*Line no.s 513 – 523 of the revised manuscript have the revised text as per the suggestion. *

Reviewer 2:-

Comment 1:- Authors describe anti-CHIKV and anti-DENV activities of herbacetin and caffeic acid phenyl ester (CAPE). The antiviral effect is not reversed buy exogenous polyamines suggesting multiple mechanisms of action. NS5-Met complex with caffeic acid phenyl ester was obtained and its structure resolved at high resolution. The resolved structure reveals two binding sites for antiviral compound overlapping with that of GTP and possibly with a site involved in binding of RNA

Other than analysis of crystal structure of NS5/CAPE complex the provided data is of low quality and is not analyzed properly. There is no evidence that data is reproducible. Authors have calculated significance from "experimental repeats" which, based on the description of experiments, are not independent experiments but technical replicates. Some key technical details are missing and some experiments are not described at all. The writing can be vastly improved and figures be made a lot more easier to understand.

• *Response :-We appreciate the reviewer's positive feedback of on the crystal structure and as pointed out towards data quality and analysis, we have tried and made significant improvements, including enhancing data representation and providing detailed protocols in the supplementary materials where necessary. Additionally, we have addressed key technical details that were previously missing and ensured that all experiments are described adequately. We acknowledge the need for clearer writing and have now mentioned clearly that independent experiments have been carried out in the study. We have made suggested revisions to the revised manuscript. *

  Comment 2:- Bad writing lines 64-65 . Viral genomes lack protein synthesis machinery. Basically correct but no genome has protein synthesis machinery

• Response:-We thank the reviewer for pointing this out. We have modified the text as follows: lines 64-65 "Viral genomes lack protein synthesis machinery, and the ability to hijack the host cell's resources for replication is crucial for all viruses". to lines 65-67 "Viral particles lack essential protein synthesis machinery. Consequently, viruses rely on the host cell's resources to replicate effectively."

  Comment 3:- line 137 flavonoids play a role in reducing the levels of nsP1 in CHIKV - what can this possibly mean? Are shown to reduce the level of nsP1 in CHIKV-infected cells?

• Response: We appreciate the reviewer for bringing this to our attention, and we acknowledge that it was due to a writing issue in English. This has now been rectified. A dose-dependent reduction of the CHIKV E2, nsP1, and nsP3 proteins was observed upon treatment with baicalein and fisetin. This finding would suggest that baicalein and fisetin might inhibit the production of CHIKV protein, especially the proteins involved in the negative-strand synthesis and part of the replicase unit (Lani et al., 2016). To account for this suggestion, we have modified the text in the revised manuscript to (line 145-147): " Moreover, flavonoids treatment has demonstrated the dose-dependent decrease in CHIKV titer due to reduced levels of CHIKV viral proteins, including nsP1*. *

__Comment 4 :-__line 250-251 - RNA was isolated from the infected cells' supernatant, used for cloning, and inserted between the NheI and XhoI restriction sites... …..It should be impossible as one cannot insert RNA into bacterial plasmid DNA.

• Response:- We thank the reviewer for pointing this out. line 250-251 – "RNA was isolated from the infected cells' supernatant……..". This has been changed to line 267-271 " RNA was isolated from the supernatant of the cells infected with DENV 3, and used for cDNA preparation, cloning of the MTase gene fragment into the pET28c (+) vector using NheI and XhoI restriction sites."

  __Comment 5 :-__Missing parts. Examples

the source of nsP1 of CHIKV is not indicated, True, there are references to previous studies, but this is extremely important point and it should have been clearly stated that it was obtained from E. coli. The issue is that authors made some predictions and modelling based on structure of nsP1 from eukaryotic expression system. It is not known does the enzyme purified from bacteria have similar structure (actually, in cited Nature paper - doi: 10.1038/s41586-020-3036-8 - attempts to purify nsP1 from bacteria were made. The protein was monomeric and had no activity)

• Response:- We thank the reviewer for the comments. In response to the reviewer's concern regarding the source of the nsP1 protein from CHIKV, we would like to clarify that the recombinant protein was expressed and purified from E. coli Rossetta cells in our laboratory. We acknowledge the importance of this point and apologize for any oversight in not explicitly stating it in the manuscript. In response to the reviewer's suggestion, we have incorporated a detailed expression and purification protocol into the manuscript supplementary methodology (line number 1068-1091).
• Response:- Alphaviruses share a high degree of sequence similarity (>80%), particularly within the nsP1 protein, with conserved active site residues (Supplementary Figure 2). Several studies investigating nsP1 proteins from alphaviruses, including Sindbis virus, Semliki Forest virus, and Venezuelan equine encephalitis virus, have successfully employed E. coli Rosetta cells for protein expression, followed by enzyme activity assays (Abdelnabi et al., 2020; Li et al., 2015; Tomar et al., 2011). Our laboratory is working on this protein for more than a decade and have conducted extensive assays on the activity of nsP1 protein purified from bacterial expression system. Our results are reproducible. These studies have been published in reputed peer reviewed research articles, including (Kaur et al., 2018; Mudgal et al., 2020). Additionally, similar assays have been demonstrated in the study by Bullard-Feibelman et al., 2016. We trust that this clarification resolves the reviewer's concern, and we are delighted to address any further inquiries.

  Comment 6:- Figure lacks quality (and figure legends are unclear) Examples:

• it is impossible to understand what exactly is shown in Figure 1J

• important information is missing, for example, it is not clear what were concentrations of antiviral compounds for panels 1F and 1I

• Response :- We thank the reviewer for the constructive comments that has helped us to improve the revised manuscript. We have revised Figure 1J and as suggested we have updated the legends accordingly. Similar revisions have been made in the revised manuscript to the TLC protocol and results to ensure clarity. We thank the reviwer for pointing out the missing information regarding the concentrations of the antiviral compounds used in panels 1F and 1I. As per your suggestion, we added the antiviral compounds concentrations for these experiments in figure legends.

Comment 7:- 4. wrong data - line 478 it is stated that there is no vaccine for DENV or CHIKV. It is correct, DENV vaccine has been in use for several years and CHIKV vaccine was approved at 2023 - line 476 refers to family alphaviridae. This does not exist, family is Togaviridae

• Response:- We appreciate the reviewer for bringing this to our attention. We have accordingly revised the sentences for accuracy. "Although human viruses belong to several viral families, Alphaviridae and Flaviviridae are the most significant burden on public health" changed to line number 505-506 "Although human viruses belong to several viral families, Togaviridae and Flaviviridae impose one of the most significant burdens on public health"
• *

Line no.. 478 “ Neither commercially available drugs nor vaccines are available for these viruses.” Changed to line number 508 to 509 “Although FDA-approved vaccines for Dengue and Chikungunya viruses are available, no antiviral therapies have been approved against these viral infections.”

Comment 8: ____5. unjustified conclusions. Example

• authors have analyzed sequences of nsP1 of alphaviruses and made conclusions regarding conservation of active site. It is probably correct but the analyzed viruses do not represent all diversity of alphaviruses, insect specific members and aquatic alphaviruses should also be analyzed (same problem with analysis performed for flaviviruses)

• Response:-Following the reviewer's recommendation, we have included Salmonid alphavirus, an aquatic virus, and Eilat virus, an insect-specific virus, in our comparison along with other human-infecting alphaviruses. Additionally, for flaviviruses, we have incorporated Palm Creek virus, an insect-specific virus, and Wenzhou shark flavivirus, an aquatic virus. As suggested, the relevant modifications have been done to the MSA protocol, results, and figure legends.

  Comment 9:- 6. Insufficient analysis of data. In some cases, there is a significant discrepancy between the results of different assays. For example, CAPE inhibits DENV at 2.5 microM (Fig 1H) but in test tube assay only small inhibition was observed even at 1000 microM. Authors should provide plausible explanation for this and similar discrepancies.

(CE and ELISA-based assays shown on figure 6 also resulted in drastically different inhibitions). It is expected assays would produce different results but there should also be explanation for this. If this is not provided one can assume that it is due to experimental errors.

• Response:- We thank the reviewers for their valuable comments. We acknowledge the importance of providing plausible explanations for such variations and are committed to addressing these concerns in our revised analysis. * Our explanation: Capillary electrophoresis (CE) offers a direct approach for detecting S-adenosylhomocysteine (SAH), the product of the methyltransferase reaction. However, this assay has a limitation in sensitivity, it is only able to detect SAH concentrations above ~ 300 µM. A previously validated CE-based assay for Chikungunya virus (CHIKV) nsP1 by Mudgal et al.,2020 addresses this limitation. Their work demonstrates that using specific concentrations of S-adenosylmethionine (SAM) at 0.3 mM and guanosine triphosphate (GTP) at 4 mM enables reliable detection of SAH in the reaction. However, *CAPE is observed to inhibit DENV at ~2.5 micro, supporting that viral inhibition not only is due to MTase inhibition but through other mechanism i.e. host cells polyamine depletion.

• *

• Therefore, this presents one plausible explanation, although we cannot currently dismiss the possibility of other mechanisms that could also contribute to viral inhibition by CAPE.*

The established ELISA assay of nsP1 utilizes an indirect detection method, which exhibits higher sensitivity. Additionally, previously published studies on alphaviral nsP1 inhibitors also report nsP1 enzyme activity inhibition by compounds at concentrations several folds higher than their respective active doses in cell culture-based studies (Delang et al., 2016; Mudgal et al., 2020; Kovacikova et al., 2020).Therefore, differing substrate concentrations and CE-based assay limitations may be attributed to discrepancies between the capillary electrophoresis (CE) and ELISA assays. Numerous studies have utilized the CE-based assay or equivalent assays based on similar principles as qualitative tools for evaluating enzyme activity.

In the revised manuscript, Figures 6B and 6C graphical representation has been transitioned from a dose-response curve IC50 format to a bar chart for enhanced clarity. This bar chart effectively conveys the key finding of a dose-dependent decrease in activity observed for both HC and CAPE.

Similarly, we again tried to reoptimize the MTase CE-based assay by reducing the GTP concentration in enzyme reaction from 4 mM to 0.3 mM. This modification resulted in slight improvement and shows clear (~50%) decrease in enzyme activity at the highest concentration, as shown in Fig. 6 F and G. Furthermore, our approach with CE based assay is centered around detecting inhibition rather than conducting quantitative analyses.

• *

The discrepancy in the in vitro vs the enzyme test tube assay could be attributed to HC and CAPE's multifaceted mechanism of action when used in vitro (i.e polyamine depletion and anti methyltransferase activity). However, only methyltransferase inhibition has been assessed in enzymatic assay. Following the reviewer's suggestion, we have revised the methyltransferase assay protocol, results, and figure legends for clarifications. Additionally, the results have been appropriately discussed in the discussion section.

• *

Comment 10 :-6. Discussion is essentially missing, it is just list of statements mostly repeating what was said in other sections

> Response: We appreciate the reviewer's suggestion regarding the discussion section; we have incorporated a comprehensive discussion in the revised manuscript.

3rd reviewer :-

The manuscript submitted by Bhutkar M. et al. details the antiviral properties of two compounds, herbacetin (HC) and caffeic acid phenethyl ester (CAPE), against Chikungunya virus (CHIKV) and Dengue virus (DENV) through cellular, bioinformatics, biochemical, biophysical, and structural studies. The authors propose a dual antiviral mechanism of action exhibited by these compounds, beginning with an evaluation of their cytotoxicity. Subsequent assessments of their antiviral efficacy against CHIKV and DENV are addressed using plaque reduction assay and other orthogonal assays such as qRT-PCR, and Immunofluorescence assay (IFA). Further, authors performed thin layer chromatography (TLC) to monitor polyamine levels in the cells treated with these compounds and concluded that these compounds leads to polyamine depletion which is also supported by previous studies. These experiments included DFMO as a control which is well established for its role in this regulation. Beyond their impact on cellular polyamine levels, the authors propose a role for these compounds in the inhibition of MTase domains in CHIKV and DENV, supported by the crystal structure of the DENV-3 NS5 MTase domain in complex with CAPE.

Comment 1:-

__Major points:- __ While the manuscript presents promising findings regarding the dual antiviral effects of the tested compounds, the authors fall short of demonstrating direct inhibition of MTase activity as a meaningful and complementary effect to polyamine depletion. Being only indirect, the enzyme inhibition data is not convincing, and the measured indirect inhibition is not precise enough in the case of CHIK nsp1 and too weak in the case of DENV NS5 (detailed below).

Conceptually, the organization of the results should be changed to first data (structural data of DENV MTase in complex with CAPE, which is a significant achievement), then interpretation/discussion with modeling, and not the other way around.

The discussion section requires more elaborate scientific justification than simply re-reporting the results.

• Response:- We express our gratitude to the reviewers for their time and insightful comments, which have significantly contributed to in the improvement of our manuscript. We believe that the thoughtful critiques and suggestions have substantially improved the overall quality of our work. The changes made in the revised manuscript are highlighted in red. Below, we provide a point-by-point response to each comment, addressing the concerns raised by the reviewers.

  Comment 2:-

It would be best to organize the ms as follows: - Crystal structure of DENV MTase in complex with CAPE - Building of a model of nsp1 by superimposition with NS5 MTase - Modeling compound binding - Inhibition assays using enzyme assays at least in the case of NS5 MTase. The direct enzyme assays are well described in the literature.

• Response :- We appreciate the reviewer's suggestion regarding the manuscript organization. We understand the value of presenting the data in a logical flow. For this study, our initial investigations focused on the polyamine depletion ability of HC and CAPE, followed by antiviral activity assays. Based on the preliminary data from cell-based polyamine depletion assay and antiviral assays, the identified molecules were used for in silico investigations, followed by biochemical and biophysical validation. the crystal structure studies were performed to gain a deeper understanding of the inhibition mechanism. Therefore, we believe this flow, approach and the current structure have merit and is request to be considered.

  Comment 3:- Inhibition assays using enzyme assays at least in the case of NS5 MTase. The direct enzyme assays are well described in the literature.

• If there is no inhibition, then discussion about possible reasons would be interesting and help the AV field. For example, CAPE could bind to other enzyme or sites, etc...

Figure 5 is problematic.

• When presenting an y IC50 data, care should be taken that the IC50 inflexion point is preceded and followed by at least two experimental points, which is not the case. The IC50 value of 7.082 and 5.156 µM are too imprecise (and there is no need to give digits after the value). Please add more low concentration experimental points.

• Panel F and G: A reduction of 25 % at the highest inhibitor concentration is a strong indication that there is no effect.

• Response:- We sincerely thank the reviewers for their valuable comments and insights regarding the discrepancies observed in our data. We acknowledge the importance of providing plausible explanations for such variations and are committed to addressing these concerns in our revised analysis. * Capillary electrophoresis (CE) offers a direct approach for detecting S-adenosylhomocysteine (SAH), the product of the methyltransferase reaction. However, this assay has a limitation in sensitivity, typically only detecting SAH concentrations exceeding ~300 µM. *

*A previously validated CE-based assay for Chikungunya virus (CHIKV) nsp1 by Rajat et al. addresses this limitation and has been mentioned in the revised manuscript with the reference. Their work demonstrates that using specific concentrations of S-adenosylmethionine (SAM) at 0.3 mM and guanosine triphosphate (GTP) at 4 mM enables reliable detection of SAH in the reaction. The established ELISA assay utilizes an indirect detection method and exhibits higher sensitivity. Also, previous studies on alphaviral nsP1 inhibitors have also reported nsP1 enzyme activity inhibition by compounds at concentrations several folds higher than their respective active doses in cell culture-based studies (Delang et al., 2016; Mudgal et al., 2020; Kovacikova et al., 2020). *

Hence, differing substrate concentrations may be attributed to discrepancies between the capillary electrophoresis (CE) and ELISA assays. Numerous studies have utilized the CE-based assay or equivalent assays based on similar principles as qualitative tools for evaluating enzyme activity.

• *In response to the reviewer's suggestion to test compounds at lower dilutions, we acknowledge that we are currently unable to perform an assay for lower dilutions as recommended due to time constraints and limited availability (screen shot below) of "MABE419 Sigma-Aldrich (Merk), Anti-m3G-cap, m7G-cap Antibody, clone H-20 antibody" used as the primary antibody (Kaur et al., 2018). Our attempts to procure this antibody from Sigma were unsuccessful.For India it shows limted availability and the vendor has given the estimated shipment time of more than 7 weeks. As per reviewers suggestion and the current limitations in the IC50 data, we have revised the graphical representation from a non-linear regression format (which estimates IC50) to a bar chart format. In the revised manuscript, Figures 6B and 6C graphical representation has been transitioned from a dose-response IC50 format to a bar chart for clarity. This bar chart effectively conveys the key finding of inhibitory activity observed for both HC and CAPE.

We tried to reoptimize the Dengue virus MTase CE-based assay by reducing the GTP concentration from 4 mM to 0.3 mM. This modification resulted in slight improvement and shows clear (~50%) decrease in enzyme activity at the highest concentration, as shown in Fig. 6 F and G. The CE-based assay for HC and CAPE data clearly indicates inhibition above >50%. Our approach with this assay is centered around detecting inhibition rather than conducting quantitative analyses. Following the reviewer's suggestion, we have revised the methyltransferase assay protocol, results, and figure legends. Additionally, the results have been appropriately discussed in the discussion section.

Comment 4- Please describe more panel D in the legend.

• Response :-We sincerely appreciate your suggestion and wish to express our gratitude. We have revised figure legend 6 D from. Line no. 791 "The CE based HC and CAPE Methyltransferase inhibition activity assay CHIKV nsP1" changed to line no. 884 to 886 "CE-based nsP1 MTase activity inhibition assay as described previously by Mudgal et al. 2020". HC and CAPE compounds were tested at a concentration of 200 µM and CAPE 1000 µM respectively.

  Minor Points/Comments/ Suggestions:

Comment 1:-

In the Introduction section, line 58: Are DENV infection numbers representative of worldwide distribution, please clarify. Also, in the case of CHIKV infection, the most affected countries are mentioned, why not follow the same pattern for DENV, please consider homogenizing the text.

Response:- Thank you for your suggestion; we have revised the text accordingly. Line no. 58 "It is estimated that ~100-400 million DENV infections occur annually" changed to line no. 58 to 61 "It is estimated that annually ~100-400 million DENV infections occur worldwide. The Philippines and Vietnam are among the most affected countries. Moreover, dengue is endemic in India, Indonesia, Myanmar, Sri Lanka, and Thailand (Bhatt et al., 2013; Lobo et al., 2011, National Center for Vector Borne Diseases Control Report 2022 (NCVBDC)."

__Comment 2:- __B. Before p. 4 (line 91), alphaviruses were not introduced. Please consider introducing them.

Response :- Thank you for your feedback; brief introduction of alphaviruses have been added.

1. • 4 (line 92) Alphaviruses belonging to the Togaviridae family include viruses such as Chikungunya, Eastern equine encephalitis, Venezuelan equine encephalitis, etc.*
2. *

Comment 3:- C. Consider introducing Dengue serotypes to help readers understand the significance of DENV-2 and DENV-3.

Additionally, ensure uniformity by referring to these serotypes as DENV-2, DENV-3 throughout. There are inconsistencies in the current text, such as 'DENV 3' in lines 39 and 152, and 'DENV3' in lines 249 and 250, among others.

• Response:-Thank you for your valuable input. Dengue serotypes have been introduced, and we have meticulously reviewed and rectified all inconsistencies regarding their nomenclature. Line no. 120 to 123 "Flaviviruses are classified within the Flaviviridae family and encompass viruses like Dengue, Zika, Japanese encephalitis, etc. Dengue virus consists of four distinct antigenic types: DENV 1, DENV 2, DENV 3, and DENV 4. DENV 2 has been India's most prevalent serotype for the past 50 years, however serotypes 3 and 4 have also appeared in some recent epidemics (Kalita et al., 2021)."

  Comment 4:- D. P. 4, 5 lines 91-134: Consider rephrasing/reorganizing the methylation process: conventional and unconventional. The current introduction doesn't clearly indicate the difference between the cap-0 capping in alphaviruses and cap-1 in flaviviruses.

• Response:-Line 100 changed from "Cellular enzyme capping mechanisms usually involve the methylation of guanosine triphosphate (GTP) after transferring it to the 5' end of the RNA. However, the molecular mechanism of viral mRNA capping in alphaviruses is distinct." To line no. 102 to 108 "Cellular enzymes use conventional capping mechanisms, usually where GTP is first transferred to RNA's 5' end, followed by its methylation. On the other hand, viral capping in the case of alphaviruses is unconventional, where GTP is first methylated, followed by the guanyltion of viral RNAs. Furthermore, Cap 0 alphaviruses feature monomethylation at the N7 position of the guanosine nucleotide, while Cap 1 in flaviviruses has additional methylation at both the N7 and 2'O positions."

  Comment 5:-

• Please consider citing the article instead of the referred link, wherever possible, for e.g., for ref. 22 PMID: 28218572 (a more recent reference for Flaviviridae taxonomy available than that mentioned in the current manuscript.)

• Response :- We have addressed the reviewer's insightful suggestion regarding the citation and included the references accordingly.

  Comment 6:- F. Homogenize the writing of taxonomic names (viral families) in the text. For example, in line 126 change Flaviviridae to Flaviviridae, and line 476 (Discussion section), alphaviridae to Alphaviridae, flaviviridae to Flaviviridae and so on. For further clarification on addressing this, one can also refer to https://ictv.global/faq/names.

• Response :-We sincerely appreciate the reviewer's input. We have incorporated the suggested changes as follows : In line 126, we changed "Flaviviridae" to "Flaviviridae".

In line 476 (Discussion section), we corrected "alphaviridae" to "Togaviridae".

We ensured consistency in the formatting of taxonomic names throughout the manuscript.

Comment 7:-

1. Please make sure to appropriately reference the corresponding supplementary information (text or figures) in the main text wherever necessary to avoid the impression of missing information. For instance, in none of the sub-sections of Materials and Methods (M&M), it is being indicated to refer to the suppl. experimental procedures for more details. Also consider not repeating the same information between the main experimental procedures text and the supplementary text.

• Response :-The reviewer's feedback has been invaluable, and we've acted upon it accordingly. In response to the suggestion, we've made it clear in the manuscript to refer to the supplementary experimental procedures for detailed protocols where appropriate. Additionally, we've listed certain protocols exclusively in the supplementary material to enhance clarity and avoid repetition.

  Comment 8:-

• M&M sub-section. 2, line 163: Which specific culture media is being referred to here? Could you provide additional details? On line 164, it mentions that polyamines were diluted in water. Is this water sterile tissue culture-grade water as indicated in line 161?

• Response :-We appreciate the reviewer's attention to detail. At the time of usage, further dilutions were prepared in 2% DMEM media. Additionally, individual polyamines (putrescine, spermidine, and spermine) stocks were diluted in sterile tissue culture-grade water from Alfa-Aeser, USA, and used as indicated. As such, we have revised the sentence to enhance clarity. Line number 173 to 175 "At the time of usage, further dilutions were prepared in culture media. Similarly, individual polyamines (put, spm, and spd) (Alfa-Aeser, USA) stocks were diluted in water and used as designated." changed to this "At the time of usage, further dilutions were prepared in 2 % DMEM media. Similarly, individual polyamines (put, spm, and spd) (Alfa-Aeser, USA) stocks were diluted in sterile tissue culture grade water and used as designated."

• *

Comment 9:-

1. M&M, line 274: What is CE? Please expand the term before using the abbreviation.

2. Response :- Thank you for bringing that to our attention. CE mentioned in line 294 stands for Capillary electrophoresis__.__

   Comment 10:-

line 306. Ref. 53: This is not a reference.

• Response :-Thank you for bringing this to our attention. We understand that reference 53 does not correspond to a valid source. We acknowledge this and want to clarify that due to the unavailability of the proper reference, we included this reference. We have now changed the reference to the Crysalis Pro software.

  Comment 11:-

• Results. 1: Didn't understand the relevance of Fig. 1C, as this data is already included in Fig. 1B.

• Response :-Thank you for bringing this to our attention. We apologize for any confusion caused by including Fig. 1C, especially since the data it presents overlaps with that of Fig. 1B. To ensure clarity, we have made modifications accordingly. Figures (A) and (C) depict the viability of Vero cells measured by an MTT assay after a total incubation of 134 hours. This protocol involved a 12-hour pre-treatment with either HC (A) or CAPE (C), followed by additional incubation steps as detailed in the legend. In contrast, figure (B) shows the cell viability of Vero cells treated with CAPE only, measured after a total incubation of 38 hours.

• To avoid further confusion figure legend has been changed from "(A) and (C) depicts the percent cell viability of Vero cells treated with HC and CAPE for 12 hr pre-treatment and 24 hr post-treatment and incubated in maintenance media for 4 days, (B) shows the percent cell viability of Vero cells treated with CAPE for 12 hr pre-treatment and 24 hr post-treatment. " to "(A) and (C) depicts the percent cell viability of Vero cells treated with HC and CAPE for 12 hr pre-treatment followed by a 2-hour incubation with maintenance media, 24 hr post-treatment, and incubated in maintenance media for 4 days, (B) shows the percent cell viability of Vero cells treated with CAPE for 12 hr pre-treatment, followed by a 2-hour incubation with maintenance media and 24 hr post-treatment."

  Comment 12:-

Fig. 1G and H are not referred to in the result text.

• Response :-Thank you for pointing out the oversight regarding Fig. 1G and H not being referred to in the results text. We have added following statement Results p.1 Line no. 354 "Likewise, HC and CAPE treatment to Vero cells has shown a decrease in viral titer DENV-infected cells in a dose-dependent manner (Figure 1 G-H)."
• *

Comment 12:-

Lines 342, 343: 'At the mentioned concentrations', where are these concentrations mentioned?

• Response:-*Thank you for bringing this to our attention. We acknowledge this mistake regarding the mentioned concentrations at lines 342 and 343. RT-PCR was conducted for CHIKV using concentrations of 200 µM for HC, 25 µM for CAPE, and 1000 µM for DFMO. Similarly, for DENV, RT-PCR was performed with concentrations of 200 µM for HC, 2.5 µM for CAPE, and 1000 µM for DFMO. To avoid further confusion, Figure legends were revised and line no. 846 to 848 "(1F) RT-PCR for CHIKV with HC 200 µM, CAPE 25 µM, DFMO 1000 µM concentration (1I) RT-PCR for DENV with HC 200 uM, CAPE 2.5 uM and DFMO 1000 µM" *
• *

Comment 13:-

qRT-PCR data is not very clear. Please consider elaborating on some details. Why were the statistics only performed between HC and DFMO and not with CAPE? How the fold reduction is being calculated? For example, the fold difference of 97 is not visibly evident.

• Response:- We regret that the clarity of the qRT-PCR data was not satisfactory. We acknowledge your feedback and understand the importance of elaborating on certain details. The statistics were performed for all treatment groups, including HC, CAPE, and DFMO. However, the representation in the graph was adjusted by replacing the "top square bracket" with a "line" to avoid confusion. The y-axis of the graph depicts the log10 fold change in target gene expression relative to a designated virus control (VC). A value of ~ -2 on this axis corresponds to a significant downregulation, reflecting a 97-fold decrease in expression compared to the VC. A comparable graphical depiction is also evident in the work by Mudgal et al. (2022).

  Comment 14:-

Line 375: 'SAM is lined by residues ... would be more appropriate than 'formed'

• Response :-Done as suggested. We have revised the sentence in question and similar ones accordingly. "In CHIKV nsP1, SAM is formed by residues Gly65, Ser66, Ala67, Pro83, Arg85, Ser86, Asp89, Thr137, Asp138…" changed to line no. 393 "In CHIKV nsP1, SAM binding site is lined by,….."

  Comment 15:-

Fig. 1J. For TLC results, consider using the term panel (left, center, right) to navigate within this figure. The representation of this result is not uniform, as the time course is shown for HC while it is not shown for DFMO and CAPE. The treatment time is not indicated for DFMO and CAPE. For better representation and significant differences, one can consider quantifying these TLC results.

• Response:- Thank you for bringing these points to our attention. Done as suggested. We have simplified the presentation of the TLC results to enhance clarity and revised the methodology, results, figure, and legend accordingly. Also, we have quantified the TLC results. * -*Polyamine determination by Thin-layer chromatography (TLC)

-Vero cells were treated with HC, CAPE and DFMO, as mentioned in the antiviral assay protocol. Similarly, HC-treated cells were collected after 12, 24, and 36 hr of treatment." Revised to " Vero cells were treated with CAPE (25 µM), HC (200 µM), and DFMO (1000 µM) for 36 hr …… Further, TLC images were quantified utilizing ImageJ software." *Figure legend 1:- (J) depicts the effect of polyamines level after treating with HC (200 µM) and CAPE (25 µM). Polyamine level of Vero treated cells at 12, 24, and 36 hr for HC and pre (12 hr) and post-treatment (24 h) for CAPE and DEMO, using untreated cells as a cell control (CC) for both of the conditions. 0.1 μM putrescine (put), spermine (spm), and spermidine (spd) as a positive control marker. changed to *

"(J) the chromatographic analysis of polyamine levels in Vero cells after 36 hr treatment with (from left) CAPE (25 µM), HC (200 µM), DFMO(1000 µM), and cell control (CC), 0.1 μM putrescine (Put), spermine (Spm), and spermidine (Spd) as a positive control marker. "

Results: Line no. 351 "Polyamine levels in cells treated with CAPE were significantly lower as compared to DFMO treatment (Figure 1J). Meanwhile, HC showed a reduction in polyamine levels with the initial 12 hr treatment; later, polyamine levels elevated gradually with time."

Revised to line no. 371 to 373"After treatment with CAPE, HC, and DFMO to Vero cells, overall residual polyamine levels are 28.33%, 29.67 %, and 46 %, respectively, compared to cell control."

