DOI: 10.24433/co.3851388.v1

Resource: None

Curator: @scibot

SciCrunch record: RRID:AB_1079227

What is this?

DOI: 10.1093/molbev/msaf042

Resource: ggplot2 (RRID:SCR_014601)

Curator: @scibot

SciCrunch record: RRID:SCR_014601

What is this?

DOI: 10.1016/j.scr.2025.103694

Resource: (BD Biosciences Cat# 560161, RRID:AB_1645540)

Curator: @scibot

SciCrunch record: RRID:AB_1645540

What is this?

DOI: 10.1016/j.celrep.2025.115409

Resource: (Millipore Cat# AB1031, RRID:AB_90460)

Curator: @scibot

SciCrunch record: RRID:AB_90460

What is this?

Developing

(dropped o)

Διαχωρισμός δουλειάς με έμφυλη διάσταση, γυναίκες στο χωράφι και άντρες κυρίως να κουβαλούν ασήκωτα φορτία στα πλοία

Nessa linha, vide a Instrução Normativa n° 40, de 15 de março de 2016 retificada, a qual aproximou a sistemática de admissibilidade recursal do RR à do REsp e RE.

A Instrução Normativa nº 39, 10 de março de 2016, por sua vez, regula as compatibilidades e incompatibilidades existentes entre o CPC e a CLT.

Putin is attempting to push the idea that the leadership of Russia is putting these ideas in place from their work rather than taking ideas from the West. Making the view of the new plans to be of the national interest.

This goes back to what I said earlier that Putin and the political leaders of the country are trying to lay this new ideology into the groundworks of society rather than making an institutionalized system due to it technically not being allowed. Putin wants the society of Russia to have one congruent goal together.

I find it to be very interesting that the Russian government is trying its best to shift to a more conservative ideology that is more replicant of a Western model, but at the same time, they are stating that it is of a non-Western model and that they are doing it with their own ideas. Russia's constitution even states that the government is not allowed to assign a congruent ideology for the government. So I would assume they are trying to lay it in the ground work of the political parties ideology.

Creo que no se entiende muy bien. A lo mejor podrías decir let's create a pre marker and a posterior marker o algo así

Compute statistics

o...

Compute descriptive statistics

más que tip pondría according to a condition o algo así

Esto si no haces un str o algo no sé ve que haya cambiado nada

O requerimento de limitação de litisconsórcio é cabível em qualquer fase do processo.

método de tratamento adequado dos conflitos.

não usar "alternativo" não usar "solução"

evolução terminológica

Reviewer #1 (Public review):

Summary

In this article, Kawanabe-Kobayashi et al., aim to examine the mechanisms by which stress can modulate pain in mice. They focus on the contribution of noradrenergic neurons (NA) of the locus coeruleus (LC). The authors use acute restraint stress as a stress paradigm and found that following one hour of restraint stress mice display mechanical hypersensitivity. They show that restraint stress causes the activation of LC NA neurons and the release of NA in the spinal cord dorsal horn (SDH). They then examine the spinal mechanisms by which LC→SDH NA produces mechanical hypersensitivity. The authors provide evidence that NA can act on alphaA1Rs expressed by a class of astrocytes defined by the expression of Hes (Hes+). Furthermore, they found that NA, presumably through astrocytic release of ATP following NA action on alphaA1Rs Hes+ astrocytes, can cause an adenosine-mediated inhibition of SDH inhibitory interneurons. They propose that this disinhibition mechanism could explain how restraint stress can cause the mechanical hypersensitivity they measured in their behavioral experiments.

Strengths:

(1) Significance. Stress profoundly influences pain perception; resolving the mechanisms by which stress alters nociception in rodents may explain the well-known phenomenon of stress-induced analgesia and/or facilitate the development of therapies to mitigate the negative consequences of chronic stress on chronic pain.

(2) Novelty. The authors' findings reveal a crucial contribution of Hes+ spinal astrocytes in the modulation of pain thresholds during stress.

(3) Techniques. This study combines multiple approaches to dissect circuit, cellular, and molecular mechanisms including optical recordings of neural and astrocytic Ca2+ activity in behaving mice, intersectional genetic strategies, cell ablation, optogenetics, chemogenetics, CRISPR-based gene knockdown, slice electrophysiology, and behavior.

Weaknesses:

(1) Mouse model of stress. Although chronic stress can increase sensitivity to somatosensory stimuli and contribute to hyperalgesia and anhedonia, particularly in the context of chronic pain states, acute stress is well known to produce analgesia in humans and rodents. The experimental design used by the authors consists of a single one-hour session of restraint stress followed by 30 min to one hour of habituation and measurement of cutaneous mechanical sensitivity with von Frey filaments. This acute stress behavioral paradigm corresponds to the conditions in which the clinical phenomenon of stress-induced analgesia is observed in humans, as well as in animal models. Surprisingly, however, the authors measured that this acute stressor produced hypersensitivity rather than antinociception. This discrepancy is significant and requires further investigation.

(2) Specifically, is the hypersensitivity to mechanical stimulation also observed in response to heat or cold on a hotplate or coldplate?

(3) Using other stress models, such as a forced swim, do the authors also observe acute stress-induced hypersensitivity instead of stress-induced antinociception?

(4) Measurement of stress hormones in blood would provide an objective measure of the stress of the animals.

(5) Results:

a) Optical recordings of Ca2+ activity in behaving rodents are particularly useful to investigate the relationship between Ca2+ dynamics and the behaviors displayed by rodents.

b) The authors report an increase in Ca2+ events in LC NA neurons during restraint stress: Did mice display specific behaviors at the time these Ca2+ events were observed such as movements to escape or orofacial behaviors including head movements or whisking?

c) Additionally, are similar increases in Ca2+ events in LC NA neurons observed during other stressful behavioral paradigms versus non-stressful paradigms?

d) Neuronal ablation to reveal the function of a cell population.

e) The proportion of LC NA neurons and LC→SDH NA neurons expressing DTR-GFP and ablated should be quantified (Figures 1G and J) to validate the methods and permit interpretation of the behavioral data (Figures 1H and K). Importantly, the nocifensive responses and behavior of these mice in other pain assays in the absence of stress (e.g., hotplate) and a few standard assays (open field, rotarod, elevated plus maze) would help determine the consequences of cell ablation on processing of nociceptive information and general behavior.

f) Confirmation of LC NA neuron function with other methods that alter neuronal excitability or neurotransmission instead of destroying the circuit investigated, such as chemogenetics or chemogenetics, would greatly strengthen the findings. Optogenetics is used in Figure 1M, N but excitation of LC→SDH NA neuron terminals is tested instead of inhibition (to mimic ablation), and in naïve mice instead of stressed mice.

g) Alpha1Ars. The authors noted that "Adra1a mRNA is also expressed in INs in the SDH".

h) The authors should comprehensively indicate what other cell types present in the spinal cord and neurons projecting to the spinal cord express alpha1Ars and what is the relative expression level of alpha1Ars in these different cell types.

i) The conditional KO of alpha1Ars specifically in Hes5+ astrocytes and not in other cell types expressing alpha1Ars should be quantified and validated (Figure 2H).

j) Depolarization of SDH inhibitory interneurons by NA (Figure 3). The authors' bath applied NA, which presumably activates all NA receptors present in the preparation.

k) The authors' model (Figure 4H) implies that NA released by LC→SDH NA neurons leads to the inhibition of SDH inhibitory interneurons by NA. In other experiments (Figure 1L, Figure 2A), the authors used optogenetics to promote the release of endogenous NA in SDH by LC→SDH NA neurons. This approach would investigate the function of NA endogenously released by LC NA neurons at presynaptic terminals in the SDH and at physiological concentrations and would test the model more convincingly compared to the bath application of NA.

l) As for other experiments, the proportion of Hes+ astrocytes that express hM3Dq, and the absence of expression in other cells, should be quantified and validated to interpret behavioral data.

m) Showing that the effect of CNO is dose-dependent would strengthen the authors' findings.

n) The proportion of SG neurons for which CNO bath application resulted in a reduction in recorded sIPSCs is not clear.

o) A1Rs. The specific expression of Cas9 and guide RNAs, and the specific KD of A1Rs, in inhibitory interneurons but not in other cell types expressing A1Rs should be quantified and validated.

(6) Methods:

It is unclear how fiber photometry is performed using "optic cannula" during restraint stress while mice are in a 50ml falcon tube (as shown in Figure 1A).

навело на думки про наші дипломи і курсові))

Це, на мою думку, фінальна крапка в усіх дебатах про квір і транс людей у фемінізмі. В принципі немає різниці чи "всі такими народжуються", чи "стають під впливом обставин", чи хочуть транс люди робити "медичний перехід", чи не хочуть, чи можуть лесбійки і геї зустрічатися з транс людьми, чи не можуть. Право будь-якої людини жити будь-як без дискримінації має біти фундаментальною метою

особливо коли суспільство зазвичай захищає багату, відому, білу Джоан Роулінг

nova modalidade de tributação. nao contradiz com o art. 21?

Author response:

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

Reviewer #1:

We thank the Reviewer for being very supportive of the work and acknowledging how important it is to understand allosteric modulation in the spike and the potential of this knowledge to contribute to the design of novel therapeutic strategies (for example, disrupting or altering the allosteric networks within the spike can be a novel strategy for drug development against COVID-19). We address their comments below: 

(1) The Reviewer states that although the strategy used to extract the responses has been "previously validated", the complexity of the interactions investigated requires "a robust statistical analysis, which is not shown quantitatively". 

As the Reviewer points out, the D-NEMD approach has been previously validated in various protein systems ranging from soluble enzymes to integral membrane proteins, including the spike (e.g. [Kamsri et al. (2024) Biochem; Beer et al. (2024) Chem Sci; Oliveira et al. (2023) J Mol Cell Biol; Chan et al. (2023) JACS Au; Castelli et al. (2023) JACS; Castelli et al. (2023) Protein Sci; Oliveira et al. (2022) Comput Struct Biotechnol J; Gupta et al. (2022) Nat Comm; Oliveira et al. (2021) JACS; Galdadas et al. (2021) eLife; Abreu et al. (2019) Proteins; Oliveira et al. (2019) JACS; Oliveira et al. (2019) Structure]. The Kubo-Onsager relation is used to extract the evolution of the protein's response to a perturbation by comparing the equilibrium and nonequilibrium trajectories at equivalent points in time. The calculated responses at individual times are then averaged over all the repeats (210 repeats in the current work), and the standard error of the mean (SEM) is used to assess the significance of the average response. The SEM indicates how much the calculated mean deviates from the true population mean. Calculating the SEM allows us to determine how accurate the measured response is as an estimate of the population response and assess the convergence of our calculations. The evolution of the average C_α displacement and corresponding SEM values for each individual monomer can be visualised in detail in Figures S7-S9. We have added a new sentence to the Materials and Methods section in the Supporting Information, explicitly stating how the convergence and statistical significance of the responses were assessed.

(2) The Reviewer considers that the evidence presented in the paper "is compelling" but suggests performing a sequence analysis to facilitate the understanding of the results by the scientific community. 

We thank the Reviewer for their excellent suggestion to perform a sequence analysis of the FA site region and its allosteric connections. Indeed, this analysis (Figure S24) clearly shows that several of the mutations, deletions and insertions in the Alpha, Beta, Gamma, Delta, and Omicron variants are located either in or near the regions of the protein shown to respond to the removal of linoleate from the FA site. These sequence changes affect the protein's responses, and are responsible for the differences in allosteric behaviour observed between variants, as described previously for the non-glycosylated spike [Oliveira et al. (2023) J Mol Cell Biol]. Furthermore, some variants, such as Beta, Gamma, and Omicron, contain residue substitutions at the FA site. For example, the lysine in position 417 in the ancestral spike is mutated to asparagine in Beta and Omicron and threonine in the Gamma variant. Another example is arginine 408 in the original protein, which has been replaced by asparagine in several Omicron sub-variants. 

To summarise, the sequence analysis (Figure S24) supports our initial 3D analysis (Figure S25), indicating that many of the changes observed in the variants of concern are indeed in or close to the allosteric networks involving the FA site. We have now included the sequence analysis results in the current paper and added a new figure to Supporting Information showing the sequence alignments between the ancestral spike and different variants (Figure S24). 

(3) The Reviewer also has "minor considerations": first, they point to a discrepancy in the presentation of residue values S325 in the plots of Chains A, B, and C of Figure S3; second, they ask why several regions, such as RBM and Furin Site in figures S6, S7, and S8 show significant changes.

To answer both points raised by the Reviewer, we need to start by explaining that the spike typically features 22 N-glycosylation and at least two O-glycans sites per monomer. These sites have been found to be heterogeneously populated in different experimental studies (e.g. [Watanabe et al. (2020) Science; Shajahan et al. (2020) Glycobiology; Zhang et al. (2021) Mol Cell Proteomics]). Given this, the spike model used as the starting point for this work reflects this heterogeneity, with asymmetric site-specific glycosylation profiles derived from the glycoanalytic data reported by Watanable et al. for N-glycans [Watanabe et al. (2020) Science] and Shajahan et al. for O-glycans [Shajahan et al. (2020) Glycobiology]. This means that the glycan occupancy and composition for each site differ between the three monomers. For example, while monomer A contains the two O-glycans sites (linked to T323 and S325, respectively) fully occupied, monomers B and C only contain the T323 O-glycan. A detailed description of the glycosylation of the spike model is given in the supporting information of [Casalino et al. (2020) ACS Cent Sci].

Regarding the Reviewer's first minor point, the discrepancy in behaviour observed in Figure S3 for S325 is related to the fact that this glycosylation site is only occupied in monomer A, with no glycans present in this site in monomers B and C. 

Regarding the second point, the differences observed in the responses between the three monomers in Figures S7-S9 are probably due to asymmetries in the protein dynamics introduced by the different glycosylation patterns in the monomers. 

We have now added a new paragraph to the materials and methods section in the Supporting Information describing the asymmetric site-specific glycosylation profiles of the monomers.

(4) Due to the complexity of the allosteric interactions observed, the Reviewer suggests including in the paper a "diagram showing the flow of allosteric interactions" or a "vector showing how the perturbation done in the FA Active site takes contact with other relevant regions". 

This is an excellent suggestion to facilitate the visualisation of the allosteric networks. We have added a new figure to Supporting Information highlighting the allosteric pathways identified from the DNEMD simulations and the direction of the propagation of the structural changes (Figure S26).

Reviewer #2:

We thank the Reviewer for their time in evaluating our manuscript and providing suggestions for improving it and ideas for further work. We are happy that the Reviewer found this to be a "nice paper" with the calculations "well done" and interesting results. We address their comments below: 

(1) The Reviewer suggests improving the paper by adding a more detailed explanation of the DNEMD simulations approach, a method that, although proposed decades ago, is still generally unfamiliar to the community. They also asked for "information on the convergence of the observables".

As stated by the Reviewer, a dynamical approach to nonequilibrium molecular dynamics (D-NEMD) was first proposed in the seventies by Ciccotti et al. [Ciccotti et al. (1975) Phys Rev Lett; Ciccotti et al. (1979) J Stat Phys]. This approach combines MD simulations in equilibrium and nonequilibrium conditions. The rationale for the D-NEMD approach is simple and can be described as follows: if an external perturbation (e.g. binding/unbinding of a ligand) is added to a simulation sampling an equilibrium state and, by doing so, a parallel nonequilibrium simulation is started, the structural response of the protein to the perturbation can be directly measured by comparing the equilibrium and nonequilibrium trajectories at equivalent points in time by using the Kubo-Onsager relation as long as enough sapling is gathered (for more details, please see the reviews [Balega et al. (2024) Mol Phys; Oliveira et al. (2021) Eur Phys J B; Ciccotti et al. (2016) Mol Simul]). This approach, although conceptually simple, is very powerful as it allows for computing the evolution of the dynamic response of the protein to the external perturbation, while assessing the convergence and statistical significance of that response. This approach also has the advantage that the convergence and significance of the response can be easily evaluated, and the associated errors can be computed and made as small as desirable by increasing the number of nonequilibrium trajectories. Determining the statistical errors associated with the responses (through, e.g., the determination of the standard error of the mean, SEM) is essential to test if the sampling gathered is sufficient. In this paper, the SEM was calculated for each average C_α displacement value at times 0.1, 1 and 10 ns after the removal of linoleate, LA (see Figures S7-S9). The SEM indicates how accurate the measured response is as an estimate of the population response and allows us to assess the convergence of the results. 

Generally, multiple (tens to hundreds) D-NEMD simulations are needed to achieve statistically significant results for biomolecular systems (for examples, see [Balega et al. (2024) Mol Phys; Oliveira et al. (2021) Eur Phys J B]). As such, the length of the D-NEMD simulations (typically 5 to 10 ns) reflects the balance between the computational resources available and the number of replicates needed to achieve statistically significant responses from the system. Following the Reviewer's suggestion, we have now added a brief description of the D-NEMD approach to the main manuscript and expanded the D-NEMD section in the Supporting Information with a more detailed description of the method, including adding a new figure showing a schematic representation of the D-NEMD approach (Figure S5) as well as explicitly stating the settings used in these simulations and how the statistical significance of the responses was assessed. 

(2) The Reviewer suggests comparing the D-NEMD results with "more traditional analysis, such as correlation analysis, or community network analysis". 

We agree with the Reviewer that this is an important comparison, which can provide a broader, more articulate and coherent picture of spike allostery and have, therefore, performed additional analysis. The dynamic cross-correlation analysis suggested by the Reviewer is a valuable tool for identifying the regions in the protein influenced by the FA site in equilibrium conditions. However, such an approach is not straightforwardly applicable to D-NEMD simulations, as these simulations are not in equilibrium. Nevertheless, as suggested by the Reviewer, we have determined the cross-correlation matrices for both the equilibrium and D-NEMD simulations (Figure S22), similar to those in our previous work [Galdadas et al. (2021) eLife] and [Oliveira et al. (2022) J Mol Cell Biol]. The analysis of these matrices can provide information about possible allosteric networks. In Figure S22, the cyan and blue regions represent moderate and high negative correlations between C_α atoms, while orange and red regions correspond to moderate and high positive correlations. Negative correlations indicate residues moving in opposite directions (moving toward or away from each other). In contrast, positive values imply that the residues are moving in similar directions. We also note that, with collaborators, we have compared D-NEMD and other nonequilibrium and equilibrium MD analysis methods for allostery [Castelli et al.  (2023) JACS].

The cross-correlation maps depicted in Figure S22 show moderate to high positive correlations between the FA sites and two of the three RBDs in the protein. This happens because each FA site sits at the interface between two neighbouring RBDs. Low to moderate negative and mildly positive correlated motions can also be observed between the FA site and the NTDs and fusion peptide surrounding regions, respectively. To facilitate the visualisation of the above-described motions, we have also mapped the statistical correlations for R408 and K417 (two FA site residues able to directly form salt-bridge interactions with the carboxylate head group of LA) on the protein's three-dimensional structure (Figure S23). Figure S23 highlights the patterns of movement described above and allows us to identify the regions whose motions are coupled to the FA site.

Interestingly, some segments forming the signal propagation pathways, such as R454-K458 in all three monomers, and C525-K537 in monomers B and C, can also be identified from the cross-correlation matrices, showing moderate to high correlations with the FA site (Figures S22-S23). The crosscorrelation maps computed from the equilibrium trajectories (with FA sites occupied with LA) show a slight increase in the dynamic correlations, mainly for the RBDs, compared to the maps obtained from the nonequilibrium trajectories (Figure S22). This indicates that the presence of LA in the FA strengthens the connections between the FA site and other parts of the protein. 

We have updated the manuscript to include the cross-correlation analysis, with two new figures added to Supporting Information: one depicting the cross-correlation maps for the D-NEMD and equilibrium simulations (Figure S22), and the other showing the statistical correlations for R408 and K417 (Figure S23). 

(3) The Reviewer considers the observed connection between the fatty acid site and the heme/biliverdin site "interesting" and suggests "exploring the impact of ligand removal on this secondary site on the protein".

Similarly to the Reviewer, we find the connection between the FA and the heme/biliverdin site fascinating and worthy of further investigation. The observed connection between these two sites shows the complexity of the allosteric effects in the spike. It would be interesting and informative to perform new equilibrium simulations of the heme/biliverdin spike complex and a new set of D-NEMD simulations in which this site is perturbed (e.g. through the removal of the heme group) to map the networks connecting this allosteric site to other functionally important regions of the spike, including the FA site and potentially other allosteric sites. These new simulations would allow us to assess the reversibility of the connection between the FA and heme/biliverdin sites and enhance our understanding of allosteric modulation in the spike and the role of the heme/biliverdin site in this process. However, due to the large size of the system and the associated computational demands, such simulations are not possible within the timeframe of the revision of this paper. These simulations would take many months to complete using our HPC resources. We also note that an experimental structure of the spike containing both heme and linoleate is not available. Further simulation analysis of the communication pathways involving the heme/biliverdin site is an excellent idea for future work.

(4) The Reviewer "liked the mapping of existing mutations on the communication pathway" and suggested a more detailed study focusing on the effect of the mutations. 

We fully agree with the Reviewer and consider that a detailed study focusing on the effect of the mutations, insertions, and deletions in the different glycosylated variants of concern (including new emerging ones) would be of great interest. Our previous work using D-NEMD on the non-glycosylated ancestral, Alpha, Delta, Delta plus and Omicron BA.1 spikes revealed significant differences in the allosteric responses to LA removal, with the changes in the variants affecting both the amplitude of the structural responses and the rates at which these rearrangements propagate within the protein [Oliveira et al. (2023) J Mol Cell Biol]. 

