A variety of backgrounds will often create a variety of views, which might not be clear for one person to the next.

A variety of backgrounds will often create a variety of views, which might not be clear for one person to the next.

falsifiability for the win

yes yes yes yes yes yes yes

Show alternative approaches to text visualization beyond analytics

Show alternative approaches to text visualization beyond analytics

Show alternative approaches to text visualization beyond analytics

Show alternative approaches to text visualization beyond analytics

Show alternative approaches to text visualization beyond analytics

Show alternative approaches to text visualization beyond analytics

Show alternative approaches to text visualization beyond analytics

Find civic text visualization systems that are explicitly named.

Find civic text visualization systems that are explicitly named.

Find civic text visualization systems that are explicitly named.

Find civic text visualization systems that are explicitly named.

Highlight all civic participation approaches

Highlight all civic participation approaches

Highlight all civic participation approaches

Highlight all civic participation approaches

I think editors would have done some preliminary QC on this?

reply to u/Vibeuel_ at https://www.reddit.com/r/typewriters/comments/1s7lfdu/i_hate_loving_typewriters/

He used Chapstick as a heavy grease to hold ball bearings in place to re-insert the carriage and ball bearings into a Royal Arrow.

Sorry you went through this insertion of ball bearings into a Royal in this manner. For the future attempts, there is a re-creation of a custom tool for doing this sort of operation which also includes some repair manual details: https://www.m-morren.nl/product/17071027/royal-bearing-guide-ball-feeder-tool

Lucas Dul also has a video that might help: https://www.youtube.com/watch?v=a_PONcT8HHI as well as some discussion of using straws at the 21:40 mark in this video: https://virtualhermans.com/lucas-dul

those from which data is mined are entitled to some of this economic value

data dividends as means to offset negative economic externalities of tech industry itself

not just equity in value of products derived but equity in the data as a product itself

data can no longer be treated as a raw resource

Online life during covid enabled consolidation by tech

equity in the value generated by resources we provide

Oh wow neuroscience just invented...Kant!

This suggests that there is an "external world" giving us information, and our perceptions are best guesses. That seems different from the idealism of QBism.

Heidegger seemed somewhat opposed to views like idealism, or even this predictive coding approach, right? He thought the problem of the external world (how we do we know anything about noumena when all we experience are phenomena) dissolves under a careful attention to how we operate. We are already part of and operating within the world - beings in the world - using and manipulating entities as they exist in the totality. It's only when things break down that we perceive objects as objects, and the problem seems to arise?

Interesting. So QBism is a form of idealism?

eLife Assessment

This study investigates how the HIV inhibitor lenacapavir influences capsid mechanics and interactions with the nuclear pore complex. It provides important insights into how drug-induced hyperstabilization of the viral shell can compromise its structural integrity during nuclear entry. The modeling is technically sophisticated, and the analyses provide solid support for the mechanistic conclusions.

Reviewer #1 (Public review):

The paper from Hudait and Voth details a number of coarse-grained simulations as well as some experiments focused on the stability of HIV capsids in the presence of the drug lenacapavir. The authors find that LEN hyperstabilizes the capsid, making it fragile and prone to breaking inside the nuclear pore complex.

I found the paper interesting. I have a few suggestions for clarification and/or improvement.

(1) How directly comparable are the NPC-capsid and capsid-only simulations? A major result rests on the conclusion that the kinetics of rupture are faster inside the NPC, but are the numbers of LENs bound identical? Is the time really comparable, given that the simulations have different starting points? I'm not really doubting the result, but I think it could be made more rigorous/quantitative.

(2) Related to the above, it is stated on page 12 that, based on the estimated free-energy barrier, pentamer dissociation should occur in ~10 us of CG time. But certainly, the simulations cover at least this length of time?

(3) At first, I was surprised that even in a CG simulation, LEN would spontaneously bind to the correct site. But if I read the SI correctly, LEN was parameterized specifically to bind to hexamers and not pentamers. This is fine, but I think it's worth describing in the main text.

Comments on revisions:

I found that the authors addressed my concerns satisfactorily. The other reviewer raised a number of important points regarding the nuances of the model and the interpretation of the simulations, which the authors rebutted. I think the paper in its current form now is a worthwhile addition to the literature.

Reviewer #3 (Public review):

I have carefully reviewed the manuscript, the two referee reports, and the authors' detailed responses. I appreciate the substantial effort the authors have invested in addressing the reviewers' comments, and I also recognize the strength and ambition of the work. This is a technically sophisticated study that integrates coarse-grained modeling with live-cell imaging to address an important and timely question regarding HIV-1 capsid inhibition by lenacapavir.

Embedded within Reviewer #2's report are several substantive points that warrant careful consideration, particularly with respect to framing, terminology, and engagement with the broader literature. I view my role here is to distinguish those issues from claims that I do not find to be supported.

First, I do not agree with Reviewer #2's central assertion that the manuscript lacks novelty or fails to present meaningful new findings. While individual elements of the system studied here-capsid docking at the NPC, lenacapavir-induced capsid hyperstabilization, capsid rupture, and competition with FG- nucleoporins-have been observed previously, this work provides a coherent, mechanistic account of how these elements are coupled. In particular, the proposed sequence linking LEN-induced lattice hyperstabilization, preferential pentamer loss at the narrow end, NPC-induced mechanical stress, and failure of nuclear import represents a nontrivial integration that goes beyond prior phenomenological observations. I therefore do not view this work as redundant with existing literature.

That said, Reviewer #2 is correct to note that the manuscript would benefit from broader and more explicit engagement with recent independent studies, including computational and hybrid modeling efforts that address capsid mechanics, nuclear entry, and LEN effects using different frameworks. While the authors' bottom-up coarse-grained approach is clearly distinct and, in many respects, more systematically derived, eLife readers would benefit from a clearer discussion of how the present results relate to, complement, or differ from these other approaches. I strongly encourage the authors to add a short discussion paragraph situating their work within this broader context, without disparaging alternative models.

Second, I find that some mechanistic claims in the manuscript would benefit from more careful language distinguishing model-conditioned interpretation from de novo prediction. This applies in particular to discussions of LEN binding heterogeneity and stoichiometry, as well as to conclusions drawn from biased enhanced-sampling simulations. While I agree with the authors that parameterization does not invalidate mechanistic insight, it is important to be precise about what aspects of the behavior emerge from the simulations versus what is constrained by prior experimental knowledge. Modest tightening/revising of language (e.g., "suggests," "is consistent with," "within the model") would address this concern without weakening the scientific conclusions.

Third, Reviewer #2 raises a legitimate semantic issue regarding the use of the term "elasticity." The manuscript infers changes in capsid mechanical response using heterogeneous elastic network models, which quantify effective stiffness and deformability rather than elasticity in the macroscopic materials sense. I recommend that the authors clarify this definition explicitly in the text to avoid confusion and unnecessary debate.

Finally, I note that several of Reviewer #2's objections-particularly those asserting circular reasoning, misuse of enhanced sampling methods, or invalidity of coarse-grained predictions-reflect a misunderstanding of contemporary bottom-up coarse-grained modeling rather than genuine methodological flaws. I do not believe these points require further rebuttal or revision beyond what the authors have already provided.

In summary, in my view, the manuscript represents a solid contribution to the field, provided that the authors undertake a limited set of targeted revisions aimed at improving framing, clarity, and engagement with the broader literature. Addressing these points will strengthen the manuscript and ensure that its contributions are clearly and fairly communicated to the community.

Author response:

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

It is important to make a few key points about our work. First, our paper is largely a computational biophysics paper, augmented by experimental results. Generally speaking, computational biophysics work intends to achieve one of two things (or both). One is to provide more molecular level insight into various behaviors of biomolecular systems that have not been (or cannot be) provided by qualitative experimental results alone. The second general goal of computational biophysics it to formulate new hypotheses to be tested subsequently by experiment. In our paper, we have achieved both of these goals and then confirmed the key computational results by experiment.

eLife Assessment

This study investigates how the HIV inhibitor lenacapavir influences capsid mechanics and interactions with the nuclear pore complex. It provides important insights into how drug-induced hyperstabilization of the viral shell can compromise its structural integrity during nuclear entry. While the modeling is technically sophisticated and the results are promising, some mechanistic interpretations rely on assumptions embedded in the simulations, leaving parts of the evidence incomplete.

Given our response below, regarding the rigor and “completeness” of our work, we do not feel that an editorial judgement of “leaving parts of the evidence incomplete” is justified.

We also note that another recent experimental paper has validated essentially every prediction made in our eLife paper: https://www.biorxiv.org/content/10.64898/2026.01.05.697065v1

We thus disagree that the evidence we have presented in our paper is incomplete.

Public Reviews:

Reviewer #1 (Public review):

The paper from Hudait and Voth details a number of coarse-grained simulations as well as some experiments focused on the stability of HIV capsids in the presence of the drug lenacapavir. The authors find that LEN hyperstabilizes the capsid, making it fragile and prone to breaking inside the nuclear pore complex.

I found the paper interesting. I have a few suggestions for clarification and/or improvement. 

(1) How directly comparable are the NPC-capsid and capsid-only simulations? A major result rests on the conclusion that the kinetics of rupture are faster inside the NPC, but are the numbers of LENs bound identical? Is the time really comparable, given that the simulations have different starting points? I'm not really doubting the result, but I think it could be made more rigorous/quantitative.

We note (also in the manuscript) that it is difficult to compare the timescales obtained from coarse-grained MD simulations and experiments (“real time”) given that, by design, the CG simulations are accelerated to greatly enhance sampling. However, we can qualitatively compare the timescales of different CG simulations (without directly comparing the corresponding experimental timescales).

We agree with the reviewer that the starting point of NPC-capsid and capsid-only simulations is different, as is the biological environment in which the rupture occurs. When analyzing the NPC-only and capsid-only simulations, what was striking to us was that at the NPC the capsid-LEN complex ruptures in a multicomponent environment, where several FG-NUPs compete to displace the LENs. It is well established in experiments that LEN has a detrimental effect on capsid integrity.

In Figure 2, we plot the number of LEN molecules as a function of CG simulation time. The initial capsid-LEN complex was equilibrated without NPC and then placed at the cytoplasmic end of the NPC for docking. The number of LEN molecules for the capsid-only simulations and the NPC-docked simulations is nearly identical, and an insignificant number of LEN molecules unbind at the NPC. Hence, we added the following clarification:

Page 10, paragraph 11

“Note that the number of LEN molecules bound to the capsid for the free capsid and NPCdocked capsids are nearly identical. Hence, the disparity in timescale of lattice rupture is not only because of the effect of LEN on capsid lattice properties.”

Is the time really comparable, given that the simulations have different starting points?

Yes, the CG timescales of both the NPC and freely diffusing capsid unbiased simulations are comparable, since they were done using identical simulation settings.

(2) Related to the above, it is stated on page 12 that, based on the estimated free-energy barrier, pentamer dissociation should occur in ~10 us of CG time. But certainly, the simulations cover at least this length of time?

Our implicit solvent CG MD simulations are designed to access timescales far beyond the capabilities of the fully atomistic simulations. We reiterate here that it is difficult to directly compare the timescales obtained from CG MD simulations and experiments.

As described in the text, there are 12 pentamers in the capsid (7 in the wide end and 5 in the narrow end). For the narrow end to rupture, all 5 pentamers should progressively dissociate. In our unbiased simulations (Fig. S5), in 25 us of CG time, we observe (partial) dissociation of one or two pentamers. Hence, our unbiased CG simulation timescales were not long enough to observe rupturing of the narrow end.

(3) At first, I was surprised that even in a CG simulation, LEN would spontaneously bind to the correct site. But if I read the SI correctly, LEN was parameterized specifically to bind to hexamers and not pentamers. This is fine, but I think it's worth describing in the main text.

We modified (see below) the main text to include the details.

Page 4, paragraph 1

“We model LEN and CA interactions such that LEN molecules can only bind to CA hexamers, and all interactions to CA pentamers are turned off, as in experiments, CA selectively associates with hexamers (25, 36).”

Reviewer #2 (Public review):

Here, Hudait et al. use CG modeling to investigate the mechanism by which Lenacapavir (LEN) treats HIV capsids that dock to the nuclear pore complex (NPC). However, the manuscript fails to present meaningful findings that were previously unreported in the literature and is thus of low impact. Many claims made in the manuscript are not substantiated by the presented data. Key mechanistic details that the work purports to reveal are artifacts of the parameterization choices or simulation/analysis design, with the simulations said to reveal details that they were specifically biased to reproduce. This makes the manuscript highly problematic, as its contributions to the literature would represent misconceptions based on oversights in modeling and thus mislead future readers. 

We strongly disagree with these statements, and they do not reflect the facts. We provide a rebuttal to these statements in the “Author Response” statements below.

(1) Considering the literature, it is unclear that the manuscript presents new scientific discoveries. The following are results from this paper that have been previously reported:

(a) LEN-bound capsid can dock to the nuclear pore (Figure 2; see e.g. 10.1016/j.cell.2024.12.008 or 10.1128/mbio.03613-24). 

(b) NUP98 interacts with the docked capsid (Figure 2; see e.g. 10.1016/j.virol.2013.02.008 or 10.1038/s41586-023-06969-7 or 10.1016/j.cell.2024.12.008). 

(c) LEN and NUP98 compete for a binding interface (Figure 2; see e.g. 10.1126/science.abb4808 or 10.1371/journal.ppat.1004459). 

(d) LEN creates capsid defects (Figure 3 and 5, see e.g. 10.1073/pnas.2420497122). 

(e) RNP can emerge from a damaged capsid (Figure 3 and 5; see e.g. 10.1073/pnas.2117781119 or 10.7554/eLife.64776). 

(f) LEN hyperstabilizes/reduces the elasticity of the capsid lattice (Figure 6; see e.g. 10.1371/journal.ppat.1012537). 

The goal of our simulations (in combination with experiments from the Pathak group) is to provide molecular-level insight into the sequence of events of NPC docking of capsid and the effect of LEN binding leading to sequential dissociation of pentamers and leading to rupturing of the narrow end of the cone-shaped capsid. We also compare the events leading to capsid rupture at the NPC with the same for a freely diffusing capsid, akin to that in cytoplasm. The reviewer should carefully read the abstract of our paper. In fact, the above are all papers that present qualitative experimental results that help validate our model, but they do not provide details on the molecule-scale events. For example, the paper (10.1073/pnas.2420497122 written by our coauthors in the Pathak group) is extensively used to compare the behavior of LEN-bound capsid in the cytoplasm.

(2) The mechanistic findings related to how these processes occur are problematic, either based on circular reasoning or unsubstantiated, based on the presented data. In some cases, features of parameterization and simulation/analysis design are erroneously interpreted as predictions by the CG models. 

We strongly disagree with this assessment. Our CG NPC model is largely a “bottomup” model derived from molecular scale interactions sampled in atomistic simulations (see our previous paper in PNAS https://doi.org/10.1073/pnas.2313737121). The reviewer appears to be ignorant of the “bottom-up” approach based on rigorous statistical mechanics to derive moleculescale model (please refer to a detailed review on bottom-up coarse-graining: J. Chem. Theory. Comput., 2022, 18. 5759-5791).

Using the “bottom-up” CG model of the NPC, we predicted several molecular-level details of capsid import and docking to the NPC. Our key predictions were that there is an intrinsic capsid lattice elasticity and also the pleomorphic nature of the NPC channel is key for successful capsid docking https://doi.org/10.1073/pnas.2313737121). Our computational predictions have benn, for example, validated in a recently published paper by an experimental group: Hou, Z., Shen, Y., Fronik, S. et al. HIV-1 nuclear import is selective and depends on both capsid elasticity and nuclear pore adaptability. Nat Microbiol 10, 1868–1885 (2025). https://doi.org/10.1038/s41564025-02054-z). Our work is an excellent example of how systematically derived “bottom-up” CG models can accurately predict molecular details of complex biological processes.

We have now added the following statement:

Page 3, Paragraph 1

“Importantly, the computational predictions of capsid docking to the NPC central channel have been recently validated in a HIV-1 core import at the NPC using cryo-ET (33), demonstrating how systematically derived “bottom-up” CG models can accurately predict molecular details of complex biomolecular processes.”

(a) Claim: LEN-bound capsids remain associated with the NPC after rupture. CG simulations did not reach the timescale needed to demonstrate continued association or failure to translocate, leaving the claim unsubstantiated.

The reviewer fails to recognize that the statement is based on the experimental results of LEN-bound capsid that remains bound to the NPC after rupture and fails to translocate to the nuclear side (from the Pathak group in the section “Ruptured LEN-viral complexes remain bound to the NPC”). The Reviewers’ comment is incorrect. 

(b) Claim: LEN contributes to loss of capsid elasticity. The authors do not measure elasticity here, only force constants of fluctuations between capsomers in freely diffusing capsids. Elasticity is defined as the ability of a material to undergo reversible deformation when subjected to stress. Other computational works that actually measure elasticity (e.g., 0.1371/journal.ppat.1012537) could represent a point of comparison but are not cited. The changes in force constants in the presence of LEN are shown in Figure 6C, but the text of the scale bar legend and units of k are not legible, so one cannot discern the magnitude or significance of the change.

The concept of elasticity can extend down to the mesoscopic scale. Many examples can be found in the large number of elastic network models (ENMs) of proteins published by many authors. The reviewer also fails to comprehend the meaning of the effective spring constants in the HeteroENM model and how they relate to the response of the capsid to stress (e.g., in the NPC). Note, in the NPC central channel, the capsid encounters several nucleoporins (including disordered FG Nucleoporins that not have specific interactions to rest of the proteins), and also a confined environment. This environment can exert inward stress to the capsid, which is also reflected in stress on the capsid lattice. Furthermore, the cited computational AFM studies are very far from a realistic in vivo or even in vitro set of conditions. In contrast, our study presents a realistic environment which the capsid will encounter in NPC, and then these predictions are validated by experimental results.

(c) Claim: Capsid defects are formed along striated patterns of capsid disorder. Data is not presented that correlates defects/cracks with striations. 

We presented the data of formation of striated patterns of lattice stress in the capsid that runs from capsid narrow end to the wide end in coarse-grained model (https://doi.org/10.1073/pnas.2313737121), and atomistic model (https://doi.org/10.1073/pnas.2117781119). Both of our papers are extensively cited in the current manuscript. Also, when the capsid is ruptured, one cannot visualize the striated patterns.

(d) Claim: Typically 1-2 LEN, but rarely 3 bind per capsid hexamer. The authors state: "The magnitude of the attractive interactions was adjusted to capture the substoichiometric binding of LEN to CA hexamers (Faysal et al., 2024). ... We simulated LEN binding to the capsid cone (in the absence of NPC), which resulted in a substoichiometric binding (~1.5 LEN per CA hexamer), consistent with experimental data (Singh et al., 2024)." This means LEN was specifically parameterized to reproduce the 1-2 binding ratio per hexamer apparent from experiments, so this was a parameterization choice, not a prediction by CG simulations as the authors erroneously claim: "This indicates that the probability of binding a third LEN molecule to a CA hexamer is impeded, likely due to steric effects that prevent the approach of an incoming molecule to a CA hexamer where 2 LEN molecules are already associated. ... Approximately 20% of CA hexamers remain unoccupied despite the availability of a large excess of unbound LEN molecules. This suggests a heterogeneity in the molecular environment of the capsid lattice for LEN binding." These statements represent gross over-interpretation of a bias deliberately introduced during parameterization, and the "finding" represents circular reasoning. Also, if "steric effects" play any role, the authors could analyze the model to characterize and report them rather than simply speculate.

Reviewer comment: “This means LEN was specifically parameterized to reproduce the 1-2 binding ratio per hexamer apparent from experiments, so this was a parameterization choice, not a prediction by CG simulations as the authors erroneously claim.” – This comment by reviewer is deeply flawed and we strongly disagree. In our CG model there is no restriction on the number of LEN molecules that can bind to a CA hexamer. We again restate that, the experimental results on LEN binding to CA hexamers and inability of LEN to bind to pentamers were used as no allatom (AA) forcefield yet exists.

The steric effect of the lack of third LEN binding to a hexamer is a likely hypothesis (which one is allowed to make). More importantly, an investigation of the steric effect of LEN binding to the CA hexamer is not the main goal of the manuscript.

(e) Claim: Competition between NUP98 and LEN regulates capsid docking. The authors state: "A fraction of LEN molecules bound at the narrow end dissociate to allow NUP98 binding to the capsid ... Therefore, LEN can inhibit the efficient binding of the viral cores to the NPC, resulting in an increased number of cores in the cytoplasm." Capsid docking occurs regardless of the presence of LEN, and appears to occur at the same rate as the LEN-free capsid presented in the authors' previous work (Hudait &Voth, 2024). The presented data simply show that there is a fluctuation of bound LEN, with about 10 fewer (<5%) bound at the end of the simulation than at the beginning, and the curve (Figure 2A) does not clearly correlate with increased NUP98 contact. In that case, no data is shown that connects LEN binding with the regulation of the docking process. Further, the two quoted statements contradict each other. The presented data appear to show that NUP outcompetes LEN binding, rather than LEN inhibiting NUP binding. The "Therefore" statement is an attempt to reconcile with experimental studies, but is not substantiated by the presented data.

We disagree with this spurious statement, and we see no real contradiction. We have now added a minor clarification that LEN can inhibit efficient capsid binding at significantly high concentration.

Page 6, Paragraph 1

“Therefore, at significantly high concentration LEN can inhibit the efficient binding of the viral cores to the NPC, resulting in an increased number of cores in the cytoplasm.”

(f) Claim: LEN binding leads to spontaneous dissociation of pentamers. The CG simulation trajectories show pentamer dissociation. However, it is quite difficult to believe that a pentamer in the wide end of the capsid would dissociate and diffuse 100 nm away before a hexamer in the narrow end (previously between two pentamers and now only partially coordinated, also in a highly curved environment, and further under the force of the extruding RNA) would dissociate, as in Figure 2B. A more plausible explanation could be force balance between pent-hex versus hex-hex contacts, an aspect of CG parameterization. No further modeling is presented to explain the release of pentamers, and changes in pent-hex stiffness are not apparent in the force constant fluctuation analysis in Figure 6C.

This is both a misrepresentation of the simulations and a failure to understand them (as well as the supporting experiments) on the part of the reviewer. In the presence of LEN, the hexameric lattice is hyperstabilized. In contrast, the pentamers are not. As a consequence, the pentamers are dissociated. The pentamers at the narrow end are dissociated first, due to high curvature. The reviewer, from a point of being uninformed, simply speculates on what they think should happen. Moreover, as emphasized earlier and which the reviewer fails to comprehend is that ours is a “bottom-up CG model” so it predicts, not builds in, these effects.

(g) Claim: WTMetaD simulations predict capsid rupture. The authors state: "In WTMetaD simulations, we used the mean coordination number (Figure S6) between CA proteins in pentamers and in hexamers as the reaction coordinate." This means that the coordination number, the number of pent-hex contacts, is the bias used to accelerate simulation sampling. Yet the authors then interpret a change in coordination number leading to capsid rupture as a discovery, representing a fundamental misuse of the WTMetaD method. Changes in coordination number cannot be claimed as an emergent property when they are in fact the applied bias, when the simulation forced them to sample such states. The bias must be orthogonal to the feature of interest for that feature to be discoverable. While the reported free energies are orthogonal to the reaction coordinate, the structural and stepwise-mechanism "findings" here represent circular reasoning.

Unfortunately, the reviewer appears to be quite uninformed on the WTMetaD method and what it does. The chosen collective variable (CV) in our case is the coordination variable and the MetaD samples along that variable (the conditional free energy) as it is designed to do. The reviewer may wish to educate themself by reading Dama et al (https://doi.org/10.1103/PhysRevLett.112.240602). We also note that “emergent properties” are not along some other, uncoupled coordinate.

(3) Another major concern with this work is the excessive self-citation, and the conspicuous lack of engagement with similar computational modeling studies that investigate the HIV capsid and its interactions with LEN, capsid mechanical properties relevant to nuclear entry, and other capsidNPC simulations (e.g., 10.1016/j.cell.2024.12.008 and 10.1371/journal.ppat.1012537). Other such studies available in the literature include examination of varying aspects of the system at both CG and all-atom levels of resolution, which could be highly complementary to the present work and, in many cases, lend support to the authors' claims rather than detract from them. The choice to omit relevant literature implies either a lack of perspective or a lack of collegiality, which the presentation of the work suffers from. Overall, it is essential to discuss findings in the context of competing studies to give readers an accurate view of the state of the field and how the present work fits into it. It is appropriate in a CG modeling study to discuss the potential weaknesses of the methodology, points of disagreement with alternative modeling studies, and any lack of correlation with a broader range of experimental work. Qualitative agreement with select experiments does not constitute model validation. 

We disagree with this statement and point out where we have cited other work, including the ones mentioned above. However, our CG model is a largely bottom-up CG model which differs from other more ad hoc CG approaches (and some well-known CG models). We do not wish to emphasize the obvious flaws in those other CG approaches and models, since that is not the focus of our manuscript.

(4) Other critiques, questions, concerns:

(a) The first Results sub-heading presents "results", complete with several supplementary figures and a movie that are from a previous publication about the development of the HIV capsid-NPC model in the absence of LEN (Hudait &Voth, 2024). This information should be included as part of the introduction or an abbreviated main-text methods section rather than being included within Results as if it represents a newly reported advancement, as this could be misleading. 

The movie in question (capsid docking to NPC without LEN) is essential for comparison of LEN-binding dynamics. Different from our previous paper, we simulated significantly longer timescales of capsid docking and performed several additional analyses that is relevant to this paper. Moreover, the first section of the result is titled “Coarse-grained modeling and simulation”, hence we only present a summary of the CG models and key validation steps in this section.

(b) The authors say the unbiased simulations of capsid-NPC docking were run as two independent replicates, but results from only one trajectory are ever shown plotted over time. It is not mentioned if the time series data are averaged or smoothed, so what is the shadow in these plots (e.g., Figures 1,2, and Supplementary Figure 5)?

These simulations are the average from two replicas. “For all the plots, the solid lines are the mean values calculated from the time series of two independent replicas, and the shaded region is the standard deviation at each timestep.” This was mentioned in the original figure caption.

(c) Why do the insets showing LEN binding in Figure 2A look so different from the models they are apparently zoomed in on? Both instances really look like they are taken from different simulation frames, rather than being a zoomed-in view.

It is difficult to discern a high curvature region of the capsid due to object overlap of different regions of the capsid. This is likely a case of “perspective distortion” in image processing.

(d) What are the sudden jerks apparent in the SI movies? Perhaps this is related to the rate at which trajectory frames are saved, but occasionally, during the relatively smooth motion of the capsidNPC complex, something dramatic happens all of a sudden in a frame. For example, significant and apparently instantaneous reorientation of the cone far beyond what preceding motions suggest is possible (SI movie 2, at timestamp 0.22), RNP extrusion suddenly in a single frame (SI movie 2, at timestamp 0.27), and simultaneous opening of all pentamers all at once starting in a single frame (SI movie 2, at timestamp 0.33). This almost makes the movie look generated from separate trajectories or discontinuous portions of the same trajectory. If movies have been edited for visual clarity (e.g., to skip over time when "nothing" is happening and focus on the exciting aspects), then the authors should state so in the captions. 

This is due to the rate at which trajectory frames are saved for movie generation for faster processing of the movies. We added the following in movie caption: 

“The movie frames correspond to snapshots every 250000 𝜏_CG.” 

(e) Figure 3c presents a time series of the degree of defects at pent-hex and hex-hex interfaces, but I do not understand the normalization. The authors state, "we represented the defects as the number of under-coordinated CA monomers of the hexamers at the pentamer-hexamer-pentamer and hexamer-hexamer interface as N_Pen-Hex and N_Hex-Hex ... Note that in N_Pen-Hex and N_Hex-Hex are calculated by normalizing by the total number of CA pentamer (12) and hexamer rings (209) respectively." Shouldn't the number of uncoordinated monomers be normalized by the number of that type of monomer, rather than the number of capsomers/rings? E.g., 12*5 and 209*6, rather than 12 and 209?