Comment 16:-

Fig. 1, figure legend, lines 750-751: instead of 'Panels D-G depicts the inhibitory effect of CHIKV and DENV infected cells on different concentrations of HC and CAPE' should be

'Panels D-G depicts the inhibitory effect of different concentrations of HC and CAPE on CHIKV and DENV infected cells'

• Response:-Thank you for the suggestion. We have updated the figure legend to ensure clarity based on your recommendation. (D,E,G,H) depicts the inhibitory effect of different concentrations of HC and CAPE on CHIKV and DENV infected cells'.

  Comment 17:-

Line 755: DFMO is wrongly written as 'DEMO'

• Response:- Thank you for bringing that to our attention. We have corrected the typo, changing Line 845 'DEMO' to 'DFMO' as appropriate.

  Comment 18:-

Fig.2. IFA. Authors must consider on elaborating the IFA data. One can also consider quantifying these data for better comparison with other assays.

• Response:- We thank reviewer for your input. As per the suggestion we have elaborated the results on IFA. The qualitative application of IFA was chosen because of the absence of dedicated paid software/hardware for image quantification on the Thermofisher EVOS platform, thereby impeding our quantification efforts.

  Comment 19:-

Result 1 (Suppl. Fig. 1). Line 359: 'After infection': please indicate the time here.

• Response:- Thank you for the feedback. Line no. 377:We have updated the line to specify the time as “ after 2 h of virus infection," and we have also revised this in the methodology section for clarity.

  Comment 20:-

Suppl. Fig.1: How was the concentration of these polyamines chosen to be 1µM?

What will be the effect on increasing concentrations?

Why were all these three polyamines added together?

What is the effect of addition of individual polyamine in the rescue of viral titer?

Will this effect vary if cells are pre-treated with these polyamines and compounds in question are added post viral infection or if both are added simultaneously?

Response:- We thank the reviewer, for raising these insightful questions. We performed an Exogenous polyamine addition assay as per Mounce et al. 2016 to maintain consistency with established practices and the research focus. The concentration of 1 µM biogenic polyamines (Putrescine, Spermidine, and Spermine) was chosen based on the findings of Mounce et al. (2016), where viral titers were restored to levels comparable to non-treated conditions at this concentration (Mounce, Cesaro, et al., 2016; Mounce, Poirier, et al., 2016)*. Furthermore, increasing the concentration of these polyamines did not yield significant additional effects on viral titer rescue, as observed in their study. *

The potential influence of pre-treating cells with the biogenic polyamines (putrescine, spermidine, spermine) prior to viral infection, compared to simultaneous addition with the compound in question, is an interesting point. While Mounce et al. (2016) suggest this order may not significantly impact the rescue effect (Mounce, Poirier, et al., 2016)*. Further investigations are warranted to address this question definitively within the context of our specific experimental design. *

Comment 20:-

It is understandable that from the data of Suppl Fig.1, authors became keen on exploring the 'other' antiviral target, but then conclusions from Fig. 1J and Suppl. Fig. 1 are contradictory. As from Fig. 1J, it is being conveyed that the tested compounds depletes polyamines level better than the control. On the other hand, in suppl fig.1, when these polyamines are supplemented, the viral titer is not rescued. Of course this might be related to the time of addition of polyamines and compounds. Authors should consider discussing these results in details.

• Response:-Thank you for your insightful suggestion. We have addressed these results in detail in the discussion section of the manuscript. We conducted an Exogenous Polyamine Addition Assay following the methodology outlined by Mounce et al. (2016) to adhere to established procedures and align with our research objectives. Treatment with DFMO in the presence of exogenous polyamines, as well as treatment with DFMO followed by polyamine addition, led to the rescue of virus titers, as indicated by Mounce et al. (2016). Therefore, according to the data, the timing of exogenous polyamine addition may not be a significant factor. In our manuscript, the timing of polyamine and compound addition was consistent across all treatments (HC, CAPE, and DFMO).

  Comment 21:-

Result 2. Suppl fig. 2. MSA. Provide complete information in the figure legend: indicate virus names to the corresponding Accession numbers and GenBank ID.

• Response:-Thank you for bringing this to our attention. We have updated the figure legend in Supplementary Figure 2 to include complete information, indicating the virus names corresponding to the Accession numbers and GenBank IDs.

  Comment 22:-

Line 392: '2 dimensions' ?

• Response:-Thank you for bringing this to our attention. As suggested, we have made the change, replacing "2 dimensions" with "2D" for clarity.

  Comment 23:-

Result 3. Authors didn't comment/discuss on the significance of these tests with GTP, SAM and difference in the Kd values: for CHIKV and DENV and other details

• Response:- We appreciate the reviewer's feedback. We have expanded upon these results in more detail in the discussion section. Discussion p.4 line no. 512 "Biophysical interactions by TFS indicate distinct red shift for nsP1 and NS5 MTase, with each compound displaying specific affinities toward the target proteins." revised to line no. 551 to 557 "The binding affinities of SAM and GTP with CHIKV nsP1 and DENV NS5 MTase were investigated and used as a reference to compare with HC and CAPE. HC has a high binding affinity for both enzymes, as evidenced by the Kd values. Conversely, CAPE demonstrates a more selective binding profile, exhibiting a significantly stronger affinity towards nsP1 than NS5 MTase. Significantly, both HC and CAPE have demonstrated a dose-dependent red shift, indicating structural changes upon interaction (Figure 5 and Supplimentary figure 5)."
• *

Comment 25 Result 4. Fig. 6A and 6E: The text does not report this result (SDS-PAGE). Fig. 6

• Response We appreciate the reviewer for bringing this to our attention. As per suggestion, we have incorporated the SDS-PAGE results in Fig. 6 in the text.line no. 467 to 468 "Single band at ~ 56 and ~ 32 kDa was observed in 12% SDS-PAGE for purified nsP1 and NS5 MTase, respectively ( Figure 6A and 6E)."

  Comment 24:-

Did authors also perform the enzymatic assays (inhibition assays) with DFMO?

• Response:- Thank you for your intriguing question. We appreciate the reviewer's interest. We opted not to conduct enzymatic assays (inhibition assays) with DFMO, as it is a known analog of ornithine, a well-established inhibitor of the polyamine pathway (ornithine decarboxylase inhibitor). This decision was made as it was deemed outside the scope of our study.

  Comment 25:-

Typographic errors: ml to mL, µl to µL, E. coli to E. coli (line 956), in multiple figures: chose titre or titer

• Response:- We thank the reviewer for their meticulous attention to detail. As per your observation, we have carefully reviewed the manuscript and made the necessary corrections, including changing "ml" to "mL", "µl" to "µL", and "E. coli" to " coli" (line no.. 1042). Additionally, we have standardized the usage of "titre" to "titer" across multiple figures. __References: __

Aljabr, W., Touzelet, O., Pollakis, G., Wu, W., Munday, D. C., Hughes, M., Hertz-Fowler, C., Kenny, J., Fearns, R., Barr, J. N., Matthews, D. A., & Hiscox, J. A. (2016). Investigating the Influence of Ribavirin on Human Respiratory Syncytial Virus RNA Synthesis by Using a High-Resolution Transcriptome Sequencing Approach. Journal of Virology, 90(10), 4876. https://doi.org/10.1128/JVI.02349-15

Arisan, E. D., Obakan, P., Coker, A., & Palavan-Unsal, N. (2012). Inhibition of ornithine decarboxylase alters the roscovitine-induced mitochondrial-mediated apoptosis in MCF-7 breast cancer cells. Molecular Medicine Reports, 5(5), 1323–1329. https://doi.org/10.3892/MMR.2012.786

Bhatt S, Gething PW, Brady OJ, Messina JP, Farlow AW, Moyes CL, Drake JM, Brownstein JS, Hoen AG, Sankoh O, Myers MF. The global distribution and burden of dengue. Nature. 2013 Apr 25;496(7446):504-7.

Bouteille, B., & Dumas, M. (2003). Human African Trypanosomiasis. Encyclopedia of the Neurological Sciences, 587–594. https://doi.org/10.1016/B0-12-226870-9/00431-7

DENGUE/DHF SITUATION IN INDIA :: National Center for Vector Borne Diseases Control (NCVBDC). [cited 2022 Jan 15].

https://nvbdcp.gov.in/index4.php?lang=1&level=0&linkid=431&lid=3715

Kalita, J. M., Aggarwal, A., Yedale, K., Gadepalli, R., & Nag, V. L. (2021). A 5-year study of dengue seropositivity among suspected cases attending a teaching hospital of North-Western region of India. Journal of Medical Virology, 93(6), 3338–3343. https://doi.org/10.1002/JMV.26592

Lani, R., Hassandarvish, P., Shu, M. H., Phoon, W. H., Chu, J. J. H., Higgs, S., Vanlandingham, D., Abu Bakar, S., Zandi, K., R, L., P, H., MH, S., WH, P., JJ, C., S, H., D, V., S, A. B., & K, Z. (2016). Antiviral activity of selected flavonoids against Chikungunya virus. Antiviral Research, 133, 50–61. https://doi.org/10.1016/J.ANTIVIRAL.2016.07.009

Lobo DA, Velayudhan R, Chatterjee P, Kohli H, Hotez PJ. The neglected tropical diseases of India and South Asia: review of their prevalence, distribution, and control or elimination. PLoS neglected tropical diseases. 2011 Oct 25;5(10):e1222.

Logiudice, N., Le, L., Abuan, I., Leizorek, Y., & Roberts, S. C. (2018). medical sciences Alpha-Difluoromethylornithine, an Irreversible Inhibitor of Polyamine Biosynthesis, as a Therapeutic Strategy against Hyperproliferative and Infectious Diseases. https://doi.org/10.3390/medsci6010012

Mounce, B. C., Cesaro, T., Moratorio, G., Hooikaas, P. J., Yakovleva, A., Werneke, S. W., Smith, E. C., Poirier, E. Z., Simon-Loriere, E., Prot, M., Tamietti, C., Vitry, S., Volle, R., Khou, C., Frenkiel, M.-P., Sakuntabhai, A., Delpeyroux, F., Pardigon, N., Flamand, M., … Vignuzzi, M. (2016). Inhibition of Polyamine Biosynthesis Is a Broad-Spectrum Strategy against RNA Viruses. Journal of Virology, 90(21), 9683–9692. https://doi.org/10.1128/JVI.01347-16

Mounce, B. C., Poirier, E. Z., Passoni, G., Simon-Loriere, E., Cesaro, T., Prot, M., Stapleford, K. A., Moratorio, G., Sakuntabhai, A., Levraud, J. P., & Vignuzzi, M. (2016). Interferon-Induced Spermidine-Spermine Acetyltransferase and Polyamine Depletion Restrict Zika and Chikungunya Viruses. Cell Host & Microbe, 20(2), 167–177. https://doi.org/10.1016/J.CHOM.2016.06.011

Mudgal, R., Bharadwaj, C., Dubey, A., Choudhary, S., Nagarajan, P., Aggarwal, M., Ratra, Y., Basak, S., & Tomar, S. (2022). Selective Estrogen Receptor Modulators Limit Alphavirus Infection by Targeting the Viral Capping Enzyme nsP1. Antimicrobial Agents and Chemotherapy, 66(3). https://doi.org/10.1128/AAC.01943-21/ASSET/CFFD4322-C11C-41A1-9D51-7D0C3242FA63/ASSETS/IMAGES/LARGE/AAC.01943-21-F006.JPG

Nazir, A., Nazir, A., & Kandel, K. (2024). Advancing neuroblastoma care: Future horizons after approval of eflornithine by FDA. International Journal of Surgery (London, England). https://doi.org/10.1097/JS9.0000000000001182

Pegg, A. E., & Casero, R. A. (n.d.). Current Status of the Polyamine Research Field. https://doi.org/10.1007/978-1-61779-034-8_1

Rao, J. N., Rathor, N., Zhuang, R., Zou, T., Liu, L., Xiao, L., Turner, D. J., & Wang, J. Y. (2012). Polyamines regulate intestinal epithelial restitution through TRPC1-mediated Ca2+ signaling by differentially modulating STIM1 and STIM2. American Journal of Physiology - Cell Physiology, 303(3), C308. https://doi.org/10.1152/AJPCELL.00120.2012

Shen, H., Yamashita, A., Nakakoshi, M., Yokoe, H., & Sudo, M. (2013). Inhibitory Effects of Caffeic Acid Phenethyl Ester Derivatives on Replication of Hepatitis C Virus. PLoS ONE, 8(12), 82299. https://doi.org/10.1371/journal.pone.0082299

Tate, P. M., Mastrodomenico, V., & Mounce, B. C. (2019). Ribavirin Induces Polyamine Depletion via Nucleotide Depletion to Limit Virus Replication. Cell Reports, 28(10), 2620-2633.e4. https://doi.org/10.1016/J.CELREP.2019.07.099

Terui, Y., Yoshida, T., Sakamoto, A., Saito, D., Oshima, T., Kawazoe, M., Yokoyama, S., Igarashi, K., & Kashiwagi, K. (2018). Polyamines protect nucleic acids against depurination. The International Journal of Biochemistry & Cell Biology, 99, 147–153. https://doi.org/10.1016/J.BIOCEL.2018.04.008

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Referee #3

Evidence, reproducibility and clarity

The manuscript submitted by Bhutkar M. et al. details the antiviral properties of two compounds, herbacetin (HC) and caffeic acid phenethyl ester (CAPE), against Chikungunya virus (CHIKV) and Dengue virus (DENV) through cellular, bioinformatics, biochemical, biophysical, and structural studies. The authors propose a dual antiviral mechanism of action exhibited by these compounds, beginning with an evaluation of their cytotoxicity. Subsequent assessments of their antiviral efficacy against CHIKV and DENV are addressed using plaque reduction assay and other orthogonal assays such as qRT-PCR, and Immunofluorescence assay (IFA). Further, authors performed thin layer chromatography (TLC) to monitor polyamine levels in the cells treated with these compounds and concluded that these compounds leads to polyamine depletion which is also supported by previous studies. These experiments included DFMO as a control which is well established for its role in this regulation. Beyond their impact on cellular polyamine levels, the authors propose a role for these compounds in the inhibition of MTase domains in CHIKV and DENV, supported by the crystal structure of the DENV-3 NS5 MTase domain in complex with CAPE.

Major points

While the manuscript presents promising findings regarding the dual antiviral effects of the tested compounds, the authors fall short of demonstrating direct inhibition of MTase activity as a meaningful and complementary effect to polyamine depletion. Being only indirect, the enzyme inhibition data is not convincing, and the measured indirect inhibition are not precise enough in the case of CHIK nsp1, and too weak in the case of DENV NS5 (detailed below).

Conceptually, the organization of the results should be changed to first data (structural data of DENV MTase in complex with CAPE, which is a significant achievement), then interpretation/discussion with modeling, and not the other way around.

The discussion section requires more elaborate scientific justification rather than simply re-reporting the results.

Specific major remarks:

It would be best to organize the ms as follows: - Crystal structure of DENV MTase in complex with CAPE - Building of a model of nsp1 by superimposition with NS5 MTase - Modeling compound binding - Inhibition assays using enzyme assays at least in the case of NS5 MTase. The direct enzyme assays are well described in the literature. - If there is no inhibition, then discussion about possible reasons would be interesting and help the AV field. For example, CAPE could bind to other enzyme or sites, etc...

Figure 5 is problematic. - When presenting an y IC50 data, care should be taken that the IC50 inflexion point is preceded and followed by at least two experimental points, which is not the case. The IC50 value of 7.082 and 5.156 µM are too imprecise (and there is no need to give digits after the value). Please add more low concentration experimental points. - Please describe more panel D in the legend. - Panel F and G: A reduction of 25 % at the highest concentration of inhibitor is a strong indication that there is no effect.

Minor Points/Comments/ Suggestions:

A. In the Introduction section, line 58: Are DENV infection numbers representative of worldwide distribution, please clarify. Also, in the case of CHIKV infection, the most affected countries are mentioned, why not follow the same pattern for DENV, please consider homogenizing the text.

B. Before p. 4 (line 91), alphaviruses were not introduced. Please consider introducing them.

C. Consider introducing Dengue serotypes to help readers understand the significance of DENV-2 and DENV-3. Additionally, ensure uniformity by referring to these serotypes as DENV-2, DENV-3 throughout. There are inconsistencies in the current text, such as 'DENV 3' in lines 39 and 152, and 'DENV3' in lines 249 and 250, among others.

D. P. 4, 5 lines 91-134: Consider rephrasing/reorganizing the methylation process: conventional and unconventional. The current introduction doesn't clearly indicates the difference between the cap-0 capping in alphaviruses and cap-1 in flaviviruses.

E. Please consider citing the article instead of the referred link, wherever possible, for e.g., for ref. 22 PMID: 28218572 (a more recent reference for Flaviviridae taxonomy available than that mentioned in the current manuscript.)

F. Homogenize the writing of taxonomic names (viral families) in the text. For example, in line 126 change Flaviviridae to Flaviviridae, and line 476 (Discussion section), alphaviridae to Alphaviridae, flaviviridae to Flaviviridae and so on. For further clarification on addressing this, one can also refer to https://ictv.global/faq/names.

G. Please make sure to appropriately reference the corresponding supplementary information (text or figures) in the main text wherever necessary to avoid the impression of missing information. For instance, in none of the sub-sections of Materials and Methods (M&M), it is being indicated to refer to the suppl. experimental procedures for more details. Also consider not repeating the same information between the main experimental procedures text and the supplementary text.

H. M&M sub-section. 2, line 163: Which specific culture media is being referred to here? Could you provide additional details? On line 164, it mentions that polyamines were diluted in water. Is this water sterile tissue culture-grade water as indicated in line 161?

I. M&M, line 274: What is CE? Please expand the term before using the abbreviation.

J. line 306. Ref. 53: This is not a reference.

K. Results. 1: Didn't understand the relevance of Fig. 1C, as this data is already included in Fig. 1B. Fig. 1G and H are not referred to in the result text. Lines 342, 343: 'At the mentioned concentrations', where are these concentrations mentioned? qRT-PCR data is not very clear. Please consider elaborating on some details. Why the statistics were only performed between HC and DFMO and not with CAPE? How the fold reduction is being calculated? For example, the fold difference of 97 is not visibly evident. Line 375: 'SAM is lined by residues ... would be more appropriate than 'formed' Fig. 1J. For TLC results, consider using the term panel (left, center, right) to navigate within this figure. The representation of this result is not uniform, as the time course is shown for HC while it is not shown for DFMO and CAPE. The treatment time is not indicated for DFMO and CAPE. For better representation and significant differences, one can consider quantifying these TLC results. Fig. 1, figure legend, lines 750-751: instead of 'Panels D-G depicts the inhibitory effect of CHIKV and DENV infected cells on different concentrations of HC and CAPE' should be 'Panels D-G depicts the inhibitory effect of different concentrations of HC and CAPE on CHIKV and DENV infected cells'. Line 755: DFMO is wrongly written as 'DEMO' Fig.2. IFA. Authors must consider on elaborating the IFA data. One can also consider quantifying these data for better comparison with other assays.

Result 1 (Suppl. Fig. 1). Line 359: 'After infection': please indicate the time here. Suppl. Fig.1: How was the concentration of these polyamines chosen to be 1µM? What will be the effect on increasing concentrations? Why were all these three polyamines added together? What is the effect of addition of individual polyamine in the rescue of viral titer? Will this effect vary if cells are pre-treated with these polyamines and compounds in question are added post viral infection or if both are added at the same time? It is understandable that from the data of Suppl Fig.1, authors became keen on exploring the 'other' antiviral target, but then conclusions from Fig. 1J and Suppl. Fig. 1 are contradictory. As from Fig. 1J, it is being conveyed that the tested compounds depletes polyamines level better than the control. On the other hand, in suppl fig.1, when these polyamines are supplemented, the viral titer is not rescued. Of course this might be related to the time of addition of polyamines and compounds. Authors should consider discussing these results in details.

Result 2. Suppl fig. 2. MSA. Provide complete information in the figure legend: indicate virus names to the corresponding Accession numbers and GenBank ID. Line 392: '2 dimensions' ?

Result 3. Authors didn't comment/discuss on the significance of these tests with GTP, SAM and difference in the Kd values: for CHIKV and DENV and other details

Result 4. Fig. 6A and 6E: This result (SDS-PAGE) is not reported in the text. Fig. 6

Did authors also perform the enzymatic assays (inhibition assays) with DFMO?

Typographic errors: ml to mL, µl to µL, E. coli to E. coli (line 956), in multiple figures: chose titre or titer

Significance

This a body of work that is very interesting and has good potential, however it lacks the correct demonstration of the additive effect of MTase inhibition to polyamine depletion.

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Referee #2

Evidence, reproducibility and clarity

Authors describe anti-CHIKV and anti-DENV activities of herbacetin and caffeic acid phenyl ester (CAPE). The antiviral effect is not reversed buy exogenous polyamines suggesting multiple mechanisms of action. NS5-Met complex with caffeic acid phenyl ester was obtained and its structure resolved at high resolution. The resolved structure reveals two binding sites for antiviral compound overlapping with that of GTP and possibly with site involved in binding of RNA

Other than analysis of crystal structure of NS5/CAPE complex the provided data is of low quality and is not analyzed properly. There is no evidence that data is reproducible. Authors have calculated significance from "experimental repeats" which, based on the description of experiments, are not independent experiments but technical replicates. Some key technical details are missing and some experiments are not described at all. The writing can be vastly improved and figures be made a lot more easier to understand.

There are several points that need to be addressed, so here I provide some examples:

1. Bad writing lines 64-65 . Viral genomes lack protein synthesis machinery. Basically correct but no genome has protein synthesis machinery line 137 flavonoids play role in reducing of the levels of nsP1 in CHIKV - what this can possibly mean? Are shown to reduce level of nsP1 in CHIKV infected cells? line 250-251 - RNA was isolated from the infected cells' supernatant, used for cloning, an inserted between the NheI and XhoI restriction sites... It should be impossible as one cannot insert RNA into bacterial plasmid DNA
2. Missing parts. Examples
   • the source of nsP1 of CHIKV is not indicated, True, there are references to previous studies but this is extremely important point and it should have been clearly stated that it was obtained from E. coli. The issue is that authors made some predictions and modelling based on structure of nsP1 from eukaryotic expression system. It is not known does the enzyme purified from bacteria have similar structure (actually, in cited Nature paper - doi: 10.1038/s41586-020-3036-8 - attempts to purify nsP1 from bacteria were made. The protein was monomeric and had no activity)
   • description of experiment shown on Figure 4 is missing
3. Figure lacks quality (and figure legends are unclear) Examples:
   • it is impossible to understand what exactly is shown on Figure 1J
   • important information is missing, for example it is not clear what were concentrations of antiviral compounds for panels 1F and 1I
4. wrong data
   • line 478 it is stated that there is no vaccine for DENV or CHIKV. It is correct, DENV vaccine has been in use for several years and CHIKV vaccine was approved at 2023
   • line 476 refers to family alphaviridae. This does not exist, family is Togaviridae
5. unjustified conclusions. Example
   • authors have analyzed sequences of nsP1 of alphaviruses and made conclusions regarding conservation of active site. It is probably correct but the analyzed viruses do not represent all diversity of alphaviruses, insect specific members and aquatic alphaviruses should also be analyzed (same problem with analysis performed for flaviviruses)
6. Insufficient analysis of data. Some cases there is significant discrepancy between results of different assays. For example, CAPE inhibits DENV at 2.5 microM (Fig 1H) but in test tube assay only small inhibition was observed even at 1000 microM. Authors should provide plausible explanation for this and similar discrepancies (CE and ELISA based assays shown on figure 6 also resulted in drastically different inhibitions). It is expected assays would produce different results but there should also be explanation for this. If this is not provided on can assume that it is due to experimental errors.
7. Discussion is essentially missing, it is just list of statements mostly repeating what was said in other sections

The reviewer is sorry for not being able to provide more specific and useful comments and suggestions. To my opinion, manuscript should have been better prepared before submitting for review. Multiple mistakes, discrepancies and lack of clarity makes it difficult (for me nearly impossible) to focus on scientific value of study and provide constructive comments

Significance

It is difficult to assess the significance of the studys findings as the data presented and writing lacks sufficient quality and depth. While some experiments that can be understood (crystal structure, some antiviral assays) show potentially interesting scientific findings, the manuscript needs a major overhaul before it can be considered relevant for the scientific community.

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

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Referee #1

Evidence, reproducibility and clarity

In this manuscript, the authors use structural and functional approaches to investigate the potential anti-DENV and anti-CHIKV activity of HC and CAPE, two naturally occurring compounds. They find that these compounds reduce cellular polyamine levels and specifically inhibit the viral methyltransferase (MTase) activity. Hence, the authors propose that HC and CAPE have anti-viral potential against DENV and CHIKV, which have been implicated in severe disease in humans.

Overall, this is a straightforward investigation and is quite suitable for publication as a "first report" on the anti-MTase activity of these compounds. The data support the conclusions. This will be of interest to researchers in the anti-virals field. A strength of this investigation is the multi-faceted approach to get to the target of these compounds, i.e., the viral MTase enzymes. This is commendable.

My main concern is that the depletion of polyamines is likely to have broad implications for host cell metabolism. Polyamines are critical for genome folding and stability. Hence, polyamine depletion will likely compromise cellular metabolic homeostasis. My suggestion is to perform a literature survey on this topic, identify appropriate assays of cellular homeostasis, and add at least one such assay in the relevant HC and CAPE concentration range to address my question.

I also suggest adding the potential negative effects of polyamine depletion on host cell metabolism in the discussion section.

Significance

Strengths- multi-faceted approach

Target audience- researchers interested in anti-virals

Author Response

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

eLife assessment

This valuable study advances our understanding of the brain nuclei involved in rapid-eye movement (REM) sleep regulation. Using a combination of imaging, electrophysiology, and optogenetic tools, the study provides convincing evidence that inhibitory neurons in the preoptic area of the hypothalamus influence REM sleep. This work will be of interest to neurobiologists working on sleep and/or brain circuitry.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

This paper identifies GABA cells in the preoptic hypothalamus which are involved in REM sleep rebound (the increase in REM sleep) after selective REM sleep deprivation. By calcium photometry, these cells are most active during REM, and show more claim signals during REM deprivation, suggesting they respond to "REM pressure". Inhibiting these cells ontogenetically diminishes REM sleep. The optogenetic and photometry work is carried out to a high standard, the paper is well-written, and the findings are interesting.

We thank the reviewer for the detailed feedback and thoughtful comments on how to improve our manuscript. To address the reviewer’s concerns, we revised our discussion and added new data. Below, we address the concerns point by point.

Points that could be addressed or discussed:

(1) The circuit mechanism for REM rebound is not defined. How do the authors see REM rebound as working from the POAGAD2 cells? Although the POAGAD2 does project to the TMN, the actual REM rebound could be mediated by a projection of these cells elsewhere. This could be discussed.

We demonstrate thatPOA GAD2→TMN cells become more frequently activated as the pressure for REMs builds up, whereas inhibiting these neurons during high REMs pressure leads to a suppression of the REMs rebound. It is not known how POA GAD2→TMN cells encodeincreased REMs pressure and subsequently influence the REMs rebound. REMsdeprivation wasshown to changethe intrinsic excitabilityof hippocampal neurons and impact synaptic plasticity (McDermott et al., 2003; Mallick and Singh, 2011 ; Zhou et al., 2020) . We speculate that increasedREMs pressure leads to an increase in the excitabilityof POA->TMN neurons, reflected inthe increased number ofcalcium peaks. The increased excitability of POA GAD2→TMN neurons in turn likely leads to stronger inhibition of downstream REM-off neurons. Consequently, as soon as REMsdeprivation stops, there is an increased chance for enteringREMs. The time coursefor how long it takes till the POA excitability resettles toits baseline consequently sets a permissive time window for increasedamounts of REMs to recover its lostamount. For future studies, it would be interesting to map how quickly the excitability ofPOA neurons increases or decays as afunction of the lost or recovered amount of REMs andunravel the cellularmechanisms underlying the elevated activity of POAGAD2 →TMN neurons during highREMs pressure, e.g., whether changes in the expression of ion channels contribute to increasedexcitability of these neurons (Donlea et al., 2014) . As we mentioned in the Discussion, the POAalso projects to other REMs regulatorybrain regions such as the vlPAG and LH. Therefore, it remains to be tested whether POA GAD2 →TMN neurons also innervate these brain regions to potentially regulate REMs homeostasis. We explicitly state this now in the revised Discussion.

(2) The "POAGAD2 to TMN" name for these cells is somewhat confusing. The authors chose this name because they approach the POAGAD2 cells via retrograde AAV labelling (rAAV injected into the TMN). However, the name also seems to imply that neurons (perhaps histamine neurons) in the TMN are involved in the REM rebound, but there is no evidence in the paper that this is the case. Although it is nice to see from the photometry studies that the histamine cells are selectively more active (as expected) in NREM sleep (Fig. S2), I could not logically see how this was a relevant finding to REM rebound or the subject of the paper. There are many other types of cells in the TMN area, not just histamine cells, so are the authors suggesting that these non-histamine cells in the TMN could be involved?

We acknowledge that other types of neurons in the TMN may also be involved in the REMs rebound, and therefore inhibition of histamine neurons by POA GAD2 →TMN neurons may not be the sole source of the observed effect. To stress that other neurons within the TMN and/or brain regions may also contribute to the REMs rebound, we have revised the Results section.

We performed complementary optogenetic inhibition experiments of TMN HIS neurons to investigate if suppression of these neurons is sufficient to promote REMs. We foundthat SwiChR++ mediated inhibition of TMNHIS neurons increased theamount of REMs compared withrecordings without laser stimulation in the same mice and eYFPmice withlaser stimulation. Thus, while TMN HIS neurons may not bethe only downstream target of GABAergic POA neurons, these data suggest that they contribute to REMs regulation. We have incorporated these results in Fig. S4 .