Using the D-NEMD approach to systematically investigate the impact of each individual mutation and their contribution to the overall allosteric response of the glycosylated variants (similar to what we have done previously for the D614G mutation in the non-glycosylated protein [Oliveira et al. (2021) Comput Struct Biotechnol J]) would provide insights into the functional modulation of the spike. However, as noted above in point 3, spike simulations are highly computationally expensive, both in terms of processing and data storage requirements, because of the large size of the protein and the need for equilibrium and D-NEMD simulations. This makes the suggested mutational study unfeasible within the timeframe of the current revisions. It is, however, an excellent idea for future research.

Reviewer #3:

We thank the Reviewer for carefully reading and critically reviewing this work and recognising that the findings reported are "based on an impressive amount of sampling" and "meticulous" analysis. We address their comments below: 

(1) The Reviewer considers that this work "does not clearly show any new findings" as it shows that the glycans do not significantly impact the internal networks in the protein.

We respectfully disagree with the Reviewer. This work identifies new allosteric effects in the spike, specifically, the connection of the FA site with the heme binding site. The equilibrium simulations alone provide the first analysis of the effects of linoleate binding in the fully glycosylated spike. The finding that glycosylation does not significantly affect the allosteric pathways in the spike is in itself an important finding. Previous D-NEMD simulations investigated only the non-glycosylated spike ([Oliveira et al. (2021) Comput Struct Biotechnol J; Oliveira et al. (2022) J Mol Cell Biol] ) leading to questions of whether the allosteric effects pathways were changed by glycosylation; our results here show that the main conclusions are reinforced, but glycosylation does have some effect on networks, and also on the speed of the dynamical response. To the best of our knowledge, our work represents the first investigation to analyse the impact of glycosylation on the allosteric networks in the spike. We show that even though the presence of glycans in the exterior of the spike does not significantly alter the internal communication pathways in the protein, in some cases (for example, the glycans linked to N234, T373 and S375), they create direct connections between different regions, which may facilitate the propagation of the structural changes. 

(2) The Reviewer suggests adding a "clear and concise description" of the D-NEMD approach to the manuscript.

We appreciate that the use of the D-NEMD method to study biomolecular systems is relatively new, and so may be unfamiliar. As explained above in our response to Reviewer 2 (point 1), a brief description of the D-NEMD approach was now included in the main manuscript. A detailed description of the method was also added to Supporting Information, including a new figure representing the rationale for the approach (Figure S5). The interested reader is directed to previous applications and reviews for more details of the method (e.g. [Balega et al. (2024) Mol Phys; Oliveira et al. (2021) Eur Phys J B; Ciccotti et al. (2016) Mol Simul; Kamsri et al. (2024) Biochem; Beer et al. (2024) Chem Sci; Oliveira et al. (2023) J Mol Cell Biol; Chan et al. (2023) JACS Au; Castelli et al. (2023) JACS; Castelli et al. (2023) Protein Sci; Oliveira et al. (2022) Comput Struct Biotechnol J; Gupta et al. (2022) Nat Comm; Oliveira et al. (2021) JACS; Galdadas et al. (2021) eLife; Abreu et al. (2019) Proteins; Oliveira et al. (2019) JACS; Oliveira et al. (2019) Structure]). 

(3) The Reviewer invites us to "discuss the robustness of the findings with respect to forcefield choices".

The Reviewer raises an important but rather complex question, and one which can, of course, be posed for any molecular dynamics simulation study. The short answer is that we have chosen state-of-the-art forcefields, which have been shown to give results for the spike that are in good agreement with experiments; glycosylated spike simulations are rather computationally expensive, and constructing the models also requires significant human time and effort. Thus, while in principle interesting, it is not practical to repeat the current simulations with different forcefields. However, as detailed below, comparison of our simulations of the glycosylated and non-glycosylated [Oliveira et al. (2022) Comput Struct Biotechnol J] spike using different forcefields indicates that our conclusions are robust and are not dependent on the choice of forcefield. 

Comparing the performance and accuracy of different force fields is not straightforward, as the results depend on the system of interest, properties simulated and sampling. In this work, the CHARMM36m all-atom additive force field was used to describe the protein and glycans. CHARMM36m is a widely used force field that has previously been validated for the simulations of biological systems [Huang et al. (2013) J Comput Chem; Guvench et al. (2009) J Chem Theory Comput], including proteins, lipids and glycans, with many of studies adopting it in the literature. Additionally, the glycosylated models of the spike used in this work have also been successfully applied and tested before (e.g. [Dommer et al. (2023) Int J High Perform Comput Appl; Sztain et al. (2021) Nat Chem; Casalino et al. (2021) Int J High Perform Comput Appl; Casalino et al. (2020) ACS Cent Sci]), with their dynamics shown to correlate well with experimental data.   

It is also worth pointing out that, despite differences in the amplitude of the responses, the allosteric networks identified using the D-NEMD approach for the non-glycosylated [Oliveira et al. (2022) Comput Struct Biotechnol J] and glycosylated spikes are generally similar (Figure S13). While the responses for the non-glycosylated protein were extracted from simulations using the AMBER99SBILDN forcefield [Oliveira et al. (2022) Comput Struct Biotechnol J], those reported in this work were obtained from trajectories using the CHARMM36m forcefield. The similarity between the responses for the two systems (which were simulated using different forcefields) is a good indication that our findings are forcefield independent. 

(4) The Reviewer suggests comparing our findings with "alternative methods of analysing allostery". 

As stated above in our response to Reviewer 2 point 2, we consider the suggested comparison an excellent idea. We have therefore performed a dynamic cross-correlation analysis to identify the regions in the protein coupled to the FA site in both equilibrium and nonequilibrium conditions (see Figures S22-S23). Overall, this analysis shows that the FA site motions are strongly coupled to the RBDs and moderately to weakly connected to the NTDs and fusion peptide surrounding regions (please see a detailed description of the results of the correlation analysis in our response to Reviewer 2 point 2). The cross-correlation analysis performed was added to the manuscript, and two new figures were included in the Supporting Information (Figures S22-S23): the first, showing the cross-correlation maps for the D-NEMD and equilibrium simulations; the second, showing the statistical correlations for R408 and K417 (two residues forming the FA site and that can directly interact with the carboxylate head group of LA). 

We agree that comparing different allosteric analysis methods is interesting, informative and important. As noted above, we have compared D-NEMD and other nonequilibrium and equilibrium MD analysis methods for allostery in the well-characterised K-Ras system [Castelli et al.  (2023) JACS].

Othello wishes to had never heard what Iago told him just to grant him peace of mind.

HDDs overwrite data easily, so freed blocks stay unchanged until reused. However, NVM flash storage requires erasing blocks before rewriting, which is slow. To manage this, file systems use commands like TRIM (for ATA drives) and unallocate (for NVMe) to mark pages as free. This prevents performance drops by allowing erasure and garbage collection to happen in advance, keeping storage efficient.

ZFS manages large-scale file systems efficiently by organizing storage into metaslabs, each with a space map that logs block activity instead of constantly updating bitmaps. When space is allocated or freed, ZFS loads the space map into memory as a balanced tree for quick access. It merges contiguous free blocks to reduce size and updates the free-space list as part of its transaction system, ensuring efficient storage management.

Some operating systems, like UNIX, treat directories as special types of files, while others, like Windows, handle files and directories separately with different system calls. Regardless of the approach, the logical file system maps directory operations to storage blocks, which are then managed by the basic file system and I/O control system.

The I/O control level transfers data between memory and storage using device drivers, which convert commands into hardware instructions. The basic file system handles read/write operations, I/O scheduling, and caching. The file-organization module manages file storage and free space. The logical file system tracks metadata, directories, and file protection. Layering reduces code duplication but may slow performance due to added overhead.

Disks are commonly used for file storage because they allow data to be rewritten in place and provide direct access to any block of data. This makes it easy to read, modify, and switch between files. Nonvolatile memory (NVM) devices are also used for storage, but they cannot rewrite data in place and have different performance features. To speed up data transfers, both disks and NVM devices use blocks of data, typically 512 or 4,096 bytes, for efficient reading and writing.

Efficiency depends on balancing storage structures, metadata management, and allocation methods. For example, UNIX’s inode system reserves space even when empty but improves file access speed. Similarly, BSD UNIX’s adaptive cluster sizes optimize storage use. Metadata tracking, such as access timestamps, adds processing overhead and should be evaluated for necessity. Pointer size decisions affect file system scalability—while 64-bit pointers enable larger files, they consume more memory. Poor early design choices can cause limitations, as seen in MS-DOS FAT’s early partition size restrictions. Modern operating systems use dynamic allocation to avoid these constraints, enhancing long-term system performance.

Counting leverages the observation that contiguous blocks are often freed together. Instead of tracking each block separately, the system stores the starting block and the number of contiguous free blocks. This reduces the number of entries needed to maintain the free-space list, leading to improved efficiency. The method is particularly useful in file systems using contiguous allocation or clustering. Additionally, the data can be stored in a balanced tree, enabling fast searches, insertions, and deletions. By reducing list size while retaining quick lookup capabilities, counting balances storage efficiency and performance for block allocation.

Space Maps ZFS employs space maps to efficiently track free space in large storage systems. Instead of modifying bitmaps or linked lists directly, ZFS logs allocation and deallocation operations in a time-ordered manner. These logs are processed into balanced-tree structures, minimizing I/O overhead. This approach optimizes performance by condensing metadata and reducing the frequency of disk updates. ZFS divides storage into metaslabs, each with its own space map, improving scalability. By combining log-structured techniques with counting, ZFS ensures efficient space management, making it well-suited for large-scale file systems that handle vast amounts of data.

Performance optimization involves caching strategies, memory management, and efficient write operations. Modern storage controllers use on-board caches, while operating systems employ buffer caches or page caches to reduce I/O latency. Unified caches prevent redundant data storage, minimizing memory waste and CPU overhead. Solaris and Linux leverage unified virtual memory for efficiency. Least Recently Used (LRU) algorithms manage cache replacement, but their implementation varies. Asynchronous writes improve speed by allowing immediate process continuation, whereas synchronous writes ensure data integrity. Balancing caching, write strategies, and metadata updates is crucial for maximizing storage performance while maintaining reliability.

The I/O control level is responsible for handling the transfer of data between memory and storage devices. It includes device drivers, which translate high-level commands into low-level instructions for hardware controllers. These drivers communicate with the I/O device by writing specific bit patterns to controller memory. The block I/O subsystem issues generic read and write commands to the appropriate driver while managing request scheduling, buffers, and caches. Buffers store temporary data blocks during transfers, and caches hold frequently accessed metadata to improve performance. Efficient buffer and cache management is crucial to maintaining system responsiveness and optimizing I/O operations.

The file-system structure is fundamental to how data is stored and retrieved on storage devices. Disks, as the primary medium for secondary storage, allow efficient data modification and direct access to any block, making them ideal for sequential and random file access. However, emerging nonvolatile memory (NVM) devices differ from traditional hard disks as they cannot be rewritten in place and exhibit distinct performance traits. File systems enhance storage efficiency by managing data organization through block-based I/O transfers. Their design involves defining user-visible structures and implementing algorithms to map logical data to physical storage, often using a layered architectural approach.

A file system tracks a file’s position using a pointer unique to each process. The system also maintains a file-open count to manage open entries and remove them when no processes are using the file. File location details are stored in memory for quick access. Each process has specific access rights, ensuring proper permissions for reading or writing. Some systems also support file locks to prevent multiple processes from modifying a file simultaneously.

A file is a type of data structure that allows various operations. The operating system provides system calls to create, open, write, read, reposition, delete, and truncate files. Creating a file requires finding space and adding an entry in the directory. Opening a file generates a file handle used for other operations. Writing and reading involve system calls that specify the file and update pointers for sequential access. Repositioning moves the file pointer without performing actual input or output. Deleting a file removes its entry and frees up space unless multiple links exist. Truncating clears the file’s content while keeping its attributes and space allocation intact.

A file is an abstract data type that supports various operations, including creation, reading, writing, repositioning, deletion, and truncation. These operations enable users and applications to interact with files in a structured manner. For instance, when a file is opened, a file handle is assigned to avoid repeated directory searches. The file system also maintains read and write pointers to track file operations efficiently. Additionally, file repositioning allows users to navigate through files without performing actual I/O operations, improving performance. Understanding these file operations is essential for programmers, as efficient file handling can optimize system performance and resource utilization. These operations form the foundation for complex file management tasks such as file compression, synchronization, and remote access.

Sequential access is the simplest and most common method of retrieving file data, where records are accessed in order, one after another. This approach is ideal for text files, logs, and programs that process data sequentially, such as compilers and text editors. Read and write operations move a file pointer forward, automatically advancing to the next record. Some systems allow limited skipping, enabling navigation through the file in predefined increments. Sequential access follows a tape-based model, where operations like rewinding reset the pointer to the beginning. While efficient for linear data processing, sequential access becomes inefficient when frequent random access is required. It remains widely used in applications where data naturally follows a fixed sequence, such as streaming media or logs.

Managing file data efficiently is crucial for operating systems, particularly in how logical records are mapped to physical storage blocks. Disk storage operates in fixed-size blocks, while logical records may vary in size. To optimize space and performance, files are packed into blocks, though this can cause internal fragmentation, where unused space in the last block is wasted. For example, UNIX treats files as streams of bytes, automatically packing them into physical blocks (e.g., 512 bytes). This system simplifies file handling but necessitates additional software logic for structured data. Internal fragmentation increases with larger block sizes, leading to inefficient space utilization. Despite these challenges, block-based allocation remains essential for efficient disk I/O operations and file system management.

Advanced access methods build upon direct access by incorporating indexing for faster data retrieval. An index acts like a book’s table of contents, pointing to specific file locations. For example, a retail-price file sorted by product codes (UPCs) can have an index of first UPCs per block, reducing search time via binary search. If the index grows too large, a hierarchical approach, such as IBM's ISAM, introduces multiple index levels, ensuring efficient lookups. OpenVMS follows a similar strategy, maintaining indexed files for rapid retrieval. These methods enhance performance for large databases, enabling quick access with minimal disk reads. While efficient, indexed access requires additional storage and maintenance, making it a trade-off between speed and system resource usage.

Memory-mapped files work by associating disk blocks with pages in memory. The system initially loads data into memory using demand paging, causing a page fault when first accessed. Instead of using traditional file I/O functions like read() and write(), subsequent access is handled as direct memory operations, improving efficiency. However, file modifications are not immediately written to disk; instead, updates occur when the file is closed or when memory pressure forces changes to be written. Some operating systems memory-map files automatically to optimize performance, such as Solaris, which maps all file I/O operations to the kernel address space. The ability for multiple processes to map the same file enables shared memory, which is critical for efficient interprocess communication (IPC).

Art. 9º - Em garantia da execução, pelo valor da dívida, juros e multa de mora e encargos indicados na Certidão de Dívida Ativa, o executado poderá:

• I - efetuar depósito em dinheiro, à ordem do Juízo em estabelecimento oficial de crédito, que assegure atualização monetária;

• II - oferecer fiança bancária ou seguro garantia;

• III - nomear bens à penhora, observada a ordem do artigo 11; ou

• IV - indicar à penhora bens oferecidos por terceiros e aceitos pela Fazenda Pública.

§ 1º - O executado só poderá indicar e o terceiro oferecer bem imóvel à penhora com o consentimento expresso do respectivo cônjuge.

§ 2º Juntar-se-á aos autos a prova do depósito, da fiança bancária, do seguro garantia ou da penhora dos bens do executado ou de terceiros.

§ 3º A garantia da execução, por meio de depósito em dinheiro, fiança bancária ou seguro garantia, produz os mesmos efeitos da penhora.

§ 4º - Somente o depósito em dinheiro, na forma do artigo 32, faz cessar a responsabilidade pela atualização monetária e juros de mora.

§ 5º - A fiança bancária prevista no inciso II obedecerá às condições pré-estabelecidas pelo Conselho Monetário Nacional.

§ 6º - O executado poderá pagar parcela da dívida, que julgar incontroversa, e garantir a execução do saldo devedor.

§ 7º As garantias apresentadas na forma do inciso II do caput deste artigo somente serão liquidadas, no todo ou parcialmente, após o trânsito em julgado de decisão de mérito em desfavor do contribuinte, vedada a sua liquidação antecipada.

Ocorre revelia acaso o contribuinte não contestar os pedidos iniciais referente à medida cautelar.

Especialmente para a geom_col( ) e geom_bar( ) o argumento position = é muito útil: position = "identity" - Mantém as posições originais dos dados. Indicado para gráficos de dispersão

position = "stack" - Empilha os elementos uns sobre os outros.

position = "dodge" - Posiciona os elementos lado a lado para facilitar a comparação.

position = "fill" - Funciona como "stack", mas normaliza os valores para que o total de cada barra seja 100%.

position = "jitter" - Adiciona aleatoriedade às posições dos pontos para evitar sobreposição. Útil em geom_point().

A interposição de agravo contra deferimento ou indeferimento de suspensão de liminar não interfere e nem condiciona o julgamento do pedido de suspenção.

Isto é, contra a liminar deferida contra o Poder Público, é cabível tanto o Agravo de Instrumento, quanto o Suspensão de Liminar.

O Presidente do Tribunal é o legitimado a apreciar o pedido de suspenção de liminar.

Com isso, acaso seja deferido ou indeferido o pleito, primeiramente, caberá Agravo.

Se o Agravo resultar no julgamento manutenção ou reestabelecimento da suspenção, daí sim caberá novo pedido de suspenção ao Presidente do STJ ou STF conforme for o caso.

Tècnicament pot parlar de risk o bé s'hauria de parlar de rate? Perquè no només tenim en compte l'event sino el time to event

afegiria que estàs comparant, to compare the risk between sex o algo així

survival times o bé surivial funcions

Author response:

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

Reviewer #1 (Public review):

Summary:

Multiple compounds that inhibit ATP-sensitive potassium (KATP) channels also chaperone channels to the surface membrane. The authors used an artificial intelligence (AI)-based virtual screening (AtomNet) to identify novel compounds that exhibit chaperoning effects on trafficking-deficient disease-causing mutant channels. One compound, which they named Aekatperone, acts as a low affinity, reversible inhibitor and effective chaperone. A cryoEM structure of KATP bound to Aekatperone showed that the molecule binds at the canonical inhibitory site.

Strengths and weaknesses:

The details of the AI screening itself are inevitably opaque, but appear to differ from classical virtual screening in not involving any physical docking of test compounds into the target site. The authors mention criteria that were used to limit the number of compounds, so that those with high similarity to known binders and 'sequence identity' (does this mean structural identity) were excluded. The identified molecules contain sulfonylurea-like moieties. How different are they from other sulfonylure4as?

We thank the reviewers for the questions. As part of the library preparation, molecules with greater than 0.5 Tanimoto similarity in ECFP4 space to any known binders of the target protein and its homologs within 70% sequence identity were excluded to increase the possibility of identifying novel hits. After scoring and ranking the molecules by the AtomNet® technology, a diversity clustering was performed using the Butina algorithm (Butina D. Unsupervised Data Base Clustering Based on Daylight’s Fingerprint and Tanimoto Similarity: A Fast and Automated Way To Cluster Small and Large Data Sets, J. Chem. Inf. Comput. Sci. 1999, 39, 747–750) with a Tanimoto similarity cutoff of 0.35 in ECFP4 space to minimize selection of structurally similar scaffolds for the final compound buy-list. We have revised the results and methods sections to make this clear.

Sulfonylureas are defined by their core structure comprising a sulfonyl group (–S(=O)₂) and a urea moiety (–NH–CO–NH–). While some compounds identified in our study contain a sulfonamide group (R-S(=O) ₂-NR₂), they differ structurally from sulfonylureas by lacking the key urea group and by incorporating unique R-group substitutions (we have now added this to Figure 1A legend). For example, compound C27 (Z2068224500) includes a sulfonamide group but not a urea moiety. Likewise, C45 (Aekatperone, Z1620764636) contains a sulfonamide group along with an aromatic, nitrogen-rich heterocyclic ring, but no urea group. Additionally, the R-groups in these compounds are more complex than the simple aromatic or alkyl chains typical of sulfonylureas. They include heterocyclic aromatic systems and nitrogen-rich structures, which likely influence their binding properties and lipophilicity. These structural differences suggest distinct functional and pharmacological profiles as supported by our biochemical and functional studies.

The experimental work confirming that Aekatperone acts to traffic mutant KATP channels to the surface and acts as a low affinity, reversible, inhibitor is comprehensive and clear, with very convincing cell biological and patch-clamp data, as is the cryoEM structural analysis, for which the group are leading experts. In addition to the three positive chaperone-effective molecules, the authors identified a large number of compounds that are predicted binders but apparently have no chaperoning effect. Did any of them have inhibitory action on channels? If so, does this give clues to separating chaperoning from inhibitory effects?

This is an interesting question. Evidence from cryo-EM, biochemical and electrophysiology studies reveal a critical role of Kir6.2 N-terminus in K_ATP channel assembly and gating, and that pharmacological chaperones like glibenclamide, repaglinide, carbamazepine, and now aekatperone exert their chaperoning and inhibitory effects by stabilizing the interaction between Kir6.2 N-terminus and the SUR1-ABC core. This stabilization, while promoting the assembly of Kir6.2 and SUR1 to “chaperone” trafficking-impaired mutant channels to the cell surface, also inhibits the channel by restricting the Kir6.2 C-terminal domain from rotating to an open state. An additional mechanism by which these compounds inhibit channel activity is by preventing SUR1-NBD dimerization, which mediates physiological activation of the channel by MgADP (see review: Driggers CM, Shyng SL. Mechanistic insights on K_ATP channel regulation from cryo-EM structures. J Gen Physiol. 2023 Jan 2;155(1): e202113046, PMID: 36441147). From our compound screening, we did find some compounds that showed mild inhibition of the channel by electrophysiology but no obvious chaperone effects by western blots. It is possible that small chaperoning effects of some compounds showing mild channel inhibition effects were missed due to the lower sensitivity of the western blot assay compared to electrophysiology. Alternatively, these compounds could inhibit channels by preventing SUR1NBD dimerization without stabilizing the Kir6.2 N-terminus, which is required for the chaperone effect based on our model. Unfortunately, we did not find any compounds that show chaperone effects but no channel inhibition effects, which is consistent with our understanding of how this type of K_ATP chaperones work (i.e. by stabilizing Kir6.2 N-terminus interaction with SUR1’s ABC core).