We prefer to continue with the current normalization, since typically in the HIV-1 literature capsids are represented as a collection of hexamers and pentamers (rather than total number of CA monomers).

(f) The authors state that "Although high computational cost precluded us from continuing these CG MD simulations, we expect these defects at the hexamer-hexamer interface to propagate the high curvature ends of the capsid." The defects being reported are apparently propagating from (not towards) the high curvature ends of the capsid. 

We corrected the statement as follows:

“Although high computational cost precluded us from continuing these CG MD simulations, we expect these defects at the hexamer-hexamer interface to propagate from the high curvature to low curvature end of the capsid.”

(g) The first half of the paper uses the color orange in figures to indicate LEN, but the second half uses orange to indicate defects, and this could be confusing for some readers. Both LEN and "defects" are simply a cluster of spheres, so highlighted defects appear to represent LEN without careful reading of captions.

We only show LEN in Figure 1, and in rest of the figures the bound LEN molecules are not shown for clarity. The defects are shown in a darker shade of orange (amber). 

(h) SI Figure S3 captions says "The CA monomers to which at least one LEN molecule is bound are shown in orange spheres. The CA monomers to which no LEN molecule is bound are shown in white spheres. " While in contradiction, the main-text Fig 2 says "The CA monomers to which at least one LEN molecule is bound are shown in white spheres. The CA monomers to which no LEN molecule is bound are shown in orange spheres. " One of these must be a typo.

We have corrected the erroneous caption in Fig. S3. The color scheme in Fig. 2 and Fig. S3 are now consistent.

(i) The authors state that: "CG MD simulations and live-cell imaging demonstrate that LEN-treated capsids dock at the NPC and rupture at the narrow end when bound to the central channel and then remain associated to the NPC after rupture." However, the live cell imaging data do not show where rupture occurs, such that this statement is at least partially false. It is also unclear that CG simulations show that cores remain bound following rupture, given that simulations were not extended to the timescale needed to observe this, again rendering the statement partially false.

We modified the statement as follows:

“CG MD simulations complemented by the outcome of live-cell imaging demonstrate that LENtreated capsids dock at the NPC and rupture at the narrow end when bound to the central channel and then remain associated with the NPC after rupture.”

(j) The authors state: "We previously demonstrated that the RNP complex inside the capsid contributes to internal mechanical strain on the lattice driven by CACTD-RNP interactions and condensation state of RNP complex (Hudait &Voth, 2024). " In that case, why do the present CG models detect no difference in results for condensed versus uncondensed RNP?

In our previous paper, the difference from condensation state of RNP complex appear only in the pill-shaped capsid, and not in the cone-shaped capsid. In this manuscript, we only investigated the cone-shaped capsid.

(k) The authors state: "The distribution demonstrates that the binding of LEN to the distorted lattice sites is energetically favorable. Since LEN localizes at the hydrophobic pocket between two adjoining CA monomers, it is sterically favorable to accommodate the incoming molecule at a distorted lattice site. This can be attributed to the higher available void volume at the distorted lattice relative to an ordered lattice, the latter being tightly packed. This also allows the drug molecule to avoid the multitude of unfavorable CA-LEN interactions and establish the energetically favorable interactions leading to a successful binding event. " What multitude of unfavorable interactions are the authors referring to? Data is not presented to substantiate the claim of increased void volume between hexamers in the distorted lattice. Capsomer distortion is shown as a schematic in Figure 6A rather than in the context of the actual model.

“What multitude of unfavorable interactions are the authors referring to?” We have now added the following sentence to clarify

“Here we denote unfavorable CA-LEN interactions as all interactions other than the electrostatic and van der Waal interactions that lead to CA-LEN binding (17).”

“In the distorted lattice, there is an increase of void volume is based on standard solid-state physics understanding. We added the word “likely” in the statement. “. This can likely be attributed to the higher available void volume at the distorted lattice relative to an ordered lattice, the latter being tightly packed (41).”

Moreover, in one of our previous manuscripts, we established that compressive or expansive strain induces more closely packed or expanded lattice (A. Yu et al., Strain and rupture of HIV-1 capsids during uncoating. Proceedings of the National Academy of Sciences 119, e2117781119 (2022)).

(l) The authors state that "These striated patterns also demonstrate deviations from ideal lattice packing. " What does ideal lattice packing mean in this context, where hexamers are in numerous unique environments in terms of curvature? What is the structural reference point?

The ideal lattice packing definition is provided in our previous manuscripts: 1. A. Yu et al., Strain and rupture of HIV-1 capsids during uncoating. Proceedings of the National Academy of Sciences 119, e2117781119 (2022), 2. A. Hudait, G. A. Voth, HIV-1 capsid shape, orientation, and entropic elasticity regulate translocation into the nuclear pore complex. Proceedings of the National Academy of Sciences 121, e2313737121 (2024).

These manuscripts are cited in the previous statement. The ideal lattice packing is defined based on lattice separations in each core (in cryo-ET and atomistic simulations) using a local order parameter, which measures the near-neighbor contacts of a particle. Moreover, the ideal packing reference is calculated from all available capsid shapes (cone, ellipsoid, and tubular), and takes into account different curvatures.

(m) If pentamer-hexamer interactions are weakened in the presence of LEN, why are differences at these interfaces not apparent in the Figure 6C data that shows stiffening of the interactions between capsomer subunits?

We have added a statement as follows:

“Based on our analysis, we hypothesize that LEN binding hyperstabilzes the CA hexamerhexamer interactions relative to CA hexamer-pentamer interaction.”

(n) The authors state: "Lattice defects arising from the loss of pentamers and cracks along the weak points of the hexameric lattice drive the uncoating of the capsid." The word rupture or failure should be used here rather than uncoating; it is unclear that the authors are studying the true process of uncoating and whether the defects induced by LEN binding relate in any way to uncoating. 

We have now changed “uncoating” to “rupture” throughout the manuscript.

(o) The authors state: " LEN-treated broken cores are stabilized by the interaction with the disordered FG-NUP98 mesh at the NPC." But no data is presented to demonstrate that capsid stability is increased by NUP98 interaction. In fact, the presented data could suggest the opposite since capsids in contact with NUP98 in the NPC appeared to rupture faster than freely diffusing capsids.

We have modified the statement as follows

“We hypothesize that LEN-treated broken cores are stabilized by the interaction with the disordered FG-NUP98 mesh at the NPC.”

(p) The authors state: "LEN binding stimulates similar changes in free capsids, but they occur with lower frequency on similar time scales, suggesting that the cores docked at the NPC are under increased stress, resulting in more frequent weakening of the hexamer-pentamer and hexamerhexamer interactions, as well as more nucleation of defects at the hexamer-hexamer Interface. ... Our results suggest that in the presence of the LEN, capsid docking into the NPC central channel will increase stress, resulting in more frequent breaks in the capsid lattice compared to free capsids." The first is a run-on sentence. The results shown support that LEN stimulates changes in free capsids to happen faster, but not more frequently. The frequency with which an event occurs is separate from the speed with which the event occurs.

We have fixed the run-on sentence.

The results shown support that LEN stimulates changes in free capsids to happen faster, but not more frequently. The frequency with which an event occurs is separate from the speed with which the event occurs.

We disagree with the reviewer. The statement was intended to provide a comparison between free capsid and NPC-bound capsid.

(q) The authors state: "A possible mechanistic pathway of capsid disassembly can be that multiple pentamers are dissociated from the capsid sequentially, and the remaining hexameric lattice remains stabilized by bound LEN molecules for a time, before the structural integrity of the remaining lattice is compromised." This statement is inconsistent with experimental studies that say LEN does not lead to capsid disassembly, and may even prevent disassembly as part of its disruption of proper uncoating (e.g., 10.1073/pnas.2420497122 previously published by the authors).

We disagree with the interpretation of the reviewer. Our interpretation based on our results is LEN binding accelerates capsid rupture (from pentamer-rich high curvature ends), and the rest of the broken hexameric lattice is hyperstabilized. Ultimately, lattice rupture will lead to release the RNP, and hence the intended goal of the drug is achieved.

(r) Finally, it remains a concern with the authors' work that the bottom-up solvent-free CG modeling software used in this and supporting works is not open source or even available to other researchers like other commonly used molecular dynamics software packages, raising significant questions about transparency and reproducibility.

The simulations were performed in LAMMPS, which is open source. This software is already stated in the Methods. Input data is provided upon request.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

(1) Figure 1: In part B, it appears the middle panel was screenshotted from a ppt, given the red line underneath Lenacapavir. You can export it to an image instead.

The figure is fixed.

(2) Figure 6: In part A, the LEN_d in the graph is illegible. Also, in the panel next to it, it also appears to have been screenshotted from a ppt.

The figure is fixed.

(3) Page 6: There's an errant quotation mark at the end of a paragraph.

Removed the errant quotation

Reviewer #2 (Recommendations for the authors):

The code used to perform bottom-up solvent-free CG modeling simulations is not made available.

This is not true. LAMMPS was used as stated in Methods.

sentence describing the concept of interaction

The US 'DSA is censorship' bs is not holding up even within the US admin itself, the regime ploughs on though.

is this a full preprint then or forthcoming?

preprint paper by [[Martijn Aslander p]] on his lls The Information Problem Was Never the Real Problem: Building a Confirmation-Driven Personal Knowledge Graph and the Four Principles That Emerge From Practice in Zotero

Exactly, for someone with a keen eye for collecting data, they probably see this and they are in complete data collection bliss.

That is quite fascinating. This is a good example of what I was just talking about with new technologies opening up for new methods that did not exist from back then they did not exist.

Ultimately, if something makes something easier, especially something as complex as measuring space, I think it is totally fine to embrace new technologies in this way.

This is fascinating since it is making me truly appreciate how much the chronicling of space and time are so heavily intertwined. This is correct, you cannot measure one without the other.

eLife Assessment

In this fundamental work Horne et al present compelling evidence that YbjP is a novel binding partner of the TolC channel protein. The YbjP is characterized using cryo-EM, and its role probed using pull-down experiments, in vivo crosslinking, functional assays along with phylogenetic analysis which are all properly performed and presented and support the main conclusions. While the study does not identify a clear role for this protein, the revised manuscript offers improved clarity and contributes invaluable insight into membrane transport and antimicrobial resistance.

Reviewer #2 (Public review):

This article focuses on the study of two E. coli tripartite efflux pumps, both using TolC as a partner in the outer membrane, namely MacAB-TolC and AcrABZ-TolC.

By preparing MacAB-TolC in Peptidiscs rather than in detergent for cryo-EM structure determination, they visualized an extra protein localized around TolC. The resolution was sufficient to build part of the structure, and using the AlphaFold2 database and DALI topology recognition program, they identified it as the lipoprotein YbjP. This protein has an anchorage in the outer membrane, and it was suggested that it could act as a support for TolC, which is the only OMF that does not have an N-terminal extension anchored in the outer membrane, which is very puzzling for the community working in this field of research.

Authors used a large number of different approaches to evaluate the importance of YbjP (structure, genomic evolution, microbiology, photocrosslink in vivo, proteomic profile), but did not succeed in finding it a clear role so far, even if it could be important depending on environmental stress. Nevertheless, their results, obtained with extreme rigour, are of main interest for the comprehension of the complexity of such systems and deserve publication.

Comments on revisions:

Thank you for clarifying the points that puzzled me concerning the crosslink experiments. This version does not need further modifications.

Author Response:

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

Reviewer #1 (Public review):

The presentation and especially main-text illustrative material seem to focus disproportionately on MacAB-TolC-YbjP complex, and the AcrABZ-TolC-YbjP is relegated to supplementary data which is somewhat confusing. There is no high-resolution side view of the AcrABZ-TolC-YbjP side-by-side to MacAB-TolC-YbjP which may be helpful to spot parallels and differences in the organisation of the two systems.

This was previously presented in Supplementary Figure S2. However, because the models were shown at a small scale, we have now included the comparison in a main manuscript (Figure 4). This figure presents AcrABZ-TolC-YbjP and MacAB-TolC-YbjP side-by-side, a structural alignment of TolC-YbjP in the two pumps, and close-up views of the interaction interface.

Supplementary Figure 2 may also be better presented in the main text, as it shows specific displacements of residues upon binding of the YbjP relative to the apo-complexes, although this can be left at the authors' discretion.

We added more text to describe the displacements of residues upon YbjP binding: ‘Nonetheless, the side chains of a few residues in TolC, which mainly correspond to positively charged amino acids (R18, R24, K214, R227, R234), reorient to interact with the YbjP lipoprotein partner (Figure 2B).’

Reviewer #1 (Recommendations for the authors):

The work is of high quality and requires minimal modifications, which are mentioned as suggestions above and are mostly connected to the illustrative material.

One additional suggestion, which is connected to the earlier BioRxiv preprint, the data seen in Fig 6 of the preprint seems to have been edited out from the current version, and perhaps can be included in a revised version, as it seems to support the "rapid adaptation under stress" role for YbjP, which currently is only speculatively mentioned in p.11, line 365 of the manuscript.

We acknowledge that the BioRxiv preprint Figure 6 can support the rapid adaptation under stress role for YbjP. However, upon sequencing the ΔybjP strain from the Keio collection used in the preprint, we identified a large deletion in the yecT-flhD region. We therefore generated a new ΔybjP strain without the yecT-flhD deletion and repeated the experiment. However, the results with the corrected strain did not support the previous conclusion, and these data were consequently removed in the current manuscript.

Reviewer #2 (Public review):

In Figure 3C, the experiment performed with AcrA is clear and the extra band appears at the proper size. On the right panel, it is clear that the crosslink doesn't work when pBPA is placed on residues too far from TolC. Only when introduced on N113 or T110 does a band appear.

This is in accordance with an interaction in vivo. Nevertheless, 17 + 54 = 71kDa, which is more than the two bands appearing on the gel. This difference in size migration can occur, but it is not clear when looking at Figure S3. In Figure S3a, the purified proteins are highlighted at approximately the expected size (≈20kDa instead of 17 for YbjP and between 56 and 60kDa in two bands for TolC instead of 54kDa). On the right panel, it seems that the bands are present exactly at the same position, instead of an upper band as expected for the crosslinked YbjP-TolC (at 71kDa). It would be clearer if having the control of the same sample without illumination, revealed by anti-TolC, to see the difference.

We thank the reviewer for pointing out this discrepancy. We identified an error in the molecular weight ladder, as one band was missing. This has now been corrected: YbjP migrates just below 17 kDa, consistent with Figure 3C. In addition, we previously reported a size of 54 kDa for TolC, whereas matured TolC, after signal peptide cleavage, is actually 52 kDa.

We believe that the differences in the apparent molecular weight observed in Figures 3A, 3C and S3 (now S2) mainly result from tagging and post-translation modifications.

In Figure 3A, we used the soluble construct His-YbjP_28-1711 (theoretical M_w ~18 kDa), as also done for the controls in Figures 3C and S3 (now S2). However, for the crosslinking samples, we used full-length His-tagged YbjP, which carries a post-translational lipid modification (theoretical M_w ~19 kDa, considering the protein lipidation). The presence of the lipid chains alters the migration as this species migrates at ~15 kDa (Fig 3A). Increased hydrophobicity, due here to YbjP lipidation, could accelerate the migration (Emmanuel et al. 2025 FEBS Open Bio).

In Figure 3A, we used the TolC-FLAG whose apparent M_w is ~52 kDa, as previously reported (Fig S3, Fitzpatrick et al. 2017). In Figure S3 (now S2), we used His-tagged TolC (theoretical M_w 55 kDa) for the control, which migrates above 56 kDa. In the crosslinking samples, however, we detect tag-free, endogenous TolC, with a theoretical M_w of ~51 kDa.

In conclusion, the crosslinked complex composed of lipidated FL YbjP (~15 kDa) and endogenous TolC (~51 kDa) would be expected to migrate at ~66 kDa, which is consistent with what is observed in Figures 3C and S3 (now S2).

A second point that could be discussed further is the comparison of the structure of the pump in the presence of the peptidoglycan with the images previously obtained by tomography. It is not totally clear to me if YbjP could have been positioned in these maps.

There is density corresponding to YbjP in the map obtained in the presence of peptidoglycan. To improve clarity, we have specified the location of the peptidoglycan relative to the pumps in the revised Figure 4, and Supplementary Figure S4, together with the position of YbjP. In both figures, the lipoprotein appears distant from the peptidoglycan density.

Reviewer #2 (Recommendations for the authors):

In addition, please add explanations in the legend of Figure 3C concerning the structures.

We added the following description of the structures: ‘As shown underneath, AcrA residues Q136 and Y137, proximal to TolC in the structure of the AcrABZ-TolC pump (PDB 5NG5), were replaced by pBPA. For YbjP, the two residues N113 and T110 proximal to TolC in the MacAB-TolC-YbjP complex (PDB 9QGY) and the three residues N43, N90 and H104 distal to TolC were mutated.’

It would be clearer if having the control of the same sample without illumination, revealed by anti-TolC, to see the difference.

As the amount of crosslinked material is low, samples were enriched via His-tag purification of YbjP prior to Western blotting. In the absence of illumination (see sample N113, UV-), no crosslink would be formed, and therefore TolC would not be co-purified.

In addition, some typo errors have been noted.

Table S1 minus is missing for the defocus range for AcrABZ-TolC-YbjP.

Thank you for noting the typo. We have added the minus sign.

Table S3, please specify what is N in the legend.

N is the stoichiometry parameter, which is now specified in the table legend.

Line 237, I suppose it has to refer to Figure S6, not S5.

Thank you for noting the error. We have verified the text matches the figures here and in the entire manuscript.

Several errors are present in the legend of Figure 6.

No letters are indicated for the different panels; line 841 must be C, F and I; the indicated colors for the differentially expressed proteins do not correspond to the volcano plots.

Thank you for suggesting the improvements for the labels. We have modified the plot accordingly.

Reference Glavier 2020 has been cited as Glacier on line 72.

We have modified the writing accordingly and checked the reference.

eLife Assessment

This is an important study that takes a key step towards understanding developmental disorders linked to mutations in the O-GlcNAc transferase enzyme by generating a mouse model harboring the C921Y mutation. While the mechanisms remain open, the study thoroughly examines behavioral and anatomical differences in these mice and provides convincing evidence for behavioral hyperactivity and learning/memory deficits, as well as phenotypic differences in skull and brain formation. This study will be of interest to those studying neurodevelopmental disorders and associated mechanisms.

Reviewer #1 (Public review):

This study established a C921Y OGT-ID mouse model, systematically demonstrating in mammals the pathological link between O-GlcNAc metabolic imbalance and neurodevelopmental disorders (cortical malformation, microcephaly) as well as behavioral abnormalities (hyperactivity, impulsivity, learning/memory deficits). Researchers comprehensively assessed the model phenotype through integrated multi-level analysis methods, including long-term behavioral monitoring, high-resolution brain structural imaging (micro-CT and MRI), histopathology, and quantitative proteomics.

The core strength of this study lies in its multimodal experimental design. The evidence chain spanning in vivo behavior, brain structure, and molecular characteristics demonstrates high consistency and correlation. Of particular note is the combination of non-invasive behavioral tracking with quantitative neuroimaging techniques, providing objective validation for the observed phenotypes. The findings support the authors' core conclusion: O-GlcNAc homeostasis imbalance correlates with neurodevelopmental deficits, including structural abnormalities in specific brain regions and altered cognitive behaviors. Furthermore, this model reproduces certain clinical features observed in human patients.

Nevertheless, several avenues remain open for further exploration. For instance, sample sizes in certain omics analyses remain relatively small, and investigations into downstream molecular mechanisms are still confined to the level of correlation-direct causal validation through genetic or pharmacological interventions is still required. Furthermore, as this model focuses on a single recurrent mutation, the generalizability of its findings to other OGT-ID variants remains to be verified.

It provides the first actionable vertebrate model for neurodevelopmental disorders with unclear mechanisms, filling a critical gap in this field. The multidimensional research methods established in the paper-such as the digital behavioral phenotyping workflow-also offer valuable references for related disease studies.

Reviewer #2 (Public review):

Summary:

The authors are trying to understand why certain mutants of O-GlcNAc transferase (OGT) appear to cause developmental disorders in humans. As an important step towards that goal, the authors generated a mouse model with one of these mutations that disrupts OGT activity. They then go on to test these mice for behavioral differences, finding that the mutant mice exhibit some signs of hyperactivity and differences in learning and memory. They then examine alterations to the structure of the brain and skull, and again find changes in the mutant mice that have been associated with developmental disorders. Finally, they identify proteins that are up or down regulated between the two mice as potential mechanisms to explain the observations.

Strengths:

The major strength of this manuscript is the creation of this mouse model, as a key step in beginning to understand how OGT mutants cause developmental disorders. This line will prove important for not only the authors but other investigators as well, enabling the testing of various hypotheses and potentially treatments. The experiments are also rigorously performed and the conclusions are well supported by the data.

Weaknesses:

The only weakness is a lack of mechanistic insight. However, this certainly may come in the future through more targeted experimentation using this mouse model. I do not recommend that these experiments need to be performed in this manuscript.

Comments on revisions:

The authors have addressed all of my suggestions proactively.

Author Response:

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

Public Reviews:

Reviewer #1 (Public review):

This study established a C921Y OGT-ID mouse model, systematically demonstrating in mammals the pathological link between O-GlcNAc metabolic imbalance and neurodevelopmental disorders (cortical malformation, microcephaly) as well as behavioral abnormalities (hyperactivity, impulsivity, learning/memory deficits). However, critical flaws in the current findings require resolution to ensure scientific rigor.

The most concerning finding appears in Figure S12. While Supplementary Figure S12 demonstrates decreased OGA expression without significant OGT level changes in C921Y mutants via Western blot/qPCR, previous reports (Florence Authier, et al., Dis Model Mech. 2023) described OGT downregulation in Western blot and an increase in qPCR in the same models. The opposite OGT expression outcomes in supposedly identical mouse models directly challenge the model's reliability. This discrepancy raises serious concerns about either the experimental execution or the interpretation of results. The authors must revalidate the data with rigorous controls or provide a molecular biology-based explanation.

We thank the reviewer for their time and effort in improving the quality of our manuscript.

We would like to point out that the results presented in the previous Fig. S12 (now Fig. S13) are from different ages of the mice and restricted to the prefrontal cortex, compared to the previous report (Florence Authier, et al., Dis Model Mech. 2023) where we showed OGT and OGA mRNA/protein expression in total brain homogenates. In this previous study, we observed a significant reduction in OGT protein levels while OGT mRNA levels were significantly increased in the brains of 3 months old mutant C921Y compared to WT controls. However, in our current study (Figure S12, now S13), OGA and OGT mRNA/protein expression have been a) restricted to the pre-frontal cortex and b) are from 4 months old male mice. Therefore, a direct comparison of findings from total brain vs. prefrontal cortex would be speculative. In our present work, OGT protein levels are not changed in the pre-frontal cortex, while OGT mRNA levels are increased (similarly to the total brain data), albeit not significantly.

It is plausible that the different levels of OGT protein expression in total brain (previous study) and prefrontal cortex (current study) potentially reflect regional differences in the regulation of OGT protein levels/stability, since OGT mRNA levels are increased in both cases. This notion is also supported by additional analyses in three other brain regions (hippocampus, striatum and cerebellum) and these data are now included in Figures S13 and S14.

A few additional comments to the author may be helpful to improve the study.

Major

(1) While this study systematically validated multi-dimensional phenotypes (including neuroanatomical abnormalities and behavioral deficits) in OGT C921Y mutant mice, there is a lack of relevant mechanisms and intervention experiments. For example, the absence of targeted intervention studies on key signaling pathways prevents verification of whether proteomics-identified molecular changes directly drive phenotypic manifestations.

We agree with the reviewer that the suggested experiments would further strengthen our work. However, the extensive nature of the suggested studies would result in considerable delay in sharing this work with the scientific and patient communities. Nevertheless, we appreciate the reviewers’ comment and will continue to work along these lines, and report in a follow up manuscript in the future.

(2) Although MRI detected nodular dysplasia and heterotopia in the cingulate cortex, the cellular basis remains undefined. Spatiotemporal immunofluorescence analysis using neuronal (NeuN), astrocytic (GFAP), and synaptic (Synaptophysin) markers is recommended to identify affected cell populations (e.g., radial glial migration defects or intermediate progenitor differentiation abnormalities).

Following the reviewers’ suggestion, we have performed additional analyses to identify the cellular composition of the observed nodular dysplasia using neuronal and glial markers. These new analyses indicate that the nodular collections in the layers II/III were predominantly neurons, for example see cresyl violet (Fig. 6E). Moreover, we have also performed immunofluorescence imaging using NeuN and GFAP (Fig. 6G-H), which reflect that the dystrophic collections are predominantly neurons. To further corroborate these findings, we have also performed multiplex IHC analyses, presented in Fig. S12, which indicate that: i) the nodular cortical malformations were populated by neurons and oligodendrocytes and ii) predominantly affected layers II-V, as reflected by the distribution of neuronal markers Reelin and POU class 3 homeobox 2 (POU3F2), and collectively (Fig. 6 and Fig. S12) reflect neuronal disorganisation due to migration defects rather than differentiation defects. We appreciate the reviewers’ suggestion to perform spatiotemporal analyses of these cellular features; however, tissue from defined stages of development is not available. 

(3) While proteomics revealed dysregulation in pathways including Wnt/β-catenin and mTOR signaling, two critical issues remain unresolved: a) O-GlcNAc glycoproteomic alterations remain unexamined; b) The causal relationship between pathway changes and O-GlcNAc imbalance lacks validation. It is recommended to use co-immunoprecipitation or glycosylation sequencing to confirm whether the relevant proteins undergo O-GlcNAc modification changes, identify specific modification sites, and verify their interactions with OGT.

We agree with the referee that these experiments would further strenghten the work. However, we respectfully point out that the inference that altered proteins must themselves be O-GlcNAc modified is not necessarily correct. For instance, O-GlcNAcylation of unknown protein kinase X, E3 ligase/DUB, Y or transcription factor Z could indirectly affect these pathways/proteins. Nevertheless, we have performed further experiments to explore whether Wnt/β-catenin and mTOR signalling are functionally affected, as pointed out by the referee. In the qPCR analyses, we did not observe significant changes in expression of Wnt target genes (Cdkn1a, Ccnd1, Myc, Ramp3, Tfrc), neither in protein levels of key proteins involved in Wnt/β-catenin (non-phosphorylated β-catenin) and mTOR (phosphorylated rpS6) signalling by western blots (data not shown). These results suggest that both pathways are not functionally deregulated in prefrontal cortex of adult OGT^C921Y mice to a significant extent.

(4) Given that OGT-ID neuropathology likely originates embryonically, we recommend serial analyses from E14.5 to P7 to examine cellular dynamics during critical corticogenesis phases.

We appreciate the reviewers’ suggestion to perform spatiotemporal analyses of these cellular dynamics; however, tissue from defined stages of development is not available. As stated above, we want to share our current findings with the scientific and patient communities in a timely manner, and the suggested experiments could form the foundation of a follow up study in the future.

(5) The interpretation of Figure 8A constitutes overinterpretation. Current data fail to conclusively demonstrate impairment of OGT's protein interaction network and lack direct evidence supporting the proposed mechanisms of HCF1 misprocessing or OGA loss.

Thank you for the comment. To avoid misleading the readers, we have removed panel A from the previous version of Figure 8 and updated the version of record.

Reviewer #2 (Public review):

Summary:

The authors are trying to understand why certain mutants of O-GlcNAc transferase (OGT) appear to cause developmental disorders in humans. As an important step towards that goal, the authors generated a mouse model with one of these mutations that disrupts OGT activity. They then go on to test these mice for behavioral differences, finding that the mutant mice exhibit some signs of hyperactivity and differences in learning and memory. They then examine alterations to the structure of the brain and skull and again find changes in the mutant mice that have been associated with developmental disorders. Finally, they identify proteins that are up- or down-regulated between the two mice as potential mechanisms to explain the observations.