We further investigated whether the activity of TMN HIS neurons changes between two REMs episodes. Assumingthat REMs pressure inhibits the activity ofREM-off histamine neurons,their firing rates should behighest right after REMs ends when REMs pressure is lowest, and progressivelydecay throughout the inter-REM interval, and reach their lowest activity right before the onset of REMs ( Park et al., 2021) , similarto the activity profile observed for vlPAG REM-off neurons (Weber et al., 2018).We indeed found that TMNHIS neurons displaya gradual decrease in their activity throughout theinter-REM interval and thus potentially reflect the build up of REM pressure ( Fig. S2F ).

(3) It is a puzzle why most of the neurons in the POA seem to have their highest activity in REM, as also found by Miracca et al 2022, yet presumably some of these cells are going to be involved in NREM sleep as well. Could the same POAGAD2-TMN cells identified by the authors also be involved in inducing NREM sleep-inhibiting histamine neurons (Chung et al). And some of these POA cells will also be involved in NREM sleep homeostasis (e.g. Ma et al Curr Biol)? Is NREM sleep rebound necessary before getting REM sleep rebound? Indeed, can these two things (NREM and REM sleep rebound) be separated?

Previous studies have demonstrated that POA GABAergic neurons, including those projecting to the TMN, are involved in NREMs homeostasis (Sherin et al., 1998; Gong et al., 2004; Ma et al., 2019) . Therefore, we predict that POA neurons that are involved in NREMs homeostasis are a subset of POA GAD2 → TMN neurons in our manuscript.

Using optrode recordings in the POA, we recently reported that 12.4% of neurons sampled have higher activity during NREMs compared with REMs; in contrast, 43.8% of neurons sampled have the highest activity during REMs compared with NREMs (Antila et al., 2022) indicating that the proportion of NREM max neurons is smaller compared with REM max neurons. These proportions of neurons are in agreement with previous results (Takahashi et al., 2009) . Considering fiber photometry monitors the average activity of a population of neurons as opposed to individual neurons, it is possible that we recorded neural activity across heterogeneous populations and therefore our findings may disguise the neural activity of the low proportion of NREMs neurons. We previously reported thespiking activity of POA GAD2 →TMN neurons at the singlecell level (Chung et al., 2017) . We have noted in themanuscript thatwhile the activity ofPOA GAD2→TMN neurons is highestduring REMs, theneural activity increases at NREMs → REMs transitions indicating these neurons also areactive during NREMs.

Using our REMs restriction protocol, we selectively restricted REMs leading to the subsequent rebound of REMs without affecting NREMs and consequently we did not find an increase in the amount of NREMs during the rebound or an increase in slow-wave activity, a key characteristic of sleep rebound that gradually dissipates during recovery sleep (Blake and Gerard, 1937; Williams et al., 1964; Rosa and Bonnet, 1985; Dijk et al., 1990; Neckelmann and Ursin, 1993; Ferrara et al., 1999) . However, during total sleep deprivation when subjects are deprived of both NREMs and REMs, isolating NREMs and REMs rebound may not be attainable.

(4) Is it possible to narrow down the POA area where the GAD2 cells are located more precisely?

POA can be subdivided into anatomically distinct regions such as medial preoptic area, median preoptic area, ventrolateral preoptic area, and lateral preoptic area (MPO, MPN, VLPO, and LPO respectively). To quantify where the virus expressing GAD2 cells and optic fibers are located within the POA, we overlaid the POA coronal reference images (with red boundaries denoting these anatomically distinct regions) over the virus heat maps and optic fiber tracts from datasets used in Figure 1A. We found that virus expression and optic fiber tracts were located in the ventrolateral POA, lateral POA, and the lateral part of medial POA, and included this description in the text.

Author response image 1.

Location of virus expression (A) and optic fiber placement (B) within subregions of POA.

(5) It would be ideal to further characterize these particular GAD2 cells by RT-PCR or RNA seq. Which other markers do they express?

Single-cell RNA-sequencing of POA neurons has revealed an enormous level of molecular diversity, consisting of nearly 70 subpopulations based on gene expression of which 43 can be clustered into inhibitory neurons (Moffitt et al., 2018) . One of the most studied subpopulation of POA sleep-active neurons contains the inhibitory neuropeptide galanin (Sherin et al., 1998; Gaus et al., 2002; Chung et al., 2017; Kroeger et al., 2018; Ma et al., 2019; Miracca et al., 2022) . Galanin neurons have been demonstrated to innervate the TMN (Sherin et al., 1998) yet, within the galanin neurons 7 distinct clusters exist based on unique gene expression (Moffitt et al., 2018) . In addition to galanin, we have previously performed single-cell RNA-seq on POA GAD2 → TMN neurons and identified additional neuropeptides such as cholecystokinin (CCK), corticotropin-releasing hormone (CRH), prodynorphin (PDYN), and tachykinin 1 (TAC1) as subpopulations of GABAergic POA sleep-active neurons (Chung et al., 2017; Smith et al., 2023) . Like galanin, these neuropeptides can also be divided into multiple subtypes as well (Chen et al., 2017; Moffitt et al., 2018) . Thus while these molecular markers for POA neurons are immensely diverse, we agree that characterizing the molecular identity of POA GAD2 → TMN neurons and investigating the functional relevance of these neuropeptides in the context of REMs homeostasis would enrich our understanding of a neural circuit involved in REMs homeostasis and can stand as a separate extension of this manuscript.

Reviewer #2 (Public Review):

Maurer et al investigated the contribution of GAD2+ neurons in the preoptic area (POA), projecting to the tuberomammillary nucleus (TMN), to REM sleep regulation. They applied an elegant design to monitor and manipulate the activity of this specific group of neurons: a GAD2-Cre mouse, injected with retrograde AAV constructs in the TMN, thereby presumably only targeting GAD2+ cells projecting to the TMN. Using this set-up in combination with technically challenging techniques including EEG with photometry and REM sleep deprivation, the authors found that this cell-type studied becomes active shortly (≈40sec) prior to entering REM sleep and remains active during REM sleep. Moreover, optogenetic inhibition of GAD2+ cells inhibits REM sleep by a third and also impairs the rebound in REM sleep in the following hour. Despite a few reservations or details that would benefit from further clarification (outlined below), the data makes a convincing case for the role of GAD2+ neurons in the POA projecting to the TMN in REM sleep regulation.

We thank the reviewer for the thorough assessment of our study and supportive comments. We have addressed your concerns in the revised manuscript, and our point by point response is provided below.

The authors found that optogenetic inhibition of GAD2+ cells suppressed REM sleep in the hour following the inhibition (e.g. Fig2 and Fig4). If the authors have the data available, it would be important to include the subsequent hours in the rebound time (e.g. from ZT8.5 to ZT24) to test whether REM sleep rebound remains impaired, or recovers, albeit with a delay.

We thank the reviewer for this comment and agree that it would be interesting to know how REMs changes for a longer period of time throughout the rebound phase. For Fig. 2, we did not record the subsequent hours. For Fig 4, we recorded the subsequent rebound between ZT7.5 and 10.5. When we compare the REMs amount during this 4 hr interval, the SwiChR mice have less REMs compared with eYFP mice with marginal significance (unpaired t-test, p=0.0641). We also plotted the cumulative REMs amount during restriction and rebound phases, and found that the cumulative amount of REMs was still lower in SwiChR mice than eYFP mice at ZT 10.5 (Author response image 2). Therefore, it will be interesting to record for a longer period of time to test when the SwiChR mice compensate for all the REMs that was lost during the restriction period.

Author response image 2.

Cumulative amount of REMs during REMs deprivation and rebound combined with optogenetic stimulation in eYFP and SwiChR groups. This data is shown as bar graphs in Figure 4.

REM sleep is under tight circadian control (e.g. Wurts et al., 2000 in rats; Dijk, Czeisler 1995 in humans). To contextualize the results, it would be important to mention that it is not clear if the role of the manipulated neurons in REM sleep regulation hold at other circadian times of the day.

Author response image 3.

Inhibiting POA GAD2→ TMN neurons at ZT5-8 reduces REMs. (A) Schematic of optogenetic inhibition experiments. (B) Percentage of time spent in REMs, NREMs and wakefulness with laser in SwiChR++ and eYFP mice. Unpaired t-tests, p = 0.0013, 0.0469 for REMs and wakeamount. (C) Duration of REMs, NREMs, and wake episodes. Unpaired t-tests, p = 0.0113 for NREMs duration. (D) Frequency of REMs, NREMs, and wake episodes. Unpaired t-tests, p = 0.0063, 0.0382 for REMs and NREMs frequency.

REMs propensity is largest towards the end of the light phase (Czeisler et al., 1980; Dijk and Czeisler, 1995; Wurts and Edgar, 2000). As a control, we therefore performed the optogenetic inhibition experiments of POA GAD2→TMN neurons during ZT5-8 (Author response image 3). Similar to our results in Figure 2, we found that SwiChR-mediated inhibition of POA GAD2 →TMN neurons attenuated REMs compared with eYFP laser sessions. These findings suggest our results are consistentat other circadian times of the day.

The effect size of the REM sleep deprivation using the vibrating motor method is unclear. In FigS4-D, the experimental mice reduce their REM sleep to 3% whereas the control mice spend 6% in REM sleep. In Fig4, mice are either subjected to REM sleep deprivation with the vibrating motor (controls), or REM sleep deprivations + optogenetics (experimental mice).

The control mice (vibrating motor) in Fig4 spend 6% of their time in REM sleep, which is double the amount of REM sleep compared to the mice receiving the same treatment in FigS4-D. Can the authors clarify the origin of this difference in the text?

The effect size for REM sleep deprivation is now added in the text.

It is important to note that these figures are analyzing two different intervals of the REMs restriction. In Fig. S4D, we analyzed the total amount of REMs over the entire 6 hr restriction interval (ZT1.5-7.5). In Fig. 4, we analyzed the amount of REMs only during the last 3 hr of restriction (ZT4.5-7.5) as optogenetic inhibition was performed only during the last 3 hrs when the REMs pressure is high. In Fig. S4D, we looked at the amount of REMs during ZT1.5-4.5 and 4.5-7.5 and found that the amount of REMs during ZT4.5-7.5 (4.46 ± 0.25 %; mean ± s.e.m.) is indeed higher than ZT 1.5-4.5 (1.66 ± 0.62 %), and is comparable to the amount of REMs during ZT4.5-7.5 in eYFP mice (5.95 ± 0.52 %) in Fig. 4. We now clearly state in the manuscript at which time points we analyzed the amount, duration and frequency of REMs.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

(1) A few further citations suggested: Discussion "The TMN contains histamine producing neurons and antagonizing histamine neurons causes sleepiness..." It would be appropriate to cite Uygun DS et al 2016 J Neurosci (PMID: 27807161) here. Using the same HDC-Cre mice as used by Maurer et al., Uygun et al found that selectively increasing GABAergic inhibition onto histamine neurons produced NREM sleep.

We apologize for omitting this important paper. In the revised manuscript, we added this citation.

(2) Materials and Methods.

Although the JAX numbers are given for the mouse lines based on researchers generously donating to JAX for others to use, please cite the papers corresponding to the GAD2-ires-Cre and HDC-ires-Cre mouse lines deposited at JAX.

GAD2-ires-Cre was described in Taniguchi H et al., 2011, Neuron (PMID: 21943598).

The construction of the HDC-ires-CRE line is described in Zecharia AY et al J Neurosci et al 2012 (PMID: 22993424).

We have now added these important citations in the revised manuscript.

(3) Similarly, for the viruses, please provide the citations for the AAV constructs that were donated to Addgene.

We have now added these citations in the revised manuscript.

Reviewer #2 (Recommendations For The Authors):

The authors rely heavily on their conclusions by using an optogenetic tool that inhibits the activity of GAD2+ neurons, however, it is not shown that these neurons are indeed inhibited as expected. An alternative approach to tackle this could be the application of a different technique to achieve the same output (e.g. chemogenetics). However, both experiments (confirmation of inhibition, or using a different technique) would require a significant amount of work, and given the numerous studies out there showing that these optogenetic tools tend to work, may not be necessary. Hence the authors could also cite a similar study that used a likewise construct and where it was indeed shown that this technique works (i.e. similar retrograde optogenetic construct with Cre depedendent expression combined with electrophysiological recordings).

This laser stimulation protocol was designed based on previous reports of sustained inhibition using the same inhibitory opsin and our prior results that recapitulate similar findings as inhibitory chemogenetic techniques (Iyer et al., 2016; Kim et al., 2016; Wiegert et al., 2017; Stucynski et al., 2022). We have now added this description in the Result section.

Fig1A - Right: the virus expression graphs are great and give a helpful insight into the variability. The image on the left (GCAMP+ cells) is less clear, the GCAMP+ cells don't differentiate well from the background. Perhaps the whole brain image with inset in POA can show the GCAMP expression more convincingly.

We have added a histology picture showing the whole brain image with inset in the POA in the updated Fig. 1A .

Statistics: The table is very helpful. Based on the degrees of freedom, it seems that in some instances the stats are run on the recordings rather than on the individual mice (e.g. Fig1). It could be considered to use a mixed model where subjects as taken into account as a factor.

Author response image 4.

ΔF/Factivity of POA GAD2→TMN neurons during NREMs. The duration of NREMs episodes was normalized in time, ranging from 0 to 100%. Shading, ± s.e.m. Pairwise t-tests with Holm-Bonferroni correctionp = 5.34 e-4 between80 and100. Graybar, intervals where ΔF/F activity was significantly different from baseline (0 to 20%, the first time bin). n = 10 mice. In Fig. 1E , we ran stats based on the recordings. In this data set, we ran stats based on the individual mice, and found that the activity also gradually increased throughout NREMs episodes.

There is an effect of laser in Fig2 on REM sleep amount, as well as an interaction effect with virus injection (from the table). Therefore, it would be helpful for the reader to also show REM sleep data from the control group (laser stimulation but no active optogenetics construct) in Fig 2.

To properly control laser and virus effect, we performed the same laser stimulation experiments in eYFP control mice (expressing only eYFP without optogenetic construct, SwiChR++) and the data is provided in Fig 2C .

Fig3B: At the start of the rebound of REM sleep, there is a massive amount of wakefulness, also reflected in the change of spectral composition. Could you comment on the text about what is happening here?

We quantified the amount of wakefulness during the first hour of REMs rebound and found that indeed there is no significant difference in wakefulness between REM restriction and baseline control conditions ( Fig. S4H ). Therefore, while the representative image in Fig 3B shows increased wakefulness at the beginning of REMs rebound, we do not think the overall amount of wakefulness is increased.

Fig 4, supplementary data: it would be helpful for the reader to have mentioned in the text the effect size of the REM sleep restriction protocol (e.g. mean and standard deviation).

Thank you for this suggestion. We have now added the effect size for the REM sleep restriction experiments in the main text.

REM sleep restriction and photometry experiment: could be improved by adding within the main body of text that, in order to conduct the photometry experiment in the last hours of REM sleep deprivation, the manual REM sleep deprivation had to be applied, because the vibrating motor technique disturbed the photometry recordings.

Thank you for this suggestion. We have added the description in the main text.

Suggestion to build further on the already existing data (not for this paper): you have a powerful dataset to test whether REM sleep pressure builds up during wakefulness or NREM sleep, by correlating when your optogenetic treatment occurs (NREM or wakefulness), with the subsequent rebound in REM sleep (see also Endo et al., 1998; Benington and Heller, 1994; Franken 2001).

We thank the reviewer for this excellent suggestion. We plan to carry out this experiment in the future.

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As we can see, one of the primary driving tasks of the OWI group was to identify best practices for online writing instruction assessment. This presentation one one way that we are working through some of these approaches in our classrooms.

The food insecurity usually starts from home, because there are families that care about their children's health and how much they have to consume so they don't over eat more at certain point.

Ok. In my leapfrog study of Chemistry over the last 40+ years, we didn't cover this in high school chemistry. I have often puzzled about orbitals but never asked about the mechanism which caused them. Now I am fresh with questions which brought me to the previous answer to my previous question, "how does a reduction of neutrons make an isotope more unstable?" I could understand more neutrons making the isotope radioactive (but I really don't). I was annotating my new Periodic Table of the Elements with group numbers, trends, and electron shell counts (not orbitals). I noticed that Uranium had an isotope with an atomic mass which was less than 238 and it was radioactive. I didn't understand how that could be. This morning I asked the question and it took me to Stable_and_Unstable_Isotopes in another course. When I looked for it again, I was in Allied Health. When I read further, I saw the reference to Quantum Physics of which my wife and I are interested. (We have an enlarged photo of the 1929 Solvay Conference over our piano in the family room.) Peace.

missing image?

How do we establish norms and expectations to avoid this problem?

What other courses use this content? (Section 21.2, for example)

Ograniczenia przeglądu badań - Geiss, L., Stemmler, M., Beck, B., Hillemacher, T., Widder, M., & Hösl, K. M. (2023). Dysregulation of the autonomic nervous system in adult attention deficit hyperactivity disorder. A systematic review. Cognitive Neuropsychiatry, 28(4), 285–306. https://doi.org/10.1080/13546805.2023.2255336

https://web.archive.org/web/20240409122434/https://www.henrikkarlsson.xyz/p/go

• In the decades before AI beat Go-worldchampion, the highest level of Go-players was stable.
• After AI beat the Go-worldchampion, there is a measurable increase in the creativity and quality of Go-players. The field has risen as a whole.
• The change is not attributable to copying AI output (although 40% of cases that happened) but to increased human creativity (60%).
• The realisation that improvement is possible, creates the improvement. This reminds me of [[Beschouw telkens je adjacent possibles 20200826185412]] wrt [[Evolutionair vlak van mogelijkheden 20200826185412]]
• Also the improvement coincides with the appearance of an open source model and tool, which allowed players to explore and interact with the AI, not just play a Go-game against it.
• Examples of plateau-ing of accomplishments and sudden changes exist in sports
• There may also be a link to how in other fields one might see the low end of an activity up their game through using AI rather than be overtaken by it.

Paper 2022 publication in Zotero

Author response:

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

Public Reviews

Reviewer #1 (Public Review):

Summary:

Dormancy/diapause/hibernation (depending on how the terms are defined) is a key life history strategy that allows the temporal escape from unfavorable conditions. Although environmental conditions do play a major role in inducing and terminating dormancy (authors call this energy limitation hypothesis), the authors test a mutually non-exclusive hypothesis (life-history hypothesis) that sex-specific selection pressures, at least to some extent, would further shape the timing of these life-history events. Authors use a metanalytic approach to collect data (mainly on rodents) on various life-history traits to test trade-offs among these traits between sexes and how they affect entry and termination of dormancy.

Strengths:

I found the theoretical background in the Introduction quite interesting, to the point and the arguments were well-placed. How sex-specific selection pressures would drive entry and termination of diapause in insects (e.g. protandry), especially in temperate butterflies, is very well investigated. Authors attempt to extend these ideas to endotherms and trying to find general patterns across ectotherms and endotherms is particularly exciting. This work and similar evidence could make a great contribution to the life-history theory, specifically understanding factors that drive the regulation of life cycle timing.

Weaknesses:

(1) I felt that including 'ectotherms' in the title is a bit misleading as there is hardly (in fact any?) any data presented on ectotherms. Also, most of the focus of the discussion is heavily mammal (rodent) focussed. I believe saying endotherms in the title as well is a bit misleading as the data is mammalfocused.

We change the title to : "Evolutionnary trade-offs in dormancy phenology". This is a hybrid article comprising both a meta-analysis and a literature review. Each of these parts brings new elements to the hypotheses presented. The statistical analyses only concern mammals and especially rodent species. But the literature review highlighted links between the evolution of dormancy in ectotherms and endotherms that have not been linked in previous studies. We feel it is important for readers to know that much of the discussion will focus on the comparison of these two groups. But we understand that placing the term ectotherms in the title might suggest a meta-analysis including these two groups.

In addition, we indicated more specifically in the abstract and at the end of the introduction that the article includes two approaches associated with different groups of animals.

We also specified in the section « review criteria » that:

Only one bird species is considered to be a hibernator, and no information is available on sex differences in hibernation phenology (Woods and Brigham 2004, Woods et al. 2019).

We have also added a "study limitations" section, which explains that although the meta-analysis is limited by the data available in the literature, the information available for the species groups not studied seems to support our results.

(2) I think more information needs to be provided early on to make readers aware of the diversity of animals included in the study and their geographic distribution. Are they mostly temperate or tropical? What is the span of the latitude as day length can have a major influence on dormancy timings? I think it is important to point out that data is more rodent-centric. Along the line of this point, is there a reason why the extensively studied species like the Red Deer or Soay Sheep and other well-studied temperate mammals did not make it into the list?

We specified in the abstract and at the end of the introduction that the species studied in the metaanalysis are mainly Holarctic species. We have also added a map showing all the study sites used in the meta-analysis. Finally, we've noted in the methods and added a "study limitation" section at the end of the discussion an explanation for those species that were not studied in the meta-analysis and the consequences for the interpretation of results

The hypotheses developed in this article are based on the survival benefits of seasonal dormancy thanks to a period of complete inactivity lasting several months. The Red Deer or Soay Sheep remain active above ground throughout the year.

The effect of photoperiod on phenology is one of the mechanisms that has evolved to match an activity with the favorable condition. In this study, we are not interested in the mechanisms but in the evolutionary pressures that explain the observed phenology. Interspecific variation in the effect of photoperiod results from different evolutionary pressures, which we are trying to highlight. It is therefore not necessary to review mechanisms and effects of photoperiod, themselves requiring a lengthy review.

We also tested the “physiological constraint hypothesis” on several variables. Temperature and precipitation are factors correlated with sex differences in phenology of hibernation. These factors allow consideration of the geographical differences that influence hibernation phenology.

(3) Isn't the term 'energy limitation hypothesis' which is used throughout the manuscript a bit endotherm-centric? Especially if the goal is to draw generalities across ectotherms and endotherms. Moreover, climate (e.g. interaction of photoperiod and temperature in temperatures) most often induces or terminates diapause/dormancy in ectotherms so I am not sure if saying 'energy limitation hypothesis' is general enough.

We renamed this hypothesis the "physiological constraint hypothesis" and we have made appropriate changes in the text so as not to focus physiological constraints solely on energy aspects.

(4) Since for some species, the data is averaged across studies to get species-level trait estimates, is there a scope to examine within population differences (e.g. across latitudes)? This may further strengthen the evidence and rule out the possibility of the environment, especially the length of the breeding season, affecting the timing of emergence and immergence.

For a given species, data on hibernation phenology are averaged for different populations, but also for the same population when measurements are taken over several years. To test these hypotheses on a population scale, precise data on reproductive effort would be needed for each population tested, but this concerns very few species (less than 5).

Testing the effects of temperature and precipitation allows us to take into account the effects of climate on phenology.

(5) Although the authors are looking at the broader patterns, I felt like the overall ecology of the species (habitat, tropical or temperate, number of broods, etc.) is overlooked and could act as confounding factors.

Yes, that's why we also tested the physiological constraints hypothesis, including the effect of temperature and precipitation. For the life-history hypothesis, we also tested reproductive effort, which takes into account the number of offspring per year.

(6) I strongly think the data analysis part needs more clarity. As of now, it is difficult for me to visualize all the fitted models (despite Table 1), and the large number of life-history traits adds to this complexity. I would recommend explicitly writing down all the models in the text. Also, the Table doesn't make it clear whether interaction was allowed between the predictors or not. More information on how PGLS were fitted needs to be provided in the main text which is in the supplementary right now. I kept wondering if the authors have fit multiple models, for example, with different correlation structures or by choosing different values of lambda parameter. And, in addition to PGLS, authors are also fitting linear regressions. Can you explain clearly in the text why was this done?

To simplify the results, we reduced the number of models to just three: one for emergence and two for immergence. In place of Table 1, we have written the structure of the models used. We have added a sentence to the statistics section: “each PGLS model produces a λ parameter representing the effect of phylogeny ranging between 0 (no phylogeny effect) and 1 (covariance entirely explained by co-ancestry)”. We have tested only three PGLS models and the estimated lambda value for these models is 0.

(7) Figure 2 is unclear, and I do not understand how these three regression lines were computed. Please provide more details.

We tested new models and modified existing figures.

Reviewer #2 (Public Review):

Summary:

An article with lots of interesting ideas and questions regarding the evolution of timing of dormancy, emphasizing mammalian hibernation but also including ectotherms. The authors compare selective forces of constraints due to energy availability versus predator avoidance and requirements and consequences of reproduction in a review of between and within species (sex) differences in the seasonal timing of entry and exit from dormancy.

Strengths:

The multispecies approach including endotherms and ectotherms is ambitious. This review is rich with ideas if not in convincing conclusions.

Weaknesses:

The differences between physiological requirements for gameatogenesis between sexes that affect the timing of heterothermy and the need for euthermy during mammalian hibernator are significant issues that underlie but are under-discussed, in this contrast of selective pressures that determine seasonal timing of dormancy. Some additional discussion of the effects of rapid climate change on between and within species phenologies of dormancy would have been interesting.

Reviewer #2 (Recommendations For The Authors):

This review provides a very interesting and ambitious among and within-species comparison of the seasonal timing of entry and exit from dormancy, emphasizing literature from hibernating mammals (sans bats and bears) and with attention to ectotherms. The authors test hypotheses related to the timing of food availability (energy) versus life history considerations (requirements for reproduction, avoiding predation) while acknowledging that these are not mutually exclusive. I offer advice for clarifications and description of the limitations of the data (accuracy of emergence and immergence times), but mainly seek more emphasis for small mammalian hibernators on the contrast for requirements for significant periods of euthermy prior to the emergence in males versus females, a contrast that has energetic and timing consequences in both the active and hibernation seasons.

A consideration alluded to but not fully explained or discussed is the differences in mammals between species and sexes in the timing of what can be called ecological hibernation, which is the seasonal duration that an animal remains sequestered in its burrow or den, and heterothermic hibernation, between the beginning and end of the use of torpor. The two are not synonymous. When "emergence" is the first appearance above ground, there is a significant missing observation key to the energetic contrasts discussed in this review, that of this costly pre-emergence behavior.

To explain the difference between heterothermic hibernation and ecological hibernation, we've added a section in review Criteria from materials and methods :

“In this study, we addressed what can be called ecological hibernation, i.e. the seasonal duration that an animal remains sequestered in its burrow or den, which is assumed to be directly linked to the reduced risk of predation. In contrast, we did not consider heterothermic hibernation, which corresponds to the time between the beginning and end of the use of torpor. So when we mention hibernation, emergence or immergence, the specific reference is to ecological hibernation.”

In arctic and other ground squirrel species, males remain at high body temperatures after immerging and remaining in their burrows in the fall for several days to a week, and more consistently and importantly, males that will attempt to breed in the spring end torpor but remain constantly in their burrows for as much as one month at great expense whilst undergoing testicular growth, spermatogenesis, spemiation, and sperm capacitation, processes that require continuous euthermy. Female arctic ground squirrels and non-breeding males do not and typically enter their first torpor bout 1-2 days after immergence and first appear above ground 1-3 days after their last arousal in spring.

The weeks spent euthermic in a cold burrow in spring by males while undergoing reproductive maturation require a significant energetic investment (can equate to the cost of the previous heterothermic period) that contrasts profoundly with the pre-mating energetic investment by females.

Males cache food in their hibernacula and extend their active season in late summer/fall in order to do so and feed from these caches in spring after resuming euthermy, often emerging at body weights similar to that at immergence. Similar between-sex differences in the timing of hibernation and heterothermy occur in golden-mantled and Columbian ground squirrels and likely most other Urocitellus spp., though less well described in other species. These differences are related to life histories and requirements for male vs. female gameatogenesis and, at the same time, energetic considerations in the costs to males for remaining euthermic while undergoing spermatogenesis and the cost related to whether males undergo gonadal development being dependent on individual body mass and cache size. These issues should be better discussed in this review.

It is the time required to complete spermatogenesis, spermiation, and maturation of sperm not the time for growth of different sizes of testes that drives the preparation time for males. This is relatively constant among rodents. I challenge the assumption that larger testes take longer to grow than smaller ones.

We took this comment into account. As we found little evidence of an increase in testicular maturation time with relative testicular size (apart from table 4 in Kenagy and Trombulak, 1986), we no longer tested the effect of relative testicular size on protandry.

We examined whether the ability to store food before hibernation might reduce protandry. Although food storage in the burrow may be favored for overcoming harsh environments or predation, model selection did not retain the food-storing factor. Thus, the ability to accumulate food in the burrow was not by itself likely to keep males of some species from emerging earlier (e.g. Cricetus cricetus, protandry : 20 day, Siutz et al., 2016). Early emerging males may benefit from consuming higher quality food or in competition with other males (e.g., dominance assertion or territory establishment, Manno and Dobson 2008).

We developed these aspects in the discussion

While it is admirable to include ectotherms in such a broad review and modelling, I can't tell what data from how many ectothermic species contributed to the models and summary data included in the figures.

Too few data on ectotherms were available to include ectotherms in the meta-analysis

Some consideration should be made to the limitations of the data extracted from the literature of the accuracy of emergence and immergence dates when derived from only observations or trapping data. The most accurate results come from the use of telemetry for location and data logging reporting below vs. above ground positioning and body temperature.

We added a "study limits" section to the discussion to address all the limits in this commentary.

L64 "favor reproduction", better to say "allow reproduction", since there is strong evolutionary pressure to initiate reproduction early, often anticipating favorable conditions for reproduction, to maximize the time available for young to grow and prepare for overwintering themselves.

Also, generally, it is not how "harsh" an environment is but rather how short the growing season is.

We took this comment into account.

L80 More simply, individuals that have amassed sufficient energy reserves as fat and caches to survive through winter may opt to initiate dormancy. This may decrease but not obviate predation, since hibernating animals are dug from their burrows and eaten by predators such as bears and ermine.