The authors suggest that the novel compound may be a promising therapeutic for treatment of congenital hyperinsulinism due to trafficking defective KATP mutations. Because they are low affinity, reversible, inhibitors. This is a very interesting concept, and perhaps a pulsed dosing regimen would allow trafficking without constant channel inhibition (which otherwise defeats the therapeutic purpose), although it is unclear whether the new compound will offer advantages over earlier low-affinity sulfonylurea inhibitor chaperones. These include tolbutamide which has very similar affinity and effect to Aekatperone. As the authors point out this (as well as other sulfonlyureas) are currently out of favor because of potential adverse cardiovascular effects, but again, it is unclear why Aekatperone should not have the same concerns.

We thank the reviewer for the comments. This is clearly an important question to address in the future. While we have not directly tested the effects of Aekatperone on cardiac functions, we did assess its inhibitory effect on cells expressing the cardiac K_ATP channel isoform (SUR2A/Kir6.2). Our results indicate that Aekatperone exhibits higher sensitivity toward the pancreatic K_ATP channel isoform (SUR1/Kir6.2) compared to the cardiac isoform. However, we acknowledge that Aekatperone could still have cardiotoxic effects through its potential action on other channels, such as the hERG channel.

It is worth noting that tolbutamide, despite its known cardiotoxic effects, does not exert these effects through cardiac K_ATP channel inhibition. This has been demonstrated in studies showing no inhibitory effect of tolbutamide on SUR2A/Kir6.2 channels and on channels formed by Kir6.2 and SUR1 harboring the S1238Y mutation (also shown as S1237Y in some studies using a different SUR1 isoform)--the amino acid substitution found in SUR2A at the corresponding position (Ashfield R, Gribble FM, Ashcroft SJ, Ashcroft FM. Identification of the high-affinity tolbutamide site on the SUR1 subunit of the K_ATP channel. Diabetes. 1999 Jun;48(6):1341-7, PMID: 10342826). This suggests that tolbutamide’s cardiotoxic effects might involve other targets like the hERG channel. Interestingly, tolbutamide contains a hydrophobic tail and aromatic rings that align well with the structural features for hERG interaction (Garrido A, Lepailleur A, Mignani SM, Dallemagne P, Rochais C. hERG toxicity assessment: Useful guidelines for drug design. Eur J Med Chem. 2020 Jun 1;195:112290, PMID: 32283295). In contrast, highaffinity sulfonylureas such as glibenclamide and glimepiride, which have additional benzamide moieties, are associated with lower cardiovascular risks (Douros A, Yin H, Yu OHY, Filion KB, Azoulay L, Suissa S. Pharmacologic Differences of Sulfonylureas and the Risk of Adverse Cardiovascular and Hypoglycemic Events. Diabetes Care. 2017, 40:1506-1513, PMID:

28864502). Given these considerations, a comprehensive assessment of Aekatperone’s potential cardiotoxicity is crucial. Future studies involving in silico modeling, in vitro, and in vivo experiments will be essential to evaluate Aekatperone’s interaction with hERG and other offtarget effects. These efforts will help clarify its safety profile. This point has now been added to the Discussion.

Reviewer #2 (Public review):

Summary:

In their study 'AI-Based Discovery and CryoEM Structural Elucidation of a KATP Channel Pharmacochaperone', ElSheikh and colleagues undertake a computational screening approach to identify candidate drugs that may bind to an identified binding pocket in the SUR1 subunit of

KATP channels. Other KATP channel inhibitors such as glibenclamide have been previously shown to bind in this pocket, and in addition to inhibition KATP channel function, these inhibitors can very effectively rescue cell surface expression of trafficking deficient KATP mutations that cause excessive insulin secretion (Congenital Hyperinsulinism). However, a challenge for their utility for treatment of hyperinsulinism has been that they are powerful inhibitors of the channels that are rescued to the channel surface. In contrast, successful therapeutic pharmacochaperones (eg. CFTR chaperones) permit function of the channels rescued to the cell membrane. Thus, a key criteria for the authors' approach in this case was to identify relatively low affinity compounds that target the glibenclamide binding site (and be washed off) - these could potentially rescue KATP surface expression, but also permit KATP function.

Strengths:

The main findings of the manuscript include:

(1) Computational screening of a large virtual compound library, followed by functional screening of cell surface expression, which identified several potential candidate pharmacochaperones that target the glibenclamide binding site.

(2) Prioritization and functional characterization of Aekatperone as a low affinity KATP inhibitor which can be readily 'washed off' in patch clamp, and cell based efflux assays. Thus the drug clearly rescues cell surface expression, but can be manipulated experimentally to permit function of rescued channels.

(3) Determination of the binding site and dynamics of this candidate drug by cryo-EM, and functional validation of several residues involved in drug sensitivity using mutagenesis and patch clamp.

The experiments are well-conceived and executed, and the study is clearly described. The results of the experiments are very straightforward and clearly support the conclusions drawn by the authors. I found the study to provide important new information about KATP chaperone effects of certain drugs, with interesting considerations in terms of ion channel biology and human disease.

Weaknesses:

I don't have any major criticisms of the study as described, but I had some remaining questions that could be addressed in a revision.

(1) The chaperones can effectively rescue KATP trafficking mutants, but clearly not as strongly as the higher affinity inhibitor glibenclamide. Is this relationship between inhibitory potency, and efficacy of trafficking an intrinsic challenge of the approach? I suspect that it may be an intractable problem in the sense that the inhibitor bound conformation that underlies the chaperone effect cannot be uncoupled from the inhibited gating state. But this might not be true (many partial agonist drugs with low efficacy can be strongly potent, for example). In this case, the approach is really to find a 'happy medium' of a drug that is a weak enough inhibitor to be washed away, but still strong enough to exert some satisfactory chaperone effect. Could some additional clarity be added in the discussion on whether the chaperone and gating effects can be 'uncoupled'.

Thank you for the suggestion. A similar question was raised by Reviewer 1, which was addressed above (public review, point 2). We have now added more discussion to clarify this point.

(2) Based on the western blots in Figure 2B, the rescue of cell surface expression appears to require a higher concentration of AKP compared to the concentration response of channel inhibition (~9 microM in Figure 3, perhaps even more potent in patch clamp in Figure 2C). Could the authors clarify/quantify the concentration response for trafficking rescue?

Thank you for bringing up this observation. Indeed, the pharmacochaperone effects of Aekatperone as well as other previously published K_ATP pharmacochaperones require higher concentrations compared to their inhibitory effects on surface-expressed channels. This difference likely stems from the necessity for these compounds to cross the cell membrane and interact with newly synthesized channels in the endoplasmic reticulum, where the trafficking rescue occurs. We estimate that effective pharmacochaperone activity for Aekatperone can be achieved at concentrations ranging from 50 to 100 µM in cells expressing trafficking-deficient K_ATP channel mutants, higher than that required for inhibition of surface-expressed channels (~9 µM IC50). Future work could focus on medicinal chemistry modifications, for example esterification of Aekatperone (Zhou G. Exploring Ester Prodrugs: A Comprehensive Review of Approaches, Applications, and Methods. Pharmacology & Pharmacy, 2024, 15, 269-284). Once inside the cell, the esters would be cleaved by endogenous esterases to release the active compound, ensuring efficient intracellular delivery. This strategy could potentially improve membrane permeability and bioavailability of the compound, which would lower the required concentrations to achieve desired chaperoning effects.

(3) A future challenge in the application of pharmacochaperones of this type in hyperinsulinism may be the manipulation of chaperone concentration in order to permit function. In experiments it is straightforward to wash off the chaperone, but this would not be the case in an organism. I wondered if the authors had attempted to rescue channel function with diazoxide ine presence of AKP, rather than after washing off (ie. is AKP inhibition insurmountable, or can it be overcome by sufficient diazoxide).

Thank you for raising this important point. We have previously shown (Martin GM et al. Pharmacological Correction of Trafficking Defects in ATP-sensitive Potassium Channels Caused by Sulfonylurea Receptor 1 Mutations. J Biol Chem. 2016, 291: 21971-21983, PMID: 27573238) that diazoxide, which stabilizes K_ATP channels in an open conformation, also reduces physical association between Kir6.2 N-terminus and SUR1 as demonstrated by reduced crosslinking of engineered azido-phenylalanine (an unnatural amino acid) at Kir6.2 N-terminal amino acid 12 position to SUR1. Incubating cells with diazoxide did not rescue the trafficking mutants but actually further reduced the maturation efficiency of trafficking mutants. For this reason, we did not include diazoxide during Aekatperone incubation and instead added diazoxide after Aekatperone washout to potentiate the activity of mutant channels rescued to the cell surface. In vivo, we envision testing alternating Aekatperone and diazoxide dosing to maximize functional rescue of K_ATP trafficking mutants.

(4) Do the authors have any information about the turnover time of KATP after washoff of the chaperone (how stable are the rescued channels at the cell surface)? This is a difficult question to probe when glibenclamide is used as a chaperone, but maybe much simpler to address with a lower affinity chaperone like AKP.

Thank you for your thoughtful comment. While we have not yet tested the duration of rescued K_ATP channels at the cell surface following Aekatperone washout, we have conducted similar studies with carbamazepine (Chen PC et al. Carbamazepine as a novel small molecule corrector of trafficking-impaired ATP-sensitive potassium channels identified in congenital hyperinsulinism. J Biol Chem. 2013, 288: 20942-20954, PMID: 23744072), another compound exhibiting reversible inhibitory and chaperone effects (apparent affinity between glibenclamide and Aekatperone). Our previous findings with carbamazepine showed that in cultured cells its chaperone effects were detectable as early as 1 hour and peaked around 6 hours after treatment. Furthermore, when carbamazepine was removed following a 16-hour treatment, the rescue effect persisted for up to 6 hours post-drug removal. These results provide a potential duration of the surface expression rescue effects of reversible pharmacochaperones.

Reviewer #1 (Recommendations for the authors):

The paper is well-written and comprehensive with only very minor essentially copy-editing needed. That said, it would be good if the authors could answer the main points raised above:

(1) What is the relevant Tanimoto parameters and sequence identity (does this mean structural identity) for the identified compounds?

As we answered above in response to the overall assessment, to facilitate the identification of novel hits, molecules with greater than 0.5 Tanimoto similarity in ECFP4 space to any known binders of the target protein and its homologs within 70% amino acid sequence identity were excluded from the commercial library. Additionally, after scoring and ranking the molecules by the AtomNet® technology, a diversity clustering was performed on the top 30,000 molecules using the Butina algorithm with a Tanimoto similarity cutoff of 0.35 in ECFP4 space to minimize selection of structurally similar scaffolds for the final compound buy-list.

(2) Did any of the identified putative binders have inhibitory action on channels? If so, does this give clues to separating chaperoning from inhibitory effects?

Please see response to the same question in the overall assessment above.

(3) Acknowledge that the identified compounds contain sulfonylurea-like moieties, and address why Aekatperone should (or perhaps does not) offer anything advantage over low affinity sulfonrylureas such as tolbutamide?

Please see response to the same question in the overall assessment above.

Reviewer #2 (Recommendations for the authors):

Thank you for assembling the interesting study, which I felt was well designed and communicated. The diverse approaches used in the study, with consistent findings, were definitely a strength. The core findings are also well distilled in the main body of the text, and although there is quite a lot of supplementary information, I felt that it was presented appropriately and well selected in terms of what would be important for readers hoping to learn more. In addition to the questions described above, I only had a few minor editorial issues that could be fixed related to presentation.

(1) Figure 1B. The colours and resolution of the chemical structures are difficult to see clearly and could be improved.

We have revised the figure accordingly.

(2) This is a minor wording point... first sentence of the discussion describes the drugs as pancreatic-selective, when it would be more clear to describe them as selective for the pancreatic isoform of KATP (Kir6.2/SUR1), or perhaps better as 'exhibiting ~4-5 fold selective for SUR1-containing KATP channels vs. SUR2A or SUR2B'.

We have changed the wording as suggested.

(3) As a curiosity (not necessary to do more experiments), but I am curious if the authors know whether there is any meaningful enhancement of trafficking of WT channels by AKP.

All pharmacochaperones we have identified to date including Aekatperone also slightly enhance WT channel surface expression (10-20%).

Reviewing editor recommendations:

(1) Given the modest resolution of the EM reconstruction, it is perhaps not entirely clear how AKP was assigned to the density observed. Specifically, it would be helpful to include a comparison of an AKP-free map and the current AKP map (filtered to a similar resolution) showing slice views of densities in the region around the inferred binding site. This would be very helpful in ascertaining whether the cryoEM reconstruction is an independent validation of the computational and functional experiments or whether the density inference depends on the additional knowledge.

We appreciate the editor’s suggestion. We have now added a Supplemental Figure (Supplementary Figure 7 in the revised manuscript) that compares our AKP-free cryoEM density deposited previously to the EMDB (EMD-26320) and the AKP-bound cryoEM density from this study, with cryoEM density (filtered to the same resolution) superimposed on the structural model.

(2) It could help to mention in brief what is a probable mechanism of AKP inhibition - that is how after binding of AKP, channel opening is restricted. Is it similar to that of other site A ligands?

Based on the strong Kir6.2 N-terminal cryoEM density observed in our AKP map, AKP most likely inhibits K_ATP channels by trapping the Kir6.2 N-terminus in the central cavity of SUR1’s ABC core thus preventing Kir6.2-C-terminal domain from rotating to an open conformation, similar to other ligands that stabilize the Kir6.2 N-terminus-SUR1 interface by binding to site A (such as tolbutamide and AKP), site B (such as repaglinide), or both site A and site B (such as glibenclamide). We have now included this in the revised Results and Discussion sections.

(3) In the context of the MD simulations, do other site A ligands (which from my understanding bind at a similar site) also exhibit similar flexibility as AKP? If there is information available on the flexibility of ligands of varying affinities, bound to the same site, maybe some correlative inferences can be drawn? However, in MD simulation trajectories it is not entirely uncommon for a ligand to simply get trapped in a local energy well. Since the authors have performed significant analysis of their MD results it could be worth mentioning/discussing such phenomena.

Previously published MD data addressing ligand dynamics, such as glibenclamide in the SUR1 pocket (Walczewska-Szewc K, Nowak W. Photo-Switchable Sulfonylureas Binding to ATPSensitive Potassium Channel Reveal the Mechanism of Light-Controlled Insulin Release. J Phys Chem B. 2021, 125: 13111-13121, PMID: 34825567), indicate a certain degree of flexibility. Unfortunately, we cannot directly compare these results, as the simulations were performed without the KNtp domain in the SUR1 cavity, which partially contributes to ligand stabilization. This is an issue we plan to investigate in the future.

In this study, we ran five independent MD simulations, each 500 ns long, resulting in a total of 2.5 μs of simulation time. Across all replicates, the ligand stayed in the same position, with variations mainly in the dynamics of the blurred segment. Considering the length of the simulations and the consistency across the runs, we believe this binding pose is stable and represents a global (or at least highly stable) energy minimum, consistent with the cryo-EM data.

(4) In electrophysiological assays, 10 uM AKP seems to inhibit all currents (Figure 2), but in the Rb+ flux assay ~10 uM appears to be the IC50. The reason for this difference is not entirely clear and it would help to comment on this.

Thank you for noticing the difference. The initial electrophysiological experiments were conducted using the very small amount of AKP provided to us from Atomwise. We estimated the concentration of the reconstituted AKP the best we could, but the concentration was likely to not be very accurate due to difficulty in handling the very small amount of the AKP powder. Subsequent Rb<sup>+>/sup> efflux experiments were conducted using a different, larger batch of AKP we purchased from Enamine. We have now stated this in the Methods section.

La retroalimentación es un punto esencial en el aprendizaje de los estudiantes ya que al recibirla permite concientizar sobre sus errores y/o potencialidades, para mejorar, cambiar o proyectar. Esta puede ser a través de sus profesores, tutores o pares.

Perfil de egreso

La educación en línea, en especial apoyado del modelo tecnopedagógico debe tener un proceso de diseño, el cuál permite la correcta formulación de un programa, curso o taller, complementando la tecnología y la pedagogía que garantice el aprendizaje de los estudiantes de acuerdo a sus necesidades, tiempos y espacios, sin presionarse. Por lo tanto la educación en línea permite esta flexibilidad, adaptación y evaluación de contenidos.

:o

PRINCÍPIO DA LEGALIDADE • CF, art. 5°, XXXIX: "não há crime sem lei anterior que o defina, nem pena sem prévia cominação legal" • CADH, art. 9°: "Ninguém pode ser condenado por ações ou omissões que, no momento em que foram cometidas, não sejam delituosas, de acordo com o direito aplicável" • DUDH, art. 11.2: "Ninguém pode ser culpado por qualquer ação ou omissão que, no momento, não constituam delito perante o direito nacional ou internacional. Também não será imposta pena mais grave do que aquela que, no momento da prática, era aplicável ao ato delituoso".

Casi siempre fiebre

A paración F recuencia E volución C ondiciones exacerbantes T ratamientos previos A tenuantes

A parición F recuencia E volución C ondiciones exacerbantes T ratamiento A tenuantes

Sin embrago, hay que considerar que la exposición a múltiples fuentes de información audiovisual inhibe la lectura y/o cantidad de lo leido.

• Tema: 816
• Processo(s): RE 882.461
• Relator: Min. Dias Toffoli

Título - a) Incidência do ISSQN em operação de industrialização por encomenda, realizada em materiais fornecidos pelo contratante, quando referida operação configura etapa intermediária do ciclo produtivo de mercadoria. - b) Limites para a fixação da multa fiscal moratória, tendo em vista a vedação constitucional ao efeito confiscatório. -

O Tribunal fixou a seguinte tese:

• 1. É inconstitucional a incidência do ISS a que se refere o subitem 14.05 da Lista anexa à LC nº 116/03 se o objeto é destinado à industrialização (IPI) ou à comercialização (ICMS);

• 2. As multas moratórias instituídas pela União, Estados, Distrito Federal e municípios devem observar o teto de 20% do débito tributário", e, no que diz respeito apenas à primeira tese fixada, atribuiu eficácia ex nunc, a contar da data de publicação da ata de julgamento do mérito, para:

• a) impossibilitar a repetição de indébito do ISS em favor de quem recolheu esse imposto até a véspera da referida data, vedando, nesse caso, a cobrança do IPI e do ICMS em relação aos mesmos fatos geradores; ,

• b) impedir que os municípios cobrem o ISS em relação aos fatos geradores ocorridos até a véspera daquela data.

Ficam ressalvadas (i) as ações judiciais ajuizadas até a véspera da mesma data, inclusive as de repetição de indébito e as execuções fiscais em que se discuta a incidência do ISS, e (ii) as hipóteses de comprovada bitributação relativas a fatos geradores ocorridos até a véspera da mencionada data, casos em que o contribuinte terá direito à repetição do indébito do ISS e não do IPI/ICMS, respeitado o prazo prescricional, independentemente da propositura de ação judicial até esse marco. No caso de não recolhimento nem do ISS nem do IPI/ICMS, o Tribunal entendeu pela incidência do IPI/ICMS em relação aos fatos geradores ocorridos até a véspera da publicação da ata de julgamento do mérito.”

Investigar si ello ya existe o existió un proyecto que lo propuso.

Explicación/Motivación ¿Ex ante o ex post?

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

The authors used a subset of a very large, previously generated 16S dataset to:<br /> (1) Assess age-associated features; and (2) develop a fecal microbiome clock, based on an extensive longitudinal sampling of wild baboons for which near-exact chronological age is known. They further seek to understand deviation from age-expected patterns and uncover if and why some individuals have an older or younger microbiome than expected, and the health and longevity implications of such variation. Overall, the authors compellingly achieved their goals of discovering age-associated microbiome features and developing a fecal microbiome clock. They also showed clear and exciting evidence for sex and rank-associated variation in the pace of gut microbiome aging and impacts of seasonality on microbiome age in females. These data add to a growing understanding of modifiers of the pace of age in primates, and links among different biological indicators of age, with implications for understanding and contextualizing human variation. However, in the current version, there are gaps in the analyses with respect to the social environment, and in comparisons with other biological indicators of age. Despite this, I anticipate this work will be impactful, generate new areas of inquiry, and fuel additional comparative studies.

Thank you for the supportive comments and constructive reviews.

Strengths:

The major strengths of the paper are the size and sampling depth of the study population, including the ability to characterize the social and physical environments, and the application of recent and exciting methods to characterize the microbiome clock. An additional strength was the ability of the authors to compare and contrast the relative age-predictive power of the fecal microbiome clock to other biological methods of age estimation available for the study population (dental wear, blood cell parameters, methylation data). Furthermore, the writing and support materials are clear, informative and visually appealing.

Weaknesses:

It seems clear that more could be done in the area of drawing comparisons among the microbiome clock and other metrics of biological age, given the extensive data available for the study population. It was confusing to see this goal (i.e. "(i) to test whether microbiome age is correlated with other hallmarks of biological age in this population"), listed as a future direction, when the authors began this process here and have the data to do more; it would add to the impact of the paper to see this more extensively developed.