Strengths:

The major strength of this manuscript is the creation of this mouse model, as a key step in beginning to understand how OGT mutants cause developmental disorders. This line will prove important for not only the authors but other investigators as well, enabling the testing of various hypotheses and potentially treatments. The experiments are also rigorously performed, and the conclusions are well supported by the data.

Weaknesses:

The only weakness identified is a lack of mechanistic insight. However, this certainly may come in the future through more targeted experimentation using this mouse model.

We agree with the reviewer that the suggested experiments would further strengthen our work. However, the extensive nature of the suggested studies would result in considerable delay in sharing this work with the scientific and patient communities. Nevertheless, we appreciate the reviewers’ comment and will continue to work along these lines, and report in a follow up manuscript in the future.

Recommendations for the authors:

Editor's note:

Should you choose to revise your manuscript, if you have not already done so, please include full statistical reporting including exact p-values wherever possible alongside the summary statistics (test statistic and df) and, where appropriate, 95% confidence intervals. These should be reported for all key questions and not only when the p-value is less than 0.05 in the main manuscript.

Statistics including exact p-values have been included in the main text for all key questions where appropriate.

Reviewer #1 (Recommendations for the authors):

(1) In Figure 1F, the y-axis labels and scale values are partially obscured by graphical elements, compromising accurate interpretation of the data range.

Panel 1F has been adjusted to make the y-axis label visible.

(2) Regarding the histological analyses in Figure 6, the current H&E staining and Luxol Fast Blue myelin staining results lack age-matched wild-type control samples processed in parallel, which undermines experimental comparability. To enhance methodological rigor, control group staining results should be displayed adjacent to each experimental group image.

The original Figure 6 already contained comparison between WT and OGT^C921Y tissues. The Figure has been updated with additional data from the WT and C921Y mutant groups shown side by side.

Reviewer #2 (Recommendations for the authors):

(1) I believe that Figures S1 and S2 were switched during the submission. The legends are correct, so the authors should just be careful with the order when they upload the final versions.

Figures S1 and S2 have been re-ordered.

(2) On page 18, the authors state, "Although no significant changes in the expression of OGT were observed in OGTC921Y cortex (Figure S12A, C), there was a significant increase in OGT/OGA protein ratio in OGTC921Y mice (Fig. S12D). As a functional consequence, global O-GlcNAcylation of proteins in the brain was drastically impaired in the OGTC921Y brain compared to WT (Figure S12E, F).

To me, this statement suggests that the incorrect ratio of OGT to OGA is responsible for the altered O-GlcNAc levels. I think this is missing important information. The authors are, I'm sure, aware that OGT and OGA expression is linked to O-GlcNAc levels. I think it would be better to describe the situation here as the tissue attempting to respond to lower OGT activity by lowering OGA levels. However, the tissue is not fully successful, resulting in lower overall O-GlcNAc levels as seen by RL2. If the difference were only driven by the OGT/OGA ratio, one would expect increased O-GlcNAc levels due to decreased OGA. I think it is important to point out more details here for non-expert readers.

Thank you for the insightful comment, we have included these aspects in the revised text, please see page 20.

(3) I am a little surprised that the authors did not explore differences in O-GlcNAc-modified proteins through a more targeted enrichment of these proteins for analysis of potential modification differences, in addition to just changes in protein abundance.

We agree that these experiments would further strengthen the work. However, it is not known yet whether OGT-CDG is caused by loss of O-GlcNAc modification on specific proteins or due to as yet to decipher mechanisms (e.g. OGT interactome, HCF1 processing, feedback on OGA levels) which we are not able to confirm in the current manuscript. Therefore, as a starting point, we have performed whole proteome analysis to establish candidate hypothesis which could lead to discovering cellular and molecular mechanisms underlying OGT-CDG. Lastly, we appreciate the reviewers’ comment and will continue to work along these lines, and report in a follow up manuscript in the future.

I like this point of view because as much as humans play a part in the extinction process, when were humans considering how animals actually live? The judgement of humans is what is holding us back from being understanding. Creating an entirely new species would be to make a shift in nature completely. We as humans do not know enough about nature to take that large of a step. Studying where we can find better ways to encourage life in the environment would be a better approach.

This section emphasizes the importance of creating inclusive curriculum and school policies that reflect diverse cultures and perspectives. It highlights the need for schools to regularly review materials and practices to ensure all students feel represented and supported.

The Umayyad army was unable to defeat Charles Martel’s strong infantry formation, and their leader was killed in battle. This stopped their expansion into Europe and helped Martel become the powerful leader of Francia until 741.

The death of Husayn at the Battle of Karbala became a powerful symbol for his followers. It inspired Persian communities to adopt Shia mourning rituals and strengthened the identity of Shia Islam.

During their time in Medina, Muhammad and his followers continued caravan trading while spreading their beliefs and gaining new followers. After fighting several battles, they returned to Mecca in 630 when their opponents stopped resisting.

After centuries of division following the Han Dynasty, China was reunified under the Sui in 581. Yang Jian took power by overthrowing the Northern Zhou emperor, eliminating rivals, and becoming Emperor Wen.

Historians once believed the Plague of Justinian was caused by the same bacteria as the Black Death. Recent genetic evidence has confirmed that it was indeed caused by Yersinia pestis.

Historians explain Christianity’s survival in Rome by looking at its inclusive nature. As a universalizing religion, it was open to people of all backgrounds, which helped it spread and endure.

This introduction challenges the traditional assumption that cancer-associated fibroblasts (CAFs) function as a single, uniform population in pancreatic cancer. Instead, the authors highlight a key gap in the field: while CAFs are known to contribute to tumor progression, extracellular matrix (ECM) deposition, and therapy resistance, it remains unclear whether all CAFs perform the same functions or whether distinct subpopulations exist. They emphasize that pancreatic ductal adenocarcinoma (PDAC) is characterized by extensive desmoplasia, where fibroblasts make up a large portion of the tumor mass and actively influence tumor behavior. Importantly, CAFs are not just structural components but secrete signaling molecules that regulate cancer progression and treatment response. This sets up the central question of the study: whether CAF heterogeneity exists, and if so, how different fibroblast subtypes contribute uniquely to the tumor microenvironment. The introduction therefore reframes fibroblasts as dynamic and functionally specialized players within the TME, rather than passive supporters of tumor growth. This introduction aligns with a broader shift in cancer biology toward understanding the tumor microenvironment as a heterogeneous and interactive system, where non-cancer cells play active and possibly opposing roles in disease progression.

Shows that these buildings had to be protected and claimed as historical landmarks or they would have been torn down decades ago. We also again here the term "Black Metropolis" and it gives us insight just how important these buildings are to the black communtiy.

The Black publications tackling race sensitive topics was one of the factors that made it so popular among African Americans. Where else would they find these topics? This might even be an issue today as digital news outlets are out there but are often overlooked or underrated.

The Author tells us that despite not having the grand scale and beautiful buildings like the Tribune or other white owned publications. African Americans still tuned into the black owned publications to get their consumption of media. I think this shows a level of "Black owned" pride and also maybe a level of distrust in White owned news publications.

The Author approaches this like a tour guide as he gives us directions and history as we start in the loop area and head across the river and south.

Endovenous ablation is contraindicated or relatively unsuitable when venous anatomy precludes catheter-based treatment, specifically: aneurysmal dilation of the GSV close to the saphenofemoral junction, subcutaneous location of truncal veins above the saphenous fascia and close to the skin, and significant tortuosity of the GSV or SSV. [1] In these scenarios, high ligation and stripping is recommended as the preferred alternative (grade 1 strong recommendation

Endovenous Thermal Ablation (RFA/EVLA)

Relative contraindications include inappropriate vein size, with veins <2 mm and >15 mm representing potential contraindications for RFA specifically. [1] A history of superficial thrombophlebitis resulting in a partially obstructed saphenous vein may preclude thermal ablation. [1] Significant tortuosity of the GSV on duplex examination can make catheter delivery difficult.

Endovenous ablation is the preferred treatment for symptomatic varicose veins with axial reflux, offering less postprocedure pain, reduced morbidity, and earlier return to activity

Endovenous thermal ablation (radiofrequency ablation [RFA] and endovenous laser ablation [EVLA]) has largely replaced surgery as the standard of care

Ultrasound-guided foam sclerotherapy (UGFS) represents a less invasive option but has higher recurrence rates

The risk of DVT or embolization is very low unless the thrombophlebitis extends into the great saphenous vein in the upper medial thigh

imaging of the deep venous system is obligatory to exclude a congenital malformation or atresia of the deep veins. Surgical treatment of varicose veins in these patients is contraindicated because the varicosities may play a significant role in venous drainage of the limb.

involved, typically the great saphenous vein and its tributaries, but the short saphenous vein (posterior lower leg) may also be affected

eLife Assessment

This important study presents a compelling link between nutrient signaling and chromosome regulation, demonstrating that reduced activity in a central nutrient-sensing pathway improves chromosome stability and alters gene expression through effects on cohesin. The convincing evidence from a combination of genetic, biochemical and cell biological approaches supports a model in which TORC1-dependent phosphorylation of Mis4 and the cohesin subunit Psm1/Smc1 can modulate cohesin loading to enhance faithful chromosome transmission. While the underlying mechanisms and biological importance of this newly described circuit are not yet fully known, the overall body of evidence is strong and supports the main conclusions.

Reviewer #1 (Public review):

Summary:

In this study, Besson et al. investigate how environmental nutrient signals regulate chromosome biology through the TORC1 signaling pathway in Schizosaccharomyces pombe. Specifically, the authors explore the impact of TORC1 on cohesin function-a protein complex essential for chromosome segregation and transcriptional regulation. Through a combination of genetic screens, biochemical analysis, phospho-proteomics, and transcriptional profiling, they uncover a functional and physical interaction between TORC1 and cohesin. The data suggest that reduced TORC1 activity enhances cohesin binding to chromosomes and improves chromosome segregation, with implications for stress-responsive gene expression, especially in subtelomeric regions.

Strengths:

This work presents a compelling link between nutrient sensing and chromosome regulation. The major strength of the study lies in its comprehensive and multi-disciplinary approach. The authors integrate genetic suppression screens, live-cell imaging, chromatin immunoprecipitation, co-immunoprecipitation, and mass spectrometry to uncover the functional connection between TORC1 signaling and cohesin. The use of phospho-mutant alleles of cohesin subunits and their loader provides mechanistic insight into the regulatory role of phosphorylation. The addition of transcriptomic analysis further strengthens the biological relevance of the findings and places them in a broader physiological context. Altogether, the dataset convincingly supports the authors' main conclusions and opens up new avenues of investigation.

Points that remain open but are appropriately discussed by the authors:

(1) The authors propose that nutrient status influences cohesin regulation. While this is not directly tested under defined nutrient conditions (e.g., by systematically examining cohesin dynamics or phosphorylation across nutrient states), the rationale is well explained in the text, and the study provides a strong foundation for addressing this question in future work.

(2) The upstream signaling cascade downstream of TORC1 remains to be fully elucidated. In particular, the identity of the relevant kinases (e.g., whether Sck1/Sck2 or other effectors are involved) and whether TORC1 directly phosphorylates Mis4 or Psm1 are not resolved. The authors acknowledge these mechanistic gaps, which represent logical next steps rather than shortcomings of the current study.

Reviewer #2 (Public review):

Summary:

In this study the authors follow up on a previous suppressor screen of a temperature-sensitive allele of mis4 (mis4-G1487D), the cohesin loading factor in S. pombe, and identify additional suppressor alleles tied to the S. pombe TORC1 complex. Their analysis suggests that these suppressor mutations attenuate TORC1 activity while enhanced TORC1 activity is deleterious in this context. Suppression of TORC1 activity also ameliorates chromosome segregation and spindle defects observed in the mis4-G1487D strain, although some more subtle effects are not reconstituted. The authors provide evidence that this genetic suppression is also tied to the reconstitution of cohesin loading. Moreover, disrupting TORC1 also enhances Mis4/cohesin association with chromatin (likely reflecting enhanced loading) in WT cells while rapamycin treatment can enhance the robustness of chromosome transmission. These effects likely arise directly through TORC1 or its downstream effector kinases as TORC1 co-purifies with Mis4 and Rad21; these factors are also phosphorylated in a TORC1-dependent fashion. Disrupting Sck2, a kinase downstream of TORC1, also suppresses the mis4-G1487D allele while simultaneous disruption of Sck1 and Sck2 enhances cohesin association with chromatin, albeit with differing effects on phosphorylation of Mis4 and Psm1/Scm1. Phosphomutants of Mis4 and Psm1 that mimic observed phosphorylation states identified by mass spectrometry that are TORC1-dependent also suppressed phenotypes observed in the mis4-G1487D background. Lastly, the authors provide evidence that the mis4-G1487D background and TORC1 mutant backgrounds display an overlap in the dysregulation of genes that respond to environmental conditions.

Overall, the authors provide compelling evidence from genetics, biochemistry and cell biology to support a previously unknown mechanism by which nutrient sensing regulates cohesin loading with implications for the stress response. The technical approaches are generally sound, well-controlled, and comprehensive.

The specific points that I raised in the first review have been addressed by changes/additions to the manuscript or have been determined to be beyond the scope of the study by the authors.

One major question that remains open is the relationship between local changes in cohesin loading and gene expression through this TORC1 regulatory signaling pathway and the details of the underlying mechanisms.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

In this study, Besson et al. investigate how environmental nutrient signals regulate chromosome biology through the TORC1 signaling pathway in Schizosaccharomyces pombe. Specifically, the authors explore the impact of TORC1 on cohesin function - a protein complex essential for chromosome segregation and transcriptional regulation. Through a combination of genetic screens, biochemical analysis, phospho-proteomics, and transcriptional profiling, they uncover a functional and physical interaction between TORC1 and cohesin. The data suggest that reduced TORC1 activity enhances cohesin binding to chromosomes and improves chromosome segregation, with implications for stress-responsive gene expression, especially in subtelomeric regions.

Strengths:

This work presents a compelling link between nutrient sensing and chromosome regulation. The major strength of the study lies in its comprehensive and multi-disciplinary approach. The authors integrate genetic suppression screens, live-cell imaging, chromatin immunoprecipitation, co-immunoprecipitation, and mass spectrometry to uncover the functional connection between TORC1 signaling and cohesin. The use of phospho-mutant alleles of cohesin subunits and their loader provides mechanistic insight into the regulatory role of phosphorylation. The addition of transcriptomic analysis further strengthens the biological relevance of the findings and places them in a broader physiological context. Altogether, the dataset convincingly supports the authors' main conclusions and opens up new avenues of investigation.

Weaknesses:

While the study is strong overall, a few limitations are worth noting. The consistency of cohesin phosphorylation changes under different TORC1-inhibiting conditions (e.g., genetic mutants vs. rapamycin treatment) is unclear and could benefit from further clarification. The phosphorylation sites identified on cohesin subunits do not match known AGC kinase consensus motifs, raising the possibility that the modifications are indirect. The study relies heavily on one TORC1 mutant allele (mip1-R401G), and additional alleles could strengthen the generality of the findings. Furthermore, while the results suggest that nutrient availability influences cohesin function, this is not directly tested by comparing growth or cohesin dynamics under defined nutrient conditions.

We thank the reviewer for his overall positive assessment and constructive criticism. We broadly agree with the few limitations he pointed out, which we will comment on below.

(1) The consistency of cohesin phosphorylation changes under different TORC1-inhibiting conditions (e.g., genetic mutants vs. rapamycin treatment) is unclear and could benefit from further clarification.

The basis of our study was to search for suppressor mutants, a situation in which an unviable strain becomes viable. It turns out that the suppressor mutants affect TORC1, necessarily in a partial manner given that TORC1 kinase activity is essential for proliferation. Likewise rapamycin partially inhibits TORC1 and does not prevent proliferation of wild-type S. pombe cells. TORC1 mutants cause a constitutive decrease in activity with possible adaptive effects, whereas rapamycin is applied for a single cell cycle. In addition, it is known that bona fide TORC1 substrates respond differently to rapamycin. Some phosphosites show acute sensitivity, while others are less sensitive or even insensitive (Kang et al., 2013, PMID: 23888043). Therefore, both hypomorphic TORC1 genetic mutants and rapamycin treatment result in partial inhibition of TORC1 kinase activity. While the lists of affected TORC1 substrates may overlap, they are unlikely to be identical. Furthermore, the phosphorylation level of the relevant substrates is not necessarily altered to the same extent. Nevertheless, both conditions suppress the heatsensitive phenotype of the mis4 mutant, although the suppressor effect of rapamycin is weaker. Consequently, some phosphorylation sites involved in mis4-ts suppression may behave similarly in rapamycin and TORC1 mutants (i.e. Psm1-S1022), while others (i.e. Mis4-183) may behave differently.

It is clear that there are phenotypic differences between the suppression of mis4-ts by rapamycin treatment or by genetic alteration of TORC1. This can be seen also in our ChIP analysis of Rad21 distribution at CARs. The trend is upward, but the pattern is not identical. We have added the following text to summarize the above considerations:

“It is important to note at this stage that, although rapamycin and TORC1 mutants both decrease TORC1 kinase activity, the two are not equivalent. The mechanisms by which TORC1 kinase activity is reduced are different, and TORC1 mutants suppress the mis4G1487D phenotype more effectively than rapamycin. It is known that bona fide TORC1 substrates respond differently to rapamycin. Some phosphosites show acute sensitivity, while others are less sensitive or even insensitive (Kang et al, 2013). TORC1 mutants cause a constitutive decrease in activity with possible adaptive effects, whereas rapamycin is applied for a single cell cycle. While the lists of affected TORC1 substrates may overlap, they are unlikely to be identical. Furthermore, the phosphorylation level of the relevant substrates is not necessarily altered to the same extent. It is therefore remarkable that negative regulation of TORC1 by rapamycin or a genetic mutation both alleviate mis4G14878D phenotypes and have a fairly similar effect on cohesin dynamics.”

(2) The phosphorylation sites identified on cohesin subunits do not match known AGC kinase consensus motifs, raising the possibility that the modifications are indirect.

The genetic and biochemical analyses provided in this study show that the AGC kinases Sck1 and Sck2 influence cohesin phosphorylation and function. Whether Sck1, Sck2 or TORC1 directly phosphorylates cohesin components are the next questions to address. The fact that the phosphorylation of Psm1-S1022 and Mis4-S183 were never abolished in the sck1-2 mutants may suggest they are indirectly involved. This should be taken with caution because we have been using deletion mutants. In this situation, cells adapt and other kinases may substitute, at least partially (Plank et al, 2020, PMID: 32102971). Asking whether cohesin components display consensus sites for AGC kinases is a complementary approach. The consensus site for Sck1 and Sck2 is unknown. If we assume some conservation with budding yeast SCH9, the consensus sequence would be RRxS/T. Psm1S1022 (DQMSP) and Mis4-S183 (QLCSP) do not fit the consensus. However, this kind of information should be taken with care as many SCH9-dependent phosphorylation sites did not fall within the consensus in a study using analogue-sensitive AGC kinases and phosphoproteomics (Plank et al, 2020, PMID: 32102971). Alternatively, Sck1-2 may regulate other kinases. Indeed Psm1-S1022 and Mis4-183 lie within CDK consensus sites and Psm1-S1022 phosphorylation is Pef1-dependent. In summary, yes, the changes may be indirect, that remains to be seen, but in any case they are influenced by TORC1 signalling. The following paragraph was added:

“The consensus site for Sck1 and Sck2 is unknown. If we assume some conservation with budding yeast SCH9, the consensus sequence would be RRxS/T. Psm1-S1022 (DQMSP) and Mis4-S183 (QLCSP) do not fit the consensus. However, this should be taken with care as many SCH9-dependent phosphorylation sites did not fall within the consensus in a study using analogue-sensitive AGC kinases and phosphoproteomics (Plank et al, 2020). Alternatively, Sck1-2 may regulate other kinases. Indeed Psm1-S1022 and Mis4-183 lie within CDK consensus sites and Psm1-S1022 phosphorylation is Pef1-dependent.”

(3) The study relies heavily on one TORC1 mutant allele (mip1-R401G), and additional alleles could strengthen the generality of the findings.

It is true that we focused our attention on mip1-R401G, which is present in all the experiments presented. That said, other alleles were used in one or more figures. Five mip1 alleles and one tor2 allele were identified as mis4-ts suppressors (Fig. 1). We have also shown that another mip1 allele, mip1-Y533A, created by another group (Morozumi et al, 2021), is also a suppressor of mis4-ts and affects the phosphorylation of Mis4-S183 and Psm1-S1022 (Fig. 1, Figure 5—figure supplement 1). To this we can add the effect of mutants that render TORC1 hyperactive (Fig. 1E, Fig. 2H) as well as AGC kinase mutants (Figure 5—figure supplement 3.). And finally, the effect of rapamycin. So yes, mip1-R401G has been used extensively, but we have still broadly covered the TORC1 signalling pathway.

(4) Furthermore, while the results suggest that nutrient availability influences cohesin function, this is not directly tested by comparing growth or cohesin dynamics under defined nutrient conditions

We agree that studying the dynamics of cohesin, genome folding and gene expression in relation to nutrient availability is a very exciting topic, and we hope to address these issues in detail in the future.

Reviewer #2 (Public review):

Summary:

In this study, the authors follow up on a previous suppressor screen of a temperaturesensitive allele of mis4 (mis4-G1487D), the cohesin loading factor in S. pombe, and identify additional suppressor alleles tied to the S. pombe TORC1 complex. Their analysis suggests that these suppressor mutations attenuate TORC1 activity, while enhanced TORC1 activity is deleterious in this context. Suppression of TORC1 activity also ameliorates chromosome segregation and spindle defects observed in the mis4-G1487D strain, although some more subtle effects are not reconstituted. The authors provide evidence that this genetic suppression is also tied to the reconstitution of cohesin loading. Moreover, disrupting TORC1 also enhances Mis4/cohesin association with chromatin (likely reflecting enhanced loading) in WT cells, while rapamycin treatment can enhance the robustness of chromosome transmission. These effects likely arise directly through TORC1 or its downstream effector kinases, as TORC1 co-purifies with Mis4 and Rad21; these factors are also phosphorylated in a TORC1-dependent fashion. Disrupting Sck2, a kinase downstream of TORC1, also suppresses the mis4-G1487D allele while simultaneous disruption of Sck1 and Sck2 enhances cohesin association with chromatin, albeit with differing effects on phosphorylation of Mis4 and Psm1/Scm1. Phosphomutants of Mis4 and Psm1 that mimic observed phosphorylation states identified by mass spectrometry that are TORC1-dependent also suppressed phenotypes observed in the mis4-G1487D background. Last, the authors provide evidence that the mis4-G1487D background and TORC1 mutant backgrounds display an overlap in the dysregulation of genes that respond to environmental conditions, particularly in genes tied to meiosis or other "stress".

Overall, the authors provide compelling evidence from genetics, biochemistry, and cell biology to support a previously unknown mechanism by which nutrient sensing regulates cohesin loading with implications for the stress response. The technical approaches are generally sound, well-controlled, and comprehensive.

Specific Points:

(1) While the authors favor the model that the enhanced cohesin loading upon diminished TORC1 activity helps cells to survive harsh environmental conditions, as starvation of S. pombe also drives commitment to meiosis, it seems as plausible that enhanced cohesin loading is related to preparing the chromosomes to mate.

(2) Related to Point 1, the lab of Sophie Martin previously published that phosphorylation of Mis4 characterizes a cluster of phosphotargets during starvation/meiotic induction (PMID: 39705284). This work should be cited, and the authors should interrogate how their observations do or do not relate to these prior observations (are these the same phosphosites?).

We agree this is a possibility and the following paragraph was added in the discussion section:

“TORC1-based regulation of cohesin may be relevant to preparing cells for meiosis. Since nitrogen deprivation stimulates meiosis initiation, subsequent TORC1 down-regulation may regulate the cohesin complex, preparing the chromosomes for fusion and meiosis. A recent phosphoproteomic study conducted by Sophie Martin's laboratory showed that Mis4-S107 phosphorylation increases during cellular fusion (Bérard et al, 2024). It is unknown whether the phosphorylation of S107 is controlled by TORC1 signalling. As the phosphorylation of Mis4-S183 and Psm1-S1022 was not detected in these experiments, the potential involvement of the TORC1-cohesin axis in the sexual programme remains to be investigated.”

(3) It would be useful for the authors to combine their experimental data sets to interrogate whether there is a relationship between the regions where gene expression is altered in the mis4-G1487D strain and changes in the loading of cohesin in their ChIP experiments.

(4) Given that the genes that are affected are predominantly sub-telomeric while most genes are not affected in the mis4-G1487D strain, one possibility that the authors may wish to consider is that the regions that become dysregulated are tied to heterochromatic regions where Swi6/HP1 has been implicated in cohesin loading

We agree that it would be interesting to see if there are correlations between cohesin positioning, heterochromatin and gene expression. That said, this would need to be done at the whole-genome level and include many other parameters (genome folding, histone modifications, Pol2 occupancy). These issues require substantial investment and may be addressed in a follow-up project.

(5) It would be helpful to show individual data points from replicates in the bar graphs - it is not always clear what comprises the data sets, and superplots would be of great help.

We verified that the figure captions clearly indicate the data sets considered, their mean, standard deviation, and statistical analysis method. As for the type of plot, we used the tools at our disposal.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

Besson et al. investigate how the nutrient-responsive TORC1 signaling pathway modulates cohesin function in S. pombe. Using a genetic screen, the authors identify TORC1 mutants that suppress the thermosensitive growth defects of a cohesin loader mutant (mis4-G1487D). They show that reducing TORC1 activity-either genetically or pharmacologically-enhances cohesin binding to chromosomal sites (CARs), improves chromosome segregation, and alters the phosphorylation state of cohesin and its loader. They also show, through coimmunoprecipitation, that TORC1 and cohesin physically associate, and that this functional interaction extends to the transcriptional regulation of stress-responsive, subtelomeric genes. Together, the data suggest that environmental cues influence chromosome stability and gene expression via a TORC1-cohesin axis.

Overall, the study is well-supported by thoughtful genetic epistasis analyses and a combination of genetic, biochemical, cell biological, and transcriptomic approaches. While not all data are equally strong, the cumulative evidence convincingly supports the authors' conclusions.

Specific Concerns and Suggestions

(1) Figure 2A - Division rates of wild-type and mip1-R401G cells are missing and should be provided for proper comparison.

This is now done in revised Figure 2A. We also made a change in the manuscript, replacing “The mip1-R401G mutation efficiently suppressed the proliferation and viability defects (Figure 2A)” by “The mip1-R401G mutation efficiently attenuated the proliferation and viability defects (Figure 2A)”, to acknowledge the fact that the proliferation rate did not return to wild-type levels.

(2) Figure 3 - Figure Supplement 1 - The authors claim that "Rapamycin treatment during a single cell cycle provoked a similar effect although less pronounced." However, for most CARs, the effect appears insignificant. This should be acknowledged in the text.

The text has been changed accordingly:

“Rapamycin treatment during a single cell cycle provoked a similar stimulation of Rad21 binding at CARs (Figure 3—figure supplement 1), albeit with noticeable differences. In mis4+ cells, both mip1-R401G and rapamycin induced a significant increase in Rad21 binding at several CARs (tRNA-left, cc2, 3323, NTS, Tel1-R). However, some CARs that exhibited increased Rad21 binding in the mip1 mutant did not respond significantly to rapamycin (dg2-R, tRNA-R). Conversely, rapamycin (but not mip1-R401G) induced a significant increase in Rad21 binding at imr2-L and CAR1806 (Figure 3D and Figure 3— figure supplement 1). In the mis4-G1487D mutant background, mip1-R401G induced a significant increase in Rad21 binding at all examined sites (Figure 3B). Similarly, rapamycin did increase Rad21 binding at all sites but only at the Tel1-R site did this reach statistical significance (Figure 3—figure supplement 1).”