In this sentence, we indicated a gap between dormancy phenology and the growing season, which suggests survival benefits of dormancy other than from a physiological point of view. We've changed the sentence to make it clearer : “However, some animals immerge in dormancy while environnemental conditions would allow them (from a physiological point of view) to continue their activity, suggesting other survival benefits than coping with a short growing season”

L88 other physiological or ecological factors.... (gameatogenesis).

In this study, we examine possible evolutionary pressures and therefore the environmental factors that may influence hibernation phenology. We focus on reproductive effort because, assuming predation pressure, we would expect a trade-off between survival and reproduction.

L113 beginning early to afford long active seasons to offspring while not compromising the survival of parents.

We added to the sentence:

“For females, emergence phenology may promote breeding and/or care of offspring during the most favorable annual period (e.g., a match of the peak in lactational energy demand and maximum food availability, Fig. 1) or beginning early to afford long active seasons to offspring while not compromising the survival of parents.”

L117 based on adequate preparation for overwintering and enter dormancy....

We modified the sentence as follows :

recovering from reproduction, and after acquiring adequate energy stores for overwintering”

L123 given that males outwardly invest the least time in reproduction yet generally have shorter hibernation seasons would seem to reject this hypothesis. This changes if you overtly include the time and energy that males expend while remaining euthermic preparing for hibernation, a cost that can be similar to energy expended during heterothermy.

Males invest a lot of time in reproduction before females emerge (whether for competition or physiological maturation) and some males seem to be subject to long-term negative effects linked to reproductive stress (see Millesi, E., Huber, S., Dittami, J., Hoffmann, I., & Daan, S. (1998). Parameters of mating effort and success in male European ground squirrels, Spermophilus citellus. Ethology, 104(4), 298-313). Both processes may contribute to reducing the duration of male hibernation.

L125 again, costs to support euthermy in males undergoing reproductive development is an investment in reproduction.

You're right, but it's difficult to quantify. We tested a model that takes into account the reproductive effort during reproduction and prior to reproduction. We also considered the hypothesis that species living in a cold climate might have a low protandry while having a high reproductive effort due to their ability to feed in the burrow (interaction effect between reproductive effort and temperature). We think these changes answer your comment.

L134 It isn't growing large testes that takes time, but instead completing spermatogenesis and maturation of sperm in the epdidymides.

We removed this part.

L140 Later immergence in male ground squirrels is related to accumulation and defense of cached food, activities that are related to reproduction the next spring. An experimental analysis that would be revealing is to compare immergence times in females that completed lactation to the independence of their litters vs. females that did not breed or lost their litters. Who immerges first?

Body mass variation from emergence to the end of mating in males seems to explain the delayed immergence of males in species that don't hide food in their burrows for hibernation. For example, in spermophilus citellus, males immege on average more than 3 weeks after females, yet they do not hide food in their burrows for the winter.

Such a study already exists and shows that non-breeding females immerge earlier than breeding females. We refer to it

L386: “In mammals, males and females that invest little or not at all in reproduction exhibit advances in energy reserve accumulation and earlier immergence for up to several weeks, while reproductive congeners continue activity (Neuhaus 2000, Millesi et al. 2008a).”

L164 So you examined literature from 152 species but included data from only 29 species? Did you include data from social hibernators (marmots) that mate before emergence?

With current models, we have 28 different species. We have few species because very few have data on both sex difference data and information on reproductive effort data (especially for males).

Data on sex differences in hibernation were not available for social hibernating species.

L169 Were these data from trapping or observation results? How reliable are these versus the use of information from implanted data loggers or collars that definitively document when euthermy is resumed and/or when immergence and first emergence occurs (through light loggers)?

We did not focus heterothermic hibernation, but in ecological hibernation. We have no idea of the margin of error for these types of data, but we have discussed these limitations in the "Study limitations" section.

L180, again, it is the time required to complete spermatogenesis and spermiation not the time for the growth of different sizes of testes that drives the preparation time for males. This is relatively constant among rodents. I challenge the assumption that larger testes take longer to grow than smaller ones.

We removed this part.

L200 Males that accumulate caches in fall and then feed from those during the spring pre-emergence euthermic interval and after will often be at their seasonal maximum in body mass. Declining from that peak may not be stressful.

It has been suggested that reproductive effort in Spermophilus citellus might induce long-term negative effects that delay male immergence.

Millesi, E., Huber, S., Dittami, J., Hoffmann, I., & Daan, S. (1998). Parameters of mating effort and success in male European ground squirrels, Spermophilus citellus. Ethology, 104(4), 298-313.

L210 How about altitude, which affects the length of the growing season at similar latitudes?

We extracted the location of each study site to determine the temperature and precipitation at that precise location (based on interpolated climate surface). We therefore take into account differences in growing season (based on temperature) in altitude between sites.

L267 How did whether males cache food or not figure into these comparisons? Refeeding before mating occurs during the pre-emergence euthermic interval.

We removed this part.

L332, 344 not a "proxy" but functionally related to advantages in mating systems with multiple mating males.

We removed this part.

L353 The need for a pre-emergence euthermic interval in male ground squirrels requires costs in the previous active season in accumulating and defending a cache and the proximal costs in spring while remaining at high body temperatures prior to emergence with resulting loss in body mass or devouring of the cache.

You're right, but in this section, we quickly explain the benefits of food catching compared with other species that don't do so.

L385 This review should discuss why females are not known to cache and contrast as "income breeders" from "capital breeder" males. What advantages of caches are females indifferent to (no need for a prolonged pre-emergence period) and what costs of accumulating caches do they avoid (prolonged activity period and defense of caches).

We clarified the case of female emergence.

L321 : “Thus, an early emergence of males may have evolved in response to sexual selection to accumulate energy reserve in anticipation of reproductive effort. Females, on the contrary, are not subject to intraspecific competition for reproduction and may have sufficient time before (generally one week after emergence) and during the breeding period to improve their body condition.”

L388 I don't understand the logic of the conclusion that "did not ...adequately explain the late male immergence" in this section. The greater mass loss in males over the mating period is afforded by the presence of a cache that requires later immergence.

We removed this part.

L412 Not just congeners that invest less in reproduction, but within species individuals that do not attempt to breed in one or more years and thus have no reproductive costs should be an interesting comparison for differences in phenology from individuals that do breed. Non-breeders are often yearlings but can be a significant overall proportion of males that fail to fatten or cache enough to afford a pre-emergence euthermic period.

L385: “In mammals, males and females that invest little or not at all in reproduction exhibit advances in energy reserve accumulation and earlier immergence for up to several weeks, while reproductive congeners continue activity (Neuhaus 2000, Millesi et al. 2008a).”

The sentence refers to individuals who reproduce little or not at all.

L445 Males that gain weight between emergence and mating may do so by feeding from a cache regardless of how "harsh" an environment is.

We observe this phenomenon even in species that are not known to hoard food

“Gains in body mass observed for some individuals, even in species not known to hoard food, may indicate that the environment allows a positive energy balance for other individuals with comparable energy demands.”

L492 Some insects retreat to refugia in mid-summer to avoid parasitism (Gynaephora).

Escape from parasites is also a benefit of dormancy.

Fig 1 - It is difficult to see the differences in black and green colors, esp if color blind.<br /> Maternal effort is front-loaded within the active season (line for "optimal period" shown in midseason).

Add "energy" underneath c) Prediction (H1) and "reproduction" underneath d) "Prediction (H2). Explain the orange vs black, green colors of triangles.

We made the necessary changes

Fig 2 - I don't buy the regression lines as significant in this figure. The red line, cannot have a regression with two sample points and without the left-hand most dot, nothing is significant.

We deleted this graph.

Fig 3 - females only?

We deleted this graph.

Reviewer #2 (Public Review):

Summary:

An article with lots of interesting ideas and questions regarding the evolution of timing of dormancy, emphasizing mammalian hibernation but also including ectotherms. The authors compare selective forces of constraints due to energy availability versus predator avoidance and requirements and consequences of reproduction in a review of between and within species (sex) differences in the seasonal timing of entry and exit from dormancy.

Strengths:

The multispecies approach including endotherms and ectotherms is ambitious. This review is rich with ideas if not in convincing conclusions. Limitations are discussed yet are impactful, namely that differences among and within species are contrast only for ecological hibernation (the duration of remaining sequestered) and not for "heterothermic hibernation" the period between first and last torpor. Differences between the two can have significant energetic consequences, especially for mammals returning to euthermic levels of body temperature whilst remaining in their cold burrows before emerging, eg. reproductively developing males in spring.

Weaknesses:

The differences between physiological requirements for gameatogenesis between sexes that affect the timing of heterothermy and need for euthermy during mammalian hibernator are significant issues that underlie, but are under discussed, in this contrast of selective pressures that determine seasonal timing of dormancy. Some additional discussion of the effects of rapid rapid climate change on between and within species phenologies of dormancy would have been interesting.

eLife assessment

This valuable and ambitious review examines seasonal dormancy in various species, including hibernating mammals (excluding bats and bears) and ectotherms. It provides a solid test of hypotheses on dormancy timing, considering energetic constraints and life history as alternative drivers. The review will be of interest to evolutionary biologists.

Laser hair removal is an advanced time and money saving treatment for unwanted and ingrown hairs. This laser-light-based treatment takes 40–45 minutes (it can vary depending on the area to be treated) to destroy the hair follicles that reside deeper in the dermal layer. As a result, one can achieve the long lasting result from laser light hair removal.

Simply taking one gen ed course does not allow you to fully develop all the skills you need. You must mix the courses "ingredients" in order to develop all skills.

Being a person that can work within multiple fields and apply multiple perspective to a singular problem is beneficial. It's like looking at a problem with a 360 degree camera view.

S: John Mur O: his personal experiences when exploring Alaska A: as one of the classic dog books, this story is geared towards people who care for dogs P: to immerse the reader in this story from his personal travels S: traveling along a glacier in Alaska Tone: anticipating, descriptive, suspenseful

Shows a comparison of them sleeping that night versus earlier that day when life was not as guaranteed to emphasize the danger of their actions

Shows a sign of caring for the dog but if he sincerely cared, why didn't he carry the dog over on his back rather than forcing this dangerous decision

Introduces a moral dilemma of whether he will leave the dog or not and transitions to the climax of the story

Personifying to add character or to imply the narrator is losing it

Shift in style for this sentence from long flowing sentences describing imagery to blatantly stating the issues at hand to make it clearly put forth for the reader

Spent half the piece describing its surroundings yet failed to mention he had company on this trip, was this a purposeful stylistic choice to bring more of a shock value when reintroducing the dog?

I wonder if Emerson or Thoreau felt these fears while out on their expeditions

Previously described as a wild and seemingly dangerous location, foreshadows an upcoming problem for the narrator

Comparison to famous location to emphasize the appeal and beauty of this area and allows for a broad audience

Connection to Emerson/Thoreau ideas that by slowing down/simplifying life, one can take the time to enjoy/appreciate/reflect about it

Implies character traits such as inefficient and unadaptable

Foreshadows an issue with him and the dog's ability to go back

Relating back to mister's little dog, even with the absence of physical presence, given the obvious power dynamic, are they ever really free?

Vivid imagery balanced with a combination of show vs tell to create mental pictures to immerse the reader in the story

Glaciers represents a connection to ancestors and historic time periods

What are the consequences for losing the dog?

Briefly provide a synopsis so that the reader doesn't need to look for the information in the image. "Pictured below is a comment from my fieldwork educator explaining my progress with communicating the OT process, as well as my exemplary performance score for articulating the value of OT"

Was the child developing OT skills, or other skills, like play skills, fine motor skills, or even developmental milestones?

This needs to appear in parentheses after the first time you spell it out and before you use it on the page.

eLife assessment

This paper addresses a fundamental issue in the field of autophagy: how is a protein responsible for autophagosome-lysosome fusion recruited to mature autophagosomes but not immature ones? The work succeeds in its ambition to provide a new conceptual advance. The evidence supporting the conclusions is convincing, with fluorescence microscopy, biochemical assays, and molecular dynamics simulations. This work will be of broad interest to cell biologists and biochemists studying autophagy, and also those focusing on lipid/membrane biology.

Reviewer #3 (Public Review):

Summary:

This manuscript by Liu et al. presents a case that CAPSL mutations are a cause of familial exudative vitreoretinopathy (FEVR). Attention was initially focused on the CAPSL gene from whole exome sequence analysis of two small families. The follow-up analyses included studies in which CAPSL was manipulated in endothelial cells of mice and multiple iterations of molecular and cellular analyses. Together, the data show that CAPSL influences endothelial cell proliferation and migration. Molecularly, transcriptomic and proteomic analyses suggest that CAPSL influences many genes/proteins that are also downstream targets of MYC and may be important to the mechanisms.

Strengths:

This multi-pronged approach found a previously unknown function for CAPSLs in endothelial cells and pointed at MYC pathways as high-quality candidates in the mechanism.

Weaknesses:

Two issues shape the overall impact for me. First, the unreported population frequency of the variants in the manuscript makes it unclear if CAPSL should be considered an interesting candidate possibly contributing to FEVR, or possibly a cause. Second, it is unclear if the identified variants act dominantly, as indicated in the pedigrees. The studies in mice utilized homozygotes for an endothelial cell-specific knockout, leaving uncertainty about what phenotypes might be observed if mice heterozygous for a ubiquitous knockout had instead been studied.

In my opinion, the following scientific issues are specific weaknesses that should be addressed:

(1) Please state in the manuscript the number of FEVR families that were studied by WES. Please also describe if the families had been selected for the absence of known mutations, and/or what percentage lack known pathogenic variants.

(2) A better clinical description of family 3104 would enhance the manuscript, especially the father. It is unclear what "manifested with FEVR symptoms, according to the medical records" means. Was the father diagnosed with FEVR? If the father has some iteration of a mild case, please describe it in more detail. If the lack of clinical images in the figure is indicative of a lack of medical documentation, please note this in the manuscript.

(3) The TGA stop codon can in some instances also influence splicing (PMID: 38012313). Please add a bioinformatic assessment of splicing prediction to the assays and report its output in the manuscript.

(4) More details regarding utilizing a "loxp-flanked allele of CAPSL" are needed. Is this an existing allele, if so, what is the allele and citation? If new (as suggested by S1), the newly generated CAPSL mutant mouse strain needs to be entered into the MGI database and assigned an official allele name - which should then be utilized in the manuscript and who generated the strain (presumably a core or company?) must be described.

(5) The statement in the methods "All mice used in the study were on a C57BL/6J genetic background," should be better defined. Was the new allele generated on a pure C57BL/6J genetic background, or bred to be some level of congenic? If congenic, to what generation? If unknown, please either test and report the homogeneity of the background, or consult with nomenclature experts (such as available through MGI) to adopt the appropriate F?+NX type designation. This also pertains to the Pdgfb-iCreER mice, which reference 43 describes as having been generated in an F2 population of C57BL/6 X CBA and did not designate the sub-strain of C57BL/6 mice. It is important because one of the explanations for missing heritability in FEVR may be a high level of dependence on genetic background. From the information in the current description, it is also not inherently obvious that the mice studied did not harbor confounding mutations such as rd1 or rd8.

(6) In my opinion, more experimental detail is needed regarding Figures 2 and 3. How many fields, of how many retinas and mice were analyzed in Figure 2? How many mice were assessed in Figure 3?

(7) I suggest adding into the methods whether P-values were corrected for multiple tests.

eLife assessment

This study explores the role of calcyphosine-like (CAPSL) in Familial Exudative Vitreoretinopathy (FEVR) via the MYC pathway, offering valuable insights into disease mechanisms that are supported by a solid, multi-pronged approach. The overall significance of the study might, however, be limited due to weak support from human genetic studies.

Reviewer #1 (Public Review):

Summary:

The author presents the discovery and characterization of CAPSL as a potential gene linked to Familial Exudative Vitreoretinopathy (FEVR), identifying one nonsense and one missense mutation within CAPSL in two distinct patient families afflicted by FEVR. Cell transfection assays suggest that the missense mutation adversely affects protein levels when overexpressed in cell cultures. Furthermore, conditionally knocking out CAPSL in vascular endothelial cells leads to compromised vascular development. The suppression of CAPSL in human retinal microvascular endothelial cells results in hindered tube formation, a decrease in cell proliferation, and disrupted cell polarity. Additionally, transcriptomic and proteomic profiling of these cells indicates alterations in the MYC pathway.

Strengths:

The study is nicely designed with a combination of in vivo and in vitro approaches, and the experimental results are good quality.

Weaknesses:

My reservations lie with the main assertion that CAPSL is associated with FEVR, as the genetic evidence from human studies appears relatively weak. Further careful examination of human genetics evidence in both patient cohorts and the general population will help to clarify. In light of human genetics, more caution needs to be exercised when interpreting results from mice and cell models and how is it related to the human patient phenotype.

Reviewer #2 (Public Review):

Summary:

This work identifies two variants in CAPSL in two-generation familial exudative vitreoretinopathy (FEVR) pedigrees, and using a knockout mouse model, they link CAPSL to retinal vascular development and endothelial proliferation. Together, these findings suggest that the identified variants may be causative and that CAPSL is a new FEVR-associated gene.

Strengths:

The authors' data provides compelling evidence that loss of the poorly understood protein CAPSL can lead to reduced endothelial proliferation in mouse retina and suppression of MYC signaling in vitro, consistent with the disease seen in FEVR patients. The study is important, providing new potential targets and mechanisms for this poorly understood disease. The paper is clearly written, and the data generally support the author's hypotheses.

Weaknesses:

(1) Both pedigrees described appear to suggest that heterozygosity is sufficient to cause disease, but authors have not explored the phenotype of Capsl heterozygous mice. Do these animals have reduced angiogenesis similar to KOs? Furthermore, while the p.R30X variant protein does not appear to be expressed in vitro, a substantial amount of p.L83F was detectable by western blot and appeared to be at the normal molecular weight. Given that the full knockout mouse phenotype is comparatively mild, it is unclear whether this modest reduction in protein expression would be sufficient to cause FEVR - especially as the affected individuals still have one healthy copy of the gene. Additional studies are needed to determine if these variants alter protein trafficking or localization in addition to expression, and if they can act in a dominant negative fashion.

(2) The manuscript nicely shows that loss of CAPSL leads to suppressed MYC signaling in vitro. However, given that endothelial MYC is regulated by numerous pathways and proteins, including FOXO1, VEGFR2, ERK, and Notch, and reduced MYC signaling is generally associated with reduced endothelial proliferation, this finding provides little insight into the mechanism of CAPSL in regulating endothelial proliferation. It would be helpful to explore the status of these other pathways in knockdown cells but as the authors provide only GSEA results and not the underlying data behind their RNA seq results, it is difficult for the reader to understand the full phenotype. Volcano plots or similar representations of the underlying expression data in Figures 6 and 7 as well as supplemental datasets showing the differentially regulated genes should be included. In addition, while the paper beautifully characterizes the delayed retinal angiogenesis phenotype in CAPSL knockout mice, the authors do not return to that model to confirm their in vitro findings.

(3) In Figure S2D, the result of this vascular leak experiment is unconvincing as no dye can be seen in the vessels. What are the kinetics for biocytin tracers to enter the bloodstream after IP injection? Why did the authors choose the IP instead of the IV route for this experiment? Differences in the uptake of the eye after IP injection could confound the results, especially in the context of a model with vascular dysfunction as here.

(4) In Figure 5, it is unclear how filipodia and tip cells were identified and selected for quantification. The panels do not include nuclear or tip cell-specific markers that would allow quantification of individual tip cells, and in Figure 5C it appears that some filipodia are not highlighted in the mutant panel.

These values and interests might be best described as those of the private sector and the profit motive. The teaching management platform Moodle seems to follow this trend as the interactivity of the platform is largely controlled by the teacher. This is one way that capitalism enforces bisected learning, and it is also an example of how bisected learning and capitalism are indirect contradiction to explorative learning and free knowledge.

This is an example of the prisoners dilemma being promoted by the way digital learning technologies are constructed. Scholia and hypothesis might present the antithesis to this problem, as these platforms constantly remind the user that the way their learning is dependent on others. Since hypothesis is always present at the side of the screen the learner is also constantly being reminded that they are able to contribute to the public knowledge database.

Author response:

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

Recommendations for the authors

Reviewer #1 (Recommendations For The Authors):

Below I summarize points that should be addressed in a revised version of the manuscript.

• Page 6, first paragraph: I don't understand by the signals average out to a single state. If the distribution is indeed randomly distributed, a broad signal with low intensity should be present.

We agree that this statement may cause confusion. We changed the text (marked in bold) to clarify the statement: The mobility of the undocked SBDs will be higher than the diffusion of the whole complex, allowing the sampling of varying interdomain distances within a single burst. However, these dynamic variations are subsequently averaged to a singular FRET value during FRET calculations for each burst, and may appear as a single low FRET state in the histograms.

• Page 6, third paragraph: how can the donor only be detected in the acceptor channel? Is this tailing out?

Donor only signal is not detected in the acceptor channel. As described in page 5 and in the Materials & Methods section, the dye stoichiometry value is defined for each burst/dwell using three types of photon counts: donor-based donor emission (FDD), donor-based acceptor emission (FDA) and acceptorbased acceptor emission (FAA).

When no acceptor fluorophore is present FAA=0 and S=1.

Some donor photons bleed through into the acceptor channel, but we correct for this by calculating the leakage and crosstalk factors as described in the Materials and Methods (page 20).

We changed the text (marked in bold) in the manuscript to address the question: The FRET data of both OpuA variants is best explained by a four-state model (Figure 2A,B; fourth and fifth panel) (Supplementary File 3). Two of the four states represent donor-only (S≈1) or acceptor-only (S≈0) dwells. The full bursts belonging to donor-only and acceptor-only molecules were excluded prior to mpH2MM. This means that some molecules transit to a donor-only or acceptor-only state within the burst period, which most likely reflects blinking or bleaching of one of the fluorophores. These donoronly and acceptor-only states were also excluded during further analysis. The other two states reflect genuine FRET dwells that were analyzed by mpH2MM. They represent different conformations of the SBDs.

• Page 7, "SBD dynamics ..": why was the V149Q mutant only analyzed in the K521C background and not also in the N414C background?

The two FRET states were best distinguished in OpuA-K521C. Therefore, we decided to focus on OpuA-K521C and not OpuA-N414C. OpuA-V149Q was used to show that reduced docking efficiency does not affect the transition rate constants and relative abundances of the two FRET states, and we regarded it sufficient to test the SBD dynamics in OpuA-K521C only.

• Page 8, second paragraph: why was the N414C mutant analyzed only from 0 - 600 mM and not also up to 1000 mM?

In line with the previous answer, our main focus was on OpuA-K521C, since the two FRET states were best distinguished in OpuA-K521C. OpuA-N414C was used to prove that similar states are observed when measuring with fluorophores on the opposite site of the SBD. We studied how the FRET states change in response to different conditions that correspond to different stages of the transport cycle and how it changes in response to different ionic strengths. Initially, 600 mM KCl was used to study the dynamics of the SBD at high ionic strength. Later in this study, we tested a very wide range of different salt concentrations for OpuA-K521C to get detailed insights into the dynamics of the SBDs over a wide ionic strength range. Note that 1 M KCl is a very high, non-physiological ionic strength for the typical habitat of L. lactis and was only used to show that the high FRET state occurs even under very extreme conditions.

• Page 8, third paragraph: why was the dimer (if it is the source of the FRET signal) only partially disrupted?

We acknowledge that this is a very good point. However, we purposely did not speculate on this point in the manuscript, because we have limited information on the molecular details of the interaction. As we highlight on page 8, the SBDs experience each other in a very high apparent concentration (millimolar range). This means that the interactions are most likely very weak (low affinity) and not very specific. Such interactions are in the literature referred to as the quinary structure of proteins and they occur at the high macromolecular crowding in the cell and in proteins with tethered domains, and thus at high local concentrations. Such interactions can be screened by high ionic strength. In the revised manuscript, we now present the partially disrupted dimer structure in the context of the quinary structure of a protein (page 11):

In other words, the high FRET state may comprise an ensemble of weakly interacting states rather than a singular stable conformation, resembling the quinary structure of proteins. The quinary structure of proteins is typically revealed in highly crowded cellular environments and describes the weak interactions between protein surfaces that contribute to their stability, function, and spatial organization (Guin & Gruebele, 2019). Despite the current study being conducted under dilute conditions, the local concentration of SBDs (~4 mM) mimics a densely populated environment and reveal quinary structure.

• Page 9, second paragraph: according to the EM data processing, only 20% of the particles were used for 3D reconstruction. Why? Does it mean that the remaining 80% were physiologically not relevant? If so, why were the 20% used relevant?

We note that it is a fundamental part of image processing of single particle cryo-EM data to remove false positives or low-resolution particles throughout the processing workflow. In particular when using a very low and therefore generous threshold during automated particle picking, as we did (t=0.01 and t=0.05 for the 50 mM KCl and 100 mM KCl datasets, respectively), the initial set of particles includes a significant amount of false positives – a tradeoff to avoid excluding particles belonging to low populated classes/orientations. It is thus common that more than 50% of ‘particles’ are excluded in the first rounds of 2D classification. In our case, only 30% and 52% of particles were retained after such first clean-up steps. Subsequently, the particle set is further refined, and additional false positives and low-resolution particles are excluded during extensive rounds of 3D classification. We also note that during the final steps, most of the data excluded represents particles of lower quality that do not contribute to a high-resolution, or belong to low population protein conformations. This does not mean that such a population is not physiological relevant. In conclusion, having only 5-20% of the initial automated picked particles contributing to the reconstruction of the final cryo-EM map is common, with the vast majority of excluded particles being false positives.

• Page 11, third paragraph: the way the proposed model is selected is also my main criticism. All alternative models do not fit the data. Therefore, the proposed model is suggested. However, I do not grasp any direct support for this model. Either I missed it or it is not presented.

Concerning the specific model in Figure 5, the reviewer is correct. We do not provide direct evidence for a side-ways interaction. However, we have evidence of transient interactions and our data rule out several scenarios of interaction, leaving 5C as the most likely model. This is also the main conclusion of this paper: In conclusion, the SBDs of OpuA transiently interact in a docking competent conformation, explaining the cooperativity between the SBDs during transport. The conformation of this interaction is not fixed but differs substantially between different conditions.

Because the interaction is very short-lived it was not possible to visualize molecular details of this interaction. We present Figure 5 to hypothesize the most likely type of interaction, since many possibilities can be excluded with the vast amount of presented data. To make our point more clear that we discuss models and rule out several possibilities but not demonstrate a specific interaction between the SBDs, we now write on page 10 (changes marked in bold): We have shown that the SBDs of OpuA come close together in a short-lived state, which is responsive to the addition of glycine betaine (Figure 4A). Although the occurrence of the state varies between different conditions, it was not possible to negate the high-FRET state completely, not even under very high or low KCl concentrations, or in the presence of 50 mM arginine plus 50 mM glutamate (Figure 4A,B). To evaluate possible interdomain interactions scenarios we consider the following: (1) The SBDs of OpuA are connected to the TMDs with very short linkers of approximately 4 nm, which limit their movement and allow the receptor to sample a relatively small volume near its docking site. (2) in low ionic strength condition OpuA-K521C displays a high FRET state with mean FRET values of 0.7-0.8, which correspond to inter-dye distances of approximately 4 nm. (3) The high FRET state is responsive to glycine betaine, which points toward direct communication between the two SBDs. (4) The distance between the density centers of the SBDs in the cryo-EM reconstructions (based on particles with a low and high FRET state) is 6 nm, which aligns with the dimensions of an SBD (length: ~6 nm, maximal width: ~4 nm). These findings collectively indicate that two SBDs interact but not necessarily in a singular conformation but possibly as an ensemble of weakly interacting states. Hence, we discuss three possible SBD-SBD interaction models to explain the highFRET state:

Reviewer #2 (Recommendations For The Authors):

In the abstract and elsewhere the authors suggest that the SBDs physically interact with one another, and that this interaction is important for the transport mechanism, specifically for its cooperativity.

I feel that this main claim is not well established. The authors convincingly demonstrate that the SBDs largely occupy two states relative to one another and that in one of these states, they are closer than in the other. Unless I have missed (or failed to understand) some major details of the results, I did not find any evidence of a physical interaction. Have the authors established that the high FRET state indeed corresponds to the physical engagement of the SBDs? I feel that a direct demonstration of an interaction is much missing.

Along the same lines, in the low-salt cryo-EM structure, where the SBDs are relatively closer together, the SBDs are still separated and do not interact.

See also our response to the final comment of reviewer 1. Furthermore, please carefully consider the following: (1) FRET values of 0.7-0.8 correspond to inter-dye distances of approximately 4 nm. (2) The high FRET state is responsive to glycine betaine, which points toward direct communication between the two SBDs. (3) The cryo-EM reconstruction is the average of all the particles in the final dataset, including both the particles with a low and high FRET state. Further, the local resolution of the SBDs in the cryo-EM map is low, indicative of high degree of flexibility. Thus, a potential interaction is possible within the observed range of flexibility. (4) The distance between the density centers is 6 nm, aligning with the dimensions of an SBD (length: 6 nm, maximal width: 4 nm). These factors collectively indicate SBD interactions, and we present these points now more explicitly in Figure 4 and the last part of the results section (page 9).

Once the authors successfully demonstrate that direct physical interaction indeed occurs, they will need to provide data that places it in the context of the transport cycle. Do the SBDs swap ligand molecules between them? Do they bind the ligand and/or the transporter cooperatively? What is the role of this interaction?

We acknowledge the intriguing nature of the posed questions, but they extend beyond the scope of this study. It is extremely challenging to obtain high-resolution structures of highly dynamic multidomain proteins, like OpuA, and to probe transient interactions as we do here for the SBDs of OpuA. We therefore combined cryo-TEM with smFRET studies and perform the most advanced and state-of-theart analysis tools as acknowledged by reviewer 1. We link our observations on the structural dynamics and interactions of the SBDs to a previous study, where we showed that the two SBDs of OpuA interact cooperatively. We do not have further evidence that connect the physical interactions to the transport cycle. In our view, the collective datasets indicate that the here reported physical interactions between the SBDs increase the transport efficiency.