Comparing the microbiome clock to other metrics of biological age in our population is a high priority (these other metrics of biological age are in Table S5 and include epigenetic age measured in blood, the non-invasive physiology and behavior clock (NPB clock), dentine exposure, body mass index, and blood cell counts (Galbany et al. 2011; Altmann et al. 2010; Jayashankar et al. 2003; Weibel et al. 2024; Anderson et al. 2021)). However, we have opted to test these relationships in a separate manuscript. We made this decision because of the complexity of the analytical task: these metrics were not necessarily collected on the same subjects, and when they were, each metric was often measured at a different age for a given animal. Further, two of the metrics (microbiome clock and NPB clock) are measured longitudinally within subjects but on different time scales (the NPB clock is measured annually while microbiome age is measured in individual samples). The other metrics are cross-sectional. Testing the correlations between them will require exploration of how subject inclusion and time scale affect the relationships between metrics.

We now explain the complexity of this analysis in the discussion in lines 447-450. In addition, we have added the NPB clock (Weibel et al. 2024) to the text in lines 260-262 and to Table S5.

An additional weakness of the current set of analyses is that the authors did not explore the impact of current social network connectedness on microbiome parameters, despite the landmark finding from members of this authorship studying the same population that "Social networks predict gut microbiome composition in wild baboons" published here in eLife some years ago. While a mother's social connectedness is included as a parameter of early life adversity, overall the authors focus strongly on social dominance rank, without discussion of that parameter's impact on social network size or directly assessing it.

Thank you for raising this important point, which was not well explained in our manuscript. We find that the signatures of social group membership and social network proximity are only detectable our population for samples collected close in time. All of the samples analyzed in  Tung et al. 2015 (“Social networks predict gut microbiome composition in wild baboons”) were collected within six weeks of each other. By contrast, the data set analyzed here spans 14 years, with very few samples from close social partners collected close in time. Hence, the effects of social group membership and social proximity are weak or undetectable. We described these findings in Grieneisen et al. 2021 and Bjork et al. 2022, and we now explain this logic on line 530, which states, “We did not model individual social network position because prior analyses of this data set find no evidence that close social partners have more similar gut microbiomes, probably because we lack samples from close social partners sampled close in time (Grieneisen et al. 2021; Björk et al. 2022).”

We do find small effects of social group membership, which is included as a random effect in our models of how each microbiome feature is associated with host age (line 529) and our models predicting microbiome Dage (line 606; Table S6).

Reviewer #2 (Public review):

Summary:

Dasari et al present an interesting study investigating the use of 'microbiota age' as an alternative to other measures of 'biological age'. The study provides several curious insights into biological aging. Although 'microbiota age' holds potential as a proxy of biological age, it comes with limitations considering the gut microbial community can be influenced by various non-age related factors, and various age-related stressors may not manifest in changes in the gut microbiota. The work would benefit from a more comprehensive discussion, that includes the limitations of the study and what these mean to the interpretation of the results.

We agree and have text to the discussion that expands on the limitations of this study and what those limitations mean for the interpretation of the results. For instance, lines 395-400 read, “Despite the relative accuracy of the baboon microbiome clock compared to similar clocks in humans, our clock has several limitations. First, the clock’s ability to predict  individual age is lower than for age clocks based on patterns of DNA methylation—both for humans and baboons (Horvath 2013; Marioni et al. 2015; Chen et al. 2016; Binder et al. 2018; Anderson et al. 2021). One reason for this difference may be that gut microbiomes can be influenced by several non-age-related factors, including social group membership, seasonal changes in resource use, and fluctuations in microbial communities in the environment”

In addition, lines 405-411 now reads, “Third, the relationships between potential socio-environmental drivers of biological aging and the resulting biological age predictions were inconsistent. For instance, some sources of early life adversity were linked to old-for-age gut microbiomes (e.g., males born into large social groups), while others were linked to young-for-age microbiomes (e.g., males who experienced maternal social isolation or early life drought), or were unrelated to gut microbiome age (e.g., males who experienced maternal loss; any source of early life adversity in females).”

Strengths:

The dataset this study is based on is impressive, and can reveal various insights into biological ageing and beyond. The analysis implemented is extensive and high-level.

Weaknesses:

The key weakness is the use of microbiota age instead of e.g., DNA-methylation-based epigenetic age as a proxy of biological ageing, for reasons stated in the summary. DNA methylation levels can be measured from faecal samples, and as such epigenetic clocks too can be non-invasive. I will provide authors a list of minor edits to improve the read, to provide more details on Methods, and to make sure study limitations are discussed comprehensively.

Thank you for this point. In response, we have deleted the text from the discussion that stated that non-invasive sampling is an advantage of microbiome clocks. In addition, we now propose a non-invasive epigenetic clock from fecal samples as an important future direction for our population (see line 450).

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

Abstract - The opening 2 sentences are not especially original or reflective of the potential value/ premise of the study. Members of this team have themselves measured variation in biological age in many different ways, and the implication that measuring a microbiome clock is easy or straightforward is not compelling. This paper is very interesting and provides unique insight, but I think overall there is a missed opportunity in the abstract to emphasize this, given the innovative science presented here. Furthermore, the last 2 sentences of the abstract are especially interesting - but missing a final statement on the broader significance of research outside of baboons.

We appreciate these comments and have revised the Abstract accordingly. The introductory sentences now read, “Mammalian gut microbiomes are highly dynamic communities that shape and are shaped by host aging, including age-related changes to host immunity, metabolism, and behavior. As such, gut microbial composition may provide valuable information on host biological age.” (lines 31-34). The last two sentences of the abstract now read, “Hence, in our host population, gut microbiome age largely reflects current, as opposed to past, social and environmental conditions, and does not predict the pace of host development or host mortality risk. We add to a growing understanding of how age is reflected in different host phenotypes and what forces modify biological age in primates.” (lines 40-43).

If possible, it would be highly useful to present some comments on concordance in patterns at different levels. Are all ASVs assessed at both the family and genus levels? Do they follow similar patterns when assessed at different levels? What can we learn about the system by looking at different levels of taxonomic assignment?

The section on relationships between host age and individual microbiome features is already lengthy, so we have not added an analysis of concordance between different taxonomic levels. However, we added a justification for why we tested for age signatures in different levels of taxa to line 171, which reads, “We tested these different taxonomic levels in order to learn whether the degree to which coarse and fine-grained designations categories were associated with host age.”

To calculate the delta age - please clarify if this was done at the level of years, as suggested in Figure 3C, or at the level of months or portion months, etc?

Delta age is measured in years. This is now clarified in lines 294, 295, and 578.

Spelling mistake in table S12, cell B4 (Octovber)

Thank you. This typo has been corrected.

Given the start intro with vertebrates, the second paragraph needs some tweaking to be appropriate. Perhaps, "At least among mammals, one valuable marker of biological aging may lie in the composition and dynamics of the mammalian gut microbiome (7-10)." Or simply remove "mammalian".

We have updated this sentence based on your suggestions in line 54. It reads, “In mammals, one valuable marker of biological aging may lie in the composition and dynamics of the gut microbiome (Claesson et al. 2012; Heintz and Mair 2014; O’Toole and Jeffery 2015; Sadoughi et al. 2022).”

A rewrite at the end of the introduction is needed to avoid the almost direct repetition in lines 115-118 and 129-131 (including lit cited). One potentially effective way to approach this is to keep the predictions in the earlier paragraph and then more clearly center the approach and the overarching results statement in the latter paragraph. (I.e., "we find that season and social rank have stronger effects on microbiome age than early life events. Further, microbiome age does not predict host development or mortality.").

Thank you for pointing this out. We have re-organized the predictions in the introduction based on your suggestion. The alternative “recency effects” model now appears in the paragraph that starts in line 110. The final paragraph then centers on the overall approach and the results statement (lines 128-140)

Be clear in each case where taxon-level trends are discussed if it's at Family, Genus, or other level. It's there most, but not all, of the time.

We have gone through the text and clarified what taxa or microbiome feature was the subject of our analyses in any places where this was not clear.

In the legend for Figure 2, add clarification for how values to right versus left of the centered value should be interpreted with respect to age (e.g. "values to x of the center are more abundant in older individuals").

We now clarify in Figure 2C and 2D that “Positive values are more abundant in older hosts”.

Figure 3 - Are Panels A, B, and C all needed - can the value for all individuals not also be overlaid in the panel showing sex differences and the same point showing individuals with "old" and "young" microbiomes be added in the same plot if it was slightly larger?

We agree and have simplified Figure 3. We reduced the number of panels from three to two, and we added the information about how to calculate delta age to Panel A. We also moved the equation from the top of Panel C to the bottom right of Panel A.

Reviewer #2 (Recommendations for the authors):

Dasari et al present an interesting study investigating the use of 'microbiota age' as an alternative to other measures of 'biological age'. The study provides several curious insights which in principle warrant publication. However, I do think the manuscript should be carefully revised. Below I list some minor revisions that should be implemented. Importantly, the authors should discuss in the Discussion the pros and cons of using 'microbiota age' as a proxy of 'biological age'. Further, the authors should provide more information on Methods, to make sure the study can be replicated.

Thank you for these important points. Based on your comments and those of the first reviewer, we have expanded our discussion of the limitations of using microbiota age as a proxy for biological age (see edits to the paragraph starting in line 395).

We have also expanded our methods around sample collection, DNA extraction, and sequencing to describe our sampling methods, strategies to mitigate and address possible contamination, and batch effects. See lines 483-490 and our citations to the original papers where these methods are described in detail.

(1) Lines 85-99: I think this paragraph could be revisited to make the assumptions clearer. For instance, the last sentence is currently a little confusing: are authors expecting males to exhibit old-for-age microbiomes already during the juvenile period?

This prediction has been clarified. Line 96 now reads, “Hence, we predicted that adult male baboons would exhibit gut microbiomes that are old-for-age, compared to adult females (by contrast, we expected no sex effects on microbiome age in juvenile baboons).”

(2) Lines 118-121: Could the authors discuss this assumption in relation to what has been observed e.g., in humans in terms of delays in gut microbiome development? Delayed/accelerated gut microbiome development has been studied before, so this assumption would be stronger if related to what we know from previous studies.

This comment refers to the sentence which originally stated, “However, we also expected that some sources of early life adversity might be linked to young-for-age gut microbiota. For instance, maternal social isolation might delay gut microbiome development due to less frequent microbial exposures from conspecifics.” We have slightly expanded the text here (line 117) to explain our logic. We now include citations for our predictions. We did not include a detailed discussion of prior literature on microbiome development in the interest of keeping the same level of detail across all sections on our predictions.

(3) As the authors discuss, various adversities can lead to old-for-age but also young-for-age microbiome composition. This should be discussed in the limitations.

We agree. This is now discussed in the sentence starting at line 371, which reads, “…deviations from microbiome age predictions are explained by socio-environmental conditions experienced by individual hosts, especially recent conditions, although the effect sizes are small and are not always directionally consistent.” In addition, the text starting at line 405 now reads, “Third, the relationships between potential socio-environmental drivers of biological aging and the resulting biological age predictions were inconsistent. For instance, some sources of early life adversity were linked to old-for-age gut microbiomes (e.g., males born into large social groups), while others were linked to young-for-age microbiomes (e.g., males who experienced maternal social isolation or early life drought), or were unrelated to gut microbiome age (e.g., males who experienced maternal loss; any source of early life adversity in females).”

(4) In various places, e.g., lines 129-131, it is a little unclear at what chronological age authors are expecting microbiota to appear young/old-for-age.

This sentence was removed while responding to the comments from the first reviewer.

(5) Lines 132-133: this statement could be backed by stating that this is because the gut microbiota can change rapidly e.g., when diet changes (or whatever the authors think could be behind this).

We have added an expository sentence at line 123, including new citations. This sentence reads, “Indeed, gut microbiomes are highly dynamic and can change rapidly in response to host diet or other aspects of host physiology, behavior, or environments”.

We now cite:

· Hicks, A.L., et al. (2018). Gut microbiomes of wild great apes fluctuate seasonally in response to diet. Nature Communications 9, 1786.

· Kolodny, O., et al. (2019). Coordinated change at the colony level in fruit bat fur microbiomes through time. Nature Ecology & Evolution 3, 116-124.

· Risely, A., et al. (2021) Diurnal oscillations in gut bacterial load and composition eclipse seasonal and lifetime dynamics in wild meerkats. Nat Commun 12, 6017.

(6) Lines 135-137: current or past season and social rank? This paragraph introduces the idea that it could be past rather than current socio-environmental factors that might predict microbiota age, so the authors should clarify this sentence.

We have clarified the information in this sentence. line 135 now reads, “In general, our results support the idea that a baboon’s current socio-environmental conditions, especially their current social rank and the season of sampling, have stronger effects on microbiome age than early life events—many of which occurred many years prior to sampling.”

(7) Lines 136-137: this sentence could include some kind of a conclusion of this finding. What might this mean?

We have added a sentence at line 138, which speculates that, “…the dynamism of the gut microbiome may often overwhelm and erase early life effects on gut microbiome age.”

(8) Use 'microbiota' or 'microbiome' across the manuscript; currently, the terms are used interchangeably. I don't have a strong opinion on this, although typically 'microbiota' is used when data comes from 16S rRNA.

We have updated the text to replace any instance of “microbiota” with “microbiome”. We use the term microbiome in the sense of this definition from the National Human Genome Research Institute, which defines a microbiome as “the community of microorganisms (such as fungi, bacteria and viruses) that exists in a particular environment”.

(9) Figure 1 legend: make sure to unify formatting; e.g., present sample sizes as N= or n=, rather than both, and either include or do not include commas in 4-digit values (sample sizes).

We have checked the formatting related to sample sizes and the use of commas in 4-digits in the main text and supplement. The formats are now consistent.

(10) Line 166: relative abundances surely?

Following Gloor et al. (2017), our analyses use centered log-ratio (CLR) transformations of read counts, which is the recommended approach for compositional data such as 16S rRNA amplicon read counts. CLR transformations are scale-invariant, so the same ratio is obtained in a sample with few read versus many reads. We now cite Gloor et al. (2017) at line 169 and in the methods in line 517, which reads “centered log ratio (CLR) transformed abundances (i.e., read counts) of each microbial phyla (n=30), family (n=290), genus (n=747), and amplicon sequence variance (ASV) detected in >25% of samples (n=358). CLR transformations are a recommended approach for addressing the compositional nature of 16S rRNA amplicon read count data (Gloor et al. 2017).”  

(11) Lines 167-172: were technical factors, e.g., read depth or sequencing batch, included as random effects?

Thank you for catching this oversight in the text. We did model sequencing depth and batch effects. The sentence starting at line 173 now reads, “For each of these 1,440 features, we tested its association with host age by running linear mixed effects models that included linear and quadratic effects of host age and four other fixed effects: sequencing depth, the season of sample collection (wet or dry), the average maximum temperature for the month prior to sample collection, and the total rainfall in the month prior to sample collection (Grieneisen et al. 2021; Björk et al. 2022; Tung et al. 2015). Baboon identity, social group membership, hydrological year of sampling, and sequencing plate (as a batch effect) were modeled as random effects.”

(12) Lines 175-180: When discussing how these alpha diversity results relate to previous findings, the authors should be clear about whether they talk about weighted or non-weighted measures of alpha diversity. - also maybe this should be included in the discussion rather than the results? Please consider this when revisiting the manuscript (see how it reads after edits).

Richness is the only unweighted metric, which we now clarify in line 181. We opted to retain the interpretation in the text in its original location to maintain the emphasis in the discussion on the microbiome clock results.

(13) Table S1 is very hard to interpret in the provided PDF format as columns are not presented side-by-side. It is currently hard to check model output for e.g., specific families. This needs to be revisited.

We agree. We believe that eLife’s submission portal automatically generates a PDF for any supplementary item. However, we also include the supplementary tables as an Excel workbook which has the columns presented side-by-side.

(14) Line 184: taxa meaning what? Unclear what authors refer to with this sentence, taxa across taxonomic levels, or ASVs, or what does the 51.6% refer to?

We have edited line 191 to clarify that this sentence refers to taxa at all taxonomic levels (phyla to ASVs).

(15) Line 191: a punctuation mark missing after ref (81).

We have added the missing period at the end of this sentence.

(16) Lines 189-197: this should go into the discussion in my opinion.

We have opted to retain this interpretation, now at line 183.

(17) Lines 215-219: Not sure what this means; do the authors mean features were not restricted to age-associated taxa, ie also e.g., diversity and other taxa-independent patterns were included? If so, the rest of the highlighted lines should be revisited to make this clear, currently to me it is very unclear what 'These could include features that are not strongly age-correlated in isolation' means. Currently, that sounds like some features included were only age-associated in combination with other features, but unclear how this relates to taxa-dependency/taxa-independency.

We agree this was not clear. We have revised line 224 to read, “We included all 9,575 microbiome features in our age predictions, as opposed to just those that were statistically significantly associated with age because removing these non-significant features could exclude features that contribute to age prediction via interactions with other taxa.”

(18) Line 403-407: There is now a paper showing epigenetic clocks can be built with faecal samples, so this argument is not valid. Please revisit in light of this publication: https://onlinelibrary.wiley.com/doi/epdf/10.1111/mec.17330

Thank you for bringing this paper to our attention. We deleted the text that describes epigenetic clocks as invasive, and we now cite this paper in line 450, which reads, “We also hope to measure epigenetic age in fecal samples, leveraging methods developed in Hanski et al. 2024.”

(19) Line 427: a punctuation mark/semicolon missing before However.

We have corrected this typo.

(20) Lines 419-428: I don't quite understand this speculation. Why would the priority of access to food lead to an old-looking gut microbiome? This paragraph needs stronger arguments, currently unclear and also not super convincing.

We agree this was confusing. We have revised this text to clarify the explanation. The text starting at line 424 now reads, “This outcome points towards a shared driver of high social status in shaping gut microbiome age in both males and females. While it is difficult to identify a plausible shared driver, one benefit shared by both high-ranking males and females is priority of access to food. This access may result in fewer foraging disruptions and a higher quality, more stable diet. At the same time, prior research in Amboseli suggests that as animals age, their diets become more canalized and less variable (Grieneisen et al. 2021). Hence aging and priority of access to food might both be associated with dietary stability and old-for-age microbiomes. However, this explanation is speculative and more work is needed to understand the relationship between rank and microbiome age.”

(21) Line 434: remove 'be'.

We have corrected this typo.

(22) Line 478: add information on how samples were collected; e.g., were samples collected from the ground? How was cross-contamination with soil microbiota minimised? Were samples taken from the inner part of depositions? These factors can influence microbiota samples quite drastically so detailed info is needed. Also what does homogenisation mean in this context? How soon were samples freeze-dried after sample collection?

We have expanded our methods with respect to sample collection. This text starts in line 483 and reads, “Samples were collected from the ground within 15 minutes of defecation. For each sample, approximately 20 g of feces was collected into a paper cup, homogenized by stirring with a wooden tongue depressor, and a 5 g aliquot of the homogenized sample was transferred to a tube containing 95% ethanol. While a small amount of soil was typically present on the outside of the fecal sample, mammalian feces contains 1000 times the number of microbial cells in a typical soil sample (Sender, Fuchs, and Milo 2016; Raynaud and Nunan 2014), which overwhelms the signal of soil bacteria in our analyses (Grieneisen et al. 2021). Samples were transported from the field in Amboseli to a lab in Nairobi, freeze-dried, and then sifted to remove plant matter prior to long term storage at -80°C.”

(23) Line 480 onwards: were negative controls included in extraction batches? Were samples randomised into extraction batches?

Yes, we included extraction blanks. These are now described in lines 495-500. This text reads, “We included one extraction blank per batch, which had significantly lower DNA concentrations than sample wells (t-test; t=-50, p < 2.2x10-16; Grieneisen et al. 2021). We also included technical replicates, which were the same fecal sample sequenced across multiple extraction and library preparation batches. Technical replicates from different batches clustered with each other rather than with their batch, indicating that true biological differences between samples are larger than batch effects.”

(24) Were extraction, library prep, and sequencing negative controls included? Is data available?

We included extraction blanks (described above) and technical replicates, which were the same sample sequenced across multiple extraction and library preparation batches. Technical replicates from different batches clustered with each other rather than with their batch, indicating that true biological differences between samples are larger than batch effects.

We have updated the data availability statement to read, “All data for these analyses are available on Dryad at https://doi.org/10.5061/dryad.b2rbnzspv. The 16S rRNA gene sequencing data are deposited on EBI-ENA (project ERP119849) and Qiita (study 12949). Code is available at the following GitHub repository: https://github.com/maunadasari/Dasari_etal-GutMicrobiomeAge”.

(25) Line 562: how were corrected microbiome delta ages calculated? Currently, the authors state x, y and z factors were corrected for, but it is unclear how this was done.

The paragraph starting at line 577 describes how microbiome delta age was calculated. We have made only a few changes to this text because we were not sure which aspects of these methods confused the reviewer. However, briefly, we calculated sample-specific microbiome Dage in years as the difference between a sample’s microbial age estimate, age_m from the microbiome clock, and the host’s chronological age in years at the time of sample collection, age_c. Higher microbiome Dages indicate old-for-age microbiomes, as age_m > age_c, and lower values (which are often negative) indicate a young-for-age microbiome, where age_c > age_m (see Figure 3).

(26) Line 579: typo 'as'.

We have corrected this typo.

Works Cited

Altmann, Jeanne, Laurence Gesquiere, Jordi Galbany, Patrick O Onyango, and Susan C Alberts. 2010. “Life History Context of Reproductive Aging in a Wild Primate Model.” Annals of the New York Academy of Sciences 1204:127–38. https://doi.org/10.1111/j.1749-6632.2010.05531.x.