(3) Figure 4 - The analysis of interactions between TORC1 and the cohesin complex is somewhat limited. The authors may wish to test interactions between Mip1 and cohesin subunits (e.g., Rad21). More interestingly, it would be valuable to explore whether MIP1 mutations that suppress cohesin mutants affect the interaction between Tor2 and Rad21.

We have added some additional data that answer this question (Figure 4—figure supplement 1) and a paragraph in the manuscript:

“Tor2, the kinase subunit of TORC1, is particularly well detected in Rad21 and Mis4 coimmunoprecipitation experiments (Figure 4 and Figure 4—figure supplement 1). To determine whether the R401G mutation in Mip1 affects these interactions, coimmunoprecipitation experiments were repeated in both the mip1-R401G and mip1+ contexts. The data obtained indicate that Tor2 co-immunoprecipitation with Mis4 and Rad21 is largely unaffected by the mip1-R401G mutation (Figure 4—figure supplement 1). If mip1-R401G affects the regulation of cohesin by TORC1, this does not appear to stem from a gross defect in their interaction, at least at this level of resolution.”

(4) Figure 5 - There appears to be a lack of correlation between cohesin subunit phosphorylation in TORC1-reducing mutants and in response to rapamycin. The reason for this discrepancy is unclear.

This point was addressed in the previous section (Public review, reviewer 1, point 1). The response is pasted below:

The basis of our study was to search for suppressor mutants, a situation in which an unviable strain becomes viable. It turns out that the suppressor mutants affect TORC1, necessarily in a partial manner given that TORC1 kinase activity is essential for proliferation. Likewise rapamycin partially inhibits TORC1 and does not prevent proliferation of wild-type S. pombe cells. TORC1 mutants cause a constitutive decrease in activity with possible adaptive effects, whereas rapamycin is applied for a single cell cycle. In addition, it is known that bona fide TORC1 substrates respond differently to rapamycin. Some phosphosites show acute sensitivity, while others are less sensitive or even insensitive (Kang et al., 2013, PMID: 23888043). Therefore, both hypomorphic TORC1 genetic mutants and rapamycin treatment result in partial inhibition of TORC1 kinase activity. While the lists of affected TORC1 substrates may overlap, they are unlikely to be identical. Furthermore, the phosphorylation level of the relevant substrates is not necessarily altered to the same extent. Nevertheless, both conditions suppress the heatsensitive phenotype of the mis4 mutant, although the suppressor effect of rapamycin is weaker. Consequently, some phosphorylation sites involved in mis4-ts suppression may behave similarly in rapamycin and TORC1 mutants (i.e. Psm1-S1022), while others (i.e. Mis4-183) may behave differently.

It is clear that there are phenotypic differences between the suppression of mis4-ts by rapamycin treatment or by genetic alteration of TORC1. This can be seen also in our ChIP analysis of Rad21 distribution at CARs. The trend is upward, but the pattern is not identical. We have added the following text to summarize the above considerations:

“It is important to note at this stage that, although rapamycin and TORC1 mutants both decrease TORC1 kinase activity, the two are not equivalent. The mechanisms by which TORC1 kinase activity is reduced are different, and TORC1 mutants suppress the mis4G1487D phenotype more effectively than rapamycin. It is known that bona fide TORC1 substrates respond differently to rapamycin. Some phosphosites show acute sensitivity, while others are less sensitive or even insensitive (Kang et al, 2013). TORC1 mutants cause a constitutive decrease in activity with possible adaptive effects, whereas rapamycin is applied for a single cell cycle. While the lists of affected TORC1 substrates may overlap, they are unlikely to be identical. Furthermore, the phosphorylation level of the relevant substrates is not necessarily altered to the same extent. It is therefore remarkable that negative regulation of TORC1 by rapamycin or a genetic mutation both alleviate mis4G14878D phenotypes and have a fairly similar effect on cohesin dynamics.”

(5) The phosphorylation sites examined on cohesin subunits are not canonical AGC kinase consensus motifs, suggesting they are unlikely to be direct targets of Sck1 or Sck2. I suggest that this point should be mentioned in the manuscript.

This is now done:

“The consensus site for Sck1 and Sck2 is unknown. If we assume some conservation with budding yeast SCH9, the consensus sequence would be RRxS/T. Psm1-S1022 (DQMSP) and Mis4-S183 (QLCSP) do not fit the consensus. However, this should be taken with care as many SCH9-dependent phosphorylation sites did not fall within the consensus in a study using analogue-sensitive AGC kinases and phosphoproteomics (Plank et al, 2020). Alternatively, Sck1-2 may regulate other kinases. Indeed Psm1-S1022 and Mis4-183 lie within CDK consensus sites and Psm1-S1022 phosphorylation is Pef1-dependent.”

(6) Figure 5 - Figure Supplement 3 - The reduction in Psm1 phosphorylation in the sck1Δ sck2Δ double mutant is not convincing without replicates and statistical analysis.

This is now done and the data are presented in Figure 5—figure supplement 3. Panel D shows the data for Psm1-S1022p and Panel E for Mis4-S183p. Each graph shows the mean ratios +/- SD from 3 experiments.

(7) Figure 5C - It would be helpful if the authors validated the effect of pef1 deletion on Mis4 phosphorylation by Western blotting, rather than relying solely on mass spectrometry data.

This is now done. The data appears in Figure 5—figure supplement 2, panel B.

(8) The statement: "The frequency of chromosome segregation defects of mis4‐G1487D was markedly reduced in a sck2‐deleted background and further decreased by the additional deletion of sck1 (Figure 5-figure supplement 3)" is not supported by the data. According to the figure, the difference between sck2Δ and sck1Δ sck2Δ is not statistically significant.

The sentence was changed to:

“The frequency of chromosome segregation defects in the mis4-G1487D strain remained unchanged in a sck1-deleted background, but was significantly reduced when either the sck2 or both the sck1 and sck2 genes were deleted (Figure 5—figure supplement 3).”

(9) Figure 6A - The data shown are not convincing. The double mutants carrying the phosphomimetic and phospho-null psm1 alleles should be shown on the same plate for direct comparison.

This is now done. The new data are shown Figure 6A.

(10) Figure 6E - The wild-type control is missing. Including it would provide an essential reference point to assess whether the mutants rescue cohesin binding to wild-type levels.

This is true that the effects were small when compared to wild-type but still significant when compared to mis4-G1487D. The comparison with wild-type is now available in Figure 6—figure supplement 1 and the paragraph was modified accordingly:

“Cohesin binding to CARs as assayed by ChIP tend to increase for the mutants mimicking the non-phosphorylated state and to decrease with the phospho-mimicking forms (Figure 6E). The rescue of mis4-G1487D by the non-phosphorylatable form was modest but significant, notably within centromeric regions (imr2-L, dg2-R) and at the telomere (Tel1-R) site (Figure 6E and see Figure 6—figure supplement 1 for comparison with wild-type levels). Conversely, the mutant mimicking the phosphorylated state displayed a significant reduction of Rad21 binding at those sites as well as to several other sites at the centromere (cc2, tRNA-R), CAR2898, and at the ribosomal non-transcribed spacer site NTS).”

Limitations of the Study (not requiring additional experiments for publication, but worth noting).

(11) The authors suggest that nutrient status affects cohesin, but this is not directly demonstrated-e.g., by comparing growth or cohesin dynamics or phosphorylation under defined nutrient conditions. That said, the paper is sufficiently detailed to allow this question to be addressed in follow-up work.

We agree that studying the dynamics of cohesin, genome folding and gene expression in relation to nutrient availability is a very exciting topic, and we hope to address these issues in detail in the future.

(12) The upstream signaling cascade remains unresolved. The identity of kinases downstream of TORC1 (e.g., whether Sck1/Sck2 or other factors are responsible) and whether TORC1 directly phosphorylates Mis4 or Psm1 are not established.

This is something we can all agree on, and it might be something we look at in a future project.

(13) The conclusions rely heavily on one TORC1 mutant allele (mip1-R401G). While this allele is informative, additional alleles or orthogonal methods could further support the generality of the findings.

It is true that we focused our attention on mip1-R401G, which is present in all the experiments presented. That said, other alleles were used in one or more figures. Five mip1 alleles and one tor2 allele were identified as mis4-ts suppressors (Fig. 1). We have also shown that another mip1 allele, mip1-Y533A, created by another group (Morozumi et al, 2021), is also a suppressor of mis4-ts and affects the phosphorylation of Mis4-S183 and Psm1-S1022 (Fig. 1, Figure 5—figure supplement 1). To this we can add the effect of mutants that render TORC1 hyperactive (Fig. 1E, Fig. 2H) as well as AGC kinase mutants (Figure 5—figure supplement 3.) and finally, the effect of a transient treatment with rapamycin. So yes, mip1-R401G has been used extensively, but we have still broadly covered the TORC1 signalling pathway.

Reviewer #2 (Recommendations for the authors):

(1) Given the lack of CTCF in fission yeast, it is worth noting that cohesin ChIP data nonetheless can predict topological domains, which reinforces its important role in dictating chromatin folding (PMID: 39543681).

We thank the reviewer for this suggestion. We now refer to this study in the discussion section.

(2) Providing context for the S. pombe nomenclature for the conserved cohesin subunits would help the reader navigate the manuscript, possibly using a cartoon as for the TORC complexes. For example, Psm1 (aka Smc1) is not introduced and therefore its phosphorylation comes into the manuscript without explanation.

Cohesin subunits and their names are given in the introduction section.

I really don't get it

sorry not trying to be mean

Summit Wellness Mental Health

eLife Assessment

This convincing study examines a novel interaction of RAB5 with VPS34 complex II. Structural data are combined with site-directed mutagenesis, sequence analysis, biochemistry, yeast mutant analysis, and prior data on RAB1-VPS34 and RAB5-VPS34 interactions to provide a new perspective on how RAB GTPases recruit related but distinct VPS34 complexes to different organelles. The judgment is that this work represents a fundamental advance in our understanding of VPS34 localization and regulation.

Reviewer #1 (Public review):

Summary:

This manuscript presents high resolution cryoEM structures of VPS34-complex II bound to Rab5A at 3.2A resolution. The Williams group previously reported the structure of VPS34 complex II bound to Rab5A on liposomes using tomography, and therefore the previous structure, although very informative, was at lower resolution.

The first new structure they present is of the 'REIE>AAAA' mutant complex bound to RAB5A. The structure resembles the previously determined one except an additional molecule of RAB5A was observed bound to the complex in a new position, interacting with the solenoid of VPS15.

Although this second binding site exhibited reduced occupancy of RAB5A in the structure, the authors determined an additional structure in which the primary binding site was mutated to prevent RAB5A binding ('REIE>ERIR'). In this structure, there is no RAB5A bound to the primary binding site on VPS34, but the RAB5A bound to VPS15 now has strong density. The authors note that the way in which RAB5A interacts with each site is distinct, though both interfaces involve the switch regions. The authors confirm the location of this additional binding site using HDX-MS.

The authors then determine multiple structures of the wild-type complex bound to RAB5A from a single sample, as they use 3D classifications to separate out versions of the complex bound to 0, 1, or 2 copies of RAB5A. Overall the structure of VPS34-Complex II does not change between the different states, and the data indicate that both RAB5A binding sites can be occupied at the same time.

The authors then design a new mutant form of the complex (SHMIT>DDMIE) that is expected to disrupt the interaction at the secondary site between VPS15 and RAB5A. This mutation had a minor impact on the Kd for RAB5A binding, but when combined with the REIE>ERIR mutation of the primary binding site, RAB5A binding to the complex was abolished.

Comparison of sequences across species indicated that the RAB5A binding site on VPS15 was conserved in yeast while the RAB5A binding site on VPS34 is not.

The authors tested the impact of a correspond yeast Vps15 mutation (SHLITY>DDLIEY) predicted to disrupt interaction with yeast Rab5/Vps21, and found this mutant Vps15 protein was mislocalized and caused defective CPY processing.

The authors then compare these structures of the RAB5A-class II complex to recently published structures from the Hurley group of the RAB1A-class I complex, and find that in both complexes the Rab protein is bound to the VPS34 binding site in a somewhat similar manner. However, a key difference is the position of VPS34 is slightly different in the two complexes because of the unique ATL14L and UVRAG subunits in the class I and class II complexes, respectively. This difference creates a different RAB binding pocket that explains the difference in RAB specificity between the two complexes.

Finally, the higher resolution structures enable the authors to now model portions of BECLIN1 and UVRAG that were not previously modeled in the cryoET structure.

Strengths:

Overall I found this to be an interesting and comprehensive study of the structural basis for interaction of RAB5A with VPS34-complex II. The authors have performed experiments to validate their structural interpretations, and they present a clear and thorough comparative analysis of the Rab binding sites in the two different VPS34 complexes. The result is a much better understanding of how two different Rab GTPases specifically recruit two different, but highly similar complexes to the membrane surface.

Weaknesses:

No significant weaknesses noted.

Reviewer #2 (Public review):

The work by Spokaite et al describes the discovery of a novel Rab5 binding site present in complex II of class III PI3K using a combination of HDX and Cryo EM. Extensive mutational and sequence analysis define this as the primordial Rab5 interface. The data presented are convincing that this is indeed a biologically relevant interface, and is important in defining mechanistically how vps34 complexes are regulated.

This paper is a very nice expansion of their previous cryo-ET work from 2021, and is an excellent companion piece on high resolution cryo-EM of the complex I class III complex bound to Rab1 from the Hurley lab in 2025. Overall, this work is of excellent technical quality, and answers important unexplained observations on some unexpected mutational analysis from the previous work.

They used their increased affinity vps34 mutant to determine the 3.2 ang structure of Rab5 bound to vps34-CII. Clear density was seen for the original Rab5 interface, but an additional site was observed. Based on this structure they mutated out the vps34 interface, allowing for a high resolution structure of the Rab5 bound at the Vps15 interface.

They extensively validated the vps15 interface in the yeast variant of vps34, showing that the Vp215-Rab5 (Vps21) interface identified is critical in controlling complex II vps34 recruitment.

The major strengths of this paper are that the experiments appear to be done carefully and rigorously and I have very few experimental suggestions.

Here is what I recommend based on some very minor weaknesses I observed

(1) My main concern has to do a little bit with presentation. My main issue is how the authors use mutant description. They clearly indicate the mutant sequence in the human isoform (for example see Fig 2A, Vps15 described as 579-SHMIT-583>DDMIE), however, when they shift to the yeast version they shift to saying vps15 mutant, but don't define the mutant, Fig 2G). I would recommend they just include the same sequence numbering and WT to mutant replacement every time a new mutant (or species) is described. It is always easier to interpret what is being shown when the authors are jumping between species when the exact mutant is included. This is particularly important in this paper, where we are jumping between both different subunits and different species, so clear description in figure/figure legends makes it much easier to read for non-specialists.

(2) The HDX data very clearly shows that Rab5 is likely able to bind at both sites, which back ups the cryo EM data nicely. I am slightly confused by some of the HDX statements described in the methods.

(3) The authors state "Only statistically significant peptides showing a difference greater than 0.25 Da and greater than 5% for at least two timepoints were kept." This seems to be confusing why they required multiple timepoints, and before they also describe that they required a p value of less than 0.05. It might be clearer to state that significant differences required a 0.25 Da, 5%, and p value of <0.05 (n=3). Also what do they mean by kept? Does this mean that they only fully processed the peptides with differences.

(4) They show peptide traces for a selection in the supplement, but it would be ideal to include the full set of HDX data as an excel file, including peptides with no differences as there is a lot of additional information (deuteration levels for everything) that would be useful to share, as recommended from the Masson et al 2019 recommendations paper. This may be attached but this reviewer could not see an example of it in the shared data dropbox folder.

Comments on revisions:

The authors have addressed all of my issues.

Reviewer #3 (Public review):

Summary:

The manuscript of Spokaite et al. focuses on the Vps34 complex involved in PI3P production. This complex exists in two variants, one (class I) specific for autophagy, and a second one (class II) specific for the endocytic system. Both differ only in one subunit. The authors previously showed that the Vps34 complexes interact with Rab GTPases, Rab1 or Rab5 (for class II), and the identified site was found at Vps34. Now, the authors identify a conserved and overlooked Rab5 binding site in Vps15, which is required for the function of the Class II complex. In support of this, they show cryo-EM data with a second Rab5 bound to Vps15, identify the corresponding residues, and show by mutant analysis that impaired Rab5 binding also results in defects using yeast as a model system.

Overall, this is a most complete study with little to criticize. The paper shows convincingly that the two Rab5 binding sites are required for Vps34 complex II function, with the Vps15 binding site being critical for endosomal localization. The structural data is very much complete. What I am missing are a few controls that show that the mutations in Vps15 do not affect autophagy. I also found the last paragraph of the results section a bit out of place, even though this is a nice observation that the N-terminal part of BECLIN has these domains. However, what does it add to the story?

Comments on revisions:

The authors answered all my questions. I have no further requests.

Author Response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

This manuscript presents high-resolution cryoEM structures of VPS34-complex II bound to Rab5A at 3.2A resolution. The Williams group previously reported the structure of VPS34 complex II bound to Rab5A on liposomes using tomography, and therefore, the previous structure, although very informative, was at lower resolution.

The first new structure they present is of the 'REIE>AAAA' mutant complex bound to RAB5A. The structure resembles the previously determined one, except that an additional molecule of RAB5A was observed bound to the complex in a new position, interacting with the solenoid of VPS15.

Although this second binding site exhibited reduced occupancy of RAB5A in the structure, the authors determined an additional structure in which the primary binding site was mutated to prevent RAB5A binding ('REIE>ERIR'). In this structure, there is no RAB5A bound to the primary binding site on VPS34, but the RAB5A bound to VPS15 now has strong density. The authors note that the way in which RAB5A interacts with each site is distinct, though both interfaces involve the switch regions. The authors confirm the location of this additional binding site using HDX-MS.

The authors then determine multiple structures of the wild-type complex bound to RAB5A from a single sample, as they use 3D classifications to separate out versions of the complex bound to 0, 1, or 2 copies of RAB5A. Overall, the structure of VPS34-Complex II does not change between the different states, and the data indicate that both RAB5A binding sites can be occupied at the same time.

The authors then design a new mutant form of the complex (SHMIT>DDMIE) that is expected to disrupt the interaction at the secondary site between VPS15 and RAB5A. This mutation had a minor impact on the Kd for RAB5A binding, but when combined with the REIE>ERIR mutation of the primary binding site, RAB5A binding to the complex was abolished.

Comparison of sequences across species indicated that the RAB5A binding site on VPS15 was conserved in yeast,while the RAB5A binding site on VPS34 is not.

The authors tested the impact of a corresponding yeast Vps15 mutation (SHLITY>DDLIEY) predicted to disrupt interaction with yeast Rab5/Vps21, and found that this mutant Vps15 protein was mislocalized and caused defective CPY processing.

The authors then compare these structures of the RAB5A-class II complex to recently published structures from the Hurley group of the RAB1A-class I complex, and find that in both complexes the Rab protein is bound to the VPS34 binding site in a somewhat similar manner. However, a key difference is that the position of VPS34 is slightly different in the two complexes because of the unique ATL14L and UVRAG subunits in the class I and class II complexes, respectively. This difference creates a different RAB binding pocket that explains the difference in RAB specificity between the two complexes.

Finally, the higher resolution structures enable the authors to now model portions of BECLIN1 and UVRAG that were not previously modeled in the cryoET structure.

Strengths:

Overall, I found this to be an interesting and comprehensive study of the structural basis for the interaction of RAB5A with VPS34-complex II. The authors have performed experiments to validate their structural interpretations, and they present a clear and thorough comparative analysis of the Rab binding sites in the two different VPS34 complexes. The result is a much better understanding of how two different Rab GTPases specifically recruit two different, but highly similar complexes to the membrane surface.

Weaknesses:

No significant weaknesses were noted.

Reviewer #2 (Public review):

Summary:

The work by Spokaite et al describes the discovery of a novel Rab5 binding site present in complex II of class III PI3K using a combination of HDX and Cryo EM. Extensive mutational and sequence analysis define this as the primordial Rab5 interface. The data presented are convincing that this is indeed a biologically relevant interface, and is important in defining mechanistically how VPS34 complexes are regulated.

This paper is a very nice expansion of their previous cryo-ET work from 2021, and is an excellent companion piece on high-resolution cryo-EM of the complex I class III complex bound to Rab1 from the Hurley lab in 2025. Overall, this work is of excellent technical quality and answers important unexplained observations on some unexpected mutational analysis from the previous work.

They used their increased affinity VPS34 mutant to determine the 3.2 ang structure of Rab5 bound to VPS34-CII. Clear density was seen for the original Rab5 interface, but an additional site was observed. Based on this structure, they mutated out the VPS34 interface, allowing for a high-resolution structure of the Rab5 bound at the VPS15 interface.

They extensively validated the VPS15 interface in the yeast variant of VPS34, showing that the Vp215-Rab5 (VPS21) interface identified is critical in controlling complex II VPS34 recruitment.

The major strengths of this paper are that the experiments appear to be done carefully and rigorously, and I have very few experimental suggestions.

Here is what I recommend based on some very minor weaknesses I observed

(1) My main concern has to do a little bit with presentation. My main issue is how the authors use mutant description. They clearly indicate the mutant sequence in the human isoform (for example, see Figure 2A, VPS15 described as 579-SHMIT-583>DDMIE); however, when they shift to the yeast version, they shift to saying VPS15 mutant, but don't define the mutant, Figure 2G). I would recommend they just include the same sequence numbering and WT to mutant replacement every time a new mutant (or species) is described. It is always easier to interpret what is being shown when the authors are jumping between species, when the exact mutant is included. This is particularly important in this paper, where we are jumping between different subunits and different species, so a clear description in the figure/figure legends makes it much easier to read for non-specialists.

The reviewer has made an excellent point here. To clarify the yeast mutation, we have revised the manuscript main text to refer to the yeast mutant as SHLITY>DDLIEY, and we have added this to the legend for Figs. 2F,G.

(2) The HDX data very clearly shows that Rab5 is likely able to bind at both sites, which back ups the cryo EM data nicely. I am slightly confused by some of the HDX statements described in the methods.

(3) The authors state, "Only statistically significant peptides showing a difference greater than 0.25 Da and greater than 5% for at least two timepoints were kept." This seems to be confusing as to why they required multiple timepoints, and before they also describe that they required a p-value of less than 0.05. It might be clearer to state that significant differences required a 0.25 Da, 5%, and p-value of <0.05 (n=3). Also, what do they mean by kept? Does this mean that they only fully processed the peptides with differences?

(4) They show peptide traces for a selection in the supplement, but it would be ideal to include the full set of HDX data as an Excel file, including peptides with no differences, as there is a lot of additional information (deuteration levels for everything) that would be useful to share, as recommended from the Masson et al 2019 recommendations paper. This may be attached, but this reviewer could not see an example of it in the shared data dropbox folder.

We have revised the HDX method description to clarify. All peptides were kept and fully processed. However, for the results displayed, we have illustrated only peptides meeting the criteria described.

The Excel file for all peptides (as recommended by Masson et al) was deposited with PRIDE, with the identifier with the dataset identifier PXD061277, in addition, we have included this excel file in our supplementary material.

Reviewer #3 (Public review):

Summary:

The manuscript of Spokaite et al. focuses on the Vps34 complex involved in PI3P production. This complex exists in two variants, one (class I) specific for autophagy, and a second one (class II) specific for the endocytic system. Both differ only in one subunit. The authors previously showed that the Vps34 complexes interact with Rab GTPases, Rab1 or Rab5 (for class II), and the identified site was found at Vps34. Now, the authors identify a conserved and overlooked Rab5 binding site in Vps15, which is required for the function of the Class II complex. In support of this, they show cryo-EM data with a second Rab5 bound to Vps15, identify the corresponding residues, and show by mutant analysis that impaired Rab5 binding also results in defects using yeast as a model system.

Overall, this is a most complete study with little to criticize. The paper shows convincingly that the two Rab5 binding sites are required for Vps34 complex II function, with the Vps15 binding site being critical for endosomal localization. The structural data is very much complete.

Weaknesses:

What I am missing are a few controls that show that the mutations in Vps15 do not affect autophagy. I am wondering if this mutant is still functional in autophagy. This can be simply tested by sorting of Atg8 to the vacuole lumen using established assays or by following PhoΔ60 sorting. This analysis would reveal that the corresponding mutant is specific for the Class II complex.

One of the first noted features of the VPS34 complexes was that the ATG14-containing complex (VPS34-CI) is important for autophagy, while the VPS38 (yeast orthologue of UVRAG) subunit characteristic of VPS34-CII is important for endocytic sorting (PMID 11157979). However, the VPS34, VPS15 and BECLIN1 subunits are required are present in both complexes, as such, mutations of them may affect both processes.

We agree with the reviewer that is an important undertaking to examine the effect of the SHLITY>DDLIEY mutation in yeast Vps15 on autophagy. However, the focus of the current manuscript is VPS34-complex II and RAB5 interaction/activation. An autophagy effect would be more relevant for VPS34 complex I and RAB1. We have not presented any results for human VPS34-complex I - RAB1 nor yeast Vps34-complex I – Ypt1 (yeast RAB1 orthologue). We are preparing another manuscript focusing entirely on this, and it is not a simple story. While we think this is an important question, we believe that this is beyond the scope of the current manuscript.

It would be helpful if the authors could clarify whether they believe that Vps34 kinase activity is stimulated by Rab binding or whether this stimulation is a consequence of better membrane localization of Vps34. In other words, is the complex active with soluble PI3P in solution, and does the activity change if Rab5 is added to the complex? This might have been addressed in the past, but I did not see evidence for this, as the authors only addressed the activity of the Vps34 complexes on membranes.

The reviewer has raised an excellent question, which was addressed briefly in the introduction to the manuscript. We have now somewhat expanded on these issues near the end of the discussion in the revised manuscript. In our previously published study, we found that soluble RAB5-GTP did not stimulate the complex II activity (supplementary figure 2b of PMID: 33692360). This is consistent with our finding in this manuscript showing that RAB5 did not cause large conformational changes in solution. However, our previous single-molecule study showed that once complex II is recruited to the membrane by RAB5, and RAB5 increases the turnover rate on membranes, indicating an additional allosteric activation (Figure 7 of PMID: 33137306). This study indicated that the primary the role of RAB5 is to anchor complex II on the membrane. Once the complex is anchored on the membrane by RAB5, the kinase domain is in the vicinity of its substrate, PI, leading to higher turnover.

The Echelon Class III PI3K ELISA Kit (Echelon, K-3000) comes with a soluble PI, diC8 to measure the VPS34 activity, and it is certainly active with this soluble substrate. However, if the substrate is in membranes, the VPS34 activity is greatly dependent on the character of the membrane.

I also found the last paragraph of the results section a bit out of place, even though this is a nice observation that the N-terminal part of BECLIN has these domains. However, what does it add to the story?

The reviewer is correct that the high-resolution features of BECLIN1 at the base of the V-shaped complex that we observed are not related to RAB5 binding, but they are characteristic of VPS34-CII and likely to be important for the specific role of VPS34-CII. This is the first high-resolution structure of the VPS34-CII that has been reported, and we believe it would be irresponsible not to briefly describe them, since they are unique to VPS34-CII. For this reason, we have placed this section at the end of the results, and we now clarify that we do not see a relevance to RAB5 function, but we describe the arrangement of a region (the BH3) that has been functionally noted in many previous studies, in the absence of a structure.

Reviewing Editor Comments:

Please address the following suggestions for minor changes to the manuscript. Use your best scientific judgment in addressing the comments and describe the modifications together with your reasoning in a cover letter. We look forward to seeing the revised version of this very nice study.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

I found a portion of the description of the cryoEM complexes on the top of page 9 to be redundant with similar descriptions near the top of page 7, and it was not clear to me at first that these were describing the same structures. Part of my confusion was due to the redundancy, including the statement near the bottom of page 7: 'Models were built and refined for all RAB5associated VPS34-CII assemblies', and then the similar statement on page 9: 'We fit and refined atomic models into both densities'. I believe these are describing the same models? To clarify for the reader, perhaps on page 9, the authors could begin this part with a statement such as "as described above", and eliminate the redundant descriptions.