As far as I understand, the smFRET data have been interpreted on the basis of a negative observation, i.e., that it is "likely" that none of the FRET states corresponds to a docked SBD. To convincingly show this, a positive observation is required, i.e., observation of a docked state.

The aim of this study was to study interdomain dynamics and not specifically docking. We have previously shown that docking can be visualized via cryo-EM (Sikkema et al., 2020), however the SBDs of OpuA appear to only dock in specific turnover conditions. We now show that the high FRET state of OpuA cannot represent a docked state, but that the SBDs transiently interact (see our response to the first comment). Importantly, a docked state was also not found in the cryo-EM reconstructions at low ionic strength, representing the smFRET conditions where we observe the interactions between the SBDs. The high FRET state occupies 30% of the dwells in this condition, and such a high percentage of molecules would have become apparent during cryo-EM 3D classification in case they would form a docked state. Therefore, we conclude that docking does not occur in low ionic strength apo condition. We discuss this point and our reasoning on page 11 of the revised manuscript.

In this respect, I find it troubling that in none of the tested conditions, the authors observed a FRET state which corresponds to the docked state. Such a state, which must exist for transport to occur (as mentioned in the authors' previous publications), needs to be demonstrated. This brings me to my next question: why have the authors not measured FRET between the SBDs and the transporter? Isn't this a very important piece that is missing from their puzzle?

We agree that investigating docking behavior under varied turnover conditions requires focused experiments on FRET dynamics between the SBDs and the transporter. As noted on page 5, OpuA exists as a homodimer, implying that a single cysteine mutation introduces two cysteines in a single functional transporter. To specifically implement a cysteine mutation in only one SBD and one transmembrane domain, it is necessary to artificially construct a heterodimer. We recently published initial attempts in this direction, and this will be a subject for future research but still requires years of work.

Additionally, I feel that important controls are missing. For example, how will the data presented in Fig1 look if the transporter is labeled with acceptor or donor only? How do soluble SBDs behave?

In the employed labeling method, donor and acceptor dyes are mixed in a 1:1 ratio and randomly attached to the two cysteines in the transporter. This automatically yields significant fractions of donor only and acceptor only transporters which are always present during the smFRET recordings. We can visualize those molecules on the basis of the dye stoichiometry, which we calculate by using three types of photon counts: donor-based donor emission (FDD), donor-based acceptor emission (FDA) and acceptorbased acceptor emission (FAA).

Unfiltered plots look as follows (a dataset of OpuA-K521C at 600 mM KCl):

Author response image 1.

Donor only and acceptor only molecules have a very well discernible stoichiometry of 1 and 0, respectively. The filtering procedure is described in the materials and methods section, and these plots can be found in the supplementary database. We did not add them to the main text or supplementary materials of the original manuscript, as this is a very common procedure in the field of smFRET. We now include such a dataset in the revised manuscript.

Soluble SBDs of OpuA have been studied previously (e.g. Wolters et al., 2010 & De Boer et al. 2019). For example, we have shown by SEC-MALLLS that soluble SBDs do not form dimers, which is consistent with our notion that the SBDs interact with low affinity. It is not possible to study interdomain dynamics between soluble SBDs by smFRET, because the measurements are carried out at picomolar concentrations (monomeric conditions). We emphasize that smFRET measurements with native complexes, with SBDs near each other at apparent millimolar concentrations, is physiologically more relevant.

Additional comments:

(1) "It could well be that cooperativity and transient interactions between SBDs is more common than previously anticipated" and a similar statement in the abstract. What evidence is there to suggest that the transient interactions between SBDs are a common phenomenon?

On page 11, we write: Dimer formation of SBPs has been described for a variety of proteins from different structural clusters of substrate-binding proteins [33–38,51–53]. We cite 9 papers that report SBD/SBP dimers. This suggest to us that the phenomenon of interacting substrate-binding proteins could be more common. Moreover, the concentration of maltose-binding protein and other SBPs in the periplasm of Gram-negative bacteria can reach (sub)millimolar concentrations, and low-affinity interactions may play a role not only in membrane protein-tethered SBDs (like in OpuA) but also be important in soluble substrate-receptors. Such low-affinity interactions are rarely studied in biochemical experiments.

(2) I think that the data presented in 1B-C better suits the supplementary information.

Figure 1B-D is already a summary of the supplementary information that describes the optimization of OpuA purification. We think it is valuable to show this part of the figure in the main text. A very clean and highly pure OpuA sample is essential for smFRET experiments. Quality of protein preparations and data analysis are key for the type of measurements we report in this paper.

(3) "the first peak in the SEC profile corresponds...." The peaks should be numbered in the figure to facilitate their identification.

We have changed the figure as suggested.

(4) "smFRET is a powerful tool for studying protein dynamics, but it has only been used for a handful of membrane proteins". With the growing list of membrane proteins studied by smFRET I find this an overstatement.

We removed this sentence in the new version of the manuscript.

(5) "We rationalized that docking of one SBD could induce a distance shift between the two SBDs in the FRET range of 3-10 nm (Figure 1E)" How and why was this assumed?

We realize that this is one of the sentences that caused confusion about the aim of this study. In this part of the manuscript, we should not have used docking as an example and we apologize for that. We replaced the sentence by: These variants are used to study inter-SBD dynamics in the FRET range of 310 nm (Figure 1E).

Also Figure 1E was adjusted to prevent confusion:

Author response image 2.

In addition, to avoid any confusion we changed the following sentence on page 4 (changes marked in bold): We designed cysteine mutations in the SBD of OpuA to study interdomain dynamics in the full length transporter.

(6) "However, the FRET distributions are broader than would be expected from a single FRET state, especially for OpuA-K521C" Have the authors established how a single state FRET of OpuA looks? Is there a control that supports this claim?

Below we compare two datasets from OpuA-K521C in 600 mM KCl with a typical smFRET dataset from the well-studied substrate-binding protein MBP from E. coli, which resides in a single state. Left: OpuA-K521C; Right: MBP

Author response image 3.

We agree that this cannot be assumed from the presented data. Therefore we rewrote this sentence: However, the FRET distributions tail towards higher FRET values, especially OpuA-K521C.

(7) "V149Q was designed as a mild mutation that would reduce docking efficiency and thereby substrate loading, but leave the intrinsic transport and ATP hydrolysis efficiency intact." I find this statement confusing: How can a mutation reduce docking efficiency yet leave the transport activity unchanged?

We rewrote the sentences (changes marked in bold): V149Q was designed as a mild mutation that would reduce docking efficiency and thereby substrate loading, but leave the ionic strength sensing in the NBD and the binding of glycine betaine and ATP intact. Accordingly, a reduced docking efficiency should result in a lower absolute glycine betaine-dependent ATPase activity. At the same time the responsiveness of the system to varying KCl, glycine betaine, or Mg-ATP concentrations should not change.

(8) Along the same lines: "whereas the glycine betaine-, Mg-ATP-, or KCl-dependent activity profiles remain unchanged" vs. "OpuA-V149Q-K521C exhibited a 2- to 3-fold reduction in glycine betainedependent ATPase activity".

See comment at point 7.

(9) In general, I find the writing wanting at places, not on par with the high standards set by previous publications of this group.

We recognize the potential ambiguity in our phrasing. We hope that after incorporating the feedback provided by the reviewers our manuscript will convey our findings in a clearer manner.

Extra changes to the text:

(1) Title changed: The substrate-binding domains of the osmoregulatory ABC importer OpuA physically transiently interact

(2) Second part of the abstract changed: We now show, by means of solution-based single-molecule FRET and analysis with multi-parameter photon-by-photon hidden Markov modeling, that the SBDs transiently interact in an ionic strength-dependent manner. The smFRET data are in accordance with the apparent cooperativity in transport and supported by new cryo-EM data of OpuA. We propose that the physical interactions between SBDs and cooperativity in substrate delivery are part of the transport mechanism.

(3) Page 6, third paragraph and Figure 2B: the wrong rate number was extracted from table 1. Changed this in the text and figure: 112 s-1  173 s-1. It did not affect any of the interpretations or conclusions.

(4) Page 8, last paragraph, changed: smFRET was also performed in the absence of KCl and with a saturating concentration of glycine betaine (100 µM). The mean FRET efficiency of the highFRET state of OpuA-K521C increased to 0.78, which corresponds to an inter-dye distance of about 4 nm. This indicates that the dyes at the two SBDs move very close towards each other (Figure 4A) (Table 1) (Supplementary File 34).

(5) Page 9, second paragraph changed: Due to the inherent flexibility of the SBDs, with respect to both the MSP protein of the nanodisc and the TMDs of OpuA, their resolution is limited. Furthermore, the cryo-EM reconstructions average all the particles in the final dataset, including those with a low and high FRET state. Nevertheless, in both conditions, the densities that correspond to the SBDs can be observed in close proximity (Figure 4D). The distance between the density centers is 6 nm and align with the dimensions of an SBD, providing further evidence for physical interactions between the SBDs.

eLife assessment

The OpuA Type I ABC importer uses two substrate binding domains to capture extracellular glycine betaine and present the substrate to the transmembrane domain for subsequent transport and correction of internal dehydration. This study presents valuable findings addressing the question of whether the two substrate binding domains of OpuA dock and physically interact in a salt-dependent manner. The single-molecule fluorescence resonance energy transfer and cryogenic electron microscopy data that are presented provide convincing support for the existence of a transient interaction between the substrate binding domains that depends on ionic strength, laying a foundation for future studies exploring how this interaction is involved in the overall transport mechanism.

Reviewer #1 (Public Review):

Summary: The type I ABC importer OpuA from Lactococcus lactis is the best studied transporter involved in osmoprotection. In contrast to most ABC import systems, the substrate binding protein is fused via a short linker to the transmembrane domain of the transporter. Consequently, this moiety is called the substrate binding domain (SBD). OpuA has been studied in the past in great detail and we have a very detailed knowledge about function, mechanisms of activation and deactivation as well as structure.

Strengths: Application of smFRET to unravel transient interactions of the SBDs. The method is applied at a superb quality and the data evaluation is excellent.

Weaknesses: The proposed model is not directly supported by experimental data. Rather alternative models are excluded as they do not fit to the obtained data. However, this is now clearly stated in the manuscript

Reviewer #2 (Public Review):

Summary:<br /> In this report the authors used solution-based single-molecule FRET and low resolution cryo-EM to investigate the interactions between the substrate-binding domains of the ABC-importer OpuA from Lactococcus lactis. Based on their results, the authors suggest that the SBDs interact in an ionic strength-dependent manner.

Strengths:<br /> The strength of this manuscript is the uniqueness and importance of the scientific question, the adequacy of the experimental system (OpuA), and the combination of two very powerful and demanding experimental approaches.

Weaknesses:<br /> A demonstration that the SBDs physically interact with one another, and that this interaction is important for the transport mechanism will greatly strengthen the claims of the authors. The relation to cooperativity is also unclear.

RE read with care

Defuturing

Decentralization

Open Data is not Enough

Cite this in PhD

Abuse of power int he internet Internet as a tool for empowerment.

Cybersyn experiment was transfered to google

Design always good at making thinks invisible...

From Semantic Web to Googles Web

This was the main reason for the server client paradigm to continue

Propietary Knoledge Graphs. how information is linked could be privatized. so even open source data is able to be captured.

Schema.org - Interesting approach on how private companies come together with a standard..

happy beginnings...

Decentralization is also accepting that nodes could disappear. also linked to the right to be forgotten. Interesting in the realm of centralization beacuse in that scenario there will be an Uthority that gives you a fine our makes you be "present"

hu, so he got rejected a paper? nice to know.

Exit to Community?

Socializing the means of computation

Knowledge economies

Control and centralization are noways in university

The goal of knowledge representation is to strive for human autonomy.

Interesting view on the relationship between autonomy, knowledge and decentralization/centralization

Centralization by definition means the loss of autonomy. In what scenarios is centralization a pharmakon?

In this definition a decntralized system is one that negotiates trust whereas distributed in this defnition takes it as if trust is already established.

Thats why the internet is decentralized

A philosophy of decentralization? Centralization is not really all too easy. It requires people, and gathering that force is usually linked to incentives

The fate of every mature industry is to be centralized? here posses the question of decentralization as a phase in the maturity of a system.

Author response:

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

We are grateful to the Editors for overseeing the review of our manuscript, and to the two reviewers for their thoughtful comments and suggestions for how it can be improved.

I submit at this time a revision, as well as a detailed response (below) to each of the points raised in the first round of review.

We feel the manuscript has been significantly improved by taking the reviewers' comments to heart. In a nutshell, we added new key pieces of data (impact of WIN site inhibition on global translation, rRNA production, as well as the requested cell biology analyses showing nucleolar stress), new analyses of the proteomics to counter potential concerns with normalization, and expanded/revised verbiage in key areas to clarify parts of the text that were confusing or problematic. The main figures have not changed; all new material is included in supplements to figures 2 and 3.

Public Reviews

Reviewer #1 (Public Review):

Building on previous work from the Tansey lab, here Howard et al. characterize transcriptional and translational changes upon WIN site inhibition of WDR5 in MLL-rearranged cancer cells. They first analyze whether C16, a newer generation compound, has the same cellular effects as C6, an early generation compound. Both compounds reduce the expression of WDR5-bound RPGs in addition to the unbound RPG RPL22L1. They then investigate differential translation by ribo-seq and observe that WIN site inhibition reduces the translational RPGs and other proteins related to biomass accumulation (spliceosome, proteasome, mitochondrial ribosome). Interestingly, this reduction adds to the transcriptional changes and is not limited to RPGs whose promoters are bound by WDR5. Quantitative proteomics at two-time points confirmed the downregulation of RPGs. Interestingly, the overall effects are modest, but RPL22LA is strongly affected. Unexpectedly, most differentially abundant proteins seem to be upregulated 24 h after C6 (see below). A genetic screen showed that loss of p53 rescues the effect of C6 and C16 and helped the authors to identify pathways that can be targeted by compounds together with WIN site inhibitors in a synergistic way. Finally, the authors elucidated the underlying mechanisms and analyzed the functional relevance of the RPL22, RPL22L1, p53, and MDM4 axis.

While this work is not conceptually new, it is an important extension of the observations of Aho et al. The results are clearly described and, in my view, very meaningful overall.

Major points:

(1) The authors make statements about the globality/selectivity of the responses in RNA-seq, ribo-seq, and quantitative proteomics. However, as far as I can see, none of these analyses have spike-in controls. I recommend either repeating the experiments with a spike-in control or carefully measuring transcription and translation rates upon WIN site inhibition and normalizing the omics experiments with this factor.

The reviewer is correct that we did not include spike-in controls in our omics experiments. We would like to emphasize that none of the omics data in this manuscript have been processed in unorthodox ways, and that the major conclusions each have independent corroborating data.

The selectivity in RPG suppression observed in RNA-Seq, for example, is supported by results from our target engagement (QuantiGene) assays; suppression of RPL22L1 mRNA levels is supported by quantitative and semi-quantitative RT-PCR, by western blotting, and by the results of our proteomic profiling; alternative splicing (and expression) of MDM4—and its dependency on RPL22—is also backed up by similar RT-PCR and western blotting data. The same applies for alternative splicing of RPL22L1.

That said, we do appreciate the point the reviewer is making here, and have done our best to respond. We do not think it is a prudent investment in resources to repeat the numerous omics assays in the manuscript. We also considered normalizing for bulk transcription and translation rates as suggested, but it is not clear in practice how this would be done, and it could introduce additional variables and uncertainties that may skew the interpretation of results. Instead, to respond to this comment, we made the following changes to the manuscript:

(1) We now explicitly state, for all omics assays, that spike-in controls were not included. These statements will prompt the reader to make their own assessment of the robustness of each of our findings and interpretations.

(2) We have added new data to the manuscript (Figure 2—figure supplement 1A–B) measuring the impact of C6 and C16 on bulk translation using the OPP labeling method. These new data demonstrate that WIN site inhibitors induce a progressive yet modest decline in protein synthesis capacity. At 24 hours, there is no significant effect of either agent on protein synthesis levels. By 48 hours, a small but significant effect is observed, and by 96 hours translation levels are ~60% of what they are in vehicle-treated control cells. These new data are important because they support the idea that normalization has not blunted the responses we observe—the magnitude of the effects are consistent between the different assays and tend to cap out at two-fold in terms of RPG suppression, translation efficiency, ribosomal protein levels, and protein synthesis capacity.

(3) We have included additional analysis regarding the LFQMS, as described below, that specifically addresses the issue of normalization in our proteomics experiments.

(2) Why are the majority of proteins upregulated in the proteomics experiment after 24 h in C6 (if really true after normalization with general protein amount per cell)? This is surprising and needs further explanation.

The reviewer is correct in noting that (by LFQMS) ~700 proteins are induced after 24 hours of treatment of MV4:11 cells with C16 (not C6, as stated). The reviewer would like us to examine whether this apparent increase in proteins is a normalization artifact. In response to this comment, we have made the following changes to the manuscript:

(1) Our new OPP labeling experiments (Figure 2—figure supplement 1A–B) show that there is no significant reduction in overall protein synthesis following 24 hours of C16 treatment. In light of this finding, it is unlikely that normalization artifacts, resulting from diminution of the pool of highly abundant proteins, create the appearance of these 700 proteins being induced. We now explicitly make this point in the text.

(2) We now clarify in the methods how we seeded identical numbers of cells for DMSO and C16-treated cultures in these experiments, and—consistent with our finding that WIN site inhibitors have little if any effect on protein synthesis or proliferation at the 24 hour timepoint— extracted comparable amounts of proteins from these two treatment conditions (DMSO: 344.75 ± 21.7 µg; C16: 366.50 ± 15.8 µg; [Mean ± SEM]).

(3) We now include in Figure 3—figure supplement 1A a plot showing the distribution of peptide intensities for each protein detected in each run of LFQMS before and after equal median normalization. This new analysis reveals that the distribution of intensities is not appreciably changed via normalization. Specifically, there is not a reduction in peptide intensities in the unnormalized data from 24 hours of C16 treatment that is reversed or tempered by normalization. This analysis provides further support for the notion that the increase we observe is not a normalization artifact.

(4) We now include in Figure 3—figure supplement 1B–D a set of new analyses examining the relationship between the initial intensity of proteins in DMSO control samples (a crude proxy for abundance) versus the fold change in response to WIN site inhibitor. This analysis shows that we have as many "highly abundant" (10th decile) proteins increasing as we do decreasing in response to WINi. Thus, it appears as though the wholesale clearance of highly abundant proteins from the cell is not occurring at this early treatment timepoint. In addition, this analysis also shows that ribosomal proteins (RP) are generally the most abundant, most suppressed, proteins and that their fold-change at the protein level at 24 hours is less than two-fold, consistent again with the magnitude of transcriptional effects of C16, as measured by RNA-Seq and QuantiGene. The fact that the drop in RP levels is consistent with expectations based on other analyses provides further empirical support for the notion that protein levels inferred from LFQMS are authentic and not skewed by global changes in the proteome.

The increase in proteins at this time point, we argue, is thus most likely genuine. It is not surprising that—at a timepoint at which protein synthesis is unaffected—several hundred proteins are induced by a factor of two. How this occurs, we do not know. It may be a transient compensatory mechanism, or it may be an early part of the active response to WIN site inhibitors. Lest the reader be confused by this finding, we have now added text to this section of the manuscript discussing and explaining the phenomenon in more detail.

(3) The description of the two CRISPR screens (GECKO and targeted) is a bit confusing. Do I understand correctly that in the GECKO screen, the treated cells are not compared with nontreated cells of the same time point, but with a time point 0? If so, this screen is not very meaningful and perhaps should be omitted. Also, it is unclear to me what the advantages of the targeted screen are since the targets were not covered with more sgRNAs (data contradictory: 4 or 10 sgRNAs per target?) than in Gecko. Also, genome-wide screens are feasible in culture for multiple conditions. Overall, I find the presentation of the screening results not favorable.

In essence, this is a single screen performed in two tiers. In Tier 1, we screened a complete GECKO library (six sgRNA/gene) with the earliest generation (less potent) inhibitor C6, and compared sgRNA representation against the time zero population. This screen would reveal sgRNAs that are specifically associated with response to C6, as well as those that are associated with general cell fitness and viability. We then identified genes connected to these sgRNAs, removed those that are pan essential, and built a custom library for the second tier using sgRNAs from the Brunello library (four sgRNA/gene). We then screened this custom library with both C6 and the more potent inhibitor C16, this time against DMSO-treated cells from the same timepoint.

We acknowledge that this is not the most streamlined setup for a screen. But our intention was to compare two inhibitors (C6 and C16) and identify high confidence 'hits' that are disconnected from general cell viability, rather than generate an exhaustive list of all genes that, when disrupted, skew the response to WIN site inhibitor. The final result of this screen (Figure 4E) is a gene list that has been validated with two chemically distinct WIN site inhibitors and up to 10 unique sgRNAs per gene. We may not have captured every gene that can modulate response to WIN site inhibitor, but those appearing in Figure 4E are highly validated.

To answer the reviewer's specific questions: (i) we cannot omit the Tier 1 screen because then there would be no rationale for what was screened in the second Tier; and (ii) the advantage of the custom Tier 2 library is that it allowed us to screen hits from the Tier 1 screen with four completely independent sgRNAs. Although there are not more sgRNAs for each gene in the Tier 2 versus the Tier 1 library, these sgRNAs are different and thus, for C6 at least, hits surviving both screens were validated with up to 10 unique sgRNAs.

We apologize that the description of the CRISPR screens was not clearer, and have reworked this section of the manuscript to make our intent and our actions clearer.

(4) Can Re-expression of RPL22 rescue the growth arrest of C6?.

We have not attempted to complement the RPL22 knock out. But we do note that evidence supporting the idea that loss of RPL22 confers resistance to WIN site inhibitor is strong—six (out of six) sgRNAs against RPL22 were significantly enriched in the Tier 1 screen, and independent knock out of RPL22 with the Synthego multi-guide system in MV4;11 and MOLM13 cells increases the GI50 for C16.

Reviewer #2 (Public Review):

Summary:

The manuscript by Howard et al reports the development of high-affinity WDR5-interaction site inhibitors (WINi) that engage the protein to block the arginine-dependent engagement with its partners. Treatment of MLL-rearranged leukemia cells with high-affinity WINi (C16) decreases the expression of genes encoding most ribosomal proteins and other proteins required for translation. Notably, although these targets are enriched for WDR5-ChIP-seq peaks, such peaks are not universally present in the target genes. High concordance was found between the alterations in gene expression due to C16 treatment and the changes resulting from treatment with an earlier, lower affinity WINi (C6). Besides protein synthesis, genes involved in DNA replication or MYC responses are downregulated, while p53 targets and apoptosis genes are upregulated. Ribosome profiling reveals a global decrease in translational efficiency due to WINi with overall ribosome occupancies of mRNAs ~50% of control samples. The magnitude of the decrements of translation for most individual mRNAs exceeds the respective changes in mRNA levels genome-wide. From these results and other considerations, the authors hypothesize that WINi results in ribosome depletion. Quantitative mass spec documents the decrement in ribosomal proteins following WINi treatment along with increases in p53 targets and proteins involved in apoptosis occurring over 3 days. Notably, RPL22L1 is essentially completely lost upon WINi treatment. The investigators next conduct a CRISPR screen to find moderators and cooperators with WINi. They identify components of p53 and DNA repair pathways as mediators of WINi-inflicted cell death (so gRNAs against these genes permit cell survival). Next, WINi are tested in combination with a variety of other agents to explore synergistic killing to improve their expected therapeutic efficacy. The authors document the loss of the p53 antagonist MDM4 (in combination with splicing alterations of RPL22L1), an observation that supports the notion that WINi killing is p53-mediated.

Strengths:

This is a scientifically very strong and well-written manuscript that applies a variety of state-ofthe art molecular approaches to interrogate the role of the WDR5 interaction site and WINi. They reveal that the effects of WINi seem to be focused on the overall synthesis of protein components of the translation apparatus, especially ribosomal proteins-even those that do not bind WDR5 by ChIP (a question left unanswered is how much the WDR5-less genes are nevertheless WINi targeted). They convincingly show that disruption of the synthesis of these proteins is accompanied by DNA damage inferred by H2AX-activation, activation of the p53pathway, and apoptosis. Pathways of possible WINi resistance and synergies with other antineoplastic approaches are explored. These experiments are all well-executed and strongly invite more extensive pre-clinical and translational studies of WINi in animal studies. The studies also may anticipate the use of WINi as probes of nucleolar function and ribosome synthesis though this was not really explored in the current manuscript.

Weaknesses:

A mild deficiency in the current manuscript is the absence of cell biological methods to complement the molecular biological and biochemical approaches so ably employed. Some microscopic observations and confirmation of nucleolar dysfunction and DNA damage would be reassuring.

We thank the reviewer for their comments. We agree that an absence of cell biological methods was a deficiency in the original manuscript. In response to this comment, we have now added immunofluorescence (IF) analyses, examining the impact of C16 on nucleolar integrity and nucleophosmin (NPM1) distribution (Figure 3—figure supplement 4). These new data clearly show that C16 induces nucleolar stress at 72 hours—as measured by the redistribution of NPM1 from the nucleolus to the nucleoplasm. These new data fill an important gap in the story, and we are grateful to the reviewer for prompting us to perform these experiments.

As part of the above study, we also probed for gamma-H2AX, expecting that we may see some signs of accumulation in the nucleoli (see comment #4 from Reviewer #2, below). We did not observe this response. Importantly, however, we did see that gamma-H2AX staining occurs only in what are overtly apoptotic cells. This is an important finding, because we had previously speculated that the induction of gamma-H2AX observed by Western blotting reflected part of a bona-fide response to DNA damage elicited by WIN site inhibitors. Instead, the IF data now leads us to conclude that this signal simply reflects the established fact that WIN site inhibitors induce apoptosis in this cell line (Aho et al., 2019). In response to this new finding, we have added additional discussion to the text and have removed or de-emphasized the potential contribution of DNA damage to the mechanism of action of WDR5 WIN site inhibitors. Again, we are grateful for this comment as it has prevented us from continuing to report/pursue erroneous observations.

Recommendations for the authors

Reviewer #1 (Recommendations For The Authors):

There is a typo in "but are are linked to mRNA instability when translation is inhibited".

Thank you for catching this typo. It has now been corrected.

Reviewer #2 (Recommendations For The Authors):

(1) The authors report that WINi initially (at 24 hrs) increases the expression of most proteins while decreasing ribosomal proteins, but at 72 hours all proteins are depressed. The transient bump-up of non-translation-related proteins seems odd. A simple resolution to this somewhat strange observation is that there is no real increase in the other proteins, but because of the loss of a large fraction of the most abundant cellular proteins (the ribosomal proteins), the relative fraction of all other proteins is increased; that is, the increase of non-ribosomal proteins may be an artifact of normalization to a lower total protein content. Can this be explored?

We are grateful to the reviewer for this comment. We have tried our best to respond, as detailed above in response to Reviewer #1 Public Comment #2.

(2) It would be really nice to assess nucleolar status microscopically. Do nucleoli get bigger? Smaller? Do they have abnormal morphology? Is there nucleolar stress? What happens to rRNA synthesis and processing?

We agree and thank the reviewer for raising this point. As noted in our response to Reviewer #2, above, we have included new IF that shows: (i) no obvious effect on nucleolar integrity, (ii) redistribution of NPM1 to the nucleoplasm (indicative of nucleolar stress), and (iii) induction of gamma-H2AX staining in apoptotic cells (indicative of apoptosis).

Additionally, in response to this comment, we also looked at the impact of WIN site inhibitors on rRNA synthesis, using AzCyd labeling. These new data appear in Figure 3—figure supplement 3. Interestingly, these new data show that there is a progressive decline in rRNA synthesis, and that by 96 hours of treatment levels of both 18S and 28S rRNAs are reduced— again by about a factor of two. Our interpretation of this finding is that in response to the progressive decline in RPG transcription there is a secondary decrease in rRNA synthesis. This result is perhaps not surprising, but it does again add an important missing piece to our characterization of WIN site inhibitors and is further support for the concept that inhibition of ribosome production is a dominant part of the response to these agents.

(3) The WINi elicited DNA damage is incompletely characterized, rather it is inferred from H2AX activation. Comet assays would help to confirm such damage.

As noted in our response to Reviewer #2, our original inference of DNA damage, prompted by gamma-H2AX activation, is erroneous, and due instead to the ability of WIN site inhibitors to induce apoptosis. We thus did not pursue comet assays, etc., and removed discussion of potential DNA damage from the manuscript.

(4) Staining and microscopic observation of H2AX would be very useful. Is the WINi provoked DNA damage nucleolar-localized? Does the deficiency of ribosomal proteins lead to localized genotoxic nucleolar stress - or alternatively does the paucity of ribosomes and decreased translation lead to imbalances in other cellular pathways, perhaps including some involved in overall genome maintenance which would provoke more global DNA damage and H2AX staining, not limited to the nucleolus.

Again, please see our response to the Public Comment from Reviewer #2.

(5) It would be important to assess the influence and effects of WINi on some p53 mutant, p53-/- and p53 wild-type cell lines. Given their prevalence, p53 status may be expected to alter WINi efficacy.

The issue of how p53 status impacts the response to WINi is interesting and important, but we feel this is beyond the scope of the current manuscript. It is likely that many factors contribute to the response of cancer cells to these agents, and thus simply surveying some cancer lines for their response and linking this to their p53 status is unlikely to be very informative. Making definitive statements about the contribution of p53, and the differences between wild-type, lossof-function mutants, gain of function mutants, and null mutants will require more extensive analyses and is fertile territory for future studies, in our opinion.

eLife assessment

This important paper reveals that one of the major roles of the WDR5 WIN site is to promote ribosome synthesis, and that by attacking the WIN site with inhibitors ribosome attrition occurs creating new vulnerabilities that can be therapeutically exploited. This deficiency of ribosomal proteins also provokes the p53 response. The data from a variety of approaches is generally very convincing, and together buttresses the authors' conclusions and interpretations quite nicely; overall, this paper will provide a justification for pre-clinical and translational studies of WDR5 interaction site inhibitors.