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Reply to the reviewers

Reviewer #1

Evidence, reproducibility and clarity

In this manuscript, the authors highlight the importance of the Golgi apparatus during SARS-CoV-2 infection. Specifically, using different compounds able to alter Golgi structure and function, the authors show a strong reduction in SARS-CoV-2 infection rate. In particular it is interesting to observe that treatments of 24 hrs with BFA strongly impair viral infection, highlithing the importance of Golgi function for this virus. Albeit the time of treatment is different. this observation is in contrast with previous studies on related coronaviruses (Ghosh et al., 2020) that did not observe any effect upon treatment with BFA. This might imply that SARS-CoV-2 relies more on conventional trafficking pathways respect to other coronaviruses which, under certain conditions, favour different trafficking routes.

We thank the reviewer for the positive comments. Indeed, our results with BFA treatment for 24 hours are inconsistent with previous studies based on the prototype coronavirus MHV (Ghosh et al., 2020). To validate this observation, we have now performed new experiments with BFA treatment for 4, 6, and 8 hours, matching the time points used in the previous study (Ghosh et al, 2020). Our new results show that BFA treatment at these early time points significantly inhibits SARS-CoV-2 assembly and secretion, as measured by immunoblotting and TCID50 assays, without reducing intracellular viral RNA levels, which serve as a marker of genome replication. This implies that Golgi function and an intact ER-to-Golgi trafficking route are required for SARS-CoV-2 assembly and secretion. These new results are now presented as new Fig. 2C-H.

The authors additionally observed that viral infection increases TGN46 levels while decreasing GRASP55 levels. To dissect the role of TGN46 and GRASPR55, the authors performed several infection studies in cells in which the levels of the two proteins were modulated either by overexpression (GRASP55) and/or siRNA-mediated knock-down (GRASP55 and TGN46). Those approaches suggest that GRASPR55 overexpression, a protein essential for Golgi stack formation, decelerates viral trafficking and inhibits viral assembly while its depletion reverses the effects. On the other hand, TGN46 knock-down impairs viral trafficking but not assembly. Overall the study clearly shows the importance of the Golgi during SARS-CoV-2 and also shows that modulation of those two factors affect viral infection.

We appreciate the reviewer's accurate summary of our work and positive comments.

However the claims that specifically the trafficking (TGN46) and trafficking and assembly (GRASP55) are not fully substantiated. Regarding GRASP55, the authors state that viral infection decreases GRASPR55 levels and this results in Golgi fragmentation. However GRASPR55 levels decrease is shown at 24 hrs post infection while Golgi fragmentation occurs as early as 5 hrs. Thus there might be no direct casual effect between the two effects.

We agree with the reviewer that GRASP55 downregulation is unlikely to be the only reason for Golgi fragmentation in the infected cells. In our results, 5- or 8-hour post infection caused only mild Golgi fragmentation (Fig. S6D), while 24 hours post infection led to severe Golgi fragmentation. On the other hand, GRASP55 is likely to play a relevant role as SARS-CoV-2 induced Golgi fragmentation can be partially rescued by exogenous GRASP55 expression (Fig S6C). We have modified the text in lines 303-305 accordingly to acknowledge the possibility that other factors also contribute to Golgi fragmentation in infected cells.

Additionally, the authors show that overexpression of GRASP55 rescue Golgi fragmentation, as observed by imaging, however is not clear if only infected cells where quantified and if they had the same level of infection.

Yes, only infected cells with either GFP or GRASP55-GFP expression were quantified. The viral infection rate was significantly lower in GRASP55-GFP expressing cells compared to GFP expressing cells (Fig 5A-B).

The authors exclude and effect on entry based on experiment on Spike expressing pseudovirus in 293-ACE2, however they also clearly observe reduction of ACE2 on the membrane of GRASPR55 expressing cells (Fig S6B). Thus how can they explain this discrepancy and how ca defect in entry can be fully marked out in these cell lines?

We thank the reviewer for pointing this out. This discrepancy is likely due to the different systems used in the two experiments.

In the pseudovirus entry assay, ACE2 was exogenously expressed in 293T cells and GRASP55 expression did not show any effect on the viral entry efficiency. In contrast, Huh7-ACE2 cells were selected for a high surface expression of ACE2. While GRASP55 expression reduces surface ACE2 levels as shown in our cell surface biotinylation assay, we believe that the surface ACE2 levels in GRASP55-expressing cells remain sufficient to support viral entry. To further investigate whether GRASP55 expression affects viral entry using authentic SARS-CoV-2, we performed RT-qPCR analysis of intracellular RNA level of the spike, N, and RdRp in both GFP and GRASP55-GFP expressing cells 4 hours post infection (new Fig 5D). Our results show that GRASP55 expression does not affect SARS-CoV-2 entry efficiency, even though it reduces ACE2 surface expression levels.

It is not clear to which process the authors refer to when they write about "viral trafficking". Is it virion trafficking or viral proteins trafficking? The two process are linked but are not the same. This oversemplification can be misleading. For instance the authors show that overexpression of GRASP55 decreases Spike protein on the plasma membrane and its depletion increases S protein incorporation into psudoviruses. However it was shown that in infected cells S protein is mainly retained at the ERGIC by M and E (Boson et al., 2021) where viral assembly occurs. Thus an increase in S trafficking on the PM does not correlate with an increase in virion trafficking,

We agree with the reviewer that our use of the term "viral trafficking" is imprecise and we have changed this throughout the manuscript to be more specific. S trafficking to the PM may not necessarily be equal to an increase in virion trafficking and thus have rephrased these terms in our writing accordingly.

We acknowledge that our cell surface biotinylation assay results only demonstrate that GRASP55 overexpression slows down spike protein trafficking to the PM. We have accordingly also examined viral protein and infectious particle secretion into the culture medium as a more direct readout of virion trafficking (new Fig 2E, 2H, 6K, and 7P).

Finally, we have removed all of the data describing spike incorporation into pseudoviruses as we acknowledge that plasma membrane assembly of lentiviruses is not a good model for SARS-CoV-2 assembly.

...and ultimately, the data provided do not fully support the authors claim on a modulation of "virion trafficking" in response to GRASP or TGN46 changes, since no experiments clearly show a change in virions secretion.

In response to the above comment, we provide the following clarification: Our Western blotting, TCID50 assay, and plaque assay results collectively demonstrate that SARS-CoV-2 virion secretion is reduced in GRASP55 expressing cells (new Fig 5E-M) and in TGN46-depleted cells (new Fig 7F-H, 7L-N). Conversely, viral assembly and secretion appear to be increased in GRASP55-depleted cells (new Fig 6A, 6E-I) at 24 hpi. Furthermore, within a single viral secretion cycle (10 hpi), GRASP55 depletion increased viral secretion (new Fig 6K), while TGN46 depletion reduced viral secretion (new Fig 7P). These findings strongly support the conclusion that GRASP55 and TGN46 modulate viral secretion.

Importantly, the authors do not rule out potential effects of their perturbations on genome replication. The only experiment that they perform in this direction is presented in Fig. S7B, where the authors show similar percentage of infected cells at early stage upon silecing of GRASPR55. The experiment suggests that productive entry is similar in these conditions, but quantification of intracellular viral genome could exclude a change in viral replication. If no changes in viral replication are observed, the authors could verify an increase in particles secretion by collecting supernatants from the early time points and performing plaque assays and quantification of viral genomes by qRT-PCR, to prove that modulation of GRASPR55 indeed promote SARS-CoV-2 trafficking.

We thank the reviewer for the excellent suggestions. In response, we performed RT-qPCR analysis in GRASP55-expressing and TGN46-depleted cells at 4 hpi to compare the viral genome replication process. Additionally, we performed western blotting analysis and released viral titer assay of the culture media from both GRASP55-depleted and TGN46-depleted cells at 10 hpi to investigate virion release. Our new results show that GRASP55 depletion increases viral secretion (new Fig. 6K), while TGN46 depletion reduces viral secretion (new Fig. 7P). Furthermore, GRASP55 expression and TGN46 depletion do not perturb viral genome replication (new Fig. 5D and new Fig. 7R).

Finally, whenever reduction of viral infection is observed upon cell partubation, a robust analysis of cell viability should be presented to exclude pleiotropic effects. Expecially in presence of multiple pertubation that might affect cell metabolism. The authors should carefully control cell viability and growth in response to depletion of TGN46 and GRASP55.

We thank the reviewer for the excellent suggestions, which were also pointed out by reviewer #3. To address this, we performed the LDH cytotoxicity assay under SARS-CoV-2 infection conditions with TGN46 depletion and GRASP55 depletion/expression (new Fig. 5C, 6L, 7Q). Our new results show that no significant cell death was induced by TGN46 depletion, GRASP55 depletion/expression, or other perturbations.

Minor: show data on viability of the drug and add the relative section in Material and Methods.

We performed LDH assays of SARS-CoV-2 infected Huh7-ACE2 cells treated with 9 small molecules, and LDH release levels were similar across all treatments (new Fig. S3C). Additionally, a CellTiter Glo viability assay of 293T-ACE2 cells did not show any significant effect of cell viability with small molecule treatment (new Fig S3F). Detailed descriptions of these assays have been included in the Material and Methods section.

Figure 3A: should read spike and not nucleocapsid eported for SARS-CoV-2

Fig. 3A labeling is correct - cells were labeled with antibodies for GRASP65 (rabbit) and for nucleocapsid (mouse).

Lack of inhibition with camostat correlates with lack of TMPRSS2 in the Huh7. The sentence seems to be too general while in this case the effect is clearly cell specific. Similarly, the importance of the lysosome in viral entry is restricted to cells lacking TMPRSS2 and cannot be generalized since CQ, for example, does not work in Calu-3 cells that express TMPRSS2 cells.

We agree with the reviewer and have added one sentence: The relative smaller effect of camostat mesylate observed here, compared to previous studies (Hoffmann et al, 2021), might be due to the use of different cell lines across studies in lines 182-184. We also discussed the discrepancy of CQ treatment between our Huh7-ACE2 cells and Calu-3 cells (Hoffmann et al, 2020) in lines 466-473.

Typo: Fig S3B - Y axis should reat viral not vrial

Thank you - we have corrected this.

S3C: concentrations of the compound used in the assay should be reported. Was a viability assay performed also in the 293T-ACE2 cell line?

We thank the reviewer for the suggestion. We have added the concentration information to the legend in Fig. S3E "Cell entry assay of 293T or 293T-ACE2 cells by SARS-CoV-2 Spike pseudotyped lentivirus for 24h in the presence of indicated molecules at the same concentrations as in Fig. 2A." Additionally, we performed a CellTiter Glo assay to assess the viability of 293T-ACE2 cells treated with the 9 molecules. The results demonstrate that treatment with these 9 molecules does not alter cell viability (Fig. S3F).

Significance

Overall, the major strenght of the manuscript is that it has clarified the importance of the Golgi during SARS-CoV-2 infection. The drugs screening demonstrate that for SARS-CoV-2 the conventional secretion seems to have major role respect to other secretory routes observed for other coronaviruses. Also it is clear that the two factors identified by the authors have a role in viral infection, however the major limitation is that the authors failed to clearly highlight which step/s of the viral life cycle are modulated upon GRASP55 and TGN46 perturbatio. Expecially the claims on "trafficking" is not fully substantiated, since the only experiment in this direction is the transport of Spike protein on the plasma membrane upon GRASPR55 overexpression. It is risky to conclude that the trafficking of a single protein reflect the intracellular trafficking of the virions.

Several of the finding presented in the first part of the manuscript have been already previously reported (for example the fragmentation of the Golgi upon SARS-CoV-2 infection), however the role of GRASP55 and TGN46 in SARS-CoV-2 infection has been reported here for the first time. This manuscript can be of interest for a broad audience considering the topic (cell biology, host-pathogen interactions and molecular virology)

My expertise reside in the field of molecular virology, expecially in the contest of the mechanisms of viral replication and host-pathogen interactions.

We thank the reviewer for the overall positive comments and excellent suggestions. We hope that our new results have convincingly demonstrated that viral trafficking is regulated by GRASP55 and TGN46.

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

Summary: In this study, Zhang and colleagues address the impact on SARS-CoV-2 infection on the morphology of the Golgi apparatus and convincingly demonstrate a fragmentation of this organelle in infected cells. Conversely, they show that the modulation of TGN46 or GRASP55 expressions, two components of this organelle impact SARS_CoV-2 replication. By monitoring the relative levels of viral Spike and nucleocapsid in the cell supernatants, they conclude that GRASP55 regulates particle assembly and trafficking while TGN46 controls only secretion. The study was generally well performed, and the quality of the microscopy and western blot data is good. It was appreciated that all the phenotypes were robustly quantified. I believe that this study is potentially interesting and relevant for the SARS-CoV-2 community since providing an extensive characterization of the interplay between SARS-CoV-2 and the Golgi apparatus.

We thank the reviewer for the positive comments.

However, as described below, I have some concerns regarding the interpretations of some of the key conclusions. Moreover, the fact that it was already described by several groups that Golgi is a key machinery used for SARS-CoV-2 virion assembly (ERGIC) and secretion dampens my enthusiasm about the study, especially without clear molecular mechanisms about the interplay between SARS-CoV-2 proteins and TNG46/GRASP55.

We rephrased some sentences following the reviewer's suggestions. Although it was believed that SARS-CoV-2 is assembled at the ERGIC, there has been significant controversy surrounding the virion secretion pathway. Our results strongly support that SARS-CoV-2 virions traffic through the Golgi apparatus and that an intact ER-to-Golgi trafficking pathway is essential for SARS-CoV-2 assembly and secretion. Manipulation of two Golgi-resident proteins, GRASP55 and TGN46, significantly regulates SARS-CoV-2 secretion. Interestingly, GRASP55 regulates both assembly and secretion of SARS-CoV-2, while TGN46 exclusively modulates viral secretion. This is consistent with their subcellular localization, as GRASP55 is localized to the medial/trans Golgi, whereas TGN46 is localized to the TGN. We hope that our new experimental results (Figs. 2C-H, 5C-D, 6J-L, and 7O-R) have addressed all concerns from the reviewer. Identification of downstream protein targets involved in TGN46/GRASP55-mediated modulation of SARS-CoV-2 trafficking will be the focus of our future studies.

Major comments: -All the assays have been performed in liver-derived Huh7 cells (overexpressing SARS-CoV-2 receptor) ACE2 (for infection) or kidney 293 cells (for pseudotyped HIV entry assays). However, no conclusion was validated in lung-derived cells (like A549-ACE2, Calu-3 or primary cells), which would be important since the respiratory tract is the main target of SARS-CoV-2

In our study, Huh7-ACE2 cells are sorted for the high expression of endogenous ACE2 protein, and we did not overexpress ACE2 protein. Also, the liver has been reported to be a site of SARS-CoV-2 infection in humans (Barnes, 2022). We did use A549 and Calu-3 cells in pilot experiments; A549 cells displayed infection rates that were too low for our purposes, and Calu-3 cells showed both low infection rates and relatively disorganized Golgi in the absence of viral infection. We were able to add new IF results from Calu-3 cells. Consistent with our findings in Huh7-ACE2 cells, SARS-CoV-2 infection disrupts Golgi structure and alters protein levels of TGN46 and GRASP55 in Calu3 cells (new Fig. S5R-W). We also confirmed GRASP55 downregulation and TGN46 upregulation in VeroE6 cells (Fig S5K-N).

-Fig2: The impact of the drugs on replication was assessed by measuring the % of infected cells. At 24 hpi, I am unsure about what this value is supposed to measure (the whole life cyle, intracellular replication or spread?), especially since it is not indicated when the drugs were added to the cells. Was it during, before or after the infection? This information should be provided.

Fig. 2 refers to infection, not replication. We agree that infection encompasses multiple steps of the viral cycle. In our experiments, cells were treated with the drugs immediately before viral infection. We have added the information into the Fig. 2 legend.

If the "Golgi" drugs impact egress only (as inferred by the genetic modulation phenotypes), I would expect that at this early time point, the % of infection would not drastically change (as well as intracellular RNA) but that the extracellular infectious titers would decrease. Plaque assays (or TCID50 assays) and RT-qPCR on intracellular viral RNA should be conducted to better understand the impact of drug treatments.

This is a great suggestion! As the reviewer expected, our new BFA time-point assay shows that at early time points, the intracellular RNA levels for S, N and RdRp are not reduced. However, the extracellular N protein (measured by WB) and virial titer (measured by TCID50 assay), which serve as readouts for virion secretion, are significantly decreased (new Fig. 2C-H).

On page 10, it is said that the virus makes three cycles of replication within 24 hours following infection. On what data is this based? This seems a lot. If this is true (and shown in Huh7-ACE2 cells), does the assay of figure 2 measure spread in general? More importantly, despite mentioned, the cell viability data are not provided. It is important to show them to ensure that these concentrations of drugs are not toxic at the tested concentrations.

It has been reported that a single cycle of SARS-CoV-2 infection is approximately 8 hours (Eymieux et al, 2021). Therefore, Fig. 2 represents a multicycle infection, reflecting a composite measure of viral infection and spread. Under the microscope, we did not observe dramatic cell death at the tested concentration. To further assess cytotoxicity, we performed a cell toxicity assay for the 9 small molecules that inhibit viral infection of Huh7-ACE2 cells. The results show that no or minor cell death was observed with all these compounds (Fig. S3C).

-I appreciated the extensive confocal microscopy analysis performed by the authors, which seems of high quality and overall, very convincing. They clearly show that SARS-CoV-2 infection induces the fragmentation of the Golgi apparatus although it was reported by others before as mentioned by the authors.

We thank the reviewer for the positive comments. We agree that Golgi fragmentation was observed during SARS-CoV-2 infection, as we mentioned. However, our study provides a comprehensive and systematic analysis of the entire host cell endomembrane system in the response to viral infection.

However, it was hard for me to make the functional link between these data and those related to GRASP55 and TGN46 overexpression/knockdown. First, the authors should assess the morphology of the Golgi apparatus in Huh7-ACE2 when GRASP55 is knocked down/out or when TGN46 is overexpressed. Second, in these 2 conditions that favor replication, it should be assessed whether this correlates with Golgi fragmentation. Even if this was probably shown before, it is relevant to show that these genetic modulations induce Golgi reshaping in this particular cell type by confocal microscopy (and ideally electron microscopy).

Thank you for the suggestion. We performed IF analysis to assess Golgi morphology in Huh7-ACE2 cells under conditions of GRASP55 knockdown or TGN46 overexpression. Our results show that GRASP55 depletion disrupts Golgi structure (Fig. S7D), whereas TGN46 expression does not significantly alter the Golgi morphology (Fig. S8D).

-The fact that GRASP55-GFP expression decreases in 293T the cell surface levels of ACE2, the receptor of Spike (Fig S6), raises concern that the effect of GRASP55 is not specific to the virus and suggests that the whole secretory pathway is altered, while an impairment of virus entry should be expected in this cell line. Is there a similar trend in Huh7-ACE2?

Reviewer 1 raised a similar question regarding viral entry efficiency. Fig. S6B, performed in Huh7-ACE2 cells, shows that GRASP55-GFP expression also decreases ACE2 surface level in these cells. To further assess whether GRASP55 expression affects viral entry, we performed RT-qPCR analysis of viral RNA at early time points of infection. We found that authentic SARS-CoV-2 entry efficiency was not altered by GRASP55 expression (new Fig. 5D). Although GRASP55 overexpression does alter the secretory pathway, we want to point out that SARS-CoV-2 infection downregulates endogenous GRASP55 expression. We have used GRASP55 overexpression as a probe to assess the effects of GRASP55 on the secretory pathway and on SARS-CoV-2 virion trafficking, but this does not actually reflect what is observed in SARS-CoV-2 infection.

In addition to addressing the functionality of the secretory machinery in Huh7-ACE2, it would be relevant to repeat the cell surface labelling in the context of pseudotyped virus production with other viral envelopes such as VSV G protein or HIV gp41/gp120. If the phenotype is specific to Spike trafficking, the cell surface abundance of these alternative viral proteins should not be impacted by GRASP55 overexpression. Otherwise, this would indicate a general effect of on the secretory pathway. Besides, since HIV Gag is directed directly to the plasma membrane during particle assembly without entering the secretory pathway, I am not convinced that upstream alteration on nucleocapsid assembly at the ERGIC should be excluded. Indeed, changes on the S/N ratios are generally mild and I feel that this cannot explain the phenotypes in the extracellular infectious titers.

We have removed the original figure because we acknowledge that HIV Gag is directed directly to the plasma membrane, which is different from the trafficking of SARS-CoV-2 spike protein. We appreciate the reviewer's recognition of the difference in extracellular infectious titers between GFP and G55-GFP expressing cells. We hypothesize that GRASP55 expression not only reduces the number of spikes on each virion but also inhibits the secretion of SARS-CoV-2, resulting in a significantly lower extracellular infectious titer. We agree that it would be interesting to test whether GRASP55 expression affects viral production with other viral envelopes. However, this is beyond the scope of the current study and represents a promising direction for future research.

More generally, the comparison between trafficking and assembly should be better assessed and not simply based on extracellular N and S levels. It was hard to see the differences between the two in terms of phenotypes. The authors should at least measure the intracellular infectivity upon TGN46 and GRASP55 knock/down and overexpression as well as intracellular vRNA abundance as a readout of RNA replication (which is anticipated to remain unchanged).

We thank the reviewer for the valuable suggestions. We performed RT-qPCR analysis of Spike, N, and RdRp at early time points of infection. The new results show that neither GRASP55 expression (new Fig. 5D) nor TGN46 depletion (new Fig. 7R) affects viral RNA abundance at an early infection timepoint (4 hpi). Also, we found that GRASP55 depletion increased intracellular infectivity (new Fig. 6J) while TGN46 depletion did not affect intracellular infectivity (new Fig. 7O), suggesting that GRASP55 modulates viral assembly but TGN46 does not.

-Finally, mechanistic insight about the viral determinants regulating the morphology of the Golgi would significantly strengthen the study.

Fig S6 shows that S expression decreases ACE2 surface levels? If so, could some S mutants be tested? Does it correlate with Golgi fragmentation? Do other viral structural proteins contribute to Golgi morphological alterations?