The reviewer is correct. Both sections describe the same set of cryo-EM classes from the same sample. The only difference is what we analysed in the two sections: number of RAB5s bound in the first section and the effect of RAB5 binding in the second section. We have revised the text to make this clear, and to make the second section more succinct.

Reviewer #3 (Recommendations for the authors):

(1) The authors show nicely that a mutation in Vps15 disrupts binding to Vps21 in vivo, with defects in the endocytic pathway as analyzed by CPY sorting. I am wondering if this mutant is still functional in autophagy. This can be simply tested by sorting of Atg8 to the vacuole lumen using established assays or by following Pho∆60 sorting. This analysis would reveal that the corresponding mutant is specific for the Class II complex. If the authors were to find evidence that this Vps15 mutant also affects autophagy, it would indicate that there is possibly also another Rab1 binding site in Vps15.

As we stated above, an autophagy effect would be more relevant for VPS34 complex I and RAB1. We have not presented any results for human VPS34-complex I - RAB1 nor yeast Vps34-complex I – Ypt1 (yeast RAB1 orthologue). We are preparing another manuscript focusing entirely on this, and it is not a simple story. While we think this is an important question, we believe that this is beyond the scope of the current manuscript.

(2) It would be helpful if the authors could clarify whether they believe that Vps34 kinase activity is stimulated by Rab binding or whether this stimulation is a consequence of better membrane localization of Vps34. In other words, is the complex active with soluble PI3P in solution, and does the activity change if Rab5 is added to the complex? This might have been addressed in the past, but I did not see evidence for this, as the authors only addressed the activity of the Vps34 complexes on membranes.

As in our response to reviewer #3 above, this point was addressed in previous publications and was described in the introduction to our manuscript.

This ignores how much of the contents of "the user–maintainer feedback loop" in the GitHub era prior to the glut (infestation?) of LLM-generated pull requests were already low quality (often aimed at projects that were of dubious character to begin with—projects like Tailwind, cited here). Hashimoto et al just seemingly had a higher threshold for tolerance and have now found where their personal threshold is (or at least established an upper bound on its location).

Previously:

• Free Software Is Not Enough: On Practical User Freedom (2020)

• Open Source Is Not Enough (2011)

Sounds like something that could have been drastically simplified by just using bookmarks (but maybe iOS makes that painful, too).

Does it? It's not obvious from the problem and proposed approach described here that the homespun service doing the LLM work (or, rather, coordinating it) and the client submitting stuff to the third-party service need to be the same.

The client really only needs to authenticate with the service and ferry data into the account. The data can be prepped elsewhere. The only real constraint is that it comes from a trusted source.

forgetting is a form of dimming, we just think it's inaccessible but it's probably not erasure. the role of both is clear though [[Forgetting by Scott A. Small]] except lls does not forget, it records.

good observation: lls does to you what bigtech already does to you, but without the ads. What does it bring yourself remains a question in both situations though.

sensing yes, our brains do it for us, measuring and recording no.

this is true for any 4 or more nodes.

Agree they matter, bc they are their own content, but content matters too, bridges to nowhere don't matter. So more not applicable, that's a sentiment stemming from their usually being more connections than nodes, and if connections are nodes of a sort, they are more plentiful than original nodes. A given, not a measure of quality / importance. #openvraag when you expand a link to be nuggets of info, how would you link to a link, i.e. link to a relation. Which is different than link to either endpoint of a relation. And by extension, how to link to a constellation [[Gestalten and Constellations above Crumbs 20200426111123]] [[Nodal point als de Gestalt zien in de data 20240327100507]]

wiki's with wikilinks brought it to more people than Obsidian does now. Wikilinks is not a very recent thing. 1990s wiki (Ward's e.g) had CamelCaseLinking, WikiPedia (2002) had wikilinks from early on, they were introduced in the UseModWiki code at the start of 2001.

Switching metaphors away from bio, diamond bc of multi-faceted. Adds 8 more aspects. - temporality - direction - valence (subjective ratio of pos/neg emotions etc.) - layer weighting (as a weight stemming from a question asked of the data, not an aspect of the actual connection) - social graph - context dependence (diff roles peers can have) - decay (a measure of temporality, with temporality being a moment, decay the distance to it) - node value, a measure of what a weak tie connects to inverse proportional to alternative routes available. replaceability, which is a overly utilitarian term when applied to peers imo.

myelin as metaphor for the completeness of info/calculated synapse strenght for a connection. Says it's not a measure of conductivity, but I wonder if you could actually use it as proper conductivity in a [[Social netwerk als filter 20060930194648]] setting, like with [[Probes in neuraal netwerk onderbrengen]].

the question is, do you need this for the connection to be usable? The example connection is part of Martijn's strong ties, and you already know, have an intuitive picture, without the data at hand. weak ties are of high interest bc of their potential bridging value, the [[Informatiewaarde ligt in verrassing surprisal 20210124072501]] they bring. In the middle ground, my [[Social Distance als ordeningsprincipe 20190612143232]], say the 150+ to 500 is where this weight may be more interesting. You have no mental image strong enough to just know them, they are not far out to have a role in wide flung connectivity, but do serve as a great curation filter for info. Is the weight here a measure of [[Contactivity 20051105150458]], for the middle part.

synapse strength (ie link weight / layers of meaning even?): how many of the 10 evidence aspects covered, how often, how complete is each of the records in each aspect. Not mentioned how this is expressed back into /logged in the data (or reconstructed every time like human memory?)

He assesses strenght of connection based on different elements of evidence, w uncertainty qualifier, and their relative weight. Intentional contact, database relationship (combo in data somewhere), bank evidence, check-ins, photo-evidence, text extraction, date coincidence, interpolation, pattern recognitions, human confirmation (for the computer derived connections from the other aspects)

Are these named later on?

starting from 184.557 data points, discounting texts he also has, you'd have over 17 million possible pairs, and many combinations will be building on over 2 data points. So yes, tens of millions of connections is a given. Not a result.

[[Martijn Aslander p]] adopts synapse as metaphor (as it is a link with (chemical) agency?). Nodes as neurons, links as synapses. Says the links outnumber the nodes. That is true very quickly also on tiny scale, not emergent coherence per se. 4 nodes woven into a tight network can have 6 links, not looking at direction. (n * (n-1)) / 2)

A few elements that can flesh out how [[Links are not binary 20201110202454]] and how to 'fill' them as [[Links als informatieobject 20201110152514]] in their own right.

Agree that in most tools a link is like that, but e.g. a link could also be just as easily carry qualifications, or be a note in itself. In complexity management adding dimensions to a link is common, lifting it out of the 2d plan of being a line between two other nodes. A unit of information itself. Same is true in [[Social software werkt in driehoeken 20060506070412]] Vgl [[Links als eersterangs burgers 20201110202222]] [[Links are not binary 20201110202454]] [[Links als informatieobject 20201110152514]]

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Referee #3

Evidence, reproducibility and clarity

Summary:

This study identifies CD81 as an anti-viral host factor that restricts EBOV infection. In-turn, EBOV encodes CD81 antagonism activity in GP and VP40. The two major lines of inquiry of this study are how CD81 restricts EBOV infection and how EBOV antagonizes CD81. These are evaluated using BSL-2 level models and authentic EBOV infection in different cell types, including human primary macrophages. This study underscores the intricate interplay between mechanisms of host innate immunity and mechanisms of virus immune evasion and antagonism.

CD81 restriction of EBOV

A previous study published by this group demonstrated that CD81 antagonizes NF-κB signaling. In this study and a very recently published study by another group, VP40 is demonstrated to activate NF-κB signaling. This study shows that VP40 activates NF-κB through a TLR4-independnet mechanism that is antagonized by CD81. This study also demonstrated that CD81 suppresses authentic EBOV infection in HEK293T cells and human primary blood monocyte derived macrophages. Using a transcription and replication-competent tetracistronic minigenome system (trVLPs), this study found that CD81 reduces the abundance of all viral RNA species (mRNA, vRNA genome, cRNA genome complement) but does not interfere with the NP-driven formation of inclusion bodies where viral RNA synthesis occurs. CD81 was also found to suppress cellular micropinocytosis activity and restrict EBOV entry. The CD81-stimulating antibody 5A6 suppressed both trVLP and authentic EBOV infection.

EBOV antagonism of CD81

CD81 (as well as the other tetraspanins CD63 and CD9) was identified to be downregulated on the cell surface of HeLa and HEK293 cells transfected with an EBOV GP expression plasmid as a part of a larger screen for EBOV GP's impact on cell surface receptor expression. Using a combination of bimolecular fluorescent complementation (BiFC), fluorescence resonance energy transfer (FRET), and proximity ligation assays, EBOV GP was shown to directly interact with CD81. Imaging analysis showed that EBOV GP colocalizes with CD81 at the cell surface. Here, GP blocks accessibility of CD81 through a glycan shielding mechanism that is reversed with PNGase F treatment. EBOV GP also reduces the total cellular abundance of CD81 protein by inducing CD81 degradation through both lysosomal and proteasome mediated degradation pathways without altering CD81 mRNA transcript levels. The GPs of other relative filoviruses (Marburg virus, Sudan virus, Reston virus, and Taï Forest virus) also downregulated accessible cell surface CD81, CD63, and CD9 in HEK293T cells. This CD81 downregulating activity appears to be both conserved amongst and specific to filovirus GPs, as none of the glycoproteins from 14 other RNA viruses tested (including arenaviruses, rhabdoviruses, influenza viruses, coronaviruses, and retroviruses) significantly altered cell surface CD81, CD63, or CD9 in HEK293T cells. In human primary blood monocyte derived macrophages, authentic EBOV infection was found to reduce the abundance of CD81 and CD9 accessible at the cell surface. EBOV VP40 was the only other EBOV structural protein that downregulated total CD81, though its effect was mild. In contrast to the mechanisms utilized by GP, VP40 was found to induce CD81 degradation mainly through the proteasome mediated degradation pathway.

Major Comments:

The claims made by the authors are appropriate and are supported by their data and their use of appropriate controls which yielded the expected results based on references from the literature. There are no new experiments that must be introduced to support the claims made by the authors. The methods section is excellent and provides extensive detail for techniques and organized lists of plasmid and antibody reagents used and their original sources. Graphed data shows individual replicates, representative flow scatter plots and images are show, and appropriate statistical analyses were used and reported. Excitingly, this study opens many new lines of question which can be addressed in future studies.

The only major comment:

• It would be valuable to add more discussion around something the data presented in Figure 7 b-d suggest, and that is that EBOV entry appears to be targeted by CD81 by multiple mechanisms. Figure 7d demonstrates CD81 suppresses cellular macropinocytosis activity, which would yield less uptake of EBOV which utilizes PS receptors to be internalized through the macropinocytosis pathway. Since PS receptors recognize the PS in the viral envelope, and not the viral GP, it makes sense that trVLPs pseudotyped with VSV-G were restricted like those with EBOV GP (Fig 7C). However, in the pseudotyped lentivirus system, EBOV GP-mediated entry was significantly suppressed by CD81 while VSV-G mediated entry was not (Fig 7B). Together this data shows that CD81 restricts EBOV entry in both viral envelope-targeted and GP-targeted mechanisms, demonstrating the vast innate immune mechanisms of CD81 against EBOV. I think this is of great impact and should be discussed more.

Minor Comments:

Some modest experimental and analysis suggestions that are not required to support the claims of the paper but would add additional depth are:

• The experiments in Figure 8E would have benefited from collection and titering of supernatants from infected cells.
• While bafilomycin treatment or MG132 treatment can partially rescue CD81 from the degradation induced by EBOV GP or VP40, neither drug is able to fully rescue this for either viral protein. It would be insightful to assess if co-treatment of bafilomycin and MG132 would yield a full rescue of total CD81. Based on the presented results, I would expect co-treatment to fully restore total CD81 in VP40-expressing cells, but I would expect co-treated GP-expressing cells to still have increased CD81 surface downregulation equivalent to the strength of which the glycans of EBOV GP shield CD81 from recognition. These experiments would give valuable insight into the relative strengths of the glycan shielding effect and induced degradation effects in EBOV GP's antagonism of CD81.
• The impact of the imaging experiments shown in Fig. 5D would be strengthened with a quantitative colocalization analysis such as Pearson's Coefficient.
• Another publication (citation 132 in this study) shows cooperativity of GP in VP40's ability to activate NF-ΚB. This study shows in Fig. 6E that CD81's suppressive activity can overcome VP40's activation of NF-ΚB. It would be valuable to assess (in the same format as the experiment done in Fig 6E) if this would remain true in cells co-expressing EBOV GP and VP40; ie, would CD81 still be able to overcome, and at a similar rate, the NF-ΚB-activating activity of VP40 in the presence of the CD81-antagonizing activity of GP.
• Understandably, the VP40 gene was used as a probe in the Fig. 6 trVLP experiments because it is encoded within the tetracistronic minigenome. However, this became mildly confusing when reading Figure 6 because other parts of the manuscript discuss and measure the effects and activities of VP40. One thought is to probe the VP24 gene. However, a simple way to reduce the initial confusion in interpreting the data in the figure is to just remove "(VP40)" from Fig.6 A-C. The methodology of using VP40 primers to probe for the viral RNAs is adequately detailed in the figure caption and methods section.

Significance

This paper gives more insight into another mechanism of innate immune responses of the host and mechanisms of evasion/antagonism by EBOV VP40 and GP. This is the first report of CD81 as an anti-viral host factor of EBOV. This study shows that CD81 interferes with EBOV viral entry and reduces the abundance of EBOV RNAs using different mechanism. Additionally, CD81 was found to have a previously unrecognized negative regulatory role in cellular micropinocytosis. In terms of how EBOV antagonizes CD81, this study demonstrates multiple mechanisms of antagonism and more broadly demonstrate the diverse mechanisms of innate immune evasion encoded by EBOV. Building on previous reports of the N-linked glycans of EBOV having a steric shielding effect that blocks accessibility of cell surface human leukocyte antigen class-1 (HLA-I) and MHC class I polypeptide-related sequence A (MICA), this study found that these glycans also sterically shield the accessibility of cell surface CD81. This opens important new lines of inquiry, such as: what else do the EBOV glycans sterically interfere with at the cell surface?

This group previously demonstrated that CD81 suppresses NF-κB activation and this study builds on those observations by demonstrating that despite the CD81-antagonizing activity of VP40, VP40's ability to activate NF-κB can be overcome by CD81. This is in line with some of the most impactful findings in this paper, namely that despite encoding CD81 antagonism activity in at least GP and VP40, EBOV remains susceptible to CD81 activation, as demonstrated by inhibition of both the trVLP system and authentic EBOV infection by the CD81-stimulating antibody 5A6.

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Referee #2

Evidence, reproducibility and clarity

The manuscript entitled "CD81 is an Ebola virus inhibiting factor that is antagonized by GP and VP40" by Hu et al. investigates the role of the tetraspanins CD81, CD63, and CD9 during Ebola virus infection. They found that CD81, among the three tetraspanins, plays a major role as a cellular antiviral factor by interfering with EBOV glycoprotein and VP40. CD81 suppressed NFkB signaling and was found to restrict EBOV replication and VLP uptake. Overall, the study design, choice of experimental approaches, and presentation of the data meet an excellent scientific standard. The figures and their legends are comprehensive and very clearly written. However, I recommend that the authors make the following clarifications:

Major:

1. I recommend including a figure that depicts the domain organization of the tetraspanins, as this would help readers better appreciate how these three tetraspanins differ from one another. Did the authors determine the minimal region of CD81 required for interaction with EBOV GP or VP40? How are tetraspanins trafficked from the plasma membrane to intracellular compartments or into extracellular vesicles, and are these trafficking pathways also altered during EBOV infection?
2. Given the clear role of VP40 in this CD81-dependent mechanism, it is important to demonstrate whether VP40 and CD81 interact directly. As the BiFC assay did not resolve this question, I recommend using a complementary approach, such as co-immunoprecipitation followed by western blotting, to address it.

Minor:

1. Please clarify how the '+' and '++' GFP categories are quantitatively defined.
2. In Figure 4b (right panels), what explains the different effects of the DMSO control on surface versus total CD81?
3. For clarity, I suggest defining the exact numerical boundaries of the individual domains shown in Figure S2C.
4. In Figure S3C, the data presentation could be improved by using different colors for the control and KO groups, or by increasing the size of the symbols representing the data points.

Significance

This article is well written, and the study underscores the critical role of the tetraspanin CD81 as a cellular antiviral factor during EBOV infection and defines its role in filoviral immune response regulation. This article can be accepted for publication after the minor revision.

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Referee #1

Evidence, reproducibility and clarity

To identify cell surface receptors modulated by GP, the authors performed a flow cytometry-based screen using the LEGENDScreen Human Cell PE Kit, which targets 332 host cell surface markers. Among the validated hits in two human cell lines, the authors focused on the tetraspanin CD81 as its expression was selectively reduced by EBOV GP and other filovirus GPs, but not by GPs from unrelated viruses, suggesting a filovirus GP-specific mechanism. This is analogous to the downregulation of CD81 expression by viral proteins such as Vpu and Gag from HIV, and NS5A from HCV. The authors also found that VP40 overexpression reduced CD81 levels, potentially enhancing VP40-mediated NF-κB activation. The authors suggest that CD81 reduction may result from degradation led by GP-CD81 interaction. CD81 downregulation was also observed in infected monocyte-derived macrophages (MDMs). Detailed analysis using CD81-KO cells and the transcription- and replication-competent VLP (trVLP) system demonstrated that CD81 is involved in EBOV entry and replication steps. While these data provide key insights, concerns remain regarding their statistical significance and interpretation.

1. Have the authors investigated the functional consequences of CD81 downregulation by GP, VP40, or viral infection? In particular, could this enable superinfection? This can be examined using the approaches used in the manuscript.
2. "Fold of modification (GP-/GP+)" in Figure 1a does not appear to match the results presented in Figure S2 and Table S2.
3. Where appropriate, please indicate 'n.s.' for comparisons that are not statistically significant. With n = 3, the results may be unreliable; increasing the number of replicates to five would be recommended, as this is critical for supporting the manuscript's conclusions.
4. Although the authors conclude that GP suppresses or counteracts CD81-mediated inhibition of viral replication (e.g., VP40 protein expression) based on experiments using trVLPs with or without GP, the presented data do not support this conclusion. In fact, higher VP40 expression was detected in trVLPΔGP-infected cells compared to trVLP-infected control cells (Fig. 3a and 3b), or no statistically significant differences was provided. These results seem inconsistent with the authors' interpretation and require clarification.
5. Increased p65 expression does not necessarily indicate activation of p65 or NF-κB signaling. Indeed, VP40-induced or infection-induced increase in p65 expression level was not significantly different between wild-type and CD81-KO cells (Fig. 6c and 6d). To properly assess the NF-κB activation, the phosphorylation status of p65 and/or nuclear translocation should be examined.
6. The authors suggest that CD81 is involved in macropinocytosis based on experiments using CD81-KO cells (Fig. 7) and anti-CD81 antibody (5A6 clone) (Fig. 8). Have the authors examined whether CD81 regulates macropinocytosis-associated signaling pathway (e.g., the PI3K/AKT1 pathway)? It is possible that AKT1 is constitutively activated in CD81-KO cells, given the increased dextran uptake. Such analysis would strengthen the authors' claim.
7. In the experiments assessing viral entry in CD81-KO and control cells, both cells were co-transfected with Tim-1. Have the authors confirmed that Tim-1 expression levels were comparable between KO and control cells?
8. The VP40-CD81 interaction was assessed only by PLA, but the results were not shown due to high background signals. Other methods, such as co-IP or the BiFC assay used in the manuscript, could yield clear data and deepen the discussion.
9. In the Fig.4b, both the number of GFP-positive cells and the GFP intensity are noticeably lower than in other similar experiments (e.g., Fig. 1c and Fig. S4a). The "GP ++" population is much smaller and difficult to define or gated. Please clarify this discrepancy.
10. The statement that "VP40-mediated downregulation of surface CD81 was strongly blocked by MG132 and partially by BafA1 (Fig. 4b)" is not supported by the data shown.
11. In Fig.4c, assessing the role of GP glycan shield, is there a statistically significant difference between GP and GPΔmucin? It appears that deletion of the mucin domain does not affect the structural shielding of CD81, whereas PNGase treatment does.
12. In Fig.5c, please enlarge the PLA image for better visibility.
13. CD81 localization in the presence of GP differs between Fig. 5c and Fig. 5d under similar conditions. In the Fig. 5d, GP redistributes CD81 to both the cytoplasm and the cell surface. Please clarify this discrepancy.
14. In Fig. 8a, the percentage of GFP-positive cells (infected cells) are very low, up to 5%. What MOI was used, and can the effect of the CD81 antibody on infection be reliably evaluated under this condition? Statistical significance should compare CD81 antibody with the isotype control, not with no antibody.
15. The authors use the term "multiple" (e.g., "multiple cell lines" and "CD81 inhibits multiple steps throughout the viral life cycle"); however, this wording feels overstated as two cell lines were used and the two steps (entry and replication steps) are inhibited.
16. Please cite the following article where relevant: Nanoscale organization of tetraspanins during HIV-1 budding by correlative dSTORM/AFM (Nanoscale, 2019).
17. What primary antibodies are used in the PLA. Please describe them in the method section.
18. Lines 468-471. Please provide the relevant references for the GP mutations tested.

Significance

To identify cell surface receptors modulated by GP, the authors performed a flow cytometry-based screen using the LEGENDScreen Human Cell PE Kit, which targets 332 host cell surface markers. Among the validated hits in two human cell lines, the authors focused on the tetraspanin CD81 as its expression was selectively reduced by EBOV GP and other filovirus GPs, but not by GPs from unrelated viruses, suggesting a filovirus GP-specific mechanism. This is analogous to the downregulation of CD81 expression by viral proteins such as Vpu and Gag from HIV, and NS5A from HCV. The authors also found that VP40 overexpression reduced CD81 levels, potentially enhancing VP40-mediated NF-κB activation. The authors suggest that CD81 reduction may result from degradation led by GP-CD81 interaction. CD81 downregulation was also observed in infected monocyte-derived macrophages (MDMs). Detailed analysis using CD81-KO cells and the transcription- and replication-competent VLP (trVLP) system demonstrated that CD81 is involved in EBOV entry and replication steps. While these data provide key insights, concerns remain regarding their statistical significance and interpretation.

eLife Assessment

This important study provides compelling evidence that fever-like temperatures enhance the export of Plasmodium falciparum transmembrane proteins, including the cytoadherence protein PfEMP1 and the nutrient channel PSAC, to the red blood cell surface, thereby increasing cytoadhesion. Using rigorous and well-controlled experiments, the authors convincingly demonstrate that this effect results from accelerated protein trafficking rather than changes in protein production or parasite development. These findings significantly advance our understanding of parasite virulence mechanisms and offer insights into how febrile episodes may exacerbate malaria severity.

Reviewer #1 (Public review):

Summary:

The manuscript from Jones and colleagues investigates a previously described phenomenon in which P. falciparum malaria parasites display increased trafficking of proteins displayed on the surface of infected RBCs as well as increased cytoadherence in response to febrile temperatures. While this parasite response was previously described, it was not uniformly accepted, and conflicting reports can be found in the literature. This variability likely arises due to differences in the methods employed and the degree of temperature increase that the parasites were exposed to. Here the authors are very careful to employ a temperature shift that likely reflects what is happening in infected humans and that they demonstrate is not detrimental to parasite viability or replication. In addition, they go on to investigate what steps in protein trafficking are affected by exposure to increased temperature and show that the effect is not specific to PfEMP1 but rather likely affects all transmembrane domain containing proteins that are trafficked to the RBC. They also detect increased rates of phosphorylation of trafficked proteins, consistent with overall increased protein export.

Strengths:

The authors used a relatively mild increase in temperature (39 degrees) that they demonstrate is not detrimental to parasite viability or replication. This enabled them to avoid potential complications of more severe heat shock that might have affected previously published studies. They employed a clever method of fractionation of RBCs infected with a var2csa-nanoluc fusion protein expressing parasite line to determine which step in the export pathway was likely accelerating in response to increased temperature. This enabled them to determine that export across the PVM is being affected. They also explored changes in phosphorylation of exported proteins and demonstrated that the effect is not limited to PfEMP1 but appears to affect numerous (or potentially all) exported transmembrane domain containing proteins.

Impact and conclusions:

The study shows that protein export, including PfEMP1 and PSAC, are accelerated in response to mild heat shock. This has implications for disease severity as well as our understanding of protein trafficking in these unique organisms. There is increasing interest in asymptomatic infections, which have been proposed to be a major reservoir for transmission and generally are not associated with fever. It will be interesting to consider whether reduced (or slower) trafficking of these proteins has a selective advantage for parasites in asymptomatic infections.

Reviewer #2 (Public review):

This manuscript describes experiments characterising how malaria parasites respond to physiologically relevant heat-shock conditions. The authors show, quite convincingly, that moderate heat-shock appears to increase cytoadherance, likely by increasing trafficking of surface proteins involved in this process.

While generally of a high quality and including a lot of data, I have a few small questions and comments, mainly regarding data interpretation.

(1) The authors use sorbitol lysis as a proxy for trafficking of PSAC components. This is a very roundabout way of doing things and does not, I think, really show what they claim. There could be a myriad of other reasons for this increased activity (indeed, the authors note potential PSAC activation under these conditions). One further reason could be a difference in the membrane stability following heat shock, which may affect sorbitol uptake, or the fragility of the erythrocytes to hypotonic shock. I really suggest that the authors stick to what they show (increased PSAC) without trying to use this as evidence for increased trafficking of a number of non-specified proteins that they cannot follow directly.

(2) Supplementary Figure 6C/D: The KAHRP signal does not look like it should. In fact, it doesn't look like anything specific. The HSP70-X signal is also blurry and overexposed. These pictures cannot be used to justify the authors' statements about a lack of colocalisation in any way.

(3) Figure 6: This experiment confuses me. The authors purport to fractionate proteins using differential lysis, but the proteins they detect are supposed to be transmembrane proteins and thus should always be found associated with the pellet, whether lysis is done using equinatoxin or saponin. Have they discovered a currently unknown trafficking pathway to tell us about? Whilst there is a lot of discussion about the trafficking pathways for TM proteins through the host cell, a number of studies have shown that these proteins are generally found in a membrane-bound state. The authors should elaborate, or choose an experiment that is capable of showing compartment-specific localisation of membrane-bound proteins (protease protection, for example).

(4) The red blood cell contains, in addition to HSP70-X, a number of human HSPs (HSP70 and HSP90 are significant in this current case). As the name suggests, these proteins non-specifically shield exposed hydrophobic domains revealed upon partial protein unfolding following thermal insult. I would thus have expected to find significantly more enrichment following heat shock, but this is not the case. Is it possible that the physiological heat shock conditions used in this current study are not high enough to cause a real heat shock?

Comments on Revision:

Although in any study there are going to be residual weaknesses, this reviewer is happy to see that the authors have gone to lengths to address many of my main concerns, and also those of other reviewers.

Reviewer #3 (Public review):

Summary:

In this paper it is established that high fever-like 39oC temperatures cause parasite infected red blood cells become stickier. It is thought that high temperatures might help the spleen to destroy parasite infected cells, so they become stickier to remain trapping in blood vessels, so they stop passing through the spleen.

Strengths:

The strength of this research is that it shows that fever-like temperatures can cause parasite infected red blood cells to stick to surfaces designed to mimic the walls of small blood vessels. In a natural infection this would cause parasite infected red blood cells to stop circulating through the spleen where the parasites would be destroyed by the immune system. It is thought that fevers could lead to infected red blood cells becoming stiffer and therefore more easily destroyed in the spleen. Parasites respond to fevers by making their red blood cells stickier, so they stop flowing around the body and into the spleen. The experiments here prove fever temperatures increase the export of Velcro-like sticky proteins onto the surface of the infected red blood cells and are very thorough and convincing.