Reviewer #1 (Public Review):

Building on previous work from the Tansey lab, here Howard et al. characterize transcriptional and translational changes upon WIN site inhibition of WDR5 in MLL-rearranged cancer cells. They first analyze whether C16, a newer generation compound, has the same cellular effects as C6, an early generation compound. Both compounds reduce the expression of WDR5-bound RPGs in addition to the unbound RPG RPL22L1. They then investigate differential translation by ribo-seq and observe that WIN site inhibition reduces the translational RPGs and other proteins related to biomass accumulation (spliceosome, proteasome, mitochondrial ribosome). Interestingly, this reduction adds to the transcriptional changes and is not limited to RPGs whose promoters are bound by WDR5. Quantitative proteomics at two time points confirmed the downregulation of RPGs. Interestingly, the overall effects are modest, but RPL22LA is strongly affected. Unexpectedly, most differentially abundant proteins seem to be upregulated 24 h after C6 (see below). A genetic screen showed that loss of p53 rescues the effect of C6 and C16 and helped the authors to identify pathways that can be targeted by compounds together with WIN site inhibitors in a synergistic way. Finally, the authors elucidated the underlying mechanisms and analyzed the functional relevance of the RPL22, RPL22L1, p53 and MDM4 axis.

Comments on revised version:

The authors have answered my points satisfactorily and the manuscript has become clearer and more meaningful as a result. In particular, the measurement of global translation rate is important and validates the upregulation of a number of proteins following WDR5 inhibitor treatment.

Reviewer #2 (Public Review):

Summary:

The manuscript by Howard et al reports the development of high affinity WDR5-interaction site inhibitors (WINi) that engage the protein to block the arginine-dependent engagement with its partners. Treatment of MLL-rearranged leukemia cells with high-affinity WINi (C16) decreases the expression of genes encoding most ribosomal proteins and other proteins required for translation. Notably, although these targets are enriched for WDR5-ChIP-seq peaks, such peaks are not universally present in the target genes. High concordance was founded between the alterations in gene expression due to C16 treatment and the changes resulting from treatment with an earlier, lower affinity WINi (C6). Besides protein synthesis, genes involved in DNA replication or MYC responses are downregulated, while p53 targets and apoptosis genes are upregulated. Ribosome profiling reveals a global decrease in translational efficiency due to WINi with overall ribosome occupancies of mRNAs ~50% of control samples. The magnitude in the decrements of translation for most individual mRNAs exceeds the respective changes in mRNA levels genome-wide. From these results and other considerations, the authors hypothesize that WINi results in ribosome depletion. Quantitative mass spec documents the decrement in ribosomal proteins following WINi treatment along with increases in p53 targets and proteins involved in apoptosis occurring over 3 days. Notably RPL22L1 is essentially completely lost upon WINi treatment. The investigators next conduct a CRISPR screen to find moderators and cooperators with WINi. They identify components of p53 and DNA repair pathways as mediators of WINi inflicted cell death (so gRNAs against these genes permit cell survival). Next, WINi are tested in combination with a variety of other agents to explore synergistic killing to improve their expected therapeutic efficacy. The authors document loss of the p53 antagonist MDM4 (in combination with splicing alterations of RPL22L1), an observation that supports the notion that WINi killing is p53-mediated.

This is a scientifically very strong and well-written manuscript that applies a variety of state-of-the art molecular approaches to interrogate the role of the WDR5 interaction site and WINi. They reveal that the effects of WINi seem to be focused on the overall synthesis of protein components of the translation apparatus, especially ribosomal proteins-even those that do not bind WDR5 by ChIP (a question left unanswered is how such the WDR5-less genes are nevertheless WINi targeted). They convincingly show that disruption of the synthesis of these proteins occurs upon activation of p53 dependent apoptosis, likely driven by unbalanced ribosomal protein synthesis leading to MDM2 inhibition. This apoptosis is subsequently followed, as expected by ɣH2AX-activation. Pathways of possible WINi resistance and synergies with other anti-neoplastic approaches are explored. These experiments are all well-executed and strongly invite more extensive pre-clinical and translational studies of WINi in animal studies. The studies also may anticipate the use of WINi as probes of nucleolar function and ribosome synthesis though this was not really explored in the current manuscript. The current version of the manuscript documents ribosomal stress revealed by leakage of NPM1 into the nucleoplasm while nucleolar integrity is preserved. A progressive loss of rRNA synthesis occurs upon drug treatment that is presumably secondary to the decrement in ribosomal protein production.

Comments on revised version:

(1) The authors to my mind, have quite nicely and professionally addressed the comments of the reviewers and are to be congratulated on an important contribution to the elucidation of WDR5 biology and pathology.

Author response:

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

eLife assessment

This is a useful study examining the determinants and mechanisms of LRMP inhibi:on of cAMP regula:on of HCN4 channel ga:ng. The evidence provided to support the main conclusions is unfortunately incomplete, with discrepancies in the work that reduce the strength of mechanis:c insights.

Thank you for the reviews of our manuscript. We have made a number of changes to clarify our hypotheses in the manuscript and addressed all of the poten:al discrepancies by revising some of our interpreta:on. In addi:on, we have provided addi:onal experimental evidence to support our conclusions. Please see below for a detailed response to each reviewer comment.

Public Reviews

Reviewer #1 (Public Review):

Summary:

The authors use truncations, fragments, and HCN2/4 chimeras to narrow down the interaction and regulatory domains for LRMP inhibition of cAMP-dependent shifts in the voltage dependence of activation of HCN4 channels. They identify the N-terminal domain of HCN4 as a binding domain for LRMP, and highlight two residues in the C-linker as critical for the regulatory effect. Notably, whereas HCN2 is normally insensitive to LRMP, putting the N-terminus and 5 additional C-linker and S5 residues from HCN4 into HCN2 confers LRMP regulation in HCN2.

Strengths:

The work is excellent, the paper well written, and the data convincingly support the conclusions which shed new light on the interaction and mechanism for LRMP regulation of HCN4, as well as identifying critical differences that explain why LRMP does not regulate other isoforms such as HCN2.

Thank you.

Reviewer #2 (Public Review):

Summary:

HCN-4 isoform is found primarily in the sino-atrial node where it contributes to the pacemaking activity. LRMP is an accessory subunit that prevents cAMP-dependent potentiation of HCN4 isoform but does not have any effect on HCN2 regulation. In this study, the authors combine electrophysiology, FRET with standard molecular genetics to determine the molecular mechanism of LRMP action on HCN4 activity. Their study shows that parts of N- and C-termini along with specific residues in C-linker and S5 of HCN4 are crucial for mediating LRMP action on these channels. Furthermore, they show that the initial 224 residues of LRMP are sufficient to account for most of the activity. In my view, the highlight of this study is Fig. 7 which recapitulates LRMP modulation on HCN2-HCN4 chimera. Overall, this study is an excellent example of using time-tested methods to probe the molecular mechanisms of regulation of channel function by an accessory subunit.

Weaknesses:

(1) Figure 5A- I am a bit confused with this figure and perhaps it needs better labeling. When it states Citrine, does it mean just free Citrine, and "LRMP 1-230" means LRMP fused to Citrine which is an "LF" construct? Why not simply call it "LF"? If there is no Citrine fused to "LRMP 1-230", this figure would not make sense to me.

We have clarified the labelling of this figure and specifically defined all abbreviations used for HCN4 and LRMP fragments in the results section on page 14.

(2) Related to the above point- Why is there very little FRET between NF and LRMP 1-230? The FRET distance range is 2-8 nm which is quite large. To observe baseline FRET for this construct more explanation is required. Even if one assumes that about 100 amino are completely disordered (not extended) polymers, I think you would still expect significant FRET.

FRET is extremely sensitive to distance (to the 6th power of distance). The difference in contour length (maximum length of a peptide if extended) between our ~260aa fragment and our ~130 aa fragments is on the order of 450Å (45nm), So, even if not extended it is not hard to imagine that the larger fragments show a weaker FRET signal. In fact, we do see a slightly larger FRET than we do in control (not significant) which is consistent with the idea that the larger fragments just do not result in a large FRET.

Moreover, this hybridization assay is sensitive to a number of other factors including the affinity between the two fragments, the expression of each fragment, and the orientation of the fluorophores. Any of these factors could also result in reduced FRET.

We have added a section on the limitations of the FRET 2-hybrid assay in the discussion section on page 20. Our goal with the FRET assay was to provide complimentary evidence that shows some of the regions that are important for direct association and we have edited to the text to make sure we are not over-interpreting our results.

(3) Unless I missed this, have all the Cerulean and Citrine constructs been tested for functional activity?

All citrine-tagged LRMP constructs (or close derivatives) were tested functionally by coexpression with HCN (See Table 1 and pages 10-11). Cerulean-tagged HCN4 fragments are of course intrinsically not-functional as they do not include the ion conducting pore.

Reviewer #3 (Public Review):

Summary:

Using patch clamp electrophysiology and Förster resonance energy transfer (FRET), Peters and co-workers showed that the disordered N-terminus of both LRMP and HCN4 are necessary for LRMP to interact with HCN4 and inhibit the cAMP-dependent potentiation of channel opening. Strikingly, they identified two HCN4-specific residues, P545 and T547 in the C-linker of HCN4, that are close in proximity to the cAMP transduction centre (elbow Clinker, S4/S5-linker, HCND) and account for the LRMP effect.

Strengths:

Based on these data, the authors propose a mechanism in which LRMP specifically binds to HCN4 via its isotype-specific N-terminal sequence and thus prevents the cAMP transduction mechanism by acting at the interface between the elbow Clinker, the S4S5-linker, the HCND.

Weaknesses:

Although the work is interesting, there are some discrepancies between data that need to be addressed.

(1) I suggest inserting in Table 1 and in the text, the Δ shift values (+cAMP; + LRMP; +cAMP/LRMP). This will help readers.

Thank you, Δ shift values have been added to Tables 1 and 2 as suggested.

(2) Figure 1 is not clear, the distribution of values is anomalously high. For instance, in 1B the distribution of values of V1/2 in the presence of cAMP goes from - 85 to -115. I agree that in the absence of cAMP, HCN4 in HEK293 cells shows some variability in V1/2 values, that nonetheless cannot be so wide (here the variability spans sometimes even 30 mV) and usually disappears with cAMP (here not).

With a large N, this is an expected distribution. In 5 previous reports from 4 different groups of HCN4 with cAMP in HEK 293 (Fenske et al., 2020; Liao et al., 2012; Peters et al., 2020; Saponaro et al., 2021; Schweizer et al., 2010), the average expected range of the data is 26.6 mV and 39.9 mV for 95% (mean ± 2SD) and 99% (mean ± 3SD) of the data, respectively. As the reviewer mentions the expected range from these papers is slightly larger in the absence of cAMP. The average SD of HCN4 (with/without cAMP) in papers are 9.9 mV (Schweizer et al., 2010), 4.4 mV (Saponaro et al., 2021), 7.6 mV (Fenske et al., 2020), 10.0 mV (Liao et al., 2012), and 5.9 mV (Peters et al., 2020). Our SD in this paper is roughly in the middle at 7.6 mV. This is likely because we used an inclusive approach to data so as not to bias our results (see the statistics section of the revised manuscript on page 9). We have removed 2 data points that meet the statistical classification as outliers, no measures of statistical significance were altered by this.

This problem is spread throughout the manuscript, and the measured mean effects are indeed always at the limit of statistical significance. Why so? Is this a problem with the analysis, or with the recordings?

The exact P-values are NOT typically at the limit of statistical significance, about 2/3rds would meet the stringent P < 0.0001 cut-off. We have clarified in the statistics section (page 10) that any comparison meeting our significance threshold (P < 0.05) or a stricter criterion is treated equally in the figure labelling. Exact P-values are provided in Tables 1-3.

There are several other problems with Figure 1 and in all figures of the manuscript: the Y scale is very narrow while the mean values are marked with large square boxes. Moreover, the exemplary activation curve of Figure 1A is not representative of the mean values reported in Figure 1B, and the values of 1B are different from those reported in Table 1.

Y-axis values for mean plots were picked such that all data points are included and are consistent across all figures. They have been expanded slightly (-75 to -145 mV for all HCN4 channels and -65 to -135 mV for all HCN2 channels). The size of the mean value marker has been reduced slightly. Exact midpoints for all data are also found in Tables 1-3.

The GV curves in Figure 1B (previously Fig. 1A) are averages with the ±SEM error bars smaller than the symbols in many cases owing to relatively high n’s for these datasets. These curves match the midpoints in panel 1C (previously 1B). Eg. the midpoint of the average curve for HCN4 control in panel A is -117.9 mV, the same as the -117.8 mV average for the individual fits in panel B.

We made an error in the text based on a previous manuscript version about the ordering of the tables that has now been fixed so these values should now be aligned.

On this ground, it is difficult to judge the conclusions and it would also greatly help if exemplary current traces would be also shown.

Exemplary current traces have been added to all figures in the revised manuscript.

(3) "....HCN4-P545A/T547F was insensitive to LRMP (Figs. 6B and 6C; Table 1), indicating that the unique HCN4 C-linker is necessary for regulation by LRMP. Thus, LRMP appears to regulate HCN4 by altering the interactions between the C-linker, S4-S5 linker, and Nterminus at the cAMP transduction centre."

Although this is an interesting theory, there are no data supporting it. Indeed, P545 and T547 at the tip of the C-linker elbow (fig 6A) are crucial for LRMP effect, but these two residues are not involved in the cAMP transduction centre (interface between HCND, S4S5 linker, and Clinker elbow), at least for the data accumulated till now in the literature. Indeed, the hypothesis that LRMP somehow inhibits the cAMP transduction mechanism of HCN4 given the fact that the two necessary residues P545 and T547 are close to the cAMP transduction centre, remains to be proven.

Moreover, I suggest analysing the putative role of P545 and T547 in light of the available HCN4 structures. In particular, T547 (elbow) points towards the underlying shoulder of the adjacent subunit and, therefore, is in a key position for the cAMP transduction mechanism. The presence of bulky hydrophobic residues (very different nature compared to T) in the equivalent position of HCN1 and HCN2 also favours this hypothesis. In this light, it will be also interesting to see whether a single T547F mutation is sufficient to prevent the LRMP effect.

We agree that testing this hypothesis would be very interesting. However, it is challenging. Any mutation we make that is involved in cAMP transduction makes measuring the LRMP effect on cAMP shifts difficult or impossible.

Our simple idea, now clarified in the discussion, is that if you look at the regions involved in cAMP transduction (HCND, C-linker, S4-S5), there are very few residues that differ between HCN4 and HCN2. When we mutate the 5 non-conserved residues in the S5 segment and the C-linker, along with the NT, we are able to render HCN2 sensitive to LRMP. Therefore, something about the small sequence differences in this region confer isoform specificity to LRMP. We speculate that this happens because of small structural differences that result from those 5 mutations. If you compare the solved structures of HCN1 and HCN4 (there is no HCN2 structure available), you can see small differences in the distances between key interacting residues in the transduction centre. Also, there is a kink at the bottom of the S4 helix in HCN4 but not HCN1. This points a putatively important residue for cAMP dependence in a different direction in HCN4. We hypothesize in the discussion that this may be how LRMP is isoform specific.

Moreover, previous work has shown that the HCN4 C-linker is uniquely sensitive to di-cyclic nucleotides and magnesium ions. We are hypothesizing that it is the subtle change in structure that makes this region more prone to regulation in HCN4.

Reviewing Editor (recommendations for the Authors):

(1) Exemplar recordings need to be shown and some explanation for the wide variability in the V-half of activation.

Exemplar currents are now shown for each channel. See the response to Reviewer 3’s public comment 2.

(2) The rationale for cut sites in LRMP for the investigation of which parts of the protein are important for blocking the effect of cAMP is not logically presented in light of the modular schematics of domains in the protein (N-term, CCD, post-CCD, etc).

There is limited structural data on LRMP and the HCN4 N-terminus. The cut sites in this paper were determined empirically. We made fragments that were small enough to work for our FRET hybridization approach and that expressed well in our HEK cell system. The residue numbering of the LRMP modules is based on updated structural predictions using Alphafold, which was released after our fragments were designed. This has been clarified in the methods section on pages 5-6 and the Figure 2 legend of the revised manuscript.

(3) Role of the HCN4 C-terminus. Truncation of the HCN4 C-terminus unstructured Cterminus distal to the CNBD (Fig. 4 A, B) partially reverses the impact of LRMP (i.e. there is now a significant increase in cAMP effect compared to full-length HCN4). The manuscript is written in a manner that minimizes the potential role of the C-terminus and it is, therefore, eliminated from consideration in subsequent experiments (e.g. FRET) and the discussion. The model is incomplete without considering the impact of the C-terminus.

We thank the reviewer for this comment as it was a result that we too readily dismissed. We have added discussion around this point and revised our model to suggest that not only can we not eliminate a role for the distal C-terminus, our data is consistent with it having a modest role. Our HCN4-2 chimera and HCN4-S719x data both suggest the possibility that the distal C-terminus might be having some effect on LRMP regulation. We have clarified this in the results (pages 12-13) and discussion (page 19).

(4) For FRET experiments, it is not clear why LF should show an interaction with N2 (residues 125-160) but not NF (residues 1-160). N2 is contained within NF, and given that Citrine and Cerulean are present on the C-terminus of LF and N2/NF, respectively, residues 1-124 in NF should not impact the detection of FRET because of greater separation between the fluorophores as suggested by the authors.

This is a fair point but FRET is somewhat more complicated. We do not know the structure of these fragments and it’s hard to speculate where the fluorophores are oriented in this type of assay. Moreover, this hybridization assay is sensitive to affinity and expression as well. There are a number of reasons why the larger 1-260 fragment might show reduced FRET compared to 125-260. As mentioned in our response to reviewer 2’s public comment 2, we have added a limitation section that outlines the various caveats of FRET that could explain this.

(5) For FRET experiments, the choice of using pieces of the channel that do not correlate with the truncations studied in functional electrophysiological experiments limits the holistic interpretation of the data. Also, no explanation or discussion is provided for why LRMP fragments that are capable of binding to the HCN4 N-terminus as determined by FRET (e.g. residues 1-108 and 110-230, respectively) do not have a functional impact on the channel.

As mentioned in the response to comment 2, the exact fragment design is a function of which fragments expressed well in HEK cells. Importantly, because FRET experiments do not provide atomic resolution for the caveats listed in the revised limitations section on page 20-21, small differences in the cut sites do not change the interpretation of these results. For example, the N-terminal 1-125 construct is analogous to experiments with the Δ1-130 HCN4 channel.

We suspect that residues in both fragments are required and that the interaction involves multiple parts. This is stated in the results “Thus, the first 227 residues of LRMP are sufficient to regulate HCN4, with residues in both halves of the LRMP N-terminus necessary for the regulation” (page 11). We have also added discussion on this on page 21.

(6) A striking result was that mutating two residues in the C-linker of HCN4 to amino acids found in HCN channels not affected by LRMP (P545A, T547F), completely eliminated the impact of LRMP on preventing cAMP regulation of channel activation. However, a chimeric channel, (HCN4-2) in which the C-linker, the CNBD, and the C-terminus of HCN4 were replaced by that of HCN2 was found to be partially responsive to LRMP. These two results appear inconsistent and not reconciled in the model proposed by the authors for how LRMP may be working.

As stated in our answer to your question #3, we have revised our interpretation of these data. If the more distal C-terminus plays some role in the orientation of the C-linker and the transduction centre as a whole, these data can still be viewed consistent with our model. We have added some discussion of this idea in our discussion section.

(7) Replacing the HCN2 N-terminus with that from HCN4, along with mutations in the S5 (MCS/VVG) and C-linker (AF/PT) recapitulated LRMP regulation on the HCN2 background. The functional importance of the S5 mutations is not clear as no other experiments are shown to indicate whether they are necessary for the observed effect.

We have added our experiments on a midpoint HCN2 clone that includes the S5 mutants and the C-linker mutants in the absence of the HCN4 N-terminus (ie HCN2 MCSAF/VVGPT) (Fig. 7). And we have discussed our rationale for the S5 mutations as we believe they may be responsible for the different orientations of the S4-S5 linker in HCN1 and HCN4 structures that are known to impact cAMP regulation.

Reviewer #1 (Recommendations For The Authors):

A) Comments:

(1) Figure 1: Please show some representative current traces.

Exemplar currents are now shown for each channel in the manuscript.

(2) Figure 1: There appears to be a huge number of recordings for HCN4 +/- cAMP as compared to those with LRMP 1-479Cit. How was the number of recordings needed for sufficient statistical power decided? This is particularly important because the observed slowing of deactivation by cAMP in Fig. 1C seems like it may be fairly subtle. Perhaps a swarm plot would make the shift more apparent? Also, LRMP 1-479Cit distributions in Fig. 1B-C look like they are more uniform than normal, so please double-check the appropriateness of the statistical test employed.

We have revised the methods section (page 7) to discuss this, briefly we performed regular control experiments throughout this project to ensure that a normal cAMP response was occurring. Our minimum target for sufficient power was 8-10 recordings. We have expanded the statistics section (page 9) to discuss tests of normality and the use of a log scale for deactivation time constants which is why the shifts in Fig. 1D (revised) are less apparent.

(3) It would be helpful if the authors could better introduce their logic for the M338V/C341V/S345G mutations in the HCN4-2 VVGPT mutant.

See response to the reviewing editor’s comment 7.

B) Minor Comments:

(1) pg. 9: "We found that LRMP 1-479Cit inhibited HCN4 to an even greater degree than the full-length LRMP, likely because expression of this tagged construct was improved compared to the untagged full-length LRMP, which was detected by co-transfection with GFP." Co-transfection with GFP seems like an extremely poor and a risky measure for LRMP expression.

We agree that the exact efficiency of co-transfection is contentious although some papers and manufacturer protocols indicate high co-transfection efficiency (Xie et al., 2011). In this paper we used both co-transfection and tagged proteins with similar results.

(2) pg 9: "LRMP 1-227 construct contains the N-terminus of LRMP with a cut-site near the Nterminus of the predicted coiled-coil sequence". In Figure 2 the graphic shows the coiledcoil domain starting at 191. What was the logic for splitting at 227 which appears to be the middle of the coiled-coil?

See response to the reviewing editor’s comment 2.

(3) Figure 5C: Please align the various schematics for HCN4 as was done for LRMP. It makes it much easier to decipher what is what.

Fig. 5 has been revised as suggested.

(4) pg 12: I assume that the HCN2 fragment chosen aligns with the HCN4 N2 fragment which shows binding, but this logic should be stated if that is the case. If not, then how was the HCN2 fragment chosen?

This is correct. This has been explicitly stated in the revised manuscript (page 14).

(5) Figure 7: Add legend indicating black/gray = HCN4 and blue = HCN2.

This has been stated in the revised figure legend.

(6) pg 17: Conservation of P545 and T547 across mammalian species is not shown or cited.

This sentence is not included in the revised manuscript, however, for the interest of the reviewer we have provided an alignment of this region across species here.

Author response image 1.

Reviewer #2 (Recommendations For The Authors):

(1) It is not clear whether in the absence of cAMP, LRMP also modestly shifts the voltagedependent activity of the channels. Please clarify.

We have clarified that LRMP does not shift the voltage-dependence in the absence of cAMP (page 10). In the absence of cAMP, LRMP does not significantly shift the voltagedependence of activation in any of the channels we have tested in this paper (or in our prior 2020 paper).

(2) Resolution of Fig. 8b is low.

We ultimately decided that the cartoon did not provide any important information for understanding our model and it was removed.

(3) Please add a supplementary figure showing the amino acid sequence of LRMP to show where the demarcations are made for each fragment as well as where the truncations were made as noted in Fig 3 and Fig 4.

A new supplementary figure showing the LRMP sequence has been added and cited in the methods section (page 5). Truncation sites have been added to the schematic in Fig. 2A.

(4) In the cartoon schematic illustration for Fig. 3 and Fig.4, the legend should include that the thick bold lines in the C-Terminal domain represent the CNBD, while the thick bold lines in the N-Terminal domain represent the HCN domain. This was mentioned in Liao 2012, as you referenced when you defined the construct S719X, but it would be nice for the reader to know that the thick bold lines you have drawn in your cartoon indicate that it also highlights the CNBD or the HCN domain.

This has been added to figure legends for the relevant figures in the revised manuscript.

(5) On page 12, missing a space between "residues" and "1" in the parenthesis "...LRMP L1 (residues1-108)...".

Fixed. Thank you.

(6) Which isoform of LRMP was used? What is the NCBI accession number? Is it the same one from Peters 2020 ("MC228229")?

This information has been added to the methods (page 5). It is the same as Peters 2020.

Reviewer #3 (Recommendations For The Authors):

(1) "Truncation of residues 1-62 led to a partial LRMP effect where cAMP caused a significant depolarizing shift in the presence of LRMP, but the activation in the presence of LRMP and cAMP was hyperpolarized compared to cAMP alone (Fig. 3B, C and 3E; Table 1). In the HCN4Δ1-130 construct, cAMP caused a significant depolarizing shift in the presence of LRMP; however, the midpoint of activation in the presence of LRMP and cAMP showed a non-significant trend towards hyperpolarization compared to cAMP alone (Fig. 3C and 3E; Table 1)".

This means that sequence 62-185 is necessary and sufficient for the LRMP effect. I suggest a competition assay with this peptide (synthetic, or co-expressed with HCN4 full-length and LRMP to see whether the peptide inhibits the LRMP effect).

We respectfully disagree with the reviewer’s interpretation. Our results, strongly suggest that other regions such as residues 25-65 (Fig. 3C) and C-terminal residues (Fig. 6) are also necessary. The use of a peptide could be an interesting future experiment, however, it would be very difficult to control relative expression of a co-expressed peptide. We think that our results in Fig. 7E-F where this fragment is added to HCN2 are a better controlled way of validating the importance of this region.

(2) "Truncation of the distal C-terminus (of HCN4) did not prevent LRMP regulation. In the presence of both LRMP and cAMP the activation of HCN4-S719X was still significantly hyperpolarized compared to the presence of cAMP alone (Figs. 4A and 4B; Table 1). And the cAMP-induced shift in HCN4-S719X in the presence of LRMP (~7mV) was less than half the shift in the absence of LRMP (~18 mV)."

On the basis of the partial effects reported for the truncations of the N-terminus of HCN4 162 and 1-130 (Fig 3B and C), I do not think it is possible to conclude that "truncation of the distal C-terminus (of HCN4) did not prevent LRMP regulation". Indeed, cAMP-induced shift in HCN4 Δ1-62 and Δ1-130 in the presence of LRMP were 10.9 and 10.5 mV, respectively, way more than the ~7mV measured for the HCN4-S719X mutant.

As you rightly stated at the end of the paragraph:" Together, these results show significant LRMP regulation of HCN4 even when the distal C-terminus is truncated, consistent with a minimal role for the C-terminus in the regulatory pathway". I would better discuss this minimal role of the C-terminus. It is true that deletion of the first 185 aa of HCN4 Nterminus abolishes the LRMP effect, but it is also true that removal of the very Cterm of HCN4 does affect LRMP. This unstructured C-terminal region of HCN4 contains isotype-specific sequences. Maybe they also play a role in recognizing LRMP. Thus, I would suggest further investigation via truncations, even internal deletions of HCN4-specific sequences.

Please see the response to the reviewing editor’s comment 3.

(3) Figure 5: The N-terminus of LRMP FRETs with the N-terminus of HCN4.

Why didn't you test the same truncations used in Fig. 3? Indeed, based on Fig 3, sequences 1-25 can be removed. I would have considered peptides 26-62 and 63-130 and 131-185 and a fourth (26-185). This set of peptides will help you connect binding with the functional effects of the truncations tested in Fig 3.

Please see the response to the reviewing editor’s comment 2 and 5.

Why didn't you test the C-terminus (from 719 till the end) of HCN4? This can help with understanding why truncation of HCN4 Cterminus does affect LRMP, tough partially (Fig. 4A).

Please see the response to the reviewing editor’s comment 3.

(4) "We found that a previously described HCN4-2 chimera containing the HCN4 N-terminus and transmembrane domains (residues 1-518) with the HCN2 C-terminus (442-863) (Liao et al., 2012) was partially regulated by LRMP (Fig. 7A and 7B)".

I do not understand this partial LRMP effect on the HCN4-2 chimera. In Fig. 6 you have shown that the "HCN4-P545A/T547F was insensitive to LRMP (Figs. 6B and 6C; Table 1), indicating that the unique HCN4 C-linker is necessary for regulation by LRMP". How can be this reconciled with the HCN4-2 chimera? HCN4-2, "containing" P545A/T547F mutations, should not perceive LRMP.

Please see the response to the reviewing editor’s comment 6.

(5) "we next made a targeted chimera of HCN2 that contains the distal HCN4 N-terminus (residues 1-212) and the HCN2 transmembrane and C-terminal domains with 5 point mutants in non-conserved residues of the S5 segment and C-linker elbow (M338V/C341V/S345G/A467P/F469T)......Importantly, the HCN4-2 VVGPT channel is insensitive to cAMP in the presence of LRMP (Fig. 7C and 7D), indicating that the HCN4 Nterminus and cAMP-transduction centre residues are sufficient to confer LRMP regulation to HCN2".

Why did you insert also the 3 mutations of S5? Are these mutations somehow involved in the cAMP transduction mechanism?

You have already shown that in HCN4 only P545 and T547 (Clinker) are necessary for LRMP effect. I suggest to try, at least, the chimera of HCN2 with only A467P/F469T. They should work without the 3 mutations in S5.