We thank the reviewer for the suggestions. These are indeed interesting experiments, but we believe that investigating viral determinants of Golgi fragmentation should be pursued by future studies.

In the same line of idea, how GRASP55 and TGN46 regulate replication. The link with Golgi morphology is unclear. Are these proteins hijacked by SARS-COV-2?

Our new data in this revised manuscript more clearly define the stages in the viral infection cycle that are modulated by GRASP55 and TGN46. New Fig. 5D and Fig. 7R show that neither GRASP55 nor TGN46 affects viral entry or early viral replication. However, GRASP55 perturbation modulates viral assembly and secretion, while TGN46 perturbation affects virion secretion but not assembly. Fig. S6C shows that GRASP55 overexpression in the presence of the virus partially rescues Golgi fragmentation. The mechanisms by which GRASP55 and TGN46 are hijacked by SARS-CoV-2 will be explored in the future studies.

Page 13 mentions some relevant mutants that could be assessed in this context and provide mechanistic insights.

It would be interesting to investigate the effects of GRASP55 mutants or specific domains on SARS-CoV-2 trafficking, which we plan to explore in future studies.

Minor comments: -The signal of calreticulin in Fig. S1 is too low to appreciate it distribution.

We have increased the intensity of calreticulin staining for both uninfected and infected cells in parallel in Fig. S1. Thank you.

-Fig 4K, Q: The differences in LC3 forms levels are not convincing. These results do not allow to draw any conclusion about autophagy, especially considering that this was done at steady-state and that the autophagic flux was not measured. Indeed, a bafilomycin A treatment control would be required to measure the real induction of autophagosomes. Lysosomal degradation inhibition allows the detection of LC3 accumulation.

We agree that additional experiments are needed to demonstrate autophagic flux alteration by SARS-CoV-2. We observed an increase in LC3II/LC3I ratio in infected cells at steady state and did not explore this further, since this is not our main focus of this study. Therefore, we have removed the LC3 blots and quantification from Figs. 4 and S5.

-In the GRASP55 overexpression and TGN46 knockdown studies, associated cell viability should be measured to control that that these genetic manipulations do not induce any cytotoxicity which may impact viral replication.

We appreciate the reviewer's suggestions. We performed the LDH cytotoxicity assay under SARS-CoV-2 infection with TGN46 depletion or GRASP55 expression. Our new results show that TGN46 depletion or GRASP55 depletion/expression did not induce significant cell death (Figs. 5C, 6L, and 7Q).

-The authors should test the impact of GRASP55 and GRASP65 knock-out on SARS-CoV-2 replication

Investigating the genetic GRASP55 knockout effect on SARS-CoV-2 replication would be valuable. However, ACE2 protein expression in our Huh7-ACE2 cells decreases with cell passages, making knockout construction on this background impractical due to low ACE2 levels and poor viral infection rates. We believe that both our GRASP55 overexpression and depletion assays sufficiently support its role in SARS-CoV-2 trafficking. Future studies will explore GRASP55 knockout in different cell lines.

-The authors should provide more details about the USA-WA1/2020 isolate in the Methods section. Is it related to the "Wuhan" strain or the variant which spread globally in early 2020 (with D614G mutation in Spike).

USA-WA1/2020 was isolated from an oropharyngeal swab from a patient who returned from China and developed COVID-19 on January 19, 2020, in Washington, USA. It is related to the "Wuhan" strain but does not have D614G mutation in spike. Additional details have been added to the Methods section.

-Fig 8: The combined modulation of GRASP55 and TGN46 expressions does not really seem additive to me since a 70% decrease of either protein modulation is observed while the combined condition brings this value to 75% in TCID50 assays. This does not bring much insight to the study in my opinion. I would suggest that the authors consider removing this figure.

We agree with the reviewer's recommendation and have removed Fig. 8.

Reviewer #2 (Significance (Required)):

General assessment and advance: The study was generally well performed, and the quality of the microscopy and western blot data is good. It was appreciated that all the phenotypes were quantified extensively. However, I have some concerns regarding the interpretations of some of the key conclusions. Moreover, the fact that it was already described by several groups that Golgi is a key machinery for SARS-CoV-2 virion assembly (ERGIC) and secretion dampens my enthusiasm about the study. In addition, the antiviral activity of several tested drugs was also reported elsewhere. A clear mechanism of how SARS-CoV-2 induces a fragmentation of the Golgi would strengthen the study. In the same line of idea, it is unclear how TGN46 and GRASP55 regulate the late steps of the life cycle. The link between SARS-CoV-2-induced Golgi fragmentation and TGN46/GRASP55 is unclear. In my opinion, the data did not allow to clearly discriminate between virion assembly and egress. I was not convinced that it was not simply due to a general disruption of the secretory pathway (as attested by ACE2 down regulation upon GRASP55 overexpression).

Targeted audience: This study will be of high interest for molecular virologists (not only working on SARS-CoV-2) but could be very well fit into the scope of molecular/cell biology-focused generalist journals

Reviewer expertise: Molecular virology, virus-host interactions (especially involving membranous organelles), SARS-CoV-2, RNA viruses

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

Summary:

Zhang et al. demonstrated in this study that the Golgi apparatus and many other organelles are disturbed by SARS-CoV-2 infection. They focused on the Golgi apparatus and especially on TGN46 and GRASP55 which are both affected differently in their level of expression by the SARS-CoV-2 infection. TGN46 is overexpressed while GRASP55 is decreased in expression. Through different methods overexpression or depletion, the authors nicely demonstrated that modulation of both proteins either increased or decreased particles production. They demonstrated that in absence of GRASP55, SARS-CoV-2 release is increased in the medium. On the contrary, depletion of TGN46 decreases the secretion of SARS-CoV-2 particles.

We thank the reviewer for the accurate summary of our work.

Major comments:

Figure 1: The authors demonstrated that SARS-CoV-2 expression affected the morphology of multiple organelles. Although the results are clear, my concern was that the MOI=1 was really high which indeed would affect the whole cell. To have a less drastic effect on the cell, I would suggest realizing the visualization of some organelles (Golgi, EEA1, Rab7 for example) at a lower MOI=0.1. In addition, it would be nice to verify with a live-dead assay with the MOI=1 if after 24h the cells are still alive, which will confirm that these disturbances are not caused by cells in process of dying.

We thank the reviewer for the excellent suggestions. Investigating how SARS-CoV-2 reshapes subcellular organelles at low MOI (e.g., 0.1) and at different time points would be interesting but is beyond the scope of our study. However, we have performed LDH assay at MOI=1, 2 and 3 for 24 hours to assess cell death. Our results show that LDH release was similar across these conditions (Fig. S5R). We also performed RT-qPCR analysis of Spike, N, and RdRp at early time points of infection. The new results show that neither GRASP55 expression (new Fig. 5D) nor TGN46 expression (Fig. 7R) affects viral RNA abundance at an early infection timepoint (4 hpi).

Figure 2: The results indicated in that panel are really nice. However, the addition of a virus with drugs could increase the proportion of cell death. For the Figure 2C, I propose that the author use a LDH assay to prove that the decrease in infection is not caused by cell death. In addition, a RT-qPCR would be more appropriate to indicate the infection rate and support the microscopy data.

We thank the reviewer for the positive feedback and suggestions. As recommended, we performed an LDH assay to assess cytotoxicity under 9 small molecules treatment of infected cells. Additionally, we performed RT-qPCR analysis for the BFA time-point treatment assay. No significant cell death was observed under these conditions (new Figs. 2D, and S3C).

Figure 3: The authors should have been consistent and add spike instead of nucleocapsid for GalT. According to the figures, Spike seemed to co-localize more with GM130 than Golgin 245. Data analysis of colocalization between Spike and GM130 should be performed to complete the observation. Are no colocalizations of Spike observed with the other Golgi markers?

We agree with the reviewer that it was ideal if spike and GalT were co-stained. Unfortunately, both our spike antibody and GalT antibody are from rabbit, so co-staining could not be done as GM130/spike. We performed colocalization analysis between Spike and GM130, and the results show that GRASP55 expression did enhance Spike and GM130 colocalization to some extent (new Fig. S6E-F). We only co-stained spike with GM130 and Golgin-245 due to the antibody availability.

Figure 4K: While all the experiments were performed at MOI=1, why is the authors using MOI=2 for the immunoblots. Did they have a different result in protein expression for MOI=1 in HuH cells? if so they should show a blot indicating this result.

We did not perform WB to assess protein expression at MOI=1, but our cell toxicity assay showed that there is no significant difference between MOI=2 and MOI=1.

Figure 5: Viral infection should be indicated using RT-qPCR data analysis to support the microscopy observations.

We performed RT-qPCR analysis (new Figs. 2F, 5D, and 7R) and found that BFA treatment did not reduce viral RNA levels at all three time points. Also, GRASP55 expression and TGN46 depletion did not inhibit viral genome RNA levels within one viral infection cycle. Additionally, our new TCID50 assay results support our microscope observation (new Fig. 7O-P). Thanks for the suggestion.

Figure 6: The authors should look at the trafficking of ACE2 and TfR in case of GRASP55 depletion like they did in case of GRASP55 overexpression. It could demonstrate if the virus is using trafficking pathways that are common to the one used by some host receptors to reach the plasma membrane.

Thanks for the excellent suggestion. We performed cell surface biotinylation assay of control and GRASP55-depleted cells. We found that ACE2 and TfR receptor displayed a similar reduction on the cell surface (Fig. S7C), consistent with previous findings that GRASP55 depletion induced Golgi fragmentation and accelerated global conventional protein secretion.

Figure 7: Viral infection assay should also be performed by RT-qPCR. Figure 7H: The immunoblots conditions were performed at MOI=3 this time. The authors should indicate why they did not keep the same MOI conditions. In that case, they should use an intracellular marker for their medium experiment to prove that they isolated proteins that are secreted and not simply released from dead cells. I will also suggest to show LDH assay at MOI=2 and 3 to monitor cell death. Is the Golgi fragmented when GRASP 55 is overexpressed in presence of the virus? Microscopy observations should be performed to reply to this question as it will support their model. The authors suggest that GRASP55 overexpression decreases spike incorporation inside the virion. Can they observe if Spike still colocalizes with GM130 when GRASP55 is overexpressed?

We showed that TGN46 depletion inhibits viral infection by both IF and WB. We further confirmed this through TCID50 assay for both cells and media (new Fig. 7O-P), strengthening our hypothesis.

As we described above, we performed morphological analysis at MOI=1 so that we could observe a significant number of infected cells but minimize cell toxicity. We performed immunoblotting (in Fig. 7H) at MOI=3 to get a good viral infection rate.

As suggested, we also performed LDH assay at MOI=2 and 3 to monitor cell death (new Fig. S2O). Fig. S6C shows that GRASP55 overexpression in the presence of the virus partially rescues Golgi fragmentation. GRASP55 expression did also enhance Spike and GM130 colocalization to some extent (new Fig. S6E-F).

Minor comments:

Figure 1P in the text: Considering that Rab7 up-regulation is equal to "growth of late endosome" is an overstatement. Rab7 is cytosolic at its inactive state and at the endosome at its active state. The authors would have to prove this statement by monitoring an increased quantity of Rab7 at the endosomes which is not enough by just monitoring protein intensity by microscopy. As Rab7 is also localized in lysosomes, and the authors used Lamp2 as a lysosomal marker, it is strange that the area of these structures is not increased. The authors should replace the term "growth" by "an increase in the area of their vesicles".

We did observe less but larger LAMP2 puncta in the infected cells. We agree with the reviewer and rephrased "growth" by an increase in the area of their vesicles". Thank you for the excellent suggestions.

Figure 1Q-T: The observations described in the text did not match the quantification, the area of lysosomes is not significantly different from the non-infected conditions.

In Fig. 1Q-T, we did observe fewer but larger LAMP2 puncta in the infected cells, which was consistent with our quantification, i.e., fewer puncta (Fig. 1R), but each punctum was larger (Fig. 1S), and total area was similar.

Figure 8: In the text, it is mentioned that there is "a dramatic reduction of spike and N in the lysate in GRASP55-expressing and TGN46 depleted cells". However, the quantification indicated that the decrease in N and S content is non-significant. Can the authors precise what was the sample of comparison in the text (siControl versus siTGN46 or siTGN46+GFP versus siTGN46+GFP-GRASP55)?

The decrease in N and S content is significant with the lysate sample comparison (siControl versus siTGN46; siControl+GFP versus siTGN46+GFP; siTGN46+GFP versus siTGN46+GFP-GRASP55). We have now removed this Figure following Reviewer #2's suggestion, since the results are consistent with single protein manipulation and more experiments are needed to confirm whether there is an additive effect.

**Referee cross-commenting**

I agree with most of the concerns of the other reviewers. I do also consider that they should have done their study on cells expressing naturally ACE2. However, at this stage, it will be a lot of work to perform all of their study in a more relevant cell type. The authors should repeat some of their key experiments in lung-derived cell types, to determine if GRASP55 and TGN46 have the same effect on SARS-CoV-2 virion secretion/production.

We thank the reviewer for the suggestions and understanding. As we mentioned before, our study utilizes Huh7-ACE2 cells, which are sorted for the high expression of endogenous ACE2 protein, without ACE2 overexpression. Actually, we also tested A549 and Calu-3 cells. While A549 cells displayed very low infection rate, Calu-3 cells displayed disorganized Golgi without viral infection. However, we did perform immunofluorescence assays in Calu-3 cells. Consistent with our findings in Huh7-ACE2 cells, SARS-CoV-2 infection disrupts Golgi structure and alters protein levels of TGN46 and GRASP55 in Calu3 cells (new Fig. S5R-W). Also, others have reported that liver can be a target for SARS-CoV-2 infection in humans. Furthermore, we confirmed GRASP55 downregulation and TGN46 upregulation in VeroE6 cells (Fig. S6K-N).

Reviewer #3 (Significance (Required)):

The study identified two Golgi proteins (TGN46 and GRASP55) that are involved in modulating the release of SARS-CoV-2 particles from the cells. As these proteins are also acting on general secretion of host proteins to the plasma membrane, the effect on SARS-CoV-2 release could just be indirect. However, it does not change the informative points of the study raised by Zhang et al. It highlights really well how the host trafficking pathway could be diverted for the purpose of the virus, which is to produce particles to maintain its survival.

Strengths: The authors performed a precise and well quantified study. Observing how SARS-CoV-2 impacts host organelles morphology and uses host trafficking proteins to produce particles, brings more clarity on some unclear parts of the life cycle of the virus. In addition, it exposes new targets for therapeutic studies.

We thank the reviewer for the positive comments.

Weakness: The paper is mostly based on microscopy analysis and need some other methods to support their data. The paper lacks some molecular mechanisms explaining the clear role of GRASP55 and TGN46 in particle production or assembly.

In the revised version, we incorporated RT-qPCR assay, cell cytotoxicity assay, and BFA time-point treatment assay. Notably, we added intracellular and extracellular viral titer assays to more precisely distinguish between effects on virion assembly and virion secretion. We also confirmed the key observation that SARS-CoV-2 infection modulates GRASP55 and TGN46 expression in the Calu-3 lung cell line. Additionally, our early time-point results clearly support the role of GRASP55 and TGN46 in viral trafficking.

• Audience: The paper will be interesting for basic research for a virology and cell biology audience.
• Field of expertise with a few keywords: Virology and host cell trafficking.

References

Barnes E (2022) Infection of liver hepatocytes with SARS-CoV-2. Nat Metab 4: 301-302

Bekier ME, 2nd, Wang L, Li J, Huang H, Tang D, Zhang X, Wang Y (2017) Knockout of the Golgi stacking proteins GRASP55 and GRASP65 impairs Golgi structure and function. Mol Biol Cell 28: 2833-2842

Eymieux S, Rouille Y, Terrier O, Seron K, Blanchard E, Rosa-Calatrava M, Dubuisson J, Belouzard S, Roingeard P (2021) Ultrastructural modifications induced by SARS-CoV-2 in Vero cells: a kinetic analysis of viral factory formation, viral particle morphogenesis and virion release. Cell Mol Life Sci 78: 3565-3576

Ghosh S, Dellibovi-Ragheb TA, Kerviel A, Pak E, Qiu Q, Fisher M, Takvorian PM, Bleck C, Hsu VW, Fehr AR et al (2020) beta-Coronaviruses Use Lysosomes for Egress Instead of the Biosynthetic Secretory Pathway. Cell 183: 1520-1535 e1514

Hoffmann M, Hofmann-Winkler H, Smith JC, Kruger N, Arora P, Sorensen LK, Sogaard OS, Hasselstrom JB, Winkler M, Hempel T et al (2021) Camostat mesylate inhibits SARS-CoV-2 activation by TMPRSS2-related proteases and its metabolite GBPA exerts antiviral activity. EBioMedicine 65: 103255

Hoffmann M, Mosbauer K, Hofmann-Winkler H, Kaul A, Kleine-Weber H, Kruger N, Gassen NC, Muller MA, Drosten C, Pohlmann S (2020) Chloroquine does not inhibit infection of human lung cells with SARS-CoV-2. Nature 585: 588-590

Xiang Y, Wang Y (2010) GRASP55 and GRASP65 play complementary and essential roles in Golgi cisternal stacking. J Cell Biol 188: 237-251

Considero que moco va enlistado esta parte, el diseño tiene un poco de todo lo que lo rodea, como su objetivo, contexto de las personas que realizaran el diseño y quien lo disfrutara

• RE 633782
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. LUIZ FUX
• Julgamento: 26/10/2020
• Publicação: 25/11/2020

EMENTA: RECURSO EXTRAORDINÁRIO. REPERCUSSÃO GERAL. TEMA 532. DIREITO CONSTITUCIONAL E ADMINISTRATIVO. PRELIMINARES DE VIOLAÇÃO DO DIREITO À PRESTAÇÃO JURISDICIONAL ADEQUADA E DE USURPAÇÃO DA COMPETÊNCIA DO SUPREMO TRIBUNAL FEDERAL AFASTADAS. PODER DE POLÍCIA. TEORIA DO CICLO DE POLÍCIA. DELEGAÇÃO A PESSOA JURÍDICA DE DIREITO PRIVADO INTEGRANTE DA ADMINISTRAÇÃO PÚBLICA INDIRETA. SOCIEDADE DE ECONOMIA MISTA. PRESTADORA DE SERVIÇO PÚBLICO DE ATUAÇÃO PRÓPRIA DO ESTADO. CAPITAL MAJORITARIAMENTE PÚBLICO. REGIME NÃO CONCORRENCIAL. CONSTITUCIONALIDADE. NECESSIDADE DE LEI FORMAL ESPECÍFICA PARA DELEGAÇÃO. CONTROLE DE ABUSOS E DESVIOS POR MEIO DO DEVIDO PROCESSO. CONTROLE JUDICIAL DO EXERCÍCIO IRREGULAR. INDELEGABILIDADE DE COMPETÊNCIA LEGISLATIVA. 1. O Plenário deste Supremo Tribunal reconheceu repercussão geral ao thema decidendum, veiculado nos autos destes recursos extraordinários, referente à definição da compatibilidade constitucional da delegação do poder de polícia administrativa a pessoas jurídicas de direito privado integrantes da Administração Pública indireta prestadoras de serviço público. 2. O poder de polícia significa toda e qualquer ação restritiva do Estado em relação aos direitos individuais. Em sentido estrito, poder de polícia caracteriza uma atividade administrativa, que consubstancia verdadeira prerrogativa conferida aos agentes da Administração, consistente no poder de delimitar a liberdade e a propriedade. 3. A teoria do ciclo de polícia demonstra que o poder de polícia se desenvolve em quatro fases, cada uma correspondendo a um modo de atuação estatal: (i) a ordem de polícia, (ii) o consentimento de polícia, (iii) a fiscalização de polícia e (iv) a sanção de polícia. 4. A extensão de regras do regime de direito público a pessoas jurídicas de direito privado (1) integrantes da Administração Pública indireta, desde que (2) prestem serviços públicos de atuação própria do Estado e em 3 (3) regime não concorrencial é admissível pela jurisprudência da Corte. (Precedentes: RE 225.011, Rel. Min. Marco Aurélio, Red. p/ o acórdão Min. Maurício Corrêa, Tribunal Pleno, julgado em 16/11/2000, DJ 19/12/2002; RE 393.032-AgR, Rel. Min. Cármen Lúcia, Primeira Turma, DJe 18/12/2009; RE 852.527-AgR, Rel. Min. Cármen Lúcia, Segunda Turma, DJe 13/2/2015). 5. A constituição de uma pessoa jurídica integrante da Administração Pública indireta sob o regime de direito privado não a impede de ocasionalmente ter o seu regime aproximado daquele da Fazenda Pública, desde que não atue em regime concorrencial. 6. Consectariamente, a Constituição, ao autorizar a criação de empresas públicas e sociedades de economia mista que tenham por objeto exclusivo a prestação de serviços públicos de atuação típica do Estado e em regime não concorrencial, autoriza, consequentemente, a delegação dos meios necessários à realização do serviço público delegado. Deveras: a) A admissão de empregados públicos deve ser precedida de concurso público, característica que não se coaduna com a despedida imotivada; b) o RE 589.998, esta Corte reconheceu que a ECT, que presta um serviço público em regime de monopólio, deve motivar a dispensa de seus empregados, assegurando-se, assim, que os princípios observados no momento da admissão sejam, também, respeitados por ocasião do desligamento; c) Os empregados públicos se submetem, ainda, aos princípios constitucionais de atuação da Administração Pública constantes do artigo 37 da Carta Política. Assim, eventuais interferências indevidas em sua atuação podem ser objeto de impugnação administrativa ou judicial; d) Ausente, portanto, qualquer incompatibilidade entre o regime celetista existente nas estatais prestadoras de serviço público em regime de monopólio e o exercício de atividade de polícia administrativa pelos seus empregados. 7. As estatais prestadoras de serviço público de atuação própria do Estado e em regime não concorrencial podem atuar na companhia do atributo da coercibilidade inerente ao exercício do poder de polícia, mormente diante da atração do regime fazendário. 8. In casu, a Empresa de Transporte e Trânsito de Belo Horizonte – BHTRANS pode ser delegatária do poder de polícia de trânsito, inclusive quanto à aplicação de multas, porquanto se trata de estatal municipal de capital majoritariamente público, que presta exclusivamente serviço público de atuação própria do Estado e em regime não concorrencial, consistente no policiamento do trânsito da cidade de Belo Horizonte. Preliminares: 9. A jurisprudência do Supremo Tribunal Federal é firme no sentido de que o princípio da fundamentação das decisões não obriga o órgão julgador a responder a todas as questões suscitadas pelas partes, mas somente aqueles que sejam suficientes para motivar o seu convencimento. Preliminar de violação do direito à prestação jurisdicional adequada afastada. 10. A alínea d, inciso III, artigo 102, da Constituição exige, para atração da competência do Supremo Tribunal Federal, declaração expressa da validade de lei local contestada em face de lei federal, o que, in casu, não se verifica. Preliminar de usurpação de competência afastada. 11. Os recursos extraordinários interpostos pela Empresa de Transporte e Trânsito de Belo Horizonte – BHTRANS e pelo Ministério Público do Estado de Minas Gerais devem ser conhecidos em razão do preenchimento de todos os requisitos de admissibilidade, notadamente o da tempestividade, prequestionamento, legitimidade e o do interesse recursal, além da repercussão geral da matéria reconhecida pelo Plenário Virtual desta Corte. 12. Ex positis, voto no sentido de (i) CONHECER e DAR PROVIMENTO ao recurso extraordinário interposto pela Empresa de Transporte e Trânsito de Belo Horizonte – BHTRANS e (ii) de CONHECER e NEGAR PROVIMENTO ao recurso extraordinário interposto pelo Ministério Público do Estado de Minas Gerais, para reconhecer a compatibilidade constitucional da delegação da atividade de policiamento de trânsito à Empresa de Transporte e Trânsito de Belo Horizonte – BHTRANS, nos limites da tese jurídica objetivamente fixada pelo Pleno do Supremo Tribunal Federal. 13. Repercussão geral constitucional que assenta a seguinte tese objetiva: “É constitucional a delegação do poder de polícia, por meio de lei, a pessoas jurídicas de direito privado integrantes da Administração Pública indireta de capital social majoritariamente público que prestem exclusivamente serviço público de atuação própria do Estado e em regime não concorrencial.”