Weaknesses:

Minor weaknesses in the original version have now been satisfactorily addressed with additional work which is very convincing.

Author Response:

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

eLife Assessment

This important study provides compelling evidence that fever-like temperatures enhance the export of Plasmodium falciparum transmembrane proteins, including the cytoadherence protein PfEMP1 and the nutrient channel PSAC, to the red blood cell surface, thereby increasing cytoadhesion. Using rigorous and well-controlled experiments, the authors convincingly demonstrate that this effect results from accelerated protein trafficking rather than changes in protein production or parasite development. These findings significantly advance our understanding of parasite virulence mechanisms and offer insights into how febrile episodes may exacerbate malaria severity.

We thank all reviewers for their constructive feedback on our manuscript.

We believe we have addressed all the questions in the rebuttal below in writing, including planned experiments we will perform to strengthen the conclusions of the manuscript.

Public Reviews:

Reviewer #1 (Public review):

Summary:

This manuscript from Jones and colleagues investigates a previously described phenomenon in which P. falciparum malaria parasites display increased trafficking of proteins displayed on the surface of infected RBCs, as well as increased cytoadherence in response to febrile temperatures. While this parasite response was previously described, it was not uniformly accepted, and conflicting reports can be found in the literature. This variability likely arises due to differences in the methods employed and the degree of temperature increase to which the parasites were exposed. Here, the authors are very careful to employ a temperature shift that likely reflects what is happening in infected humans and that they demonstrate is not detrimental to parasite viability or replication. In addition, they go on to investigate what steps in protein trafficking are affected by exposure to increased temperature and show that the effect is not specific to PfEMP1 but rather likely affects all transmembrane domain-containing proteins that are trafficked to the RBC. They also detect increased rates of phosphorylation of trafficked proteins, consistent with overall increased protein export.

Strengths:

The authors used a relatively mild increase in temperature (39 degrees), which they demonstrate is not detrimental to parasite viability or replication. This enabled them to avoid potential complications of a more severe heat shock that might have affected previously published studies. They employed a clever method of fractionation of RBCs infected with a var2csa-nanoluc fusion protein expressing parasite line to determine which step in the export pathway was likely accelerating in response to increased temperature. This enabled them to determine that export across the PVM is being affected. They also explored changes in phosphorylation of exported proteins and demonstrated that the effect is not limited to PfEMP1 but appears to affect numerous (or potentially all) exported transmembrane domain-containing proteins.

Weaknesses:

All the experiments investigating changes resulting from increased temperature were conducted after an increase in temperature from 16 to 24 hours, with sampling or assays conducted at the 24 hr mark. While this provided consistency throughout the study, this is a time point relatively early in the export of proteins to the RBC surface, as shown in Figure 1E. At 24 hrs, only approximately 50% of wildtype parasites are positive for PfEMP1, while at 32 hrs this approaches 80%. Since the authors only checked the effect of heat stress at 24 hrs, it is not possible to determine if the changes they observe reflect an overall increase in protein trafficking or instead a shift to earlier (or an accelerated) trafficking. In other words, if a second time point had been considered (for example, 32 hrs or later), would the parasites grown in the absence of heat stress catch up?

We did not assess cytoadhesion at later stages, but in the supplementary figures we show that at 40 hours post infection both heat stress and control conditions have comparable proportions of VAR2CSA-positive iRBCs, whilst they differ at 24h. This is true for the DMSO (control wildtype resembling) HA-tagged lines of HSP70x and PF3D7_072500 (Supplementary Figures 9 and 12 respectively). In the light that protein levels appear not changed, we conclude that trafficking is accelerated during these earlier timepoints, but remains comparable at later stages. This would still increase the overall bound parasite mass as parasites start to adhere earlier during or after a heat stress.

Reviewer #2 (Public review):

This manuscript describes experiments characterising how malaria parasites respond to physiologically relevant heat-shock conditions. The authors show, quite convincingly, that moderate heat-shock appears to increase cytoadherance, likely by increasing trafficking of surface proteins involved in this process.

While generally of a high quality and including a lot of data, I have a few small questions and comments, mainly regarding data interpretation.

(1) The authors use sorbitol lysis as a proxy for trafficking of PSAC components. This is a very roundabout way of doing things and does not, I think, really show what they claim. There could be a myriad of other reasons for this increased activity (indeed, the authors note potential PSAC activation under these conditions). One further reason could be a difference in the membrane stability following heat shock, which may affect sorbitol uptake, or the fragility of the erythrocytes to hypotonic shock. I really suggest that the authors stick to what they show (increased PSAC) without trying to use this as evidence for increased trafficking of a number of non-specified proteins that they cannot follow directly.

This is a valid point, however, uninfected RBCs do not lyse following heat stress, nor do much younger iRBCs, indicating that the observed effect is specific to infected RBCs at a defined stage. The sorbitol sensitivity assay is performed at 37°C under normal conditions after cells are returned to non–heat stress temperatures, so the effect is not due to transient changes in membrane permeability at elevated temperature.

Planned experiment: However, to increase the strength of our conclusions and further test our hypothesis, we will perform sorbitol sensitivity assays on >20 hours post infection iRBCs following heat stress in the presence and absence of furosemide, a PSAC inhibitor. If iRBC lysis is abolished with furosemide present, this would confirm that the effect is PSAC-dependent. However, the effect could also possibly be due to altered PSAC activity during heat stress which is maintained at lower temperatures, as outlined in the discussion.

New Results:

We performed sorbitol sensitivity assays on >20 hours post-infection iRBCs following heat stress in the presence and absence of the PSAC inhibitor furosemide. These additional experiments were added to the supplementary figures (Supplementary Figure 3). Importantly, sorbitol-mediated lysis of iRBCs, with or without prior heat stress, was reduced when furosemide was present, demonstrating that the observed effect is likely PSAC-dependent. We also observed that uninfected RBCs did not lyse with sorbitol, regardless of heat stress, confirming that the effect is specific to infected cells.

(2) Supplementary Figure 6C/D: The KAHRP signal does not look like it should. In fact, it doesn't look like anything specific. The HSP70-X signal is also blurry and overexposed. These pictures cannot be used to justify the authors' statements about a lack of colocalisation in any way.

Planned experiment: We agree that the IFAs are not the best as presented and will include better quality supplementary images in a revised version.

New Results:

Immunofluorescence microscopy, including the localisation of the two HA-tagged proteins (PF3D7_1039000 and PF3D7_0702500), has been repeated and higher-quality images are now included in the updated manuscript (Supplementary Figures 9 and 11). These images include co-staining with the P. falciparum proteins KAHRP and SPB1 to assess possible co-localisations. Furthermore, following the reviewer’s suggestion, we have softened the statement regarding PF3D7_1039000-HA to better reflect the data, changing “...does not colocalise” to “...does not strongly colocalise”.

(3) Figure 6: This experiment confuses me. The authors purport to fractionate proteins using differential lysis, but the proteins they detect are supposed to be transmembrane proteins and thus should always be found associated with the pellet, whether lysis is done using equinatoxin or saponin. Have they discovered a currently unknown trafficking pathway to tell us about? Whilst there is a lot of discussion about the trafficking pathways for TM proteins through the host cell, a number of studies have shown that these proteins are generally found in a membrane-bound state. The authors should elaborate, or choose an experiment that is capable of showing compartment-specific localisation of membrane-bound proteins (protease protection, for example).

We do not believe we identified a novel trafficking pathway, but that we capture trafficking intermediates of PfEMP1 between the PVM and the RBC periphery, in either small vesicles, and possibly including Maurer’s clefts. These would still be membrane embedded, but because of their small size, not be pelleted using the centrifugation speeds in our study (we did not use ultracentrifugation). This explanation, we believe, is in line with the current hypothesis of PfEMP1 and other exported TMD protein trafficking to the periphery or the Maurer’s clefts.

(4) The red blood cell contains, in addition to HSP70-X, a number of human HSPs (HSP70 and HSP90 are significant in this current case). As the name suggests, these proteins non-specifically shield exposed hydrophobic domains revealed upon partial protein unfolding following thermal insult. I would thus have expected to find significantly more enrichment following heat shock, but this is not the case. Is it possible that the physiological heat shock conditions used in this current study are not high enough to cause a real heat shock?

As noted by the reviewer, we do not see enrichment of red blood cell heat shock proteins following heat stress, either with FIKK10.2-TurboID or in the phosphoproteome. We used a physiologically relevant heat stress that significantly modifies the iRBC, as shown by our functional assays. While a higher temperature might induce an association of red blood cell heat shock proteins, such conditions may not accurately reflect the most commonly found in the context of malaria infection.

Reviewer #3 (Public review):

Summary:

In this paper, it is established that high fever-like 39 C temperatures cause parasite-infected red blood cells to become stickier. It is thought that high temperatures might help the spleen to destroy parasite-infected cells, and they become stickier in order to remain trapped in blood vessels, so they stop passing through the spleen.

Strengths:

The strength of this research is that it shows that fever-like temperatures can cause parasite-infected red blood cells to stick to surfaces designed to mimic the walls of small blood vessels. In a natural infection, this would cause parasite-infected red blood cells to stop circulating through the spleen, where the parasites would be destroyed by the immune system. It is thought that fevers could lead to infected red blood cells becoming stiffer and therefore more easily destroyed in the spleen. Parasites respond to fevers by making their red blood cells stickier, so they stop flowing around the body and into the spleen. The experiments here prove that fever temperatures increase the export of Velcro-like sticky proteins onto the surface of the infected red blood cells and are very thorough and convincing.

Weaknesses:

A minor weakness of the paper is that the effects of fever on the stiffness of infected red blood cells were not measured. This can be easily done in the laboratory by measuring how the passage of infected red blood cells through a bed of tiny metal balls is delayed under fever-like temperatures.

Previous work by Marinkovic et al. (cited in this manuscript) reported that all RBCs, both infected and uninfected, increase in stiffness at 41 °C compared with 37 °C, with trophozoites and schizonts exhibiting a particularly pronounced increase. We agree that it would be interesting to determine whether similar changes occur at physiological fever-like temperatures, and whether this increase in stiffness coincides with the period of elevated protein trafficking. However, here we focused on enhanced protein export using multiple complementary approaches, and have chosen to address rigidity questions in a different study.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

As mentioned above, a second time point in many of the assays (for example, 36 hrs or later) would be useful to determine if heat stress simply accelerates trafficking of proteins to the RBC or if instead it results in an overall increase in trafficking.

As mentioned earlier: We did not assess cytoadhesion at later stages, but in the supplementary figures we show that at 40 hours post infection both heat stress and control conditions have comparable proportions of VAR2CSA-positive iRBCs. This is true for the DMSO (control wildtype resembling) HA-tagged lines of HSP70x and PF3D7_072500 (Supplementary Figures 9 and 12 respectively). The end level of VAR2CSA is the same in both conditions, but at 24 hours post infection it is higher following heat stress, indicating that trafficking is accelerated.

In the text, the authors frequently mention changes in the parasites' phenotype in response to heat stress; however, the way it is described is a bit ambiguous and can be confusing. For example, on page 3, they state that "Following heat stress, significantly more iRBCs (57.6% +/-19.4%) cytoadhered.....". From this sentence, it is not initially clear if the end result is cytoadherence of 57.6% of iRBCs or if this refers to an increase of 57.6%. This could be stated explicitly (e.g., "an increase of 57.6% +/- 19.4%") to avoid confusion. Similar descriptions of the results are found throughout the paper.

We agree this is confusing and altered the text accordingly.

The authors might consider citing and discussing the paper from Andrade et al (Nat Med, 2020, 26:1929-1940), which describes longer circulation times (less cytoadherence) by parasites in the dry season (asymptomatic patients) than in febrile patients in the wet season (stronger cytoadhesion of younger stages). This would seem to be consistent with the data presented here.

We are aware of the Andrade study, but chose not to cite it in this context since the reported differences in cytoadhesion appear more consistent with PfEMP1 expression levels, as hypothesized by the authors, than with altered trafficking.

Reviewer #2 (Recommendations for the authors):

General comments on the text:

(1) "Approximately 10% of the proteins encoded by P. falciparum are predicted to be exported beyond the parasite plasma membrane (PPM) into the parasitophorous vacuole lumen (PVL) and subsequently across the parasitophorous vacuole membrane (PVM) into the RBC cytosol."

To my knowledge, it has not been really demonstrated that all exported proteins take this route (transfer step in the PVL), and how transmembrane proteins transfer from the parasite to the erythrocyte is still poorly understood. I recommend that the authors rephrase this for precision.

We agree with this reviewer and will change the statement.

Changes:

We have clarified these statements to accurately reflect the current understanding of protein export. Approximately 10% of P. falciparum encoded proteins are predicted to be exported beyond the parasite plasma membrane, with many thought to pass through the parasitophorous vacuole lumen (PVL) and parasitophorous vacuole membrane (PVM) into the RBC cytosol, although the exact routes for transmembrane proteins are not fully understood.”

(2) "Charnaud et al. 25, but not Cobb et al. 26, found HSP70x to be essential for normal PfEMP1 trafficking, although both studies concluded that HSP70x is dispensable for intraerythrocytic parasite growth at 37 {degree sign}C."

The trafficking block in Charnaud is likely due to a delay in parasite development and cannot thus really be directly related to PfEMP1 trafficking.

Charnaud et al., report: “Microscopy of Giemsa stained IE indicated that ΔHsp70-x appeared similar to CS2 with no obvious abnormalities (Fig 2c). To more accurately quantify changes in maturation through the cell cycle, the DNA content of parasites stained with ethidium bromide was measured by flow cytometry (Fig 2d). This indicated that most parasites had the same DNA content at each timepoint and were maturing at the same rate.”

Thus, we cannot conclude that the trafficking phenotype reported in the Charnaud study can be attributed to a growth delay. This is also supported by only minor changes in the transcriptome, which would likely be more widely perturbed if there was a significant growth delay. However, we will change the statement “Charnaud et al., found HSP70x to be essential for normal PfEMP1 trafficking”, to ”…important for PfEMP1 trafficking” to more precisely reflect the data.

(3) "NanoLuciferase (NanoLuc) fusion proteins and compartment-specific isolation confirmed a greater abundance of PfEMP1 in the RBC cytosol following heat stress."

Please see my comments about the differentiation between soluble and TM-containing proteins. One would expect that PfEMP1 is membrane-integrated, and thus should not be found in the cytosol (implying a soluble form).

See our response above.

(4) "Importantly, heat stress did not accelerate parasite development through the asexual life cycle (Supplementary Figure 1)."

The authors should constrain this statement to the time frame in which the heat-shock was given. Previous publications have shown a speeded-up development only in younger-stage parasites, which the authors did not study.

We will re-phrase.

Changes:

We have rephrased the sentence to clarify the time window of heat stress: ”Importantly, heat stress between 16-24 hours post-invasion did not accelerate parasite development through the asexual life cycle (Supplementary Figure 1).” The supplementary figure title has also been updated to match.

(5) I recommend that the authors include line numbers. This makes the reviewers' lives much easier.

We agree and apologize for this oversight.

We now added line numbers.

Reviewer #3 (Recommendations for the authors):

(1) All the experiments have been performed to a very high standard, and I have no major questions about the results. However, the paper would go up to the next level if the effect of fever temperatures on the stiffness of the iRBCs had been investigated by measuring the passage of iRBCs through an artificial spleen where a bed of metal spheres mimics interendothelial splenic slits.

See our comment from above.

(2) With respect to Figures 5E, 6C, and 6E, why was there not a decrease in bioluminescence levels at 39 {degree sign}C for Sap and NP40 to match the increase in EqtII?

The assay is not performed as a sequence of permeabilisation steps. Instead, samples are split into three parallel treatments: one with EqtII, one with Saponin, and one with NP40. The protein measured in each case reflects the total released under that specific condition rather than being cumulative. Therefore, the NP40 fraction includes proteins from the Saponin-accessible compartment, the EqtII-accessible compartment, and the parasite cytosol.

(3) In the Supplementary gene maps, I could not read the white text on the black gene boxes.

We apologize: these have not converted well and will be altered with the revised version.

Changes

We have significantly increased the size of all fonts within the gene maps and improved the resolution of the figures to improve readability.

(4) In Figure S6, why does HSP70-x look different between parts C and D IFAs, with the latter showing much more export?

We agree these IFAs are not optimal and we will provide better images.

New Results:

Immunofluorescence microscopy, including the localisation of the two HA-tagged proteins (PF3D7_1039000 and PF3D7_0702500), has been repeated and higher-quality images are now included in the updated manuscript (Supplementary Figures 9 and 11). These figures now include multiple images of HA-tagged staining to more accurately represent the observed localisation and export patterns.

(5) Would the authors care to comment on what kinase might be additionally phosphorylating at 39 {degree sign}C?

We presume these are Maurer’s clefts FIKK kinases as most of the hyperphosphorylated proteins are MC residents. However, without directly testing for this using conditional KO parasite lines, we cannot exclude that host kinases are also playing a role.

(6) Could the additional assembly of PSAC at the iRBC membrane be important for survival at 39 {degree sign}C?

We have tested to see if nutrient uptake helps parasite survival during heat stress in the presence of furosemide and lower nutrient concentrations, but did not see a difference in growth following heat stress compared to control temperature conditions.

New Results:

We have added a new supplementary figure (Supplementary Figure 4) detailing experiments testing parasite growth under altered nutrient availability using two approaches (sub-lethal furosemide concentrations or reduced-nutrient RPMI) and with or without a 40°C heat stress applied between 16-24 hpi.

The main text now references this data: “Culturing parasites in sub-lethal furosemide concentrations or in reduced nutrient media lead to reduced parasitaemia (Supplementary Figure 4). However, the parasitaemia is not further reduced following heat stress. This shows that increased PSAC levels/activity do not enhance parasite survival under conditions of limited nutrient availability either from furosemide-induced nutrient deprivation or a reduced nutrient media composition.”

These experiments show that nutrient uptake does not improve parasite survival during heat stress compared to control temperature conditions.

(7) Would the authors like to speculate on how higher temperatures increase the transport of exported proteins with TMDs?

There are many possible explanations, one of which is that unfolding of the hydrophobic TMD domains is favoured at elevated temperatures. However, we have no data to support this hypothesis and therefore refrained from particularly stating this possibility.

eLife Assessment

Du et al. present a valuable study examining neural activation in medial prefrontal cortex (mPFC) subpopulations projecting to the basolateral amygdala (BLA) and nucleus accumbens (NAc) during behavioral tasks assessing anxiety, social preference, and social dominance. The strength of the evidence linking in vivo neural physiology to behavioral outcomes was considered solid; however, the electrophysiology data and their interpretation were less well received. Overall, the reviewers felt that the revised work provides insight into how distinct mPFC→BLA and mPFC→NAc pathways influence anxiety, exploration, and social behaviors.

Reviewer #1 (Public review):

Summary:

It is well known that neurons in the medial prefrontal cortex (mPFC) are involved in higher cognitive functions such as executive planning, motivational processing and internal state mediated decision-making. These internal states often correlate with the emotional states of the brain. While several studies point to the role of mPFC in regulating behavior based on such emotional states, the diversity of information processing in its sub-populations remains a less explored territory. In this study, the authors try to address this gap by identifying and characterizing some of these sub-populations in mice using a combination of projection-specific imaging, function-based tagging of neurons, multiple behavioral assays and ex-vivo patch clamp recordings.

Strengths:

The authors targeted mPFC projections to the nucleus accumbens (NAc) and basolateral amygdala (BLA). Using the open field task (OFT), the authors identified four relevant behavioral states as well as neurons active while the animal was in the center region ("center-ON neurons"). By characterizing single unit activity and using dimensionality reduction, the authors show differentiated coding of behavioral events at both the projection and functional levels. They further substantiate this effect by showing higher sensitivity of mPFC-BLA center-ON neurons during time spent in the open arms of the elevated plus maze (EPM). The authors then pivoted to the three-chamber social interaction (SI) assay to show the different subsets of neurons encode preference of social stimulus over non-social. This reveals an interesting diversity in the function of these sub-populations on multiple levels. Lastly, the authors used the tube test as a manipulation of the anxiety state of mice and compared behavioral differences before/after in the OFT and social interaction tasks. This experiment revealed that "losers" of the tube test spend less time in the center of the open field while "winners" show a stronger preference for the familiar mouse over the object. Using patch-clamp experiments, the authors also found that "winners" exhibit stronger synaptic transmission in the mPFC-NAc projection while "losers" exhibit stronger synaptic transmission in the mPFC-BLA projection. Given the popularity of the tube test assay in rank determination, this provides useful insights into possible effects on anxiety levels and synaptic plasticity. Overall, the many experiments performed by the authors reveal interesting differences in mPFC neurons relative to their involvement in high or low anxiety behaviors, social preference and social rank.

Weaknesses:

The authors focused primarily on female mice limiting generalizability and leaving the readers with questions about the impact of sex differences on their results. The tube test is used as a manipulation of the "emotional state" in several of the experiments. While the authors show the changes to corticosterone levels as a consequence of win/loss in the tube test, stronger claims might be made with comparisons to other gold standard stressors such as forced social defeat or social isolation.

Reviewer #2 (Public review):

Summary:

The goal of this proposal was to understand how two separate projection neurons from the medial prefrontal cortex, those innervating the basolateral amygdala (BLA) and nucleus accumbens (NAc), contribute to the encoding of emotional behaviors. The authors record the activity of these different neuron classes across three different behavioral environments. They propose that, although both populations are involved in emotional behavior, the two populations have diverging activity patterns in certain contexts. A subset of projections to the NAc appear particularly important for social behavior. They then attempt to link these changes to the emotional state of the animal and changes in synaptic connectivity.

Strengths:

The behavioral data builds on previous studies of these projection neurons supporting distinct roles in behavior and extend upon previous work by looking at the heterogeneity within different projection neurons across contexts, this is important to understand the "neural code" within the PFC that contributes to such behaviours and how it is relayed to other brain structures.

Weaknesses:

The diversity of neurons mediating these projections and their targeting within the BLA and NAc is not explored. These are not homogeneous structures and so one possibility is that some of the diversity within their findings may relate to targeting of different sub-structures within BLA or NAc or the diversity of projection neuron subtypes that mediate these pathways. This is an important future direction for this work but does not detract from the main finding as reported. The electrophysiological data in Figure 7 have significant experimental confounds that makes their interpretation challenging.

Author Response:

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

Public review:

Reviewer #1 (Public review):

Weaknesses:

The authors focused primarily on female mice without commenting on the effect that sex differences would have on their results.

We agree that sex is an important biological variable. Our experiments were performed primarily in female mice to align with the higher prevalence of affective disorders in females and to maintain consistency across experiments. We now explicitly acknowledge this as a limitation in the Discussion and note that future studies will be needed to determine whether the projection-specific coding principles identified here generalize to male animals. Relevant literature on sex-specific mPFC→BLA/NAc function has also been incorporated.

While the authors have identified relevant behavioral states across the various behavioral tasks, there is still a missing link between them and "emotional states" - the phrase used by them emphatically throughout the manuscript. The authors have neither provided adequate references to satisfy this gap nor shared any data pertaining to relevant readouts such as cortisol levels.

We appreciate the reviewer’s concern regarding the use of the term “emotional states.” In the revised manuscript, we have clarified our terminology and now use “behavioral states associated with affective valence” where appropriate. We have also added references supporting the use of open field center vs. corner occupancy, elevated plus maze performance, and social interaction assays as established proxies for anxiety-like and affect-related behaviors.

Importantly, to provide physiological support for these interpretations, we now include data showing that repeated win/loss outcomes in the tube test are associated with increased corticosterone levels in loser mice. These results indicate that the behavioral manipulations used in this study are accompanied by measurable physiological changes linked to stress-related processes.

Both the projection-specific recordings and patch-clamp experiments, including histology reports in the manuscript, would provide essential information for anyone trying to replicate the results, especially since it's known that sub-populations in the BLA and NAc can have vastly different functions.

We agree that detailed reporting of projection targeting is important for reproducibility. We have expanded the Methods and Results to more clearly describe viral targeting, recording locations, and histological verification of mPFC projections to the lateral BLA and NAc shell. We also now explicitly acknowledge the anatomical and cellular heterogeneity within these regions as a limitation and discuss this as an important direction for future work.

The population-level analysis in the manuscript requires more rigor to reduce bias and statistical controls for establishing the significance of their results.

We have strengthened the statistical analyses throughout the manuscript. Specifically, we have incorporated permutation-based controls for key analyses, clarified how behavioral and neural features were defined, and provided additional details on dimensionality reduction and clustering approaches. Exact p values, sample sizes, and statistical tests are now reported throughout the manuscript and figure legends.

Lastly, the tube test is used as a manipulation of the "emotional state" in several of the experiments. While the tube test can cause a temporary spike in anxiety of the participating mice, it is not known to produce a sustained effect - unless there are additional interventions such as forced social defeat. Thus, additional controls for these experiments are essential to support claims based on changes in the emotional state of mice.

We agree that the tube test is not a classical chronic stress paradigm such as social defeat. In our study, the tube test was used to establish social hierarchy rather than to model sustained stress. We have revised the manuscript to clarify this point and have tempered our language accordingly. At the same time, our corticosterone measurements indicate that repeated social competition induces measurable physiological changes, suggesting that the paradigm captures aspects of social hierarchy–related stress. We now frame these effects conservatively and acknowledge the need for future studies using additional stress paradigms.

Apart from the methodology, the manuscript could also be improved with the addition of clear scatter points in all the plots along with detailed measures of the statistical tests such as exact p values and size of groups being compared.

We have revised all figures to include individual data points (scatter overlays) wherever appropriate and have improved reporting of statistical details, including exact p values and group sizes, to enhance transparency and reproducibility.

Taken together, these revisions clarify our interpretations, improve methodological transparency, and strengthen the rigor of the analyses while preserving the main conclusions of the study.

Reviewer #2 (Public Review):

Weaknesses:

The diversity of neurons mediating these projections and their targeting within the BLA and NAc is not explored. These are not homogeneous structures and so one possibility is that some of the diversity within their findings may relate to targeting of different sub-structures within each region.

We agree that both the basolateral amygdala (BLA) and nucleus accumbens (NAc) are highly heterogeneous. Our study was designed to focus on projection-defined mPFC outputs (presynaptic activity) rather than resolving postsynaptic subregional or cell-type diversity. We have now:

- Clarified targeting strategies (PL→NAc shell and PL→BLA basal region)

- Added histological descriptions of injection and recording sites

- Expanded the Discussion to acknowledge how subregional and cellular heterogeneity may contribute to the observed variability

We also highlight this as an important direction for future work.

The electrophysiological data have significant experimental confounds and more methodological information is required to support other conclusions related to these data.

We have significantly strengthened the electrophysiological component by:

- Providing detailed recording conditions (access resistance, membrane properties, inclusion criteria)

- Clarifying stimulus protocols and normalization procedures

- Including representative traces and quantification of exclusion rates

- Addressing potential confounds such as viral expression variability and stimulation parameters

These revisions improve both interpretability and reproducibility of the electrophysiological findings.

Reviewer #3 (Public Review):

Major Weaknesses:

(1) The manuscript does not clearly and consistently specify the sex of the mice used for behavioral and imaging experiments. Given the known influence of sex on emotional behaviors and neural activity, this omission raises concerns about the generalizability of the findings. The authors should make clear throughout the manuscript whether male, female, or mixed-sex cohorts were used and provide a rationale for their choice. If only one sex was used, the potential limitations of this approach should be explicitly discussed.

We agree that sex is an important biological variable. We have now clearly specified throughout the manuscript that experiments were performed primarily in female mice and have added a rationale for this choice in the Methods. Briefly, we focused on females to align with the higher prevalence of affective disorders in females and to maintain consistency across experiments. We now explicitly acknowledge this as a limitation in the Discussion and note that future studies will be needed to determine whether these findings generalize to male animals.

(2) Mice lacking "center-ON" neurons were excluded from analysis, yet the manuscript draws broad conclusions about the encoding of emotional states by mPFC pathways. It is critical to justify this exclusion and discuss how it may limit the generalizability of the findings. The inclusion of data or contextualization for animals without center-ON neurons would strengthen the interpretation.