Please see the response to the reviewing editor’s comment 7.

eLife assessment

This study identifies the molecular determinants of LRMP co-regulation of HCN 4 activity. The evidence supporting the conclusions, which is compelling, is backed by rigorous electrophysiological and spectroscopic analysis. The work is important because it greatly enhances our understanding of the mechanisms of HCN channel regulation in a tissue-specific manner and highlights a functional role for more disordered regions that have yet to be structurally resolved.

Reviewer #1 (Public Review):

Summary:

The authors use truncations, fragments, and HCN2/4 chimeras to narrow down the interaction and regulatory domains for LRMP inhibition of cAMP-dependent shifts in the voltage dependence of activation of HCN4 channels. They identify the N-terminal domain of HCN4 as a binding domain for LRMP, and highlight two residues in the C-linker as critical for the regulatory effect. Notably, whereas HCN2 is normally insensitive to LRMP, putting the N-terminus and 5 additional C-linker and S5 residues from HCN4 into HCN2 confers LRMP regulation in HCN2.

Strengths:

The work is excellent, the paper well written, and the data convincingly support the conclusions which shed new light on the interaction and mechanism for LRMP regulation of HCN4, as well as identifying critical differences that explain why LRMP does not regulate other isoforms such as HCN2.

Reviewer #2 (Public Review):

Summary:

HCN-4 isoform is found primarily in sino-atrial node where it contributes to the pacemaking activity. LRMP is an accessory subunit which prevents cAMP-dependent potentiation of HCN4 isoform but does not have any effect on HCN2 regulation. In this study, the authors combine electrophysiology, FRET with standard molecular genetics to determine the molecular mechanism of LRMP action on HCN4 activity. Their study shows parts of N- and C-termini along with specific residues in C-linker and S5 of HCN4 are crucial for mediating LRMP action on these channels. Furthermore, they show that the initial 224 residues of LRMP are sufficient to account for most of the activity. In my view, the highlight of this study is Fig. 7 which recapitulates LRMP modulation on HCN2-HCN4 chimera. Overall, this study is an excellent example of using time-tested methods to probe the molecular mechanisms of regulation of channel function by an accessory subunit.

The authors adequately addressed my earlier concerns.

Reviewer #3 (Public Review):

Summary:

Using patch clamp electrophysiology and Förster resonance energy transfer (FRET), Peters and co-workers showed that the disordered N-terminus of both LRMP and HCN4 are necessary for LRMP to interact with HCN4 and inhibit the cAMP-dependent potentiation of channel opening. Strikingly, they identified two HCN4-specific residues, P545 and T547 in the C-linker of HCN4, that are close in proximity to the cAMP transduction centre (elbow Clinker, S4/S5-linker, HCND) and account for the LRMP effect.

Strengths:

Based on these data, the Authors propose a mechanism in which LRMP specifically binds to HCN4 via its isotype-specific Nterminal sequence and thus prevents the cAMP transduction mechanism by acting at the interface between the elbow Clinker, the S4S5-linker, the HCND.

Weaknesses:

Although the work is interesting, there are some discrepancies between data that need to be addressed.

- I suggest inserting in Table 1 and in the text, the Δ shift values (+cAMP; + LRMP; +cAMP/LRMP). This will help readers.

- Figure 1 is not clear, the distribution of values is anomalously high. For instance, in 1B the distribution of values of V1/2 in the presence of cAMP goes from - 85 to -115. I agree that in the absence of cAMP, HCN4 in HEK293 cells shows some variability in V1/2 values, that nonetheless cannot be so wide (here the variability spans sometimes even 30 mV) and usually disappears with cAMP (here not).<br /> This problem is spread throughout the ms, and the measured mean effects indeed always at the limit of statistical significance. Why so? Is this a problem with the analysis, or with the recordings?<br /> There are several other problems with Figure 1 and in all figures of the ms: the Y scale is very narrow while the mean values are marked with large square boxes. Moreover, the exemplary activation curve of Fig 1A is not representative of the mean values reported in Figure 1B, and the values of 1B are different from those reported in Table 1.<br /> On this ground it is difficult to judge the conclusions and it would also greatly help if exemplary current traces would also be shown.

- "....HCN4-P545A/T547F was insensitive to LRMP (Figs. 6B and 6C; Table 1), indicating that the unique HCN4 C-linker is necessary for regulation by LRMP. Thus, LRMP appears to regulate HCN4 by altering the interactions between the C-linker, S4-S5 linker, and N-terminus at the cAMP transduction centre."

Although this is an interesting theory, there are no data supporting it. Indeed, P545 and T547 at the tip of the C-linker elbow (fig 6A) are crucial for LRMP effect, but these two residues are not involved in the cAMP transduction centre (interface between HCND, S4S5 linker and Clinker elbow), at least for the data accumulated till now in the literature. Indeed, the hypothesis that LRMP somehow inhibits the cAMP transduction mechanism of HCN4 given the fact that the two necessary residues P545 and T547 are close to the cAMP transduction centre, awaits to be proven.

Moreover, I suggest analysing the putative role of P545 and T547 in the light of the available HCN4 structures. In particular, T547 (elbow) point towards the underlying shoulder of the adjacent subunit and, therefore, it is in a key position for the cAMP transduction mechanism. The presence of bulky hydrophobic residues (very different nature compared to T) in the equivalent position of HCN1 and HCN2 is also favouring this hypothesis. In this light, it will also be interesting to see whether single T547F mutation is sufficient to prevent LRMP effect.

Madagascar debt

Author response:

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

Reviewer #1 (Public Review):

Summary:

In this study, Pan DY et al. discovered that the clearance of senescent osteoclasts can lead to a reduction in sensory nerve innervation. This reduction is achieved through the attenuation of Netrin-1 and NGF levels, as well as the regulation of H-type vessels, resulting in a decrease in pain-related behavior. The experiments are well-designed. The results are clearly presented, and the legends are also clear and informative. Their findings represent a potential treatment for spine pain utilizing senolytic drugs.

Strengths:

Rigorous data, well-designed experiments as well as significant innovation make this manuscript stand out.

Weaknesses:

Quantification of histology and detailed statistical analysis will further strengthen this manuscript.

I have the following specific comments.

(1) Since defining senescent cells solely based on one or two markers (SA-β-gal and p16) may not provide a robust characterization, it would be advisable to employ another wellestablished senescence marker, such as γ-H2AX or HMGB1, to corroborate the observed increase in senescent osteoclasts following LSI and aging.

We value the comments provided by the reviewer. In accordance with your suggestion, we have performed co-staining of HMGB1 with Trap in Supplementary Figure 1 to corroborate the observed augmentation of senescent osteoclasts following LSI and aging.

Author response image 1.

(2) The connection between heightened Netrin-1 secretion by senescent osteoclasts following LSI or aging and its relevance to pain warrants thorough discussion within the manuscript to provide a comprehensive understanding of the entire narrative.

We appreciate the reviewer's insightful comments. We have thoroughly addressed the entire narrative in the revised manuscript, as outlined below:

During lumbar spine instability (LSI) or aging, endplates undergo ossification, leading to elevated osteoclast activity and increased porosity1-4. The progressive porous transformation of endplates, accompanied by a narrowed intervertebral disc (IVD) space, is a hallmark of spinal degeneration4,5. Considering that pain arises from nociceptors, it is plausible that low back pain (LBP) may be attributed to sensory innervation within endplates. Additionally, porous endplates exhibit higher nerve density compared to normal endplates or degenerative nucleus pulposus6. Netrin-1, a crucial axon guidance factor facilitating nerve protrusion, has been implicated in this process7-9. The receptor mediating Netrin-1-induced neuronal sprouting, deleted in colorectal cancer (DCC), was found to co-localize with CGRP+ sensory nerve fibers in endplates after LSI surgery10,11. In summary, during LSI or aging, osteoclastic lineage cells secrete Netrin-1, inducing extrusion and innervation of CGRP+ sensory nerve fibers within the spaces created by osteoclast resorption. This Netrin-1/DCC-mediated pain signal is subsequently transmitted to the dorsal root ganglion (DRG) or higher brain levels.

(3) It appears that the quantitative data for TRAP staining in Figure 1j is missing.

We appreciate the reviewer's comments. We have added the statistical data of TRAP staining (Figure. 1p) to Figure 1 in the revised manuscript.

Author response image 2.

(4) Regarding Figure 6, could you please specify which panels were analyzed using a t-test and which ones were subjected to ANOVA? Alternatively, were all the panels in Figure 6 analyzed using ANOVA?

We appreciate the reviewer’s comments here. Upon careful review, we have ensured that quantitative data in panels b, c, and f are analyzed using t-tests, while panels d, e, and g are subjected to one-way ANOVA. These updates have been reflected in the revised figure legend.

Reviewer #2 (Public Review):

Summary:

This manuscript examined the underlying mechanisms between senescent osteoclasts (SnOCs) and lumbar spine instability (LSI) or aging. They first showed that greater numbers of SnOCs are observed in mouse models of LSI or aging, and these SnOCs are associated with induced sensory nerve innervation, as well as the growth of H-type vessels, in the porous endplate. Then, the deletion of senescent cells by administration of the senolytic drug Navitoclax (ABT263) results in significantly less spinal hypersensitivity, spinal degeneration, porosity of the endplate, sensory nerve innervation, and H-type vessel growth in the endplate. Finally, they also found that there is greater SnOCmediated secretion of Netrin-1 and NGF, two well-established sensory nerve growth factors, compared to non-senescent OCs. The study is well conducted and data strongly support the idea. However, some minor issues need to be addressed.

(1) In Figure 2C, "Number of SnCs/mm2", SnCs should be SnOCs.

We apologize for the oversight. This has been rectified in the revised manuscript.

Author response image 3.

(2) In Figure 3A-E, is there any statistical difference between groups Young and Aged+PBS?

We appreciate the reviewer's comments. Following your recommendation, we conducted additional statistical analyses to compare the young and PBS-treated aged mice, and we have incorporated these findings into the revised manuscript. The data reveals a significant increased paw withdrawal frequency (PWF) in aged mice treated with PBS compared with young mice, particularly at 0.4g instead of 0.07g (Figure 3a, 3b). Moreover, aged mice treated with PBS exhibited a significant reduction in both distance traveled and active time when compared to young mice (Figure. 3d, 3e). Additionally, PBS-treated aged mice demonstrated a significantly shortened heat response time relative to young mice (Figure. 3c).

Author response image 4.

(3) Again, is there any statistical difference between the Young and Aged+PBS groups in Figure 4F-K?

We appreciate the reviewer's comments. As per your suggestion, we conducted a thorough analysis to determine the statistical differences between the young and aged+PBS groups, and these statistical results have been implemented in the revised manuscript. The caudal endplates of L4/5 in PBS-treated aged mice exhibited a significant increase in endplate porosity (Figure. 4f) and trabecular separation (Tb.Sp) (Figure. 4g) compared to young mice.

Additionally, PBS-treated aged mice showed a significant elevation in endplate score (Figure. 4h), as well as an increased distribution of MMP13 and ColX within the endplates when compared to young mice (Figure. 4i, 4j). Furthermore, TRAP staining revealed a significant increase in TRAP+ osteoclasts within the endplates of PBS-treated aged mice as compared to young mice (Figure. 4k).

Author response image 5.

(4) What is the figure legend of Figure 7?

The legend for Figure 7 (as below) is included in a separate PDF file labeled 'Figures and Legends.' We have carefully checked the revised manuscript and made sure all the legends are included.

“Fig. 7. (a) Representative images of immunofluorescent analysis of CD31, an angiogenesis marker (green), Emcn, an endothelial cell marker (red) and nuclei (DAPI; blue) of adult sham, LSI and aged mice injected with PBS or ABT263. (b) Quantitative analysis of the intensity mean value of CD31 per mm2 in sham, LSI mice treated with PBS or ABT263. (c) Quantitative analysis of the intensity mean value of CD31 per mm2 in aged mice treated with PBS or ABT263. (d) Quantitative analysis of the intensity mean value of Emcn per mm2 in sham, LSI mice treated with PBS or ABT263. (e) Quantitative analysis of the intensity mean value of Emcn per mm2 in aged mice treated with PBS or ABT263. n ≥ 4 per group. Statistical significance was determined by one-way ANOVA, and all data are shown as means ± standard deviations. “

(5) In "Mice" section, an Ethical code is suggested to be added.

We appreciate the reviewer's comments. In accordance with your suggestion, we have included the Johns Hopkins University animal protocol number in the revised manuscript. The relevant paragraph has been updated to read: “All mice were maintained at the animal facility of The Johns Hopkins University School of Medicine (protocol number: MO21M276).”

(6) In "Methods" section, please indicate the primers of GAPDH.

We apologize for the absence of the GAPDH primers. Upon review, the GAPDH primers used were as follows: forward primer 5'-ATGTGTCCGTCGTGGATCTGA-3' and reverse primer 5'-ATGCCTGCTTCACCACCTTCTT-3'. These primer sequences have been included in the revised manuscript.

(7) Preosteoclasts are regarded to be closely related to H-type vessel growth, so do the authors have any comments on this? Any difference or correlation between SnCs and preosteoclasts?

The pre-osteoclast plays a crucial role in secreting anabolic growth factors that facilitate H-type vessel formation, osteoblast chemotaxis, proliferation, differentiation, and mineralization. The osteoclast represents the terminal differentiation phase, ultimately leading to the induction of resorption.

Senescent cells, including senescent osteoclasts, are characterized by permanent cell cycle arrest and changes in their secretory profile, which can impact their function. In the context of osteoclasts, senescence can lead to a reduction in bone resorption capacity and impaired bone remodeling. Senescent osteoclasts are believed to contribute to age-related bone loss and bonerelated diseases, such as osteoporosis.

Reviewer #3 (Public Review):

Summary:

This research article reports that a greater number of senescent osteoclasts (SnOCs), which produce Netrin-1 and NGF, are responsible for innervation in the LSI and aging animal models.

Strengths:

The research is based on previous findings in the authors' lab and the fact that the IVD structure was restored by treatment with ABT263. The logic is clear and clarifies the pathological role of SnOCs, suggesting the potential utilization of senolytic drugs for the treatment of LBP. Generally, the study is of good quality and the data is convincing.

Weaknesses:

There are some points that can be improved:

(1) Since this work primarily focuses on ABT263, it resembles a pharmacological study for this drug. It is preferable to provide references for the ABT263 concentration and explain how the administration was determined.

Thank you for your comment. ABT263 has been extensively employed in diverse research studies12-15. The concentration and administration of ABT263 followed the protocol outlined in the published paper13. The reference on how to use ABT263 is cited in the method section: “ABT263 was administered to mice by gavage at a dosage of 50 mg per kg body weight per day (mg/kg/d) for a total of 7 days per cycle, with two cycles conducted and a 2-week interval between them39”.

(2) It would strengthen the study to include at least 6 mice per group for each experiment and analysis, which would provide a more robust foundation.

Thank you for your comment here. In response, we conducted a new set of experiments, augmenting the majority of the sample size to six, and updated the corresponding statistical data in the revised manuscript.

(3) In Figure 4, either use "adult" or "young" consistently, but not both. Additionally, it's important to define "sham," "young," and "adult" explicitly in the methods section.

Thank you for your comment. We have addressed the inconsistency in the labeling of Figure 4. Additionally, we have explicitly defined "sham," "young," and "adult" in the methods section as follows: The control group (sham group) for the LSI group refers to C57BL/6J mice that did not undergo LSI surgery, while the control group (young group) for the Aged group refers to 4-month-old C57BL/6J mice.

Author response image 6.

(4) Assess the protein expression of Netrin 1 and NGF.

Thank you for your comment here. We employed ELISA to assess the protein expression of Netrin-1 and NGF in the L3 to L5 endplates. The data revealed that compared to the young sham mice, LSI was associated with significantly greater protein expression of Netrin1 and NGF, which was substantially attenuated by ABT263 treatment in LSI mice (Supplementary Fig. 2a, 2b)

Author response image 7.

Reference

(1) Bian, Q. et al. Excessive Activation of TGFbeta by Spinal Instability Causes Vertebral Endplate Sclerosis. Sci Rep 6, 27093, doi:10.1038/srep27093 (2016).

(2) Bian, Q. et al. Mechanosignaling activation of TGFbeta maintains intervertebral disc homeostasis. Bone Res 5, 17008, doi:10.1038/boneres.2017.8 (2017).

(3) Papadakis, M., Sapkas, G., Papadopoulos, E. C. & Katonis, P. Pathophysiology and biomechanics of the aging spine. Open Orthop J 5, 335-342, doi:10.2174/1874325001105010335 (2011).

(4) Rodriguez, A. G. et al. Morphology of the human vertebral endplate. J Orthop Res 30, 280-287, doi:10.1002/jor.21513 (2012).

(5) Taher, F. et al. Lumbar degenerative disc disease: current and future concepts of diagnosis and management. Adv Orthop 2012, 970752, doi:10.1155/2012/970752 (2012).

(6) Fields, A. J., Liebenberg, E. C. & Lotz, J. C. Innervation of pathologies in the lumbar vertebral end plate and intervertebral disc. Spine J 14, 513-521, doi:10.1016/j.spinee.2013.06.075 (2014).

(7) Hand, R. A. & Kolodkin, A. L. Netrin-Mediated Axon Guidance to the CNS Midline Revisited. Neuron 94, 691-693, doi:10.1016/j.neuron.2017.05.012 (2017).

(8) Moore, S. W., Zhang, X., Lynch, C. D. & Sheetz, M. P. Netrin-1 attracts axons through FAK-dependent mechanotransduction. J Neurosci 32, 11574-11585, doi:10.1523/JNEUROSCI.0999-12.2012 (2012).

(9) Serafini, T. et al. Netrin-1 is required for commissural axon guidance in the developing vertebrate nervous system. Cell 87, 1001-1014, doi:10.1016/s0092-8674(00)81795-x (1996).

(10) Forcet, C. et al. Netrin-1-mediated axon outgrowth requires deleted in colorectal cancer-dependent MAPK activation. Nature 417, 443-447, doi:10.1038/nature748 (2002).

(11) Shu, T., Valentino, K. M., Seaman, C., Cooper, H. M. & Richards, L. J. Expression of the netrin-1 receptor, deleted in colorectal cancer (DCC), is largely confined to projecting neurons in the developing forebrain. J Comp Neurol 416, 201-212, doi:10.1002/(sici)1096-9861(20000110)416:2<201::aid-cne6>3.0.co;2-z (2000).

(12) Born, E. et al. Eliminating Senescent Cells Can Promote Pulmonary Hypertension Development and Progression. Circulation 147, 650-666, doi:10.1161/CIRCULATIONAHA.122.058794 (2023).

(13) Chang, J. et al. Clearance of senescent cells by ABT263 rejuvenates aged hematopoietic stem cells in mice. Nat Med 22, 78-83, doi:10.1038/nm.4010 (2016).

(14) Lim, S. et al. Local Delivery of Senolytic Drug Inhibits Intervertebral Disc Degeneration and Restores Intervertebral Disc Structure. Adv Healthc Mater 11, e2101483, doi:10.1002/adhm.202101483 (2022).

(15) Yang, H. et al. Navitoclax (ABT263) reduces inflammation and promotes chondrogenic phenotype by clearing senescent osteoarthritic chondrocytes in osteoarthritis. Aging (Albany NY) 12, 12750-12770, doi:10.18632/aging.103177 (2020).

eLife assessment

This fundamental study advances our understanding of the role of senescent osteoclasts (SnOCs) in the pathogenesis of spine instability. The authors provide compelling evidence for the SnOCs to induce sensory nerve innervation. Accordingly, reduction of SnOCs by the senolytic drug Navitoclax markedly reduces spinal pain sensitivity. This work will be of broad interest to regenerative biologists working on spinal pain.

Reviewer #1 (Public Review):

Summary:

In this study, Pan DY et al. discovered that the clearance of senescent osteoclasts can lead to a reduction in sensory nerve innervation. This reduction is achieved through the attenuation of Netrin-1 and NGF levels, as well as the regulation of H-type vessels, resulting in a decrease in pain-related behavior. The experiments are well-designed. The results are clearly presented, and the legends are also clear and informative. Their findings represent a potential treatment for spine pain utilizing senolytic drugs.

Strengths:

Rigorous data, well-designed experiments as well as significant innovation make this manuscript stand out.

Weaknesses:

All my concerns have been well addressed, no further comments.

Reviewer #2 (Public Review):

Summary:

This manuscript examined the underlying mechanisms between senescent osteoclasts (SnOCs) and lumbar spine instability (LSI) or aging. They first showed that greater numbers of SnOCs are observed in mouse models of LSI or aging, and these SnOCs are associated with induced sensory nerve innervation, as well as the growth of H-type vessels, in the porous endplate. Then, the deletion of senescent cells by administration of the senolytic drug Navitoclax (ABT263) results in significantly less spinal hypersensitivity, spinal degeneration, porosity of the endplate, sensory nerve innervation, and H-type vessel growth in the endplate. Finally, they also found that there is greater SnOC-mediated secretion of Netrin-1 and NGF, two well-established sensory nerve growth factors, compared to non-senescent OCs. The study is well conducted and data strongly support the idea.

Reviewer #3 (Public Review):

Summary:

This research article reports that a greater number of senescent osteoclasts (SnOCs), which produce Netrin-1 and NGF, are responsible for innervation in the LSI and aging animal models.

Strengths:

The research is based on previous findings in the authors' lab and the fact that the IVD structure was restored by treatment with ABT263. The logic is clear and clarifies the pathological role of SnOCs, suggesting the potential utilization of senolytic drugs for the treatment of LBP. Generally, the study is of good quality and the data is convincing.

Weaknesses:

All my concerns have been well addressed, no further comments.

Author response:

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

Reviewer #1

The authors should include experiments such as Cryo-EM and genetically modified animals to demonstrate the physiological importance of the TMEM81 complex.

While we intend to pursue cryo-EM studies of the putative complex (or subcomplexes thereof), this is clearly not a straightforward endeavor and goes beyond the scope of the present manuscript. Concerning the generation of genetically modified animals, we would like to underline that the majority of the proteins that we used for AlphaFold-Multimer complex predictions were precisely chosen based on the fact that - as detailed in the publications referenced in the Introduction - ablation of the respective genes caused sex-specific infertility due to defects in gamete fusion (the other criterion used for inclusion being structural similarity to IZUMO1 coupled with expression in the testis (IZUMO2-4 and TMEM81), or evidence from other kinds of experiments in the case of human-specific MAIA). Concerning TMEM81, experimental evidence for a direct involvement in gamete fusion is described in the referenced preprint by Daneke et al., which was submitted to bioRxiv concomitantly with the present work.

Reviewer #2

I believe that the manuscript would benefit from the authors providing more information about the systematic search (Figure 4). For example, by indicating for each pair tested the average pDock score in a 2D plot (or table) and as raw data in the supplementary information.

Figure 4 has been modified to report both the top and the mean ranking scores for every interaction. Furthermore, additional metrics for the systematic search summarized in Figure 4, including pDockQ scores, are provided in this manuscript revision as supplementary Table S1.

A global search, such as including all membrane proteins expressed in eggs or sperm, could not only be more informative but could also allow the reader to understand the pDock score discrimination power for this particular subset.

The possibility of carrying out a global search was evaluated by performing preliminary computational experiments on an extended ensemble of sperm and egg proteins. In order to do so, we compiled a list of sperm membrane proteins by referring to 4 proteomic datasets (PMIDs 36384108, 36896575, 31824947, 24082039) and identifying ~600 proteins that were found in at least two of them; among these, 250 were single-pass type I or type II membrane proteins, or GPI-anchored proteins. Similarly, a list of 160 egg surface membrane proteins, excluding multipass and secreted ones, was obtained by comparing oocyte cDNA library NIH_MGC_257_N (Express Genomics, USA) with 4 proteomic datasets (PMIDs 35809850, 36042231, 29025019, 27215607). As we briefly commented at the beginning of the section “Prediction of interactions between human proteins associated with gamete fusion” of the revised manuscript, the tests carried out using the resulting list of sperm and egg proteins suggested that interpreting the results of a global search would be severely complicated by a relatively large number of putative false positives. Moreover, the tests showed that performing a complete systematic search would be beyond our current access to computing power. Based on these observations, we preferred to maintain the present study limited to proteins that had been previously clearly implicated in gamete fusion and/or matched specific structural features of IZUMO1.

Figure 5 could be improved in clarity by schematically indicating to which cell each protein is anchored.

This has been done in the revised version of the manuscript.

Reviewer #3

Major comments

(1) In Figure 1, how the protein of mouse/human IZUMO1 and JUNO is purified is not mentioned in the main text nor in the Methods. Are the mouse IZUMO1-His and mouse JUNO-His transfected together or separately? Are human JUNO-His and human IZUMO1-Myc transfected together into HEK293 cells? And purified by IMAC?

Transfection information has been included in the Methods section “Protein expression, purification and analysis” (previously “Protein expression and purification”). Concerning the purification procedure, we had already stated in the legend of Figure 1 that human JUNOE-His/IZUMO1E-Myc had been purified by IMAC before SEC, and have now done the same for mouse JUNOE-His and IZUMO1E-His.

(2) It would be easier to understand the figure if the author could run a WB to indicate which band above JUNO is specifically IZUMO1-Myc in Figure 1.

This has been done and reported in a new Figure S1 (with the original Figure S1 having now become Figure S2). Details about the antibodies used for immunoblot have been included in both Methods section “Protein expression, purification and analysis” and the Key Resources Table.

(3) Figure 4: Analysis of more proteins that have been suggested as possible candidates for sperm-egg interaction will help to highlight the following results. Also, providing a score for the possibility of interaction might help in selecting those proteins in Figures 5 and 6.

Please refer to the answer to the first question of Reviewer #2.

(4) Figure 7: The authors take advantage of the latest developments in protein structure and interaction to model protein complex formation. However, some experimental experiments such as Co-IP, pull down to support the prediction to verify some of this predicated interaction is necessary.

We agree with the reviewer; however, for the reasons we discussed during our comparison of the biochemical properties of the JUNO/IZUMO1 interaction between mouse and human, pursuing this line of inquiry will likely necessitate an extensive set of parallel experiments using proteins from different species. This work is being planned and will be the focus of future studies. However, as we mentioned at the end of the Abstract, one should also consider that some of these complexes are likely to be highly transient. Because of this, while they may have important regulated roles in vivo (function at a specific time and place), they could be very challenging to detect using standard approaches in vitro. We thus see this as a significant advance that structural modeling could contribute to the identification of such functionally important but transient interactions.

Minor points

(1) In the abstract, "three sperm (IZUMO1, SPACA6 and TMEM81) "should be "three sperm proteins."

The Abstract has been condensed to fit within the suggested 200-word limit and, as part of this, the sentence has been changed to “complex involving sperm IZUMO1, SPACA6, TMEM81 and egg JUNO, CD9”.

(2) How do the predictions of the binary complex IZUMO1/CD9 (Figure S1B) or IZUMO1/CD81 (Figure S1C) suggest "the two egg tetraspanins are interchangeable"? Was it because they are quite similar? Please provide more explanation for this speculation. Interchangeable by function or for complex formation? To support the conclusion, biochemical data is required. Otherwise, it needs to be toned down.

This is because, in the AlphaFold-Multimer predictions of the pentameric complex, CD9 and CD81 are placed in essentially the same way relative to the other subunits.

We have now clarified this at the end of page 6:

“(...) suggest that the two egg tetraspanins are interchangeable because they are predicted to bind to the same region of IZUMO1; (...)”

(3) It would be more reader-friendly if the author could label the name of each protein in the figure in Figure S1, especially when the name is not written in the figure legend.

This has been done in Figure S2 of the revised manuscript (corresponding to original Figure S1).

eLife assessment

This study offers valuable insights into the structural architecture of the mammalian egg-sperm fusion synapse, shedding light on the role of specific proteins in fertilization. The significance of the findings lies in the potential identification of a pentameric complex involved in gamete fusion by AlphaFold Multimer. The strength of evidence for the approach/methodology is solid, while the experimental validation is incomplete in supporting these interactions. This work will be of interest to biomedical researchers working on fertility and reproductive health.

Reviewer #2 (Public Review):

Summary:

Fertilization is a crucial event in sexual reproduction, but the molecular mechanisms underlying egg-sperm fusion remain elusive. Elofsson A et al. used AlphaFold to explore possible synapse-like assemblies between sperm and egg membrane proteins during fertilization. Using a systematic search of protein-protein interactions, the authors proposed a pentameric complex of three sperm (IZUMO1, SPACA6, and TMEM81) and two egg (JUNO and CD9) proteins, providing a new structural model to be used in future structure-function studies.

Strengths:

(1) The study uses the AlphaFold algorithm to predict higher-order assemblies. This approach could offer insights into a highly transient protein complex, which are challenging to detect experimentally.<br /> (2) The article predicts a pentameric complex between proteins involved in fertilization, shedding light on the architectural aspects of the egg-sperm fusion synapse.

Weaknesses:

The proposed model, which is a prediction from a modeling algorithm, lacks experimental validation of the identity of the components and the predicted contacts.

It is noteworthy that in an independent study, Deneke et al. provides experimental evidence of the interaction between IZUMO1/SPACA6/TMEM81 in zebrafish. This is an important element that supports the findings presented in this manuscript

Regarding the authors response on the question of a global search:<br /> I understand that a global search might be difficult to interpret because a large number of putative false positives. But it is this type of information that is needed to assess the validity of the model and the scoring power in the absence of any experimental validation. At minimum, the search should include a negative control set of proteins known to be unrelated to sperm fertilization or homologous egg-sperm fusion complexes from incompatible species to account for species-specific interactions.

I acknowledge that experimentally validating highly transient complexes presents technical hurdles. However, a high-confidence structural model could enable the design of point mutations specifically disrupting the predicted interactions. Subsequent rescue experiments could then validate the directionality of these interactions. Ultimately, such experiments are crucial for robust model validation.