Tema - 532 - Aplicação de multa de trânsito por sociedade de economia mista.

Tese - É constitucional a delegação do poder de polícia, por meio de lei, a pessoas jurídicas de direito privado integrantes da Administração Pública indireta de capital social majoritariamente público que prestem exclusivamente serviço público de atuação própria do Estado e em regime não concorrencial.

• Informativo nº 790
• 10 de outubro de 2023.
• PRIMEIRA TURMA
• Processo: REsp 1.950.332-RJ, Rel. Ministro Gurgel de Faria, Primeira Turma, por unanimidade, julgado em 26/9/2023, DJe 2/10/2023.

Ramo do Direito DIREITO ADMINISTRATIVO

Paz, Justiça e Instituições Eficazes Parcerias e meios de implementaçãoTema Poder de polícia. Função sancionadora. Delegação. Câmara de comercialização de energia elétrica - CCEE. Associação de natureza privada. Impossibilidade.

DESTAQUE - Não é possível delegar a função sancionadora do exercício do poder de polícia à Câmara de Comercialização de Energia Elétrica - CCEE por ser uma associação privada que não integra a Administração Pública.

INFORMAÇÕES DO INTEIRO TEOR - No plano da jurisprudência, o Supremo Tribunal Federal já se manifestou sobre a questão relativa à delegação de poder de polícia administrativa a entidades privadas no julgamento da ADI n. 1.717, de relatoria do Ministro Sydney Sanches, quando concluiu pela "indelegabilidade, a uma entidade privada, de atividade típica de Estado, que abrange até poder de polícia, de tributar e de punir, no que concerne ao exercício de atividades profissionais regulamentadas".

• O Superior Tribunal de Justiça, ao examinar o mesmo tema de fundo do presente processo, também consagrou a tese de que, em relação às fases do "ciclo de polícia", somente os atos relativos ao consentimento e à fiscalização são delegáveis, seguindo o entendimento de que aqueles referentes à legislação e à sanção derivam do poder de coerção do Poder Público, este indelegável às pessoas jurídicas de direito privado.

• Acontece que, contra a supracitada decisão do STJ, houve a interposição de Recurso Extraordinário (633782/MG), tendo sido o recurso afetado como representativo de controvérsia. Na ocasião do julgamento daquele apelo, houve a revisão parcial do entendimento do STF sobre a possibilidade de delegação da função de polícia, cristalizando o Supremo a tese de que "é constitucional a delegação do poder de polícia, por meio de lei, a pessoas jurídicas de direito privado integrantes da Administração Pública indireta de capital social majoritariamente público que prestem exclusivamente serviço público de atuação própria do Estado e em regime não concorrencial".

No caso, porém, o precedente não se aplica, pois: a) a CCEE é associação privada que não integra a Administração Pública; b) não há permissão constitucional para que atue como agente delegada da função administrativa de infligir sanções; c) os integrantes não gozam de qualquer estabilidade no emprego; d) embora a Câmara seja associação civil sem fins lucrativos, o fato é que ela é integrada "por titulares de concessão, permissão ou autorização" e "por outros agentes vinculados aos serviços e às instalações de energia elétrica", ou seja, ela é essencialmente composta por pessoas jurídicas que, como fim principal, visam o lucro.

• Ademais, não há lei formal autorizando direta e expressamente que a CCEE aplique diretamente multas aos particulares, e depois as cobre por conta própria, na medida em que essa atribuição só é mencionada no Decreto n. 5.177/2004 c/c Resolução Normativa ANEEL n. 109.

Author response:

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

Reviewer #1 (Public review):

Summary:

Wang et al. created a series of specific FLIM-FRET sensors to measure the activity of different Rab proteins in small cellular compartments. They apply the new sensors to monitor Rab activity in dendritic spines during induction of LTP. They find sustained (30 min) inactivation of Rab10 and transient (5 min) activation of Rab4 after glutamate uncaging in zero Mg. NMDAR function and CaMKII activation are required for these effects. Knockdown of Rab4 reduced spine volume change while knockdown of Rab10 boosted it and enhanced functional LTP (in KO mice). To test Rab effects on AMPA receptor exocytosis, the authors performed FRAP of fluorescently labeled GluA1 subunits in the plasma membrane. Within 2-3 min, new AMPARs appear on the surface via exocytosis. This process is accelerated by Rab10 knock-down and slowed by Rab4 knock-down. The authors conclude that CaMKII promotes AMPAR exocytosis by i) activating Rab4, the exocytosis driver and ii) inhibiting Rab10, possibly involved in AMPAR degradation.

Strengths:

The work is a technical tour de force, adding fundamental insights to our understanding of the crucial functions of different Rab proteins in promoting/preventing synaptic plasticity. The complexity of compartmentalized Ras signaling is poorly understood and this study makes substantial inroads. The new sensors are thoroughly characterized, seem to work very well, and will be quite useful for the neuroscience community and beyond (e.g. cancer research). The use of FLIM for read-out is compelling for precise activity measurements in rapidly expanding compartments (i.e., spines during LTP).

Thank you for the evaluation.

Weaknesses:

The interpretation of the FRAP experiments (Figure 5, Ext. Data Figure 13) is not straightforward as spine volume and surface area greatly expand during uncaging. I appreciate the correction for the added spine membrane shown in Extended Data Figure 14i, but shouldn't this be a correction factor (multiplication) derived from the volume increase instead of a subtraction?

We thank the reviewer for this question. The fluorescence change should reflect a subtraction of surface area, as SEP-GluA1 is only fluorescent on the cell surface, unlike cytosolic mCherry, whose fluorescence intensity is proportional to spine volume. Therefore, the overall fluorescence change (ΔF) should be the addition of the contribution from AMPAR trafficking (ΔF_t) and the change in surface area (ΔS) multiplied by the remaining SEP-GluA1 fluorescence per unit area (f):

ΔF = ΔF_t + fΔS

Since fluorescence immediately after photobleaching (before AMPAR trafficking happens), F_o, is given by fS (S is the surface area of the spine):

ΔF/F_o = ΔF_t/ F_o + fΔS / fS

\= ΔF_t/fS + ΔS/S

Assuming that the surface area change (ΔS/S) is the volume change (ΔV/V) to the power of 2/3, the contribution of the AMPAR trafficking can be calculated as:

ΔF_t/F = ΔF/F – (Δ<sup>V/V)^2/3

This is the reason that we subtracted the contribution of the spine surface area. We have discussed this in the updated method section.

Also, experiments were not conducted or analyzed blind, risking bias in the selection/exclusion of experiments for analysis. This reduces my confidence in the results.

We acknowledge the reviewer's concern regarding the lack of blinding in our experiments. However, it is challenging to conduct blinded experiments for certain types of studies, such as sensor screening for a protein family, where we do not have expected results or a specific hypothesis prior to the experiments. In these cases, our primary readout is whether the sensor indicates any activity change upon stimulation.

To address this concern, after identifying that Rab10 is inactivated during structural LTP (sLTP) and is likely important for inhibiting spine structural LTP, we performed blinded electrophysiology experiments and obtained similar results (deletion of Rab10 from Camk2a-positive neurons leads to enhanced LTP; Fig. 4k, 4l).

Reviewer #2 (Public review):

Summary:

Wang et al. developed a set of optical sensors to monitor Rab protein activity. Their investigation into Rab activity in dendritic spines during structural long-term plasticity (sLTP) revealed sustained Rab10 inactivation (>30min) and transient Rab4 activation (~5 min). Through pharmacological and genetic manipulation to constitutively activate or inhibit Rab proteins, they found that Rab10 negatively regulates sLTP and AMPA receptor insertion, while Rab4 positively influences sLTP but only in the transient phase. The optical sensors provide new tools for studying Rab activity in cells and neurobiology. However, a full understanding of the timing of Rab activity will require a detailed characterization of sensor kinetics.

Strengths:

(1) Introduction of a series of novel sensors that can address numerous questions in Rab biology.

(2) Multiple methods to manipulate Rab proteins to reveal the roles of Rab10 and rab4 in LTP.

(3) Discovery of Rab4 activation and Rab10 inhibition with different kinetics during sLTP, correlating with their functional roles in the transient (Rab4) and both transient and sustained (Rab10) phases of sLTP.

Thank you for the positive evaluation.

Weaknesses:

(1) Lack of characterization of sensor kinetics, making it difficult to determine if the observed Rab kinetics during sLTP were due to sensor behavior or actual Rab activity.

We estimated that the kinetics of the sensors for Rab4 and Rab10 are within a few minutes. For Rab4, we observed rapid increase and decrease of the activation in response to glutamate uncaging. Thus, this would be the upper limit of the ON/OFF time constants of Rab4. For Rab10, we observed a rapid dissociation of the sensor in response to sLTP induction within ~1 min. This means that the donor and acceptor molecules are quickly dissociated during the process. Thus, the off kinetics of the sensor is within the range of minute. Meanwhile, we have the on-kinetics from Rab10 activation (donor/accepter association) in response to NMDA application and again this is within a few minutes. Given these rapid sensor kinetics in neurons, our observation of the sustained inactivation of Rab10 should reflect the true behavior of Rab10, rather than just the sensor’s response.

We revised our manuscript discussion session as follows:

“Understanding the kinetics of Rab4 and Rab10 sensors is essential for interpreting their actual activity during sLTP. The Rab4 sensor exhibits a rapid rise and fall in activation (Fig. 3), indicating ON/OFF times of less than a few minutes. In contrast, the Rab10 sensor rapidly dissociates during sLTP induction (Fig. 2), with OFF kinetics occurring within one minute and fast ON kinetics in response to NMDA (Fig. 1j). Given these rapid kinetics, the observed sustained inactivation of Rab10 likely reflects its true behavior rather than sensor dynamics.”

(2) It is crucial to assess whether the overexpression of Rab proteins as reporters, affects Rab activity and cellular structure and physiology (e.g. spine number and size).

While we did not measure the effects of Rab sensor overexpression on Rab activity or cellular structure and physiology, we showed that sLTP is similar in neurons expressing sensors. This suggests that the overexpression of Rab sensors does not significantly disrupt signaling required for sLTP.

(3) The paper does not explain the apparently different results between NMDA receptor activation and glutamate uncaging. NMDA receptor activation increased Rab10 activity, while glutamate uncaging decreased it. NMDA receptor activation resulted in sustained Rab4 activation, whereas glutamate uncaging caused only brief activation of about 5 minutes. A potential explanation, ideally supported by data, is needed.

It is a long-standing question in the field why simple NMDA receptor activation by bath application of NMDA does not induce LTP, but instead induce LTD. Rab proteins are regulated by many GEFs and GAPs and identifying different mechanisms requires completely different techniques, such as molecular screening. While our manuscript provides some insights into this question by showing that they provide opposing signals for Rab10, we believe that identifying exact mechanisms would be out of the scope of this manuscript.

(4) There is a discrepancy between spine phenotype and sLTP potential with Rab10 perturbation. Rab10 perturbation affected spine density but not size, suggesting a role in spinogenesis rather than sLTP. However, glutamate uncaging affected sLTP, and spinogenesis was not examined. Explaining the discrepancy between spine size and sLTP potential is necessary. Exploring spinogenesis with glutamate uncaging would strengthen these results. Additionally, Figure 4j shows no change in synaptic transmission with Rab10 knockout, despite an increase in spine density. An explanation, ideally supported by data, is needed for the unchanged fEPSP slope despite an increase in spine density.

We thank the reviewer for raising these important questions. In our findings, shRNA-mediated knockdown of Rab10 did not alter spine size but did increase spine density in the basal state (Extended Data Fig. 11i). This suggests that Rab10 may restrict spinogenesis without affecting spine size. Conversely, sLTP induction via glutamate uncaging is an activity-dependent process that may involve different molecular mechanisms. The signal interplay between spinogenesis and sLTP and how the exact roles of Rab signaling in different modalities of plasticity would remain elusive for the future study.

The lack of change in synaptic transmission with Rab10 knockout, despite the increase in spine density from Rab10 shRNA knockdown, may be due to different preparation and developmental stages: spine density measurements were conducted with shRNA knockdown in organotypic slices (sliced at P6-8, DIV 9-13), while electrophysiological recordings were performed in knockout mice in acute slices from adult animals (P30-60).

(5) Spine volume was imaged using acceptor fluorophores (mCherry, or mCherry/Venus) at 920nm, where the two-photon cross-section of mCherry is minimal. 920nm was also used to excite the donor fluorophore, hence the spine volume measurement based on total red channel fluorescence is the sum of minimal mCherry fluorescence from direct 920nm excitation, bleed-through from the green channel, and FRET. This confounded measurement requires correction and clarification.

We assumed that the most of fluorescence is from direct excitation of mCherry at 920 nm. The contribution from the bleed-through from mEGFP-Rab (~3%) and from FRET changes (~20%) may influence the volume measurements. However, since we observed similar fluorescence changes in the green and red channels, these factors would have only a minor impact on our results (Extended Data Fig. 6a, 6d). Also, please note that the volume change in neurons expressing sensors is just to check if the volume change is normal, and not a major point of this manuscript.  We clarified this in the method section as:

“For the sensor experiments, we used mCherry as a volume indicator. We acknowledge that contributions from bleed-through from mEGFP-Rab (approximately 3%) and FRET changes (around 20%) could affect the volume measurements. However, since we observed similar fluorescence changes in both the green and red channels, we believe these factors have a minimal impact on our results (Extended Data Fig. 6a, 6d).”

Reviewer #3 (Public review):

Summary:

This study examines the roles of Rab10 and Rab4 proteins in structural long-term potentiation (sLTP) and AMPA receptor (AMPAR) trafficking in hippocampal dendritic spines using various different methods and organotypic slice cultures as the biological model.

The paper shows that Rab10 inactivation enhances AMPAR insertion and dendritic spine head volume increase during sLTP, while Rab4 supports the initial stages of these processes. The key contribution of this study is identifying Rab10 inactivation as a previously unknown facilitator of AMPAR insertion and spine growth, acting as a brake on sLTP when active. Rab4 and Rab10 seem to be playing opposing roles, suggesting a somewhat coordinated mechanism that precisely controls synaptic potentiation, with Rab4 facilitating early changes and Rab10 restricting the extent and timing of synaptic strengthening.

Strengths:

The study combines multiple techniques such as FRET/FLIM imaging, pharmacology, genetic manipulations, and electrophysiology to dissect the roles of Rab10 and Rab4 in sLTP. The authors developed highly sensitive FRET/FLIM-based sensors to monitor Rab protein activity in single dendritic spines. This allowed them to study the spatiotemporal dynamics of Rab10 and Rab4 activity during glutamate uncaging-induced sLTP. They also developed various controls to ensure the specificity of their observations. For example, they used a false acceptor sensor to verify the specificity of the Rab10 sensor response.

This study reveals previously unknown roles for Rab10 and Rab4 in synaptic plasticity, showing their opposing functions in regulating AMPAR trafficking and spine structural plasticity during LTP.

Thank you for the positive evaluation.

Weaknesses:

In sLTP, the initial volume of stimulated spines is an important determinant of induced plasticity. To address changes in initial volume and those induced by uncaging, the authors present Extended Data Figure 2. In my view, the methods of fitting, sample selection, or both may pose significant limitations for interpreting the overall results. While the initial spine size distribution for Rab10 experiments spans ~0.1-0.4 fL (with an unusually large single spine at the upper end), Rab4 spine distribution spans a broader range of ~0.1-0.9 fL. If the authors applied initial size-matched data selection or used polynomials rather than linear fitting, panels a, b, e, f, and g might display a different pattern. In that case, clustering analysis based on initial size may be necessary to enable a fair comparison between groups not only for this figure but also for main Figures 2 and 3.

We thank the reviewer for these questions. For sensor uncaging experiments, we usually uncaged glutamate at large mushroom spines because we need to have a good signal-to-noise ratio. We just happen to choose these spines with different initial sizes for Rab4 sensor and Rab10 sensor uncaging experiments.

Another limitation is the absence of in vivo validation, as the experiments were performed in organotypic hippocampal slices, which may not fully replicate the complexity of synaptic plasticity in an intact brain, where excitatory and inhibitory processes occur concurrently. High concentrations of MNI-glutamate (4 mM in this study) are known to block GABAergic responses due to its antagonistic effect on GABA-A receptors, thereby precluding the study of inhibitory network activity or connectivity [1], which is already known to be altered in organotypic slice cultures.

(1) https://www.frontiersin.org/journals/neural-circuits/articles/10.3389/neuro.04.002.2009/full

We appreciate the reviewer's comments and would like to clarify that we have conducted experiments in acute slices for LTP using conditional Rab10 knockout (Fig. 4k, 4l), and we obtained similar results. Additionally, we have recently published findings on the behavioral deficits observed in heterozygous Rab10 knockout mice (PubMed 37156612). These studies further support our conclusions and provide additional context for our findings.

Recommendations for the authors:

From the Senior/Reviewing Editor:

I apologize that this took longer than intended. As you will see from the reviews there was some disagreement on several points. There was some disagreement among reviewers as to the strength of the evidence with some characterizing it as "compelling," "convincing," or "solid" while others felt the characterization of the sensors was "incomplete" and that this could have affected some of the conclusions. After extensive discussion, reviewers agreed that there was a valid concern that the conclusion that Rab10 activation is sustained could reflect a feature of the sensor. If Rab10/RBD dissociation rate were very low, and the affinity of binding were very high, this could lead to an incorrect estimate of the sustained binding due to sensor kinetics, not Rab10 activation. It was noted that this has been seen in other sensors previously (e.g. first generation PKA activity sensors), which the developers altered in later generations to increase reversibility and off kinetics of the sensor.

There was also discussion of how this might be addressed and we would be interested in your comments on this issue. It was suggested that it might be helpful to revise Figure 2b to show binding fraction dynamics separately for each spine (to determine whether any actually return to baseline). Subsequently, clustering of these binding dynamics into two groups could be summarized in a version of Fig. 2e for each cluster. Differences in spine volume dynamics between these clusters would provide a measure of how strongly Rab10 binding correlates with spine volume. If they never go back to baseline, some extra experiments with longer post-plasticity induction (150mins instead of 35), might show if any reversible Rab10 binding exists post-LTP induction.

An alternative suggestion was to measure the time course in the presence of a GAP or GEF, which should alter the kinetics.

Thanks for the comments. It is important that the inactivation is observed as the dissociation of the donor and acceptor of the sensor.  Thus, the fact that the sensor rapidly decreases in response to uncaging means that they have rapid off kinetics. In addition, we provide evidence of a rapid increase of Rab10 in response to NMDA application, suggesting that kinetics is also rapid. We added discussion about this in the revised manuscript as:

“Understanding the kinetics of Rab4 and Rab10 sensors is essential for interpreting their actual activity during sLTP. The Rab4 sensor exhibits a rapid rise and fall in activation (Fig. 3), indicating ON/OFF times of just a few minutes. In contrast, the Rab10 sensor rapidly dissociates during sLTP induction (Fig. 2), with OFF kinetics occurring within one minute and fast ON kinetics in response to NMDA (Fig. 1j). Given these rapid kinetics, the observed sustained inactivation of Rab10 likely reflects its true behavior rather than sensor dynamics.”