We thank the reviewer for raising this important point. Mice lacking identifiable center-ON neurons were excluded from analyses that specifically relied on this functional classification, as inclusion of such datasets would preclude meaningful comparison of this neuronal population. We have now clarified this criterion in the Methods and Results. Importantly, this exclusion does not affect analyses performed at the population level or those not dependent on center-ON classification. We now explicitly discuss this limitation and note that variability in the presence of center-ON neurons may reflect biological heterogeneity across animals.

(3) The manuscript lacks baseline activity comparisons for mPFC→BLA and mPFC→NAc pathways across subjects. Providing baseline data would contextualize the observed activity changes during behavior testing and help rule out inter-individual variability as a confounding factor.

We have added baseline comparisons of mPFC→BLA and mPFC→NAc activity across subjects to control for inter-individual variability and better contextualize behavior-related changes.

(4) Extensive behavioral testing across multiple paradigms may introduce stress and fatigue in the animals, which could confound the induction of emotional states. The authors should describe the measures taken to minimize these effects (e.g., recovery periods, randomized testing order) and discuss their potential impact on the results.

We now provide detailed descriptions of experimental design, including habituation, randomized testing order, and recovery periods between assays. We also discuss potential cumulative stress effects as a limitation.

(5) Grooming is described as a "non-anxiety" behavior, which conflicts with its established role as a stress-relieving behavior that may indicate anxiety. This discrepancy requires clarification, as the distinction is central to the conclusions about the mPFC→BLA pathway's role in differentiating anxiety-related and non-anxiety behaviors.

We thank the reviewer for this important clarification. We agree that grooming can be associated with both stress-related and self-soothing behaviors. In the revised manuscript, we have clarified that grooming is not strictly a “non-anxiety” behavior but instead represents a distinct behavioral state that may reflect stress regulation or internal state transitions. We have revised the text accordingly to avoid oversimplification and to better align with the literature.

(6) While the study highlights pathway-specific neural activity, it lacks a cohesive integration of these findings with the behavioral data. Quantifying the overlap or decorrelation of neuronal activity patterns across tasks would solidify claims about the specialization of mPFC→NAc and mPFC→BLA pathways. Likewise, the discussion should be expanded to place these findings in light of prior studies that have probed the roles of these pathways in social/emotion/valence-related behaviors.

We agree that stronger integration between neural and behavioral findings would strengthen the manuscript. In the revised version, we have added quantitative analyses examining the similarity and divergence of activity patterns across behavioral contexts (e.g., cross-context comparisons and correlation-based analyses). We have also expanded the Discussion to better integrate our findings with prior studies on mPFC→NAc and mPFC→BLA pathways in reward, aversion, and social behavior, thereby providing a more cohesive interpretation of pathway-specific functions.

Minor Weaknesses:

(1) The manuscript does not explicitly state whether the same mice were used across all behavioral assays. This information is critical for evaluating the validity of group comparisons. Additionally, more detail on sample sizes per assay would improve the manuscript's transparency.

(2) In Figure 2G, the difference between BLA and NAc activity during exploratory behaviors (sniffing) is difficult to discern. Adjusting the scale or reformatting the figure would better illustrate the findings.

(3) While the characteristics of the first social stimulus (M1) are specified, there is no information about the second social stimulus (M2). This omission makes it difficult to fully interpret the findings from the three-chamber test.

(4) The methods section lacks detailed information about statistical approaches and animal selection criteria. Explicitly outlining these procedures would improve reproducibility and clarity.

We have addressed all these minor concerns, including:

- Clarifying whether the same mice were used across assays

- Reporting sample sizes for each experiment

- Improving figure clarity (e.g., scaling, labeling, scatter points)

- Providing details for social stimuli (M1 vs. M2)

- Expanding statistical methods and animal selection criteria

Summary

In summary, we have made substantial revisions to:

- Improve conceptual precision (behavior vs. emotional state)

- Increase methodological transparency and statistical rigor

- Strengthen physiological validation

- Clarify experimental design and limitations

- Enhance integration with existing literature

We believe these revisions significantly improve the clarity, rigor, and interpretability of the manuscript, and we are grateful for the reviewers’ guidance in strengthening this work.

14:55 "agenda 2030 ... depopulation ... destroy old wise people ... destroy resistance through chemical warfare"

depopulation will kill the 95% useless eaters<br /> the remaining 5%... if anyone is stupid enough to fight "the evil system" then he will be shot at sight

The given link https://human-organ-atlas.fr/ is broken. Instead, the link should probably be https://human-organ-atlas.esrf.fr/

eLife Assessment

This is a detailed and well-designed simulation study of the utility of replication metrics in animal-to-human study translations in bridging the gap between laboratory discoveries and health practice, a critical consideration in turning laboratory scientific research findings into tangible, real-world applications, to directly help human health. The study approaches are solid, and the findings are important, as they offer insights into clinical research translations to advance health decision-making. There is some potential for the strength and applicability of the presented evidence to be improved upon revision.

Reviewer #1 (Public review):

A well-designed and preregistered simulation study investigating whether replication-success metrics can be applied to assess animal-to-human translation. The study is comprehensive, uses realistic parameter settings, and provides valuable insights into how different metrics behave under varied conditions.

Strengths:

(1) Methodologically rigorous and transparently preregistered.

(2) Comprehensive simulation design covering a wide range of plausible scenarios.

(3) Clear description of metrics and decision rules.

(4) Valuable contribution to understanding the limitations of applying replication metrics to translation questions.

Weaknesses:

(1) The conceptual distinction between replication and translation could be more clearly emphasized.

(2) Interpretation of results is dense and can be challenging to follow without a clear and summarized.

(3) Some simulation parameters (effect sizes, heterogeneity, and number of animal studies) require more substantial justification.

(4) Practical recommendations could be more explicit to guide applied researchers.

Reviewer #2 (Public review):

Summary:

The authors attempt to address the issue of high rates of translation failure from animal studies to humans in the literature, where promising results in animal studies fail when conducting human clinical trials. Using parameters from a previous meta-analysis on prenatal amino acid supplementation and the effects it has on maternal blood pressure, the authors assessed the performance of the metrics used and whether they can quantify translation success. Performing a simulation study, the authors compared nine translation success metrics and found that no one method was uniformly optimal. The authors list several limitations of the study, such as comparability of effect sizes between animal and human studies, different goals of animal studies versus human studies, and the focus of the study on one aspect (statistics of translation) is part of a broader, more complex decision-making process before proceeding to human trials. The authors recommend using multiple metrics in combination while taking into consideration their strengths and weaknesses to assess the translation of animal studies to human outcomes. The paper achieves the aim of providing a model with several metrics to evaluate translation success from animal studies to humans.

Strengths:

(1) Utilizing 9 different translation success metrics in combination provides strong flexibility in evaluating whether results in animal studies can translate to humans. This would allow researchers to evaluate translation success using multiple different metrics according to the context of the study.

(2) The authors accommodated for the limited sample size in animal studies, which are typically underpowered, and also caution that special attention should be given to heterogeneity when interpreting translation results.

(3) Overall, this approach has the potential to be applied to other biomedical studies, provided the limitations for each of the metrics are considered. It would provide a useful tool in assessing translation from animals to humans, in addition to other factors such as safety, pharmacokinetics, etc.

Weaknesses:

While the study has several strengths, there are some limitations.

(1) Preclinical animal study sizes tend to be much smaller than human studies, which results in underpowered results. The authors adjusted for this by pooling animal study data. However, high heterogeneity in the animal studies can affect translation results.

(2) The study focuses only on evaluating the statistical component of translation, which is only one aspect of the decision-making process to move on to human trials. The study does not take into account safety and toxicological profiles, pharmacokinetics, or genetics, which are important considerations that influence the overall effect in humans.

Reviewer #3 (Public review):

Summary:

This paper focused on how to navigate the complex decision-making process of whether to go into human trials. This is a critical topic considering the well-documented challenges in replicating and translating findings. While these are two distinct topics (i.e., replication and translation), they are related, and the authors simulated many conditions to assess the utility of replication assessment metrics.

Strengths:

A major strength of the study is the detailed approach to identifying relevant conditions and metrics, and to providing rich results that outline the strengths and weaknesses of each metric. Any simulation study is challenged by trying to identify the most relevant variables of interest, and this study provided sound justification for its chosen variables of interest. While this study does not make a strong recommendation (which I see as a strength), it does provide a comprehensive overview of the various metrics and conditions that were investigated.

Weaknesses:

The weaknesses of the study are the limited focus on specific metrics, the assumptions, particularly in the limited number of human study variables, and the less-than-ideal approachable summary of findings for a non-technical audience.

Conclusion:

This paper provides a much-needed investigation and discussion of how decisions are made when assessing whether to go into human trials. This is an important topic that productively challenges the status quo, considering documented challenges in replication and translation in biomedical research.

eLife Assessment

The study investigates how CD1c-restricted T cells respond to Mtb-infected APCs, leading to increased cytokine production and cytotoxic activity that may help control Mtb infection. While the work is important and will interest researchers in the field, the supporting evidence is incomplete and could be strengthened by additional experiments. Experiments would: (i) evaluate THP1-CD1c cells to determine whether MHC surface expression is reduced or entirely abolished, (ii) enhance confidence in the purity of the CD1c-specific T cell population isolated from blood, and (iii) suggest what additional signal THP1-CD1c cells treated with Mtb express that is absent from the untreated cells.

Reviewer #1 (Public review):

Summary:

T cells that recognize lipids - CD1c - are frequent in circulation; however, their role in infection is unclear. This study aims to understand how Mtb infection can shape the responses of CD1c-specific T cells. CD1c is expressed in MTB granuloma, but in lower amounts than in nearby inflamed tissue. Mtb infection downregulates the expression of CD1c on monocyte-derived DCs. Single-cell RNA sequencing revealed the cytotoxic program inherent to the lipid-CD1c-specific T cells. Using an in vitro APC system where CD1c expression remains intact upon Mtb infection, the authors suggest that these T cells react better to Mtb-infected than uninfected Cd1c-expressing APC and reduce Mtb burden in infected cells. Therefore, Cd1c downregulation could be an immune evasion strategy used by Mtb.

Strengths:

This study asks an important question. The single-cell transcription analysis suggests the inherent cytotoxic program of lipid-CD1c cells and provides insights into their phenotypic and potential functional profiles. Function experiments suggest that these autoreactive T cells can react to Mtb infection, adding to the paradigm of infection control by these non-conventional T cell populations.

Weaknesses:

The study lacks sufficient rigor; conclusions may be strengthened with the incorporation of more controls, and some deeper characterization of the THP1 system and the CD1c-specific T cells isolated from blood. Crucial conclusions are drawn from the cell mixing experiments involving the engineered THP-1 system and CD1c-lipid-specific T cells from blood. These cells need more in-depth characterization. The expression of MHC-I/II is clearly reduced in THP1-CD1c cells. However, it is important to ensure that it is completely abolished, since a residual expression can skew the result with activation of conventional T cells in the blood or low levels of conventional T cells that may be present in the CD1c-tetra/multimer sorted T cells. CD1c-tetra/multimer sorting should include more markers than used in this study.

Figure 2: The immunohistochemistry appears to be shown only for one biopsy; it may be worth quantifying the immunohistochemistry of all five. The expression of CD1 molecules goes up during the differentiation of MoDC. And Mtb infection prevents or dampens the upregulation. Does Mtb infection downregulate the CD1 expression of mature DCs? Can the effect of Mtb on the expression of CD1a,b,c molecules be investigated using CD1c-expressing DCs from blood? What could be the reason THP-1 cells do not downregulate CD1 molecules upon Mtb infection, and how about the expression of CD1a and b?

Figure 3: (F) What does the X-axis read for the no infection group? The value for MOI = 0 should be incorporated for the infected T cell group.

Figure 4: In the lysis assay, THP1-CD1c cells (uninfected and infected) incubated alone should be incorporated.

Figure 5: A quantitative brief on the single cell TCR sequencing - including how many T cells were sequenced and the frequency of different clone including EM1 and EM2 - should be shown.

Reviewer #2 (Public review):

Summary:

The study by Milton et al titled "Human CD1c-autoreactive T cells recognise Mycobacterium tuberculosis-infected antigen-presenting cells and display cytotoxic effector programmes" characterises CD1c-restricted autoreactive T cells and their potential role in controlling Mtb infection. The authors develop a well-controlled system to assay for the functioning/activation of autoreactive T cells. They report the presence of CD1c-restricted autoreactive T cells in the circulating blood of healthy donors. They show that these T cells respond to CD1c and get activated even in the absence of any exogenous antigen. They next show that CD1c, along with CD1a and b, are typically downregulated on APCs during Mtb infection. These autoreactive T cells are cytotoxic, indicating they respond to Mtb treatment and/or to changes in the T cell ratio. The autoreactive T cells could effectively lyse Mtb-infected or PAMP-stimulated CD1c+APCs. Next, using TCR sequencing, they show that T cell responses were mediated by specific TCR clones with common sequence features. They show that these autoreactive T cells could curtail Mtb growth as measured by luminescence. Finally, using scRNAseq, they selectively identify the CD1c-reactive T cell pool and detect enrichment of typical effector memory CD4 and CD8 cells expressing cytolytic markers such as Granzyme, granulolysin, etc. The lung biopsy staining, along with the other data presented here, suggests that while CD1c-restricted T cells could have potential anti-bacterial roles, Mtb downregulation effectively shuts down this mechanism for TB control.

Strengths:

The study is designed well and has developed many exciting tools to generate specific information.

Weaknesses:

The study has weaknesses in two important parameters - novelty and relevance in controlling TB. Further, the results could be better presented and discussed to allow easy understanding of the experimental design. For example, at several places, UV-killed or live Mtb were used. What is the rationale behind that? Why use irradiated THP1-CD1c cells for activating T cells?

While functional assays identified only CD4+ cells as CD1c-restricted, scRNAseq shows that both CD4+ and CD8+ cells exhibit this phenotype. Identifying the specific lipid antigen presented by CD1c could add greater value to the study.

Since autoreactivity was independent of exogenous antigen, the cytotoxic activity should also be independent of exogeneous antigens? What additional signal a THP1-CD1c cells treated with UV-killed Mtb express that is absent from the untreated cells?

The relative Mtb growth assay is confusing. CD1c cells with Mtb infection triggers massive lytic response, as shown in Figure 4. Under similar conditions, in Figure 6, the authors report a significant decline in Mtb growth in these cells. The problem is that with the kind of lytic response observed, a lot more Mtb could be present extracellularly and would evade killing. How do we reconcile the two observations?

Reviewer #3 (Public review):

Summary:

Despite the rising global prevalence of TB, the role of non-classical T-cell pathways in host immunity remains unclear. The present study by Milton et al. is a novel contribution to the field of unconventional T-cell immunity in Mtb infection. The study addresses the role of CD1c-autoreactive T-cells and demonstrates that upon Mtb infection, these cells are significantly activated, resulting in increased cytokine production and cytotoxicity, and a reduction in the bacterial burden, specifically against Mtb-infected CD1c+ APCs (antigen-presenting cells). This defines their role as a plausible candidate for lipid-directed immunity in TB, complementary to MHC-restricted responses.

Strengths:

The manuscript is well written, and the novelty, impact, and limitations of this study are precisely highlighted by the authors.

Weaknesses:

The authors mention that they did not identify any specific lipids presented by CD1c on Mtb-infected APCs, making it unclear whether they are of host or bacterial origin. This leaves a gap in understanding why the response is enhanced in Mtb-infected cells, whether it is through altered self-presentation of lipids arising from Mtb-induced changes, infection-induced stress signals, or Mtb lipids, or through CD1c-dependent co-stimulation/infection signals. Direct lipid identification via lipidomics/MS of CD1c-bound lipids from Mtb-infected APCs would clarify whether the enhancement arises from altered self-lipids or subtle Mtb lipids.

Picking Return Period Thresholds is the most reliable way to label river discharge at certain date for a certain location as Flood.

The 'following file' should be called compose.yaml

progressinve co-evolutionary mutual arising permanent content with meaningful organization and the possibility of

re - use - purpose - contextualize - conpleualize

toDo

add lbilink to WikiNizer Paper information wants to be reorganized

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Reply to the reviewers

We provide current Revision Plan and will generate full point-by-point responses upon final resubmission.

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Referee #2

Evidence, reproducibility and clarity

Quantitative interactome mapping of skeletal muscle insulin resistance Ng et al present a series of proteomics/interactomics studies in skeletal muscle to identity insulin regulated complexes/interactions and changes ot these in insulin resistant muscle. More mechanistically, the Authors focus on changes in interactions involving chaperones in the ER/SR, presenting interesting data on the effect of PDIA6 overexpression alters insulin sensitivity in muscle ex vivo.

Major Comments:

The section entitled "Validating the regulation of PPIs with insulin resistance in C2C12 myotubes with quantitative XL-MS". This is not really a validation of th previous data as presented, but more an orthologous assay that helped pinpoint the interest in the ER. Suggest adjusting the title.

Figure 3B - the "decrease" in AS160 pS588 regulation appears to be due to increased basal, not decreased phosphorylation in after insulin. This should be commented on or clarified.

PDIA6 is down-regulated in muscle from people with T2D - so why did the authors decide to overexpress PDIA6? I note this rationale is explained in the discussion, and could be articulated better in the results.

Figure 5J and K. The TA muscles are substantially larger from PDIA6 OE mice. Are the muscle fibres also larger? Tbhis relates to the normalisation of data in K. This appears to be normalised to g tissue. If so, is the difference between control, and OE mice being driven by the increase in muscle mass - with uptake per muscle or per fibre the same?

Minor Comments:

For the PCP-MS data form C2C12 cells. The authors use an analysis of AUC to assess protein abundance, which, as they state, is important for chronic treatments if total protein is not separately quantified. However, the analysis of changes in protein distribution is less clear from the text in the results section. Intuitively, a profile that is normalised to total intensity in all fractions would provide a protein abundance-independent read-out for changes in protein distribution. Does the "local analysis" capture this same information? Could the Authors provide a little more information here?

Figure 1M - are the Authors sure that VPS41 should be in this panel. It doesn't seem to be insulin regulated, and the arrow appears to refer to movement between insulin sensitive and insulin resistant.

Figure 1N - "This includes an array of TBC1 domain-containing proteins (TBC1D15, 195 TBC1D17, TBC1D8B) that are consistently reduced with IR". Do the Authors mean the abundance was less, or that complex formation was reduced?

Optional. In general, there is a lot of text discussing the literature around proteins highlighted in the analysis. This is useful to an extent, but the Authors might consider streamlining this a little (perhaps moving some of the information ot supp tables?).

Why do the Authors think the crosslinking MS was not able to capture acute PPI changes like the PCP-MS was?

For the EDL crosslinking data. Are the Authors able to provide a comparison with C2C12 data - to highlight the differences and similarities between tissue and the cell model? This may be a challenge if the authors think most differences may be technical.

Please check - "reduces free-glycerol levels essential for fatty acid synthesis". Glycerol does not directly contribute to FA synthesis. But is needed for triglyceride synthesis.

Do the Authors think that the change in PDIA6 interactions may be a general/indirect indication of changes in ER redox and/or protein misfolding in insulin resistance?

Is PDIA6 an ER luminal protein? If so, it being phosphorylated is interesting.

Referees cross-commenting

Similarly, reviewer #1 raises important points on the description of key parts of the analysis, that will need to be addressed. I think we agree that the manuscript emcpmpasses a great deal of data, and that it is somewhat difficult to follow why PDIA6 was selected for validation. Overall, the reviews pick up on different aspects of the manuscript that could be improved.

Significance

Overall, the strength of the paper is in the underlaying proteomics workflows and analysis. The work presented of very high technical quality, and I have no doubt the data presented will be of use to the field beyond the analysis in this current publication.

However, a weakness is doubts over the relevance of the data on PDIA6 overexpression in muscle insulin resistance.

This will be of interest to those in the proteomics, interactomics and metabolism fields.

My expertise is in glucose metabolism, insulin signalling and insulin resistance.

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Referee #1

Evidence, reproducibility and clarity

Summary

Ng et al. use a combination of quantitative (structural) proteomics tools to study the effect of insulin resistance (IR) on protein interactions in mouse muscle cells (C2C12 myotubes) and muscle tissue. First, the authors use protein correlation profiling (PCP; co-fractionation) to separate protein complexes from C2C12 cells with and without induced insulin resistance. The PCP data is then used to reconstruct protein complexes and networks based on binary interactions and to uncover changes upon IR, this is illustrated by several examples (Fig. 1). As an orthogonal method quantitative cross-linking (XL) is used to study protein interactions in C2C12 cells. A multidimensional enrichment/fractionation scheme is designed to increase the number of cross-links/protein-protein interactions (PPIs), and again interaction networks are generated and changes between no IR vs. IR conditions assessed (Fig. 2). The XL approach is then carried forth into a mouse model of IR. Muscle tissue of mice fed on regular chow or on a high-fat diet are compared (Fig. 3). Selected XL data is validated on known structures and quantitative data from PCP and XL is compared to integrate the different interactome data (Fig. 4). Finally, the interactions of PDIA6, a key protein found to be affected by IR, are studied in more detail in the mouse model. The effect of overexpression of PDIA6 is studied using different readouts, including redox proteomics (Fig. 5). These data connect the redox imbalance / cysteine oxidation with the role of PDIA6 in insulin resistance.

Overall, the manuscript is impressive with respect to the methodological effort undertaken, generating and combining many large-scale proteomics data sets, both from a cell line and from mouse tissue. The large amount of data accumulated can be seen as a strength and as a weakness, because it is impossible to follow up on all findings (changes of interactions induced by IR observed in any sample and with any method). Nevertheless, the PDIA6 example was evaluated in more detail and with dedicated follow-up experiments. The conclusions from this experiment are plausible and presented logically. It is, however, difficult for a reviewer to quickly judge whether there would have been more promising leads for validation experiments than PDIA6.

Given the large amount of data already generated, I do not have suggestions for additional experiments. Instead, some analysis and interpretation of existing data need further clarification.

Major comments

The search strategy and statistical treatment of the cross-linking data need to be explained more clearly. The authors write that the data were searched with pLink2 and the FDR was controlled at 1%. However, it remains unclear whether the FDR was controlled at the level of peptide/cross-link-spectral-matches (PSMs/CSMs), or at the level of non-redundant site pairs or even protein-protein interactions for inter-protein links, which would be more appropriate. Controlling the FDR only at the PSM/CSM level will lead to an inflation of false positives when aggregating results at the interaction level. Moreover, it remains unclear whether all data sets were searched together or whether individual data sets or subsets of the entire data were searched individually. For example, if single files or only files belonging to a certain higher-order fraction would be searched individually, this would again lead to an underestimation of false positives. What is particularly noteworthy in this context is that in a typical dataset inter-protein links are underrepresented compared to intra-protein links for statistical reasons, while the numbers in this manuscript are much more balanced (almost equal numbers in the cell line data set, and even more inter-protein links than intra-protein links in the tissue). The authors should consider articles that discuss FDR control in XL-MS, for example by the Rappsilber group. Minimally, more details about what was searched together and at what level the FDR was controlled need to be provided.

On a similar note, it has been discussed in the literature that validating large-scale XL data on selected structures of complexes is a poor proxy for accurate FDR control, as such complexes are commonly not representative for more transient or substoichiometric PPIs, or PPIs involving low-abundant proteins.

Minor comments

Figure 1: CCT is a complex composed of eight subunits, but only seven are shown. What happened to the remaining one (CCT6)?

The authors performed a redox proteomics experiment in a PDIA6 overexpression system. However, the statement in the Conclusion section that PDIA6 overexpression promotes disulfide bond formation in interacting proteins is not directly justified because the method only quantifies cysteine oxidation, not S-S bond formation directly.

All supplementary data is not provided in an independent repository, but in a repository of the authors' institution. It is unclear whether the data could be accessed anonymously. Proteomics data need to be provided in an independent, community accepted repository such as from the members of the proteomeXchange consortium (PRIDE etc.).

A clear description of what is shown in the SI tables is missing, e.g. in the form of figure legends. In their present form, SI data are difficult to interpret. For example, I did not find information about cross-link identifications, only quantitative data on cross-link changes. However, if the identification of a cross-link is not confident in the first place (see my comments above), then the quantification will be irrelevant.

Referees cross-commenting

I trust the expertise of reviewer #2 on matters related to insulin resistance. It seems that we both agree that the PDIA6 example might require a more consistent justification throughout the manuscript.

Significance

The study is one of only a few so far that combines PCP and XL on such a large scale for a mammalian system. There are also very few studies of cross-linking on tissue. Therefore, from a methodological point of view, the study is highly innovative. The application to the muscle cell system and insulin resistance as a biological research question is furthermore very novel. As such, the study is valuable to different communities - those developing and refining experimental methods and those using them to uncover regulatory mechanisms. Another strength is that the authors made serious efforts at each step to optimize the XL method and adapt it to their sample types of interest.

The wealth of data is both a strength and a weakness of the work. Inevitably, a reviewer might argue that some aspects of the work could have been done differently. Unless someone spends a lot of time going deep into the result tables, it will be difficult to make constructive suggestions on additional targets for further investigation. Nevertheless, some statistical aspects of data analysis need to be clarified, and parts of the data analysis might need to be repeated. This, in turn, may require some reinterpretation of findings related to the XL data.

Advances: Conceptual, methodological, mechanistic

Audience: Specialized, basic research, translational

Reviewer expertise

My background is in proteomics, structural proteomics, mass spectrometry, analytical sciences, experimental methodology, and computational data analysis. I have general knowledge of biological processes, but I am not an expert on insulin resistance.