Reviewer #3 (Public Review):

Summary:

Sperm-egg fusion is a critical step in successful fertilization. Although several proteins have been identified in mammals that are required for sperm-egg adhesion and fusion, it is still unclear whether there are other proteins involved in this process and how the reported proteins complex co-operate to complete the fusion process. In this study, the authors first identified TMEM81 as a structural homologue of IZUMO1 and SPACA6, and predicted the interactions with a pool of human proteins associated with gamete fusion, using AlphaFold-Multimer, a recent advance in protein complex structure prediction. The prediction is compelling and well discussed, and the experimental evidence to verify this interaction is lacking in this study but supported by a complementary and independent study by another group.

Strengths:

The authors present a pentameric complex formation of four previously reported proteins involved in egg/sperm interaction together with TMEM181 using a deep learning tool, AlphaFold-Multimer.

Weaknesses:

It is intriguing to see that some of the proteins involved in sperm-egg interaction are successfully predicted to be assembled into a single multimeric structure by AlphaFold-Multimer. The experimental validation of the interactions is not directly supported in this study. As there are more candidate proteins in the process, testing other possible protein interactions more comprehensively will provide more rationale for the current 3D multi-protein modeling.

remove these tables. New formulas can be used:

In general, the relation between extraterrestrial and shortwave radiation can be expressed as:

(Hendriks, 2010). For the Netherlands, typical values for the constants are and (De Bruin and Stricker, 2000), while globally often and (Allen et al., 1998) are recommended. Other estimates can be found in (Iqbal, 1983).

Some examples of parasocial relationships I see on social media are when celebrities/artists make videos to their fans, addressing anything from the release of content, or to just thank their fanbase for something. The celebrity will often refer to their fans with a certain name (ex: Swifties, Directioners, Little Monsters), which instills a feeling of closeness on the fanbase's part because it feels as though the celebrity is really talking to them. Another example I often see is when fans will learn so much about their favorite celebrity to the point where it feels like they know them personally, and then proceed to act like they do. Sometimes it can even be a little invasive, especially if they act upon what they learn (address leaks, etc).

From this article, I learned that Donald Trump typically wrote the more controversial and angrier-toned tweets on his page, whereas, his staff and campaign managers wrote the more calm and 'educational' tweets. This was determined after looking at what device the tweet was posted from; Trump tweeted from an Android while his staff tweeted from an iPhone. This detection isn't necessarily groundbreaking, as a celebrity's team will often post/monitor the celebrity's page for them. Even though the tweets from the iPhone were generally more 'positive', those sets of tweets were trying to mimic Trump's behavior. It's interesting to think about how his staff would go about that, since Trump has a pattern of being pretty hostile on the internet.

In the article "How Code-Switching Explains The World" from NRP, they talks about how different people "code switch" depending on the scenario. In the article, they showed famous celebrities such as Obama, Beyonce and other YouTube videos changing their tone of voice while still being themselves. This is because code switching can help benefit you depending on where you are and what situation you are in. The article also states that code switching is how we can "try to feel each other out." Code switching is a valuable skill to develope as it can give you an advantage in certain scenarios.

The series gained significant attention for its blending of reality and fiction, pioneering the use of social media platforms for narrative storytelling.

I think people are so angry because they can't accept that their favorite YouTuber is a fake. A lot of people may be disgusted with using fake things to get attention. There are also people who are jealous that the YouTuber can get a lot of views or make a lot of money and say "it's fake."

eLife assessment

This study presents an important methodology to increase the efficiency and precision of gene editing in human hematopoietic stem and progenitor cells. The evidence supporting the claims is convincing in that primitive LTC-ICs were minimally affected as a result of the editing procedure and the lack of edits at predicted off-target sites. The work will be of interest to biologists studying hematopoietic stem and progenitor cells and genome editing for potential clinical applications.

Reviewer #2 (Public Review):

Summary:

This work by Cloarec-Ung et al. sets out to uncover strategies that would allow for the efficient and precision editing of primitive human hematopoietic stem and progenitor cells (HSPCs). Such effective editing of HSPCs via homology directed repair has implications for the development of tractable gene therapy approaches for monogenic hematopoietic disorders as well as precise engineering of these cells for clinical regenerative and/or cell therapy strategies. In the setting of experimental hematology, precision introduction of disease relevant mutations would also open the door to more robust disease modeling approaches. It has been recognized that to encourage HDR, NHEJ as the dominant mode of repair in quiescent HSPCs must be inhibited. Testing editing of human cord blood HSPCs the authors first incorporate a prestimulation phase then identify optimal RNP amounts and donor types/amounts using standard editing culture conditions identifying optimal concentrations of AAV and short single-stranded oligonucleotide donors (ssODNs) that yield minimal impacts to cell viability while still enabling heightened integration efficiency. They then demonstrate the superiority of AZD7648, an inhibitor of NHEJ-promoting DNA-PK, in allowing for much increased HDR with toxicities imparted by this compound reduced substantially by siRNAs against p53 (mean targeting efficiencies at 57 and 80% for two different loci). Although AAV offered the highest HDR frequencies, differing from ssODN by a factor by ~2-fold, the authors show that spacer breaking sequence mutations introduced into the ssODN to better mimic the disruption of the spacer sequence provided by the synthetic intron in the AAV backbone yielded ssODN HDR frequencies equal to that attained by AAV. By examining editing efficiency across specific immunophenotypically identified subpopulations they further suggest that editing efficiency with their improved strategy is consistent across stem and early progenitors and use colony assays to quantify an approximate 4-fold drop in total colony numbers but no skewing in the potentiality of progenitors in the edited HSPC pool. Finally, the authors provide a strategy using mutation-introducing AAV mixed with different ratios of silent ssODN repair templates to enable tuning of zygosity in edited CD34+ cells.

Strengths:

The methods are clearly described and the experiments for the most part also appropriately powered. In addition to using state-of-the-art approaches, the authors also provided useful insights into optimizing the practicalities of the experimental procedures that will aid bench scientists in effectively carrying out these editing approaches, for example avoiding longer handling times inherent when scaling up to editing over multiple conditions.

The sum of the adjustments to the editing procedure have yielded important advances towards minimizing editing toxicity while maximizing editing efficiency in HSPCs. In particular, the significant increase in HDR facilitated by the authors' described application of AZD7648 and the preservation of a pool of targeted progenitors is encouraging that functionally valuable cell types can be effectively edited.

The discovery of the effectiveness of spacer breaking changes in ssODNs allowing for substantially increased targeting efficiency is a promising advance towards democratizing these editing strategies given the ease of designing and synthesizing ssODNs relative to the production of viral donors.

The ability to zygosity tune was convincingly presented and provides a valuable strategy to modify this HDR procedure towards more accurate disease modelling.

Weaknesses:

Despite providing convincing evidence that functional progenitors can be successfully edited by their procedure, as the authors acknowledge it remains to be verified to what degree the survival/self-renewal capacity and in vivo regenerative potential of the more primitive fractions is maintained with their strategy. That said the inclusion of LTC-IC assays that verify the lack of effect on these quite primitive cells is encouraging that functionality of stem cells will be similarly spared.

I think it is one of the most common way of nowadays website starting up, it provides significant flows and creates incomes

eLife assessment

This important study sheds light on the mechanisms underlying a rare brain disease, offering insight into the role of microglia in this complex pathophysiology. The evidence presented is solid, utilizing state-of-the-art laboratory models to explore cellular interactions and disease development. While further research is needed, this study will be of interest to neuroscientists and clinicians aiming to understand and combat similar neurodegenerative disorders.

Reviewer #1 (Public Review):

Here, using an organoid system, Wong et al generated a new model of hereditary diffuse leukoencephalopathy with axonal spheroids, with which they investigated how CSF1R-mutaions affect the phenotypes of microglia/macrophages, and revealed metabolic changes in microglia/macrophages associated with a proinflammatory phenotype.

In general, this paper is interesting and well-written, and tackles important issues to be addressed.

This study suffers from several major concerns and limitations that dampen the value of the study. As the authors also mentioned, models that perfectly recapitulate the complexity of the HDLS brain the models would be required to better understand the molecular mechanisms of the disease. In this regard, it is unclear how nicely the organoid system in this study can recapitulate the condition in patients with HDLS (e.g. reduced microglia density, downregulated expression of P2YR12, pathological alterations). In addition, the authors used two different models with distinct mutations that could produce different readouts in CSF1R-mediated cellular responses.

Although the reviewer does understand the importance of providing several options/tools to study rare diseases like HDLS and the difficulty of generating stable organoids with less variation, it is unclear if the different outcomes between HD1 and HD2 are generated through different mutations or simply due to different differentiation efficiency from iMacs (e.g. Figure 2B), which needs to be confirmed. Lastly, there is an over-interpretation regarding the results in Figure 6A. There is no difference between isoHD1 iMac control and HD1 Mut iMac.

Reviewer #2 (Public Review):

Summary:

This paper investigates a rare and severe brain disease called Hereditary Diffuse Leukoencephalopathy with Axonal Spheroids (HDLS). The authors aimed to understand how mutations in the gene CSF-1R affect microglia, the resident immune cells in the brain, and which alterations and factors lead to the specific pathophysiology. To model the human brain with the pathophysiology of HDLS, they used the human-specific model system of induced pluripotent stem cell (iPSC)-derived forebrain organoids with integrated iPSC-derived microglia (iMicro) from patients with the HDLS-causing mutation and an isogenic cell line with the corrected genome. They found that iPSC-derived macrophages (iMac) with HDLS mutations showed changes in their response, including increased inflammation and altered metabolism. Additionally, they studied these iMacs in forebrain organoids, where they differentiate into iMicro, and showed transcriptional differences in isolated iMicro when carrying the HDLS mutation. In addition, the authors described the influence of the mutation within iMicro on the transcriptional level of neurons and neural progenitor cells (NPCs) in the organoid. They observed that the one mutation showed implications for impaired development of neurons, possibly contributing to the progression of the disease.

Overall, this study provides valuable insights into the mechanisms underlying HDLS and emphasizes the importance of studying diseases like these with a suitable model system. These findings, while promising, represent only an initial step towards understanding HDLS and similar neurodegenerative diseases, and thus, their direct translation into new treatment options remains uncertain.

Strengths:

The strength of the work lies in the successful reprogramming of two HDLS patient-derived induced pluripotent stem cells (iPSCs) with different mutations, which is crucial for the study of HDLS using human forebrain organoid models. The use of corrected isogenic iPSC lines as controls increases the validity of the mutation-specific observations. In addition, the model effectively mimics HDLS, particularly concerning deficits in the frontal lobe, mirroring observations in the human brain. Obtaining iPSCs from patients with different CSF1R mutations is particularly valuable given the limitations of rodent and zebrafish models when studying adult-onset neurodegenerative diseases. The study also highlights significant metabolic changes associated with the CSF1R mutation, particularly in the HD2 mutant line, which is confirmed by the HD1 line. In addition, the work shows transcriptional upregulation of the proinflammatory cytokine IL-1beta in cells carrying the mutation, particularly when they phagocytose apoptotic cells, providing further insight into disease mechanisms.

Weaknesses:

The authors have not elucidated the significance of the increased CSF1 dosage in Figure 2F, aside from its effect on cell viability, lacking a thorough discussion of this result. Additionally, while transcriptomic and metabolic alterations related to the mutation were demonstrated in iMac models, similar investigations in iMicros are absent, necessitating further experiments to validate the findings across cell models. The conclusion drawn regarding cytokine levels lacks robust support from the data, particularly considering the varied responses observed in different mutant lines. Further analysis of the secretome (e.g. via ELISA) could provide additional insights. Moreover, the characterization of iMicros is incomplete, with limited protein-level analysis (e.g. validate RNA-seq via flow cytometry). Additionally, the claim of microglial-like morphology lacks adequate evidence, as the provided image is insufficient for such an assessment. RNA-seq experiments should be represented better, it is not possible to read the legends or gene names in the figures. Maybe the data sets can be combined into PCAone and one overall analysis, e.g. via WGCNA-like analyses? This would make it easier for the reader to compare the two cell lines side by side. Furthermore, inaccuracies and omissions in the figure legends compromise the clarity of data representation. Statistical test information is missing. Finally, inconsistent terminology usage throughout the paper may confuse readers (iMac versus iMicros).

eLife assessment

In this study, the authors provide valuable evidence that the LGE is not a significant source of oligodendrocytes for the cortex. The reviewers did find some technical considerations that call for some modulation of the strength of the authors' conclusions and also pointed out some aspects of the data that were incomplete as presented.

Reviewer #1 (Public Review):

Summary:

In this study, the authors generated a novel transgenic mouse line OpalinP2A-Flpo-T2A-tTA2 to specifically label mature oligodendrocytes, and at the same time their embryonic origins by crossing with a progenitor cre mouse line. With this clever approach, they found that LGE/CGE-derived OLs make minimum contributions to the neocortex, whereas MGE/POA-derived OLs make a small but lasting contribution to the cortex. These findings are contradictory to the current belief that LGE/CGE-derived OPCs make a sustained contribution to cortical OLs, whereas MGE/POA-derived OPCs are completely eliminated. Thus, this study provides a revised and more comprehensive view on the embryonic origins of cortical oligodendrocytes. To specifically label mature oligodendrocytes, and at the same time their embryonic origins by crossing with a progenitor cre mouse line. With this clever approach, they found that LGE/CGE-derived OLs make minimum contributions to the neocortex, whereas MGE/POA-derived OLs make a small-but-lasting contribution to to cortex. These findings are contradictory to the current belief that LGE/CGE-derived OPCs make a sustained contribution to cortical OLs, whereas MGE/POA-derived OPCs are completely eliminated. Thus, this study has provided a revised and updated view on the embryonic origins of cortical oligodendrocytes.

Strengths:

The authors have generated a novel transgenic mouse line to specifically label mature differentiated oligodendrocytes, which is very useful for tracing the final destiny of mature myelinating oligodendrocytes. Also, the authors carefully compared the distribution of three progenitor cre mouse lines and suggested that Gsh-cre also labeled dorsal OLs, contrary to the previous suggestion that it only marks LGE-derived OPCs. In addition, the author also analyzed the relative contributions of OLs derived from three distinct progenitor domains in other forebrain regions (e.g. Pir, ac). Finally, the new transgenic mouse lines and established multiple combinatorial genetic models will facilitate future investigations of the developmental origins of distinct OL populations and their functional and molecular heterogeneity.

Weaknesses:

Since OpalinP2A-Flpo-T2A-tTA2 only labels mature oligodendrocytes but not OPCs, the authors can not suggest that the lack of LGE/CGE-derived-OLs in the neocortex is less likely caused by competitive postnatal elimination, but more likely due to limited production and/or allocation (line 118-9). It remains possible that LGE/CGE-derived OPCs migrate into the cortex but are later eliminated.

Reviewer #2 (Public Review):

Summary:

In this manuscript, Cai et al use a combination of mouse transgenic lines to re-examine the question of the embryonic origin of telencephalic oligodendrocytes (OLs). Their tools include a novel Flp mouse for labelling mature oligodendrocytes and a number of pre-existing lines (some previously generated by the last author in Josh Huang's lab) that allowed combinatorial or subtractive labelling of oligodendrocytes with different origins. The conclusion is that cortically-derived OLs are the predominant OL population in the motor and somatosensory cortex and underlying corpus callosum, while the LGE/CGE generates OLs for the piriform cortex and anterior commissure rather than the cerebral cortex. Small numbers of MGE-derived OLs persist long-term in the motor, somatosensory and piriform cortex.

Strengths:

The strength and novelty of the manuscript lies in the elegant tools generated and used and which have the potential to elegantly and accurately resolve the issue of the contribution of different progenitor zones to telencephalic regions.

Weaknesses:

(1) Throughout the manuscript (with one exception, lines 76-78), the authors quantified OL densities instead of contributions to the total OL population (as a % of ASPA for example). This means that the reader is left with only a rough estimation of the different contributions.

(2) All images and quantifications have been confined to one level of the cortex and the potential of the MGE and the LGE/CGE to produce oligodendrocytes for more anterior and more posterior cortical regions remains unexplored.

(3) Hence, the statement that "In summary, our findings significantly revised the canonical model of forebrain OL origins (Figure 4A) and provided a new and more comprehensive view (Figure 4B )." (lines 111, 112) is not really accurate as the findings are neither new nor comprehensive. Published manuscripts have already shown that (a) cortical OLs are mostly generated from the cortex [Tripathi et al 2011 (https://doi.org/10.1523/JNEUROSCI.6474-10.2011), Winker et al 2018 (https://doi.org/10.1523/JNEUROSCI.3392-17.2018) and Li et al (https://doi.org/10.1101/2023.12.01.569674)] and (b) MGE-derived OLs persist in the cortex [Orduz et al 2019 (https://doi.org/10.1038/s41467-019-11904-4) and Li et al 2024 (https://doi.org/10.1101/2023.12.01.569674)]. Extending the current study to different rostro-caudal regions of the cortex would greatly improve the manuscript.

Reviewer #3 (Public Review):

In the manuscript entitled "Embryonic Origins of Forebrain Oligodendrocytes Revisited by Combinatorial Genetic Fate Mapping," Cai et al. used an intersectional/subtractional strategy to genetically fate-map the oligodendrocyte populations (OLs) generated from medial ganglionic eminence (NKX2.1+), lateral ganglionic eminences, and dorsal progenitor cells (EMX1+). Specifically, they generated an OL-expressing reporter mouse line OpalinP2A-Flpo-T2A-tTA2 and bred with region-specific neural progenitor-expressing Cre lines EMX1-Cre for dOL and NKX2.1-Cre for MPOL. They used a subtractional strategy in the OpalinFlp::Emx1Cre::Nkx2.1Cre::RC::FLTG mouse line to predict the origins of OLs from lateral/caudal ganglionic eminences (LC). With their genetic tools, the authors concluded that neocortical OLs primarily consist of dOLs. Although the populations of OLs (dOLs or MP-OLs) from Emx1+ or Nkx2.1+ progenitors are largely consistent with previous findings, they observed that MP-OLs contribute minimally but persist into adulthood without elimination as in the previous report (PMID: 16388308).

Intriguingly, by using an indirect subtraction approach, they hypothesize that both Emx1-negative and Nkx2.1-negative cells represent the progenitors from lateral/caudal ganglionic eminences (LC), and conclude that neocortical OLs are not derived from the LC region. This is in contrast to the previous observation for the contribution of LC-expressing progenitors (marked by Gsx2-Cre) to neocortical OLs (PMID: 16388308). The authors claim that Gsh2 is not exclusive to progenitor cells in the LC region (PMID: 32234482). However, Gsh2 exhibits high enrichment in the LC during early embryonic development. The presence of a small population of Gsh2-positive cells in the late embryonic cortex could originate/migrate from Gsh2-positive cells in the LC at earlier stages (PMID: 32234482). Consequently, the possibility that cortical OLs derived from Gsh2+ progenitors in LC could not be conclusively ruled out. Notably, a population of OLs migrating from the ventral to the dorsal cortical region was detected after eliminating dorsal progenitor-derived OLs (PMID: 16436615).

The indirect subtraction data for LC progenitors drawn from the OpalinFlp-tdTOM reporter in Emx1-negative and Nkx2.1-negative cells in the OpalinFlp::Emx1Cre::Nkx2.1Cre::RC::FLTG mouse line present some caveats that could influence their conclusion. The extent of activity from the two Cre lines in the OpalinFlp::Emx1Cre::Nkx2.1Cre::RC::FLTG mice remains uncertain. The OpalinFlp-tdTOM expression could occur in the presence of either Emx1Cre or Nkx2.1Cre, raising questions about the contribution of the individual Cre lines. To clarify, the authors should compare the tdTOM expression from each individual Cre line, OpalinFlp::Emx1Cre::RC::FLTG or OpalinFlp::Nkx2.1Cre::RC::FLTG, with the combined OpalinFlp::Emx1Cre::Nkx2.1Cre::RC::FLTG mouse line. This comparison is crucial as the results from the combined Cre lines could appear similar to only one Cre line active.

Overall, the authors provided intriguing findings regarding the origin and fate of oligodendrocytes from different progenitor cells in embryonic brain regions. However, further analysis is necessary to substantiate their conclusion about the fate of LC-derived OLs convincingly.

The Smithsonian article on 19th-century American politics discusses how conspiracy theories were prevalent during that era. This historical context is relevant to the study of social media, as it highlights the longstanding human tendency to spread and believe in unverified information, a behavior that has been amplified in the digital age through platforms like social media.

Indeed, the way users form connections on social media platforms varies greatly. Some platforms, like bulletin boards, allow for casual interactions without formal connections. Others, like Facebook, emphasize reciprocal relationships, fostering a sense of mutual agreement and shared experiences. Platforms like Twitter and YouTube, on the other hand, enable one-way connections, allowing users to tailor their feeds according to their interests, without the need for reciprocation. This diversity in connection types caters to different user needs and preferences, making social media a versatile tool for communication and content consumption.

https://www.carbonbrief.org/factcheck-why-the-recent-acceleration-in-global-warming-is-what-scientists-expect/

old world diseases as primaryv killer

soils improved by human activity

I never knew this act was called Sockpuppet. This is very interesting to me because I can't imagine someone going out of their way and creating a burner account to argue. I wonder what category fan pages are under. I find it interesting how different people use social media, and how much influence it has on our society to where people create certain types of accounts.

A lo largo de la lectura consideré el uso de distintos métodos para la realización de mi investigación. Inicialmente consideré el trabajo de gabinete, ya que el tema de estudio no requiere recolección de datos a modo de trabajo de campo, ya que analizaré ejemplos ya existentes de animación y sus distintos usos en diferentes filmes a los cuales tengo un fácil acceso desde mi escritorio, siendo la película Across The Spiderverse el ejemplo principal al cual se le puede realizar un análisis completo con elementos que se pueden recopilar fácilmente. A su vez, creí apropiado el método cualitativo al basarse en el estudio de un número limitado de casos para una profunda investigación del tema a tratar, recolectando información de distintas formas, que es precisamente lo que pretendo conseguir, un análisis profundo de las distintas técnicas de animación aplicadas a un cierto número de ejemplos. En general, pude identificar los objetivos de mi investigación en este proceso de triangulación gracias a que se basa más que nada en el análisis y la observación de los distintos tipos de animación y sus aplicaciones en los filmes más recientes, destacando la película Spiderman Across The Spiderverse por su cuidadosa selección en distintas técnicas de animación, permitiendo el uso de distintas metodologías para analizar cada una de sus partes.

He is known for being the pioneer of applied psychology in law, medicine and education. When he was getting his doctoral degree at University of Leipzig he was under the guidance of Wilhelm Wundt. Was the president of APA in 1897 and was in charge of the experimental psychology lab at Harvard were he would spend the rest of his career. His work that he did about how psychology is applied in different fields would influence years of research.

The types of memory have changed over the years but there are still types of memory that revolve around the things that he mentioned: echoic memory and haptic memory.

There is still conflicting evidence about this topic and how well the memories of events hold up over time. Some say that memory is like a game of telephone, it constantly changing, and some say it stays the same and you can remember everything about an event.

The outcome of this experiment is that among most of the participants there were intrusion errors which is when there are certain things that are recalled that were never there to being with. For them to remember a hat but there being no hat all is one of the major example of this.

This show another reason why eye witness testimonies aren't reliable in court. This is also another example of illusion of truth.

This is an example of selective attention which was found in 1964 by Anne Treisman. Selective attention is still very important in cognitive psychology today and is still widely studied.

This has changed a lot over the years. Psychology in general is very present in the court room, wither its for elevations or behavioral profiling. Behavioral profiling was first used in the Jake the Ripper case but didn't gain traction until the late 20th century.

When eye witness testimony is used there is a lot of uncertainties that surround it. These issues haven't been as in prominent in the past because there wasn't research that was against the use of eye witness testimonies. However with the knowledge that we now know about false memory and bias, it shows that the mind can't always be trusted when trying to recall information especially when it comes to the justice system.

This could be seen as him filling in the blanks but when discussing cognitive psychology it can be seen as illusion of truth. Which stems from implicit memory and it's when the thing that seems more familiar is the more plausible answer. It made more sense for the clock to be wrapped in wrapping paper because it might of been before so that is what his memory remembers.

This point is also what a lot of research is later based on. Being told that his house was just broken into can lead to the misremembering of details and could be seen has a form of priming. Priming would be introduce around the 1970s-1980s

As years have past, there has been an increased number of eye witness testimonies that has been proven wrong. The reason of why has been examined to great length by the information that is available to us now about the cognitive processes.

The concept of code-switching is not just limited to personal interactions but is also prevalent in professional environments. It is similar to how companies rebrand themselves in different markets, showcasing the versatility and strategic thinking individuals employ in various aspects of life.

I think caring about and verifying the authenticity of online posts reflects a person's concern for the quality of information and a desire to make informed judgments. But this needs to be balanced, because excessive skepticism about online information can lead to excessive doubt and unnecessary anxiety. In general, I feel that seeking authenticity is a beneficial habit, but it should also be based on the ability to efficiently find and process information.

When reading analyzing a novel these are the steps that are considered to help understand it or may give the reader a better understanding. A novel should catch a readers attention from the beginning. When I am interested in a book, I will lose focus on what I'm reading and later find myself lost.

Each person appreciates art and sees their own beauty in it.

two people can see the same thing in a different way.

art has different meanings of beauty however I don't think anyone should be allowing others to hurt them for "art"

"The devil is ready to continue his eternal joke."

circling back to a previous connection from the first article I read for this class

When I first started this class I read an article talking about the modern immigration problem and how the cause of the Mexican Revolution was also created from US companies. Even after many months there a many connections to that event from some of these readings.

Home of the Aztec people. I actually did not know about this information.

People of Mexican decent born in the US

I remember reading Neuromancer. Just reading the first page will confuse you, it has so many "techno babble" ( made up words for the tech in the cyberpunk genre) that it really makes the book hard to read. Diction is very important

I feel that in certain books pace is very important and some stories need set up. Its very important

Like how the author broke down what we the readers look ( or should look for) for in the first pages of a new book

In a way the author is trying to seduce its reader in the beginning of the book. For slow burner starts this might be harder to achieve.

Like this sentence, i find literature to be the best way to sort of travel back in time. An example would be Odyssey by Homer, Odysseus by our terms of the modern day would be someone who we wouldn't see as a "pure" hero but back then it was different.

Showing and reading about humans and their experience can also give us meaning of what it means to be human. ( the good and the bad)

I can assume that the audiences would 1st try to understand this sexualized culture as it was something we had never seen before. I'm sure that there were discussions, debates, arguments held that tried to justify/make sense of this all.

The Big Three have had a huge impact on media culture today, it's the sole reason why we consume media the way we do.

Interesting to see how almost 60 years has gone by and these three networks are still afloat and thriving

From my understanding this means that TV history hasn't had clear path. Since it's discovery things liek development and technology have been short of easy

"Free World", I feel like i hear this term a lot in an instance where there's a debate on whether we're actually free or we just consume what we see. In response to the highlighted question I believe we were complacent consumers.

The situation that the Big Three were in is very similar to the situation we have today with social media, where there are only a handful of apps that the vast majority of people use. However, instead of relying on brand differentiation, I feel that a lot of social media apps today just copy other features from eachother, with some examples inlcuding YouTube copying TikTok's short form content with YouTube shorts.

While the Vietnam war was America's first televised war, the media would take a similar approach with the war in Iraq as television evolved, making it the most televised war in America's history so far.

This sentences sheds light on the beginning of news programming and how it would eventually lead to news being consumed from television to social media.

This sentence highlights some of the earlier examples of counter programming, which would continue to evolve in many different ways across media from then to present day.

Even if you aren’t one of the more than 40 million people in the path of totality, Nye says, “most of North America will get a little bit of the eclipse.

Section on what it means to be a man at the 15:00 minute mark in this 1937 industrial.

I guess dogs do have a good sense about the lives of people

I can see how there are many issues with advocating for dogs as if they are people, since even then people that say that still take charge of their lives and make decisions for them.

This is interesting to think about, traits and personalities that we assign to certain dog breeds are just something that we pinned on them, they aren't the emotions that dogs actually possess.

People have their own interpretations of who are and what their personalities reflect.

Now reading about this theory, in which wolves evolved themselves to be tamer over time, in combination with settlements being created over time, makes sense.

Logos it more of the facts and would just give it straight to you and not trying to appeal too much to your emotions.

A tour in the United States of America : containing an account of the present situation of that country ... by Stuart, John Ferdinand Smyth, 1745-1814

https://archive.org/details/tourinunitedstat00stua_0/page/n5/mode/2up

https://archive.org/details/williambyrdshist00byrd/mode/2up<br /> William Byrd's histories of the dividing line betwixt Virginia and North Carolina by Byrd, William, 1674-1744; Boyd, William Kenneth, 1879-1938; North Carolina. State Dept. of Archives and History

This has broadly been implemented by Tumblr and is a first class feature within the IndieWeb.

Existence of an RSS feed could be used as a filter to remove large swaths of social media content which don't have them.

[[Marisa Kabas]] in The Handbasket - Here's the column Meta doesn't want you to see

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ᔥ[[Ben Werdmuller]] in Mastodon @ben@werd.social on Apr 06, 2024, 10:45 AM

On Thursday I reported that Meta had blocked all links to the Kansas Reflector from approximately 8am to 4pm, citing cybersecurity concerns after the nonprofit published a column critical of Facebook’s climate change ad policy. By late afternoon, all links were once again able to be posted on Facebook, Threads and Instagram–except for the critical column." Here it is. #Media<br /> https://www.thehandbasket.co/p/kansas-reflector-meta-facebook-column-censored

Here's the column Meta doesn't want you to see by [[Marisa Kabas]]

repost with comment of:<br /> When Facebook fails, local media matters even more for our planet’s future By [[Dave Kendall]]