There was also further discussion of the nature of the "spine volume" signal, given the fact that the two-photon cross-section of mCherry is minimal at 920nm. It was suggested that this could be due to direct acceptor excitation rather than FRET, but there was agreement that further clarity on this issue would be valuable.

We assumed that the most of fluorescence is from direct excitation of mCherry at 920 nm. The contribution from the bleed-through from mEGFP-Rab (~3%) and from FRET changes (~20%) may influence the volume measurements. However, since we observed similar fluorescence changes in the green and red channels, these factors would have only a minor impact on our results (Extended Data Fig. 6a, 6d). Also, please note that the volume change in neurons expressing sensors is just to check if the volume change is normal, and not a major point of this manuscript.  We clarified this in the method section as:

“For the sensor experiments, we used mCherry as a volume indicator. We acknowledge that contributions from bleed-through from mEGFP-Rab (approximately 3%) and FRET changes (around 20%) could affect the volume measurements. However, since we observed similar fluorescence changes in both the green and red channels, we believe these factors have a minimal impact on our results (Extended Data Fig. 6a, 6d).”

The equations in the methods section differ from other papers by the same lab (e.g. Laviv et al, Neuron 2020, Tu et al. Sci Adv. 2023, Jain et al. Nature 2024). Please clarify which equations are correct.

Thanks for pointing this out. In fact, some of the equations in this manuscript were wrong, and we have corrected them in the method session.

Reviewer #1 (Recommendations for the authors):

The effects of Rab knockdown affect both spine volume expansion and AMPAR recovery in a very similar fashion. To explain this tight coupling, the authors suggest that the availability of membrane could be a limiting factor for spine enlargement. However, some Rabs are known to affect actin dynamics, which could also explain the dual effects on AMPAR exocytosis and spine enlargement. It is not easy to come up with an experiment to differentiate between these alternative explanations, as blocking actin polymerization would likely affect exocytosis, too. The authors should consider/discuss the possibility that all of the observed Ras effects result from altered actin dynamics and that the lipid bilayer is sufficiently fluid to form a minimal surface around the expanding cytoskeleton.

Thanks for the suggestions. We included the discussion about the potential impact on the actin cytoskeleton by Rab10.

Typos: heterougenous, compartmantalization, chemaical, ballistically/biolistically (chose one).

Thanks for pointing out these typos. We have corrected them in the revised manuscript.

Reviewer #2 (Recommendations for the authors):

(1) Venus shows pH sensitivity, which can be significant at synapses due to pH changes. Characterizing the pH sensitivity of the sensors is essential.

Thanks for the suggestions. We did not measure pH dependence, but the PKa of these fluorophores has already been published. PKa for EGFP and Venus are both 6.0, and it is unlikely that it influenced our measurements.

(2) Presenting individual data points within all bar graphs (e.g. Fig. 2c, 2d) would enhance data transparency.

Thanks for the suggestions. We now provide individual data points in the revised main figures.

(3) In Figure 1f: Rab5 GAP expression increased the binding fraction against expectations. In addition, clarifying the color scheme in Figure 1 is needed. Are GAPs supposed to be blue/green, and GEFs red/orange? Figure 1f seems to contradict this color scheme.

Thanks for the suggestions. We clarified these issues.

(4) Quantification of the point spread function of the uncaging laser, response/settle time of the scan mirror during uncaging, and reason for changes in neighboring spines in many example images (e.g. Figure 2a, especially at 240 s; Figure 4a) would be important.

The laser is controlled by Pockels cells, which changes the laser intensity with microsecond resolution. The laser is parked for milliseconds during uncaging, much longer than the settling time of the mirror (~0.1 milliseconds). The point spread function of the uncaging laser is limited by the diffraction (~0.5 um). The uncaging spot size is mostly limited by the diffusion of uncaged glutamate, but our calcium imaging and CaMKII imaging show that the signaling is induced mostly in the stimulated spines (Lee et al., 2009; Chang et al., 2017, 2019).

(5) Please include traces for "false" sensors in stimulated spines in Figures 2b, 2e, 3b, and 3e.

The traces for the false sensors have been presented in Extended Data Fig. 3 and Extended Data Fig. 8.

(6) The traces in Figure 4k (fEPSP slope in response to theta burst stimulation, where there is a decrease in fEPSP slope followed by a gradual increase) differ from prior publications (e.g. PMID: 1359925, 3967730, 19144965, 20016099). An investigation and explanation for these differences are necessary.

We appreciate the reviewer’s comments. We performed the experiments blindly and did not try to find a condition providing control data similar to previous publications. The variations in fEPSP responses compared to prior publications may be attributed to several factors, including differences in experimental conditions such as the genetic background of the animals used, the specific protocols for theta burst stimulation, and variations in the preparation of the hippocampal slices.

(7) The title and text state that Rab10 inactivation promotes AMPAR insertion. It is unclear if this is a direct effect on AMPAR insertion or an indirect effect through membrane remodeling. Providing data to distinguish these possibilities or adjusting the title/text to reflect alternative interpretations would be beneficial.  

We appreciate the reviewer's feedback. To clarify, we have revised our terminology to use "AMPAR trafficking" instead of "AMPAR insertion", as it includes both insertion and other mechanisms of AMPAR movement within the cell.

(8) Please provide an explanation for the initial Rab10 inactivation observed in Figure 1j upon NMDA application.

The application of NMDA in Fig. 1j is similar to the commonly used chemical LTD induction protocol. We used this broad stimulation approach to test whether our sensors could report Rab activity changes in neurons upon strong stimulation. However, it is an entirely different stimulation approach from the sLTP induction protocol, thus resulting in different sensor activity changes.  We describe the phenomenon in the revised manuscript, but we believe that detailed analyses of Rab10 activation in response to NMDA application are beyond the scope of this manuscript.

(9) Please explain why the study focuses on Rab4 and Rab10 instead of other Rab proteins.

During our initial screening of sensors for various Rab proteins, we observed significant activity changes in the sensors for Rab4 and Rab10 upon sLTP induction. This suggested their potential relevance in synaptic processes, leading us to focus on understanding their specific roles in structural long-term potentiation.

Reviewer #3 (Recommendations for the authors):

(1) Although it might seem trivial, the definition of adjacent spine has not been made in the text. It would be nice to have it in the Methods section.

We included it in the Methods section as follows:

"The adjacent spine refers to the first or second spine located next to the stimulated spine, typically positioned opposite the stimulated spine. Additionally, the size of the adjacent spine must be sufficiently large for imaging."

(2) The transfection method has been mentioned as "ballistic" and "biolistic" transfection. You might want to use only one term. Additionally, you can add the equipment used (Bio-rad?) and pressure (psi) in the Methods section.

We use “biolistic” throughout the manuscript now. We also added the equipment and conditions used.

Perquè no quedi tan pobre podries afegir un length(x) o un str(x)

Afegiria que és on surt l'output del que writes en el source

Did you consider using your vIL-4 structure(s) and using the FoldSeek web interface/server t(https://search.foldseek.com/search) o search for structural homologs? Because there is relatively low sequence identity and viruses evolve so quickly, I would be curious if you uncover more homologs via a structural search. I think the results from the BFVD (viral) and AFDB50 databases would be particularly interesting to see.

DA FUNÇÃO SOCIAL DA EMPRESA PÚBLICA E DA SOCIEDADE DE ECONOMIA MISTA

Art. 27. A empresa pública e a sociedade de economia mista terão a função social de realização do interesse coletivo ou de atendimento a imperativo da segurança nacional expressa no instrumento de autorização legal para a sua criação.

§ 1º A realização do interesse coletivo de que trata este artigo deverá ser orientada para o alcance do bem-estar econômico e para a alocação socialmente eficiente dos recursos geridos pela empresa pública e pela sociedade de economia mista, bem como para o seguinte:

• I - ampliação economicamente sustentada do acesso de consumidores aos produtos e serviços da empresa pública ou da sociedade de economia mista;

• II - desenvolvimento ou emprego de tecnologia brasileira para produção e oferta de produtos e serviços da empresa pública ou da sociedade de economia mista, sempre de maneira economicamente justificada.

§ 2º A empresa pública e a sociedade de economia mista deverão, nos termos da lei, adotar práticas de sustentabilidade ambiental e de responsabilidade social corporativa compatíveis com o mercado em que atuam.

§ 3º A empresa pública e a sociedade de economia mista poderão celebrar convênio ou contrato de patrocínio com pessoa física ou com pessoa jurídica para promoção de atividades culturais, sociais, esportivas, educacionais e de inovação tecnológica, desde que comprovadamente vinculadas ao fortalecimento de sua marca, observando-se, no que couber, as normas de licitação e contratos desta Lei.

the 5 senses allow something to manifest, but words also can cause something to manifest

animalele o sa prefere mediul natural mereu, plus ca un mediu cu enrichment o sa ofere oportunitatea animalului de a alege unde si ce sa faca. plus ca preferinta se schimba in timp in functie de sezon, perioada de imperechere, varsta, temperatura, etc. enrichmentul, adica un mediu variat constant ar trebui sa fie standardul.

DOI: 10.1016/j.xcrm.2025.101986

Resource: (BD Biosciences Cat# 556604, RRID:AB_396479)

Curator: @scibot

SciCrunch record: RRID:AB_396479

What is this?

I noticed that while reading this article that our working definition of equity places emphasis and focus on how an individual is behaving in relation to their determined success. It is nice to see this framework again shifts focus and gives power to change o the system and not the student

Author response:

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

Reviewer #1 (Public review):

Summary:

The manuscript by Cao et al. examines an important but understudied question of how chronic exposure to heat drives changes in affective and social behaviors. It has long been known that temperature can be a potent driver of behaviors and can lead to anxiety and aggression. However, the neural circuitry that mediates these changes is not known. Cao et al. take on this question by integrating optical tools of systems neuroscience to record and manipulate bulk activity in neural circuits, in combination with a creative battery of behavior assays. They demonstrate that chronic daily exposure to heat leads to changes in anxiety, locomotion, social approach, and aggression. They identify a circuit from the preoptic area (POA) to the posterior paraventricular thalamus (pPVT) in mediating these behavior changes. The POA-PVT circuit increases activity during heat exposure. Further, manipulation of this circuit can drive affective and social behavioral phenotypes even in the absence of heat exposure. Moreover, silencing this circuit during heat exposure prevents the development of negative phenotypes. Overall the manuscript makes an important contribution to the understudied area of how ambient temperature shapes motivated behaviors.

Strengths:

The use of state-of-the-art systems neuroscience tools (in vivo optogenetics and fiber photometry, slice electrophysiology), chronic temperature-controlled experiments, and a rigorous battery of behavioral assays to determine affective phenotypes. The optogenetic gain of function of affective phenotypes in the absence of heat, and loss of function in the presence of heat are very convincing manipulation data. Overall a significant contribution to the circuit-level instantiation of temperature-induced changes in motivated behavior, and creative experiments.

Weaknesses：

(1) There is no quantification of cFos/rabies overlap shown in Figure 2, and no report of whether the POA-PVT circuit has a higher percentage of Fos+ cells than the general POA population. Similarly, there is no quantification of cFos in POA recipient PVT cells for Figure 2 Supplement 2.

Thanks for the comment. The quantification results of c-Fos signal have been provided in the main text and figures.  

(2) The authors do not address whether stimulation of POA-PVT also increases core body temperature in Figure 3 or its relevant supplements. This seems like an important phenotype to make note of and could be addressed with a thermal camera or telemetry.

Thanks for raising this point. We did indeed monitor the core body temperature during stimulation of POA-PVT pathway, but we did not observe any significant changes. We have included this finding in the revised manuscript.

(3) In Figure 3G: is Day 1 vs Day 22 "pre-heat" significant? The statistics are not shown, but this would be the most conclusive comparison to show that POA-PVT cells develop persistent activity after chronic heat exposure, which is one of the main claims the authors make in the text. This analysis is necessary in order to make the claim of persistent circuit activity after chronic heat exposure.

Figure 3G does compare the Day 1 preheat to Day22 preheat, and the difference was significant. The wording has been corrected to avoid confusion. Also, we have modified Figure 3D to 3H in our revised manuscript to improve the clarity of these plots.

(4) In Figure 4, the control virus (AAV1-EYFP) is a different serotype and reporter than the ChR2 virus (AAV9-ChR2-mCherry). This discrepancy could lead to somewhat different baseline behaviors.

Thanks for bringing out this issue. We acknowledge that using AA1-EGFP (a different serotype and reporter compared to the AAV9-ChR2-mCherry) as our control virus is not ideal. But based on our own prior experiments, we observed no significant differences in baseline behaviors between animals injected with AAV1 and AAV9 EYFP as well as control mice without virus injection. Therefore, we believe that the baseline behaviors of the animals were unaffected.

(5) In Figure 5G, N for the photometry data: the authors assess the maximum z-score as a measure of the strength of calcium response, however the area under the curve (AUC) is a more robust and useful readout than the maximum z score for this. Maximum z-score can simply identify brief peaks in amplitude, but the overall area under the curve seems quite similar, especially for Figure 5N.

Thanks for the comment. We agree with the reviewer that the area under the curve (AUC) is an alternative readout for measurement of the strength of calcium response. However, the reason why we chose the maximum z-score is based on the observation that we found POA recipient pPVT neurons after chronic heat treatment exhibited a higher calcium peak corresponding to certain behavioral performances when compared to pre-heat conditions. We thus applied the maximum z-score as a representative way to describe the neuronal activity changes of mice during certain behaviors before and after chronic heat treatment. The other consideration is that we want to reflect that POA recipient pPVT neurons become more sensitive and easier to be activated after chronic heat exposure under the same stressful situations compared to control mice. The maximum z score represented by peak in combination with particular behavioral performances is considered more suitable to highlight our findings in this study.

(6) For Fig 5V: the authors run the statistics on behavior bouts pooled from many animals, but it is better to do this analysis as an animal average, not by compiling bouts. Compiling bouts over-inflates the power and can yield significant p values that would not exist if the analysis were carried out with each animal as an n of 1.

Thanks for the comment and suggestion. We had tried both methods and the statistical results were similar. As suggested, we have updated Fig 5V, as well as Fig. 5H and 5O by comparing animal average in our revised manuscript.

(7) In general this is an excellent analysis of circuit function but leaves out the question of whether there may be other inputs to pPVT that also mediate the same behavioral effect. Future experiments that use activity-dependent Fos-TRAP labeling in combination with rabies can identify other inputs to heat-sensitive pPVT cells, which may have convergent or divergent functions compared to the POA inputs.

Thanks for the valuable suggestion, which would enhance the conclusion. We will consider adopting this approach in future investigations into this question.

Reviewer #2 (Public review):

Summary

The study by Cao et al. highlights an interesting and important aspect of heat- and thermal biology: the effect of repetitive, long-term heat exposure and its impact on brain function.

Even though peripheral, sensory temperature sensors and afferent neuronal pathways conveying acute temperature information to the CNS have been well established, it is largely unknown how persistent, long-term temperature stimuli interact with and shape CNS function, and how these thermally-induced CNS alterations modulate efferent pathways to change physiology and behavior. This study is therefore not only novel but, given global warming, also timely.

The authors provide compelling evidence that neurons of the paraventricular thalamus change plastically over three weeks of episodic heat stimulation and they convincingly show that these changes affect behavioral outputs such as social interactions, and anxiety-related behaviors.

Strengths

(1) It is impressive that the assessed behaviors can be (i) recruited by optogenetic fiber activation and (ii) inhibited by optogenetic fiber inhibition when mice are exposed to heat. Technically, when/how long is the fiber inhibition performed? It says in the text "3 min on and 3 min off". Is this only during the 20-minute heat stimulation or also at other times?

Thanks for pointing out the need for clarification. Our optogenetic inhibition had been conducted for 21 days during the heat exposure period (90 mins) for each mouse. And to avoid the light-induced heating effect, we applied the cyclical mode of 3 minutes’ light on and 3 minutes’ light off only during the process of heat exposure but not other time. The detailed description has been supplemented in the Method part of our revised manuscript.

(2) It is interesting that the frequency of activity in pPVT neurons, as assessed by fiber photometry, stays increased after long-term heat exposure (day 22) when mice are back at normal room temperature. This appears similar to a previous study that found long-term heat exposure to transform POA neurons plastically to become tonically active (https://www.biorxiv.org/content/10.1101/2024.08.06.606929v1). Interestingly, the POA neurons that become tonically active by persistent heat exposure described in the above study are largely excitatory, and thus these could drive the activity of the pPVT neurons analyzed in this study.

Thanks for pointing out this study that suggests similar plasticity of POA neurons under long-term heat exposure serving a different purpose. We have included this information in our discussion as well.  

(3) How can it be reconciled that the majority of the inputs from the POA are found to be largely inhibitory (Fig. 2H)? Is it possible that this result stems from the fact that non-selective POA-to-pPVT projections are labelled by the approach used in this study and not only those pathways activated by heat? These points would be nice to discuss.

Thanks for raising these important questions. Although it is not our primary focus, we are aware of the substantial inhibitory inputs from POA to pPVT which suggests an important function. However, we do not think that this pathway, which would exert an opposite effect on POA-recipient pPVT neurons compared to the excitatory input, contributes to the long-term effect of chronic heat exposure. This is due to the increased, rather than decreased, excitability of the neurons. There is a possibility that this inhibitory input serves as a short-term inhibitory control for other purpose. Further work is needed to fully address this question.

(4) It is very interesting that no LTP can be induced after chronic heat exposure (Figures K-M); the authors suggest that "the pathway in these mice were already saturated" (line 375). Could this hypothesis be tested in slices by employing a protocol to extinguish pre-existing (chronic heat exposure-induced) LTP? This would provide further strength to the findings/suggestion that an important synaptic plasticity mechanism is at play that conveys behavioral changes upon chronic heat stimulation.

We agree with the reviewer that the results of the suggested experiment would further strengthen our hypothesis. We will try to confirm this in future studies.

(5) It is interesting that long-term heat does not increase parameters associated with depression (Figure 1N-Q), how is it with acute heat stress, are those depression parameters increased acutely? It would be interesting to learn if "depression indicators" increase acutely but then adapt (as a consequence of heat acclimation) or if they are not changed at all and are also low during acute heat exposure.

Based on our observations, we did not find increased depression parameters after acute heat stress in our experiments (data not shown), which was consistent with other two previous studies (Beas et al., 2018; Zhang et al., 2021). It appears that acute heat stress is more associated with anxiety-like behavior and may not be sufficient to induce depression-like phenotypes in rodents, aligning with our observation during experiments.

Beas BS, Wright BJ, Skirzewski M, Leng Y, Hyun JH, Koita O, Ringelberg N, Kwon HB, Buonanno A, Penzo MA (2018) The locus coeruleus drives disinhibition in the midline thalamus via a dopaminergic mechanism Nat Neurosci 21:963-973.

Zhang GW, Shen L, Tao C, Jung AH, Peng B, Li Z, Zhang LI, Whit Tao HZ (2021) Medial preoptic area antagonistically mediates stress-induced anxiety and parental behavior Nat Neurosci 24:516-528.

Weaknesses/suggestions for improvement.

(1) The introduction and general tenet of the study is, to us, a bit too one-sided/biased: generally, repetitive heat exposure --heat acclimation-- paradigms are known to not only be detrimental to animals and humans but also convey beneficial effects in allowing the animals and humans to gain heat tolerance (by strengthening the cardiovascular system, reducing energy metabolism and weight, etc.).

Thanks for the suggestion. We have modified the introduction in our revised manuscript to make it more balanced.

(2) The point is well taken that these authors here want to correlate their model (90 minutes of heat exposure per day) to heat waves. Nevertheless, and to more fully appreciate the entire biology of repetitive/chronic/persistent heat exposure (heat acclimation), it would be helpful to the general readership if the authors would also include these other aspects in their introduction (and/or discussion) and compare their 90-minute heat exposure paradigm to other heat acclimation paradigms. For example, many past studies (using mice or rats)m have used more subtle temperatures but permanently (and not only for 90 minutes) stimulated them over several days and weeks (for example see PMID: 35413138). This can have several beneficial effects related to cardiovascular fitness, energy metabolism, and other aspects. In this regard: 38{degree sign}C used in this study is a very high temperature for mice, in particular when they are placed there without acclimating slowly to this temperature but are directly placed there from normal ambient temperatures (22{degree sign}C-24{degree sign}C) which is cold/coolish for mice. Since the accuracy of temperature measurement is given as +/- 2{degree sign}C, it could also be 40{degree sign}C -- this temperature, 40{degree sign}C, non-heat acclimated C57bl/6 mice will not survive for long.

The authors could consider discussing that this very strong, short episodic heat-stress model used here in this study may emphasize detrimental effects of heat, while more subtle long-term persistent exposure may be able to make animals adapt to heat, become more tolerant, and perhaps even prevent the detrimental cognitive effects observed in this study (which would be interesting to assess in a follow-up study).

Thanks for pointing out the important aspect regarding the different heat exposure paradigms and their potential impacts. We have incorporated these points into both the Introduction and Discussion sections of the revised manuscript.

(3) Line 140: It would help to be clear in the text that the behaviors are measured 1 day after the acute heat exposure - this is mentioned in the legend to the figure, but we believe it is important to stress this point also in the text. Similarly, this is also relevant for chronic heat stimulation: it needs to be made very clear that the behavior is measured 1 day after the last heat stimulus. If the behaviors had been measured during the heat stimulus, the results would likely be very different.

Thanks for the suggestion, and we have clarified the procedure in the revised manuscript.

(4) Figure 2 D and Figure 2- Figure Supplement 1: since there is quite some baseline cFos activity in the pPVT region we believe it is important to include some control (room temperature) mice with anterograde labelling; in our view, it is difficult/not possible to conclude, based on Fig 2 supplement 2C, that nearly 100% of the cfos positive cells are