R warning visible

"to identify VHF tags and record detection data"

Current wording reads as if the receiver is directly storing the VHF signal, rather than converting it into detection data

"respectively" usually comes at the end of the sentence:

"with eight and three stations respectively"

温度与缺陷生成明确相关，这很可能源于高温下调节剂与无机金属-氧簇之间 Zr-O 键的热诱导不稳定性。因此，较高温度对应较少缺陷，从而促进调节剂封端配体从无机簇上脱离。随后，对苯二甲酸（BDC）配体可与 Zr 节点配位，导致结构缺陷减少 42.C. S. Cox , E. Slavich , L. K. Macreadie , L. K. McKemmish and M. Lessio , Understanding the Role of Synthetic Parameters in the Defect Engineering of UiO-66: A Review and Meta-analysis, Chem. Mater., 2023, 35 , 3057 —3072 43。X. Qiu and R. Wang , From construction strategies to applications: Multifunctional defective metal-organic frameworks, Coord. Chem. Rev., 2025, 526 , 216356

这反映了不同竞争物种在缺陷调控中的协同作用，然而，竞争物种间的协同机制仍不明确。 K. Tan , H. Pandey , H. Wang , E. Velasco , K.-Y. Wang , H.-C. Zhou , J. Li and T. Thonhauser , Defect Termination in the UiO-66 Family of Metal–Organic Frameworks: The Role of Water and Modulator, J. Am. Chem. Soc., 2021, 143 , 6328 —6332

双组分调节 M. A. Artsiusheuski , N. P. M. Casati , A. H. Clark , M. Nachtegaal , R. Verel , J. A. van Bokhoven and V. L. Sushkevich , Controlling the Mechanism of Nucleation and Growth Enables Synthesis of UiO-66 Metal–Organic Framework with Desired Macroscopic Properties, Angew. Chem., Int. Ed., 2024, e202415919 40.X. Chen , Y. Lyu , Z. Wang , X. Qiao , B. C. Gates and D. Yang , Tuning Zr12O22 Node Defects as Catalytic Sites in the Metal–Organic Framework hcp UiO-66, ACS Catal., 2020, 10 , 2906 —2914

单组分策略 X. Feng , H. S. Jena , C. Krishnaraj , D. Arenas-Esteban , K. Leus , G. Wang , J. Sun , M. Rüscher , J. Timoshenko , B. Roldan Cuenya , S. Bals and P. V. D. Voort , Creation of Exclusive Artificial Cluster Defects by Selective Metal Removal in the (Zn, Zr) Mixed-Metal UiO-66, J. Am. Chem. Soc., 2021, 143 , 21511 —21518 CrossRef CAS PubMed . X. Feng、H. S. Jena、C. Krishnaraj、D. Arenas-Esteban、K. Leus、G. Wang、J. Sun、M. Rüscher、J. Timoshenko、B. Roldan Cuenya、S. Bals 和 P. V. D. Voort，通过选择性金属移除在 (Zn, Zr) 混合金属 UiO-66 中创建专属人工簇缺陷，《J. Am. Chem. Soc.》，2021 年，第 143 卷，第 21511—21518 页 CrossRef CAS PubMed 。Journal of The American Chemical Society化学TOPESI学科分类：化学JCI 2.64IF(5) 15.6EI检索SCI升级版 化学1区SCI基础版 化学1区SCI Q1IF 15.7 S. Yuan , J. Peng , Y. Zhang and Y. Shao-Horn , Stability Trend of Metal–Organic Frameworks with Heterometal-Modified Hexanuclear Zr Building Units, J. Phys. Chem. C, 2019, 123 , 28266 —28274 CrossRef CAS . S. Yuan, J. Peng, Y. Zhang 和 Y. Shao-Horn, 异金属修饰六核锆构筑单元对金属有机框架稳定性的影响趋势, J. Phys. Chem. C, 2019, 123, 28266—28274 CrossRef CAS 。Journal of Physical Chemistry cESI学科分类：化学JCI 0.51IF(5) 3.5EI检索SCI升级版 化学3区SCI基础版 化学2区SCI Q3IF 3.2 Y. Ma , A. Li and C. Wang , Experimental study on adsorption removal of SO2 in flue gas by defective UiO-66, Chem. Eng. J., 2023, 455 , 140687 CrossRef CAS . Y. Ma、A. Li 和 C. Wang，缺陷型 UiO-66 吸附去除烟气中 SO2 的实验研究，Chem. Eng. J.，2023，455，140687 CrossRef CAS 。

S. Zhuang and J. Wang , Adsorptive removal of pharmaceutical pollutants by defective metal organic framework UiO-66: Insight into the contribution of defects, Chemosphere, 2021, 281 , 130997 33.X. Feng , H. S. Jena , C. Krishnaraj , K. Leus , G. Wang , H. Chen , C. Jia and P. Van Der Voort , Generating Catalytic Sites in UiO-66 through Defect Engineering, ACS Appl. Mater. Interfaces, 2021, 13 , 60715 —60735

26.L. Zhou , N. Li , G. Owens and Z. Chen , Simultaneous removal of mixed contaminants, copper and norfloxacin, from aqueous solution by ZIF-8, Chem. Eng. J., 2019, 362 , 628 —637 27.G. Chaturvedi , A. Kaur , A. Umar , M. A. Khan , H. Algarni and S. K. Kansal , Removal of fluoroquinolone drug, levofloxacin, from aqueous phase over iron based MOFs, MIL-100(Fe), J. Solid State Chem., 2020, 281 , 121029 28.R. Yu and Z. Wu , High adsorption for ofloxacin and reusability by the use of ZIF-8 for wastewater treatment, Microporous Mesoporous Mater., 2020, 308 , 110494 29.X. Fang , S. B. Wu , Y. H. Wu , W. Yang , Y. L. Li , J. Y. He , P. D. Hong , M. X. Nie , C. Xie , Z. J. Wu , K. S. Zhang , L. T. Kong and J. H. Liu , High-efficiency adsorption of norfloxacin using octahedral UIO-66-NH2 nanomaterials: Dynamics, thermodynamics, and mechanisms, Appl. Surf. Sci., 2020, 518 , 146226 30.H. Wang , X. Pei , M. J. Kalmutzki , J. Yang and O. M. Yaghi , Large Cages of Zeolitic Imidazolate Frameworks, Acc. Chem. Res., 2022, 55 , 707 —721

This comment is for the graph (couldn't select / comment on directly)

I think "Nb" is unclear for number, I've not personally seen the abbreviation used before - maybe "No." or something like "Good Records (N)" or "Good Records (Count)" would be clearer

Edit: This makes sense, I've just learned the French word for number!

This heading isn't very informative (I didn't know what was going to be in it until I read the section)

If it stays as 'Add Key Variables' adding subheadings could work (e.g. "Tide Data Processing", "Add to Dataset")

(This is very pedantic / not important if it's just us reading the site)

Technically is band number rather than ID

As above, could be simplified to "Band ID's are available in the Shorebird Tracker spreadsheet"

This is confusing / not informative - I think a very brief description or statement of the file would make sense

e.g. (see Shorebird Tracker spreadsheet)

unclear

(sorry for no actual suggested improvement; for the comments where I just say "unclear" it's where I am not 100% sure the meaning you're trying to convey)

Needs grammar fix - "Avoids to use high memory" is unclear

Maybe something like

"motus::dbConnect() links your .sql to your environment as a lazy table, preventing the entire database from being written straight to hardware memory."

This could maybe come earlier in the caption. Before reading the entire caption I didn't understand that the values for Total of days was a mean across the species.

Actually I just realised what I think is confusing me - "First day" and "Last day" are properties of the species, but "Total of days" and the other columns are average properties of individual birds.

Updated column / variable names could work, something like - First day (species) - Last day (species) - Active days

'Total' is a bit ambiguous, what do you think of 'Active'? Or something like "Tracking period" or "Monitoring period"? idk - Detected days (this one is clear) - Sites per day - Unique sites

(again trying to replace the word "total")

The term 'conditions' is a bit confusing here. When I first read "detection conditions", it sounded like conditions that impact whether a bird is detected or not, but it's really the conditions that the birds were detected in (I'm struggling to word this well too 😅).

Suggested alternatives - (about the) detection context (of each species) - (about the) tidal-diel conditions that each species is detected for

Or using plain language - about how species are detected across high and low tides, and day and night

I find it very Interesting that writing essays manually is linked to developing long-term career skills. In my cybersecurity studies, logic and analysis are everything. If I let AI do the thinking for me now, I might lose the ability to solve complex security problems later in my professional life. Learning is about the process, not just the final grade

The fact that AI 'hallucinates' and provides false information is very Troubling. This is especially dangerous for students who might trust these tools without verifying the facts. In technology, accuracy is vital, and seeing that major companies released these tools without enough public warning about their flaws is a serious concern for academic integrity and safety.

This source is largely informative and unbiased but this quote stuck out to me because I interpreted it to be slightly persuasive. The author points out that Florida is in the minority in terms of felon voting policies. To me this reads as if the author is encouraging Florida to adopt laws that already exist in other states. Although they do not explicitly attempt to sway our opinions, the framing of the facts may still have a big impact on how you interpret the voting policies in Florida and proposed changes.

Ballotpedia is produced by a nonprofit organization that specializes in neutral, encyclopedic coverage of American politics, elections, and ballot measures. Therefore, the “author” here is an institutional reference source and not a single named expert. Florida Amendment 4 page includes official election results, constitutional text, campaign finance disclosure, quotes from supporters and opponents, and separate sections for “Support”, “Opposition” and “Arguments” helps to limit overt bias while including content that is undeniably political in nature. Although the article is not a peer-reviewed scholarly article, it cites primary state documents and links to original sources, making it useful for fact-checking what Amendment 4 did and who supported it. I would select this source if I want a short factual overview of the measure and implementation history (including SB 7066 and litigation) before moving on to more analytical or academic treatments of felony disenfranchisement.

This source is clearly biased towards reenfranchisement of voting rights, although this bias does not prevent if from fairly analyzing the legal issues regarding voting rights in the US. It uses philosophical reasonings to support the idea that all citizens should have the ability to vote AND logical reasoning as well (benefits of increased voter turnout). The source explores reenfranchisement of different groups as well, expanding what we might expect to hear when discusing reenfranchisement.

(Solution) training + bilingual staff improve care quality

(Gap) not enough research on what CLR programs actually exist

language differences affect trust + safety in care

(Problem) patients may lose language ability or not share staff language

language barriers reduce quality of care

Evidence: many older adults don’t speak the main country language

scoping review used to organize research, not test results

There youth represent the Dine values and have characteristics taught to them from the Dine language.

This is the core foundation of what the US stands for and how it operates .

This represents the effects on Indigenous education and how it was more forced upon than it was converging.

How do social workers grapple with this possibility of NO improvement in well-being?

Urgent global intervention is required to fight this massive social injustice!

Training

List

Nicomachean Ethics

Definition

Theater is alive and always fluctuating and morphing

Dramaturgy is used to achieve these three things

Plato - on the dude who ONLY knows homer

this cannot be emphasised enough for reflexive learning and ethical approaches to improving well-being.

how training should look like, reflexive, context-informed supervision

we shouldn't do this as this is 'power over' rather than 'power with'.

the distant observer and the foreign social / humanitarian worker? Understanding leads to ethical and context sensitive support grounded in local agency and rights.

A solution model grounded in reality

conceptual tools for use by social workers during interventions and practise.

highlights the resilience and resistance to ongoing injustice.

We avoid pathologizing as social workers so liberation psychology and decolonial mental health frameworks should be used in the Palestinian context to improve well-being. Other papers also confirm the difficulty of data collection.

Résumé de la vidéo [00:00:00][^1^][1] - [00:22:20][^2^][2]:

Cette vidéo présente une matinale dédiée à la vie affective et sexuelle des jeunes, avec un accent sur la prévention des risques et la promotion de pratiques positives. Delphine Soulignac, référente régionale pour la thématique sexuelle, introduit l'événement et les intervenants, qui partageront leurs connaissances et expériences dans le domaine de la santé sexuelle des jeunes.

Points forts: + [00:00:05][^3^][3] Introduction de la matinale * Présentation par Delphine Soulignac * Focus sur la vie affective et sexuelle des jeunes * Importance de la prévention des risques + [00:01:17][^4^][4] Présentation des intervenants * Philippe Martin, docteur en santé publique * Bénédicte Allard et Pascal Faget de l'Université de Toulouse * Ingrid Desla, infirmière conseillère technique + [00:07:02][^5^][5] Cycle santé des jeunes * Discussion sur la santé sexuelle des jeunes * Événements et formations à venir * Importance de l'éducation à la sexualité + [00:11:37][^6^][6] Projet Sexpert * Présentation par Philippe Martin * Recherche sur la santé sexuelle et reproductive des jeunes * Utilisation des communautés en ligne pour l'éducation à la sexualité Résumé de la vidéo [00:22:22][^1^][1] - [00:46:38][^2^][2] : La vidéo aborde les perspectives des jeunes sur l'éducation sexuelle et la santé sexuelle en France. Elle met en lumière leurs expériences, leurs besoins en matière d'information et la manière dont ils naviguent dans les ressources disponibles. Les jeunes expriment le besoin d'une approche plus holistique et moins alarmiste, qui tienne compte des aspects émotionnels et relationnels de la sexualité, plutôt que de se concentrer uniquement sur les aspects scientifiques.

Points forts : + [00:22:22][^3^][3] Contextes de santé sexuelle des jeunes * Difficultés de la jeunesse * Besoin d'aide pour les tâches de la vie adulte * Attentes en matière d'éducation sexuelle + [00:26:03][^4^][4] Utilisation d'Internet et des réseaux sociaux * Accès quotidien à Internet * Compartimentation des sphères sociales * Recherche d'intimité et d'informations sur la sexualité + [00:29:36][^5^][5] Stratégies des jeunes face à la santé sexuelle * Diversité des questionnements et pressions * Importance de la communication et de l'identité sexuelle * Expériences variées avec l'éducation sexuelle + [00:32:00][^6^][6] Propositions pour l'éducation sexuelle en ligne * Besoin de fonctionnalités accessibles et ludiques * Espace sécurisé et anonyme pour les discussions * Interaction avec des professionnels spécialisés + [00:37:02][^7^][7] Contenus éducatifs créés par et pour les jeunes * Importance de la confiance et de la participation des jeunes * Aborder des sujets émotionnels et relationnels * Déconstruction des normes de genre et communication sur le plaisir + [00:41:27][^8^][8] Développement futur de l'éducation sexuelle numérique * Propositions concrètes des jeunes pour des actions en ligne * Importance de contenus valides et crédibles * Réflexions sur l'implication des jeunes et la personnalisation des actions Résumé de la vidéo [00:46:41]¹[1] - [01:08:27]²[2]:

La vidéo présente une discussion sur les initiatives de santé sexuelle et reproductive menées par des professionnels de la santé en France. Elle met en lumière l'importance de l'éducation et de la sensibilisation à la santé sexuelle, notamment à travers des consultations de sexologie, des interventions éducatives et des ressources comme des podcasts et des outils interactifs.

Points forts: + [00:46:41]³[3] Présentation du projet seexpert * Recherche sur la santé sexuelle * Liens entre santé sexuelle et mentale * Importance de l'éducation et de la sensibilisation + [00:47:49]⁴[4] Actions du Sims * Promotion de la santé au sein des universités * Consultations de sexologie disponibles * Interventions éducatives pour les étudiants + [00:51:26]⁵[5] Outils et ressources * Utilisation de modèles anatomiques pour l'éducation * Livres et manuels disponibles pour les étudiants * Podcasts sur divers sujets de santé sexuelle + [00:55:34]⁶[6] Vidéo sur le consentement * Importance du consentement dans les relations * Métaphore de la tasse de thé pour expliquer le concept * Diffusion systématique lors des rentrées universitaires + [01:03:19]⁷[7] Partage des compétences * Formation des collègues sur la santé sexuelle * Utilisation d'outils pédagogiques pour faciliter le dialogue * Sensibilisation à l'importance de discuter de la sexualité + [01:07:55]⁸[8] La licorne du genre * Outil pour lutter contre l'homophobie et la transphobie * Explication des concepts de sexe assigné et d'identité de genre * Importance de l'éducation pour le respect de tous les genres

Source : conversation avec Bing, 14/03/2024 (1) undefined. https://www.planning-familial.org/sites/default/files/2023-11/LIVRE_BLANC_WEB.pdf. (2) undefined. https://www.education.gouv.fr/education-la-sexualite-en-milieu-scolaire-341103. (3) undefined. https://soseducation.org/docs/notes-etudes-entretiens-tribunes/education-a-la-sexualite-danger-ou-prevention-final.pdf. (4) undefined. https://www. Résumé de la vidéo [01:08:28][^1^][1] - [01:32:31][^2^][2] : La vidéo aborde la sensibilisation à la diversité des identités et orientations sexuelles, l'importance de l'éducation à la sexualité dans les écoles, et les stratégies de promotion de la santé selon la Charte d'Ottawa. Elle met en lumière les outils pédagogiques utilisés pour expliquer les concepts de genre et d'attirance, ainsi que les formations destinées au personnel éducatif pour aborder ces sujets avec bienveillance et compétence.

Points forts : + [01:08:28][^3^][3] Diversité des identités et orientations sexuelles * Utilisation de métaphores pour expliquer le genre et l'orientation * Importance de la représentation et de l'acceptation de toutes les identités * Exemples d'outils pédagogiques comme les licornes pour illustrer ces concepts + [01:09:41][^4^][4] Éducation à la sexualité dans les écoles * Présentation des défis et de la nécessité de l'éducation à la sexualité * Discussion sur les attitudes homophobes et la nécessité de les combattre * Importance de l'intervention éducative pour ouvrir des discussions sur la sexualité + [01:10:00][^5^][5] Promotion de la santé selon la Charte d'Ottawa * Explication de la stratégie de promotion de la santé en prévention primaire, secondaire et tertiaire * Mise en place d'actions ancrées dans les textes de stratégie nationale et régionale * Importance de la réorientation des services de santé vers la prévention et la promotion de la santé + [01:12:46][^6^][6] Formation du personnel éducatif * Détails sur le programme de formation pour le personnel de l'Éducation nationale * Discussion sur l'importance de former divers profils professionnels * Objectif de créer un climat de confiance et de respecter la diversité des élèves Résumé de la vidéo [01:32:35][^1^][1] - [01:54:41][^2^][2]:

Cette vidéo discute de l'engagement des jeunes dans une communauté en ligne pour améliorer leurs connaissances et comportements, notamment à travers une plateforme ouverte à tous. Elle aborde également l'importance de la recherche-action impliquant les jeunes, produisant des données sur leur vie affective et sexuelle, et évaluant les actions éducatives actuelles.

Points forts: + [01:32:35][^3^][3] Communauté en ligne et plateforme éducative * Ouverte à tous les jeunes * Vise à être pérenne et inclusive * Implique les jeunes dans la recherche-action + [01:33:51][^4^][4] Étude sur les séries Netflix et la sexualité des jeunes * Analyse des messages éducatifs et de promotion de la santé sexuelle * Études en cours sur les perceptions des jeunes * Différences notables entre les séries comme "Sex Education" et "Elite" + [01:35:41][^5^][5] Accès aux ressources et outils éducatifs * Disponibilité équivalente pour tous les étudiants * Importance de la communication et de la sensibilisation * Utilisation de divers canaux pour informer les étudiants + [01:39:10][^6^][6] Formation des personnels éducatifs * Formations volontaires sur l'éducation à la sexualité * Limitations budgétaires affectant la disponibilité des formations * Importance de l'éducation à la sexualité pour le bien-être des élèves Résumé de la vidéo [01:54:43]¹[1] - [01:56:36]²[2]:

Cette partie de la vidéo aborde l'éducation à la sexualité dans le système éducatif, soulignant son importance dans la protection et l'éducation des jeunes. Elle met en lumière la nécessité d'aborder divers aspects tels que la prévention des violences, le consentement, l'expression des émotions et le développement des compétences psychosociales.

Points forts: + [01:54:43]³[3] L'importance de l'éducation à la sexualité * Protège et éduque les jeunes * Aborde la prévention des violences et le consentement * Encourage l'expression et la gestion des émotions + [01:55:38]⁴[4] Les stratégies pour gérer les oppositions * Respecte les limites individuelles * S'inscrit dans un cadre réglementaire rassurant * Intègre l'éducation à la sexualité dans le cursus global + [01:56:00]⁵[5] La conclusion de la présentation * Remerciements pour l'expertise et la bienveillance * Mention de la contribution des participants * Engagement à continuer le développement des ressources éducatives

Source : conversation avec Bing, 14/03/2024 (1) undefined. https://www.planning-familial.org/sites/default/files/2023-11/LIVRE_BLANC_WEB.pdf. (2) undefined. https://www.education.gouv.fr/education-la-sexualite-en-milieu-scolaire-341103. (3) undefined. https://soseducation.org/docs/notes-etudes-entretiens-tribunes/education-a-la-sexualite-danger-ou-prevention-final.pdf. (4) undefined. https://www.

Résumé de la vidéo [00:00:00][^1^][1] - [00:23:26][^2^][2]:

Cette vidéo est une conférence sur la puberté, la sexualité et le consentement, organisée par la CAF Touraine. Elle aborde le développement de l'enfant, de la petite enfance à l'adolescence, et comment les parents peuvent accompagner leurs enfants dans la découverte de leur corps, les relations amoureuses et la sexualité.

Points forts: + [00:00:45][^3^][3] Introduction de la conférence * Présentation des intervenants et de leurs rôles * Objectifs de la conférence et interaction avec le public + [00:04:10][^4^][4] Développement psychosexuel de l'enfant * Exploration de la sexualité dès la naissance * Importance de la découverte du corps et des sensations + [00:14:00][^5^][5] Comportements sexuels chez les jeunes enfants * Différenciation entre sexualité et acte sexuel * Jeux d'imitation et curiosité naturelle des enfants + [00:19:53][^6^][6] Consentement et respect du corps * Enseignement de l'intimité et du respect de soi * Prévention des abus et importance de la communication ouverte

Résumé de la vidéo [00:23:27][^1^][1] - [00:46:51][^2^][2] : Cette vidéo aborde la puberté, la sexualité et le consentement, en expliquant les changements physiques et émotionnels qui surviennent pendant l'adolescence. Elle souligne l'importance de l'éducation sexuelle et du respect du consentement dès le plus jeune âge.

Points forts : + [00:23:27][^3^][3] Les changements de la puberté * Description des signes physiques comme la croissance des poils et le développement de la poitrine * L'arrivée des premières règles et les premières éjaculations comme indicateurs de la capacité de reproduction * Les défis émotionnels et sociaux rencontrés par les adolescents + [00:29:00][^4^][4] L'éducation sexuelle et le rôle des parents * L'importance d'informer les enfants sur les changements à venir * Comment aborder les sujets délicats comme les éjaculations nocturnes et les règles * La nécessité pour les parents de communiquer ouvertement et de fournir des ressources adaptées + [00:37:01][^5^][5] L'introduction à la sexualité et au consentement * Comment et quand commencer à parler de sexualité avec les enfants * Utiliser le langage approprié pour nommer les parties génitales et expliquer les sensations * L'importance de respecter l'intimité et les limites personnelles pour prévenir les violences + [00:45:00][^6^][6] Le consentement dans les relations affectives et sexuelles * Le consentement comme concept non exclusivement sexuel, débutant dans l'enfance * Enseigner aux enfants que leur corps leur appartient et qu'ils peuvent refuser les marques d'affection * Préparer les adolescents à exprimer leurs limites et à refuser les actes sexuels non désirés

Résumé de la vidéo [00:46:55][^1^][1] - [01:10:14][^2^][2]:

La vidéo aborde la puberté, la sexualité et le consentement, soulignant l'importance de l'éducation sexuelle dès le plus jeune âge. Elle met en lumière la nécessité d'apprendre aux enfants à comprendre leur propre désir et à respecter leurs émotions, ce qui est essentiel pour des relations saines et consensuelles à l'avenir.

Points forts: + [00:46:55][^3^][3] Éducation à la sexualité * Importance d'écouter son corps et ses émotions * Respecter les émotions des enfants * Apprendre aux enfants à comprendre leur propre désir + [00:50:00][^4^][4] Consentement et respect * Comparaison avec le consentement à boire du thé * Importance de ne pas forcer le consentement * Enseigner aux enfants à dire non et aux autres à respecter ce non + [00:57:02][^5^][5] Ressources et accompagnement * Présentation de livres et de ressources pour les parents et les adolescents * Importance de l'accès à des informations fiables sur la sexualité * Structures d'accueil pour les questions et le soutien des adolescents et des parents

Résumé de la vidéo [01:10:16][^1^][1] - [01:22:28][^2^][2]:

Cette partie de la vidéo aborde la puberté, la sexualité et le consentement, en mettant l'accent sur l'importance de la communication entre les adolescents et les professionnels de santé. Il est souligné que les adolescents devraient avoir la possibilité de s'exprimer sans la présence de leurs parents pour préserver leur intimité. La discussion couvre également les risques associés aux réseaux sociaux, tels que le sexting et le harcèlement sexuel, et l'importance de l'éducation pour aider les adolescents à naviguer dans ces espaces en toute sécurité.

Points forts: + [01:10:16][^3^][3] Communication avec les adolescents * Importance de parler sans les parents * Respecter l'intimité et le consentement * Écouter les demandes spécifiques des adolescents + [01:11:26][^4^][4] Risques des réseaux sociaux * Sexting et harcèlement sexuel dès le collège * Difficulté de répondre en peu de temps * Nécessité d'une éducation aux dangers d'internet + [01:13:19][^5^][5] Confiance et relations amoureuses * Adolescents partageant des photos personnelles * Différenciation entre relation amoureuse et sexuelle * Engagement amoureux via les réseaux sociaux + [01:16:01][^6^][6] Éducation précoce et prévention * Aborder la sexualité dès la petite enfance * Enseigner le respect du corps et de l'intimité * Prévenir les comportements à risque liés à la sexualité

Sommaire minuté des temps forts du webinaire "Comment discuter avec les ados de l'impact des "vidéos pour adultes" | Crips IDF"

0:00 - 2:00 Introduction et présentation des intervenantes et du Crips Ile-de-France

• Chloé Ancelin et Fanie Forestier se présentent et décrivent leur rôle au Crips Ile-de-France.
• Elles expliquent les missions du Crips, notamment la prévention du VIH/SIDA et l'éducation à la sexualité auprès des jeunes.

Le webinaire se divise en deux parties : * - la première définit la pornographie et ses impacts, * - la seconde explore les moyens d'aborder ce sujet avec les jeunes.

2:00 - 10:00 Définition et historique de la pornographie

• La pornographie est définie comme une représentation obscène visant à provoquer l'excitation sexuelle.
• L'historique de la pornographie est retracée, des premières images datant de plus de 1000 ans avant J-C à l'essor d'Internet.
• L'impact du cinéma, de la VHS, d'Internet et du streaming sur la diffusion de la pornographie est mis en avant.
• L'accessibilité accrue à la pornographie via Internet est soulignée, avec un pic de consommation observé pendant le confinement lié au COVID-19.

10:00 - 20:00 Consommation de la pornographie par les mineurs et impacts sur la santé

• L'âge moyen de la première exposition à la pornographie est de 10 ans, souvent accidentelle via des pop-ups.
• La majorité des jeunes regardent de la pornographie par curiosité et pour apprendre.
• L'accessibilité facile à la pornographie, malgré l'interdiction aux moins de 18 ans, est démontrée.
• La culture pornographique véhicule des stéréotypes de genre, la culture du viol et des représentations racistes.
• Les impacts négatifs de la pornographie sur la santé mentale (perte de confiance, anxiété), la santé sexuelle (baisse de libido, problèmes d'érection) et les comportements sexuels à risque sont expliqués.

20:00 - 30:00 La culture du viol dans les médias et l'éducation à la sexualité

• La culture du viol est présente dans d'autres médias que la pornographie, comme les films, les séries et les réseaux sociaux.
• Des exemples concrets comme le "coach en séduction" Adé Laurent, le film 365 jours et le livre Captif sont analysés.
• La circulaire de 2001 sur l'éducation à la sexualité en milieu scolaire est présentée, soulignant l'obligation de trois séances annuelles.
• Malgré cette obligation, 67% des jeunes déclarent ne pas avoir bénéficié de ces séances.
• L'importance d'une éducation à la sexualité complète, incluant les aspects biologiques, psychoaffectifs et sociaux, est mise en avant.

30:00 - 40:00 Posture à adopter et outils pour discuter de la pornographie avec les jeunes

• L'importance de l'écoute, de la suspension du jugement et de la création d'un espace bienveillant est soulignée.
• Trois outils d'animation sont présentés : "Milcheck de plaisir" (pour reconstruire la chronologie d'un rapport sexuel), un tableau de discussion sur le vrai/faux de la pornographie, et "OK Hor Not OK" (pour travailler sur le consentement).

40:00 - 50:00 Ressources pour les jeunes et les adultes

• Des ressources alternatives à la pornographie mainstream, comme la série Sex Education et des sites d'information fiables, sont recommandées.
• Des livres, des podcasts et des comptes Instagram sur l'éducation à la sexualité sont présentés.
• Le guide "The P Conversation" d'Erica Lust, réalisatrice de films pornographiques éthiques, est particulièrement mis en avant.

50:00 - Fin Questions/Réponses et conclusion

• Les questions des participants portent sur la disponibilité des statistiques, l'adaptation des outils à différents publics, la formation des professionnels et la gestion des situations difficiles en animation.
• L'importance d'une approche pragmatique et bienveillante, basée sur l'écoute et la discussion, est réaffirmée.

Ce sommaire résume les points clés du webinaire "Comment discuter avec les ados de l'impact des "vidéos pour adultes". Il met en évidence l'omniprésence de la pornographie, ses impacts potentiellement néfastes et la nécessité d'une éducation à la sexualité ouverte et positive.

The model minority

Highlight all civic participation approaches

Highlight all civic participation approaches

Highlight all civic participation approaches

Highlight all civic participation approaches

Highlight all civic participation approaches

Highlight all civic participation approaches

Highlight all civic participation approaches

Highlight all civic participation approaches

Ask the community what it is that they want and need.

everything is rooted in slavery when we think about it.

This would help

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trust and integrity

machine-self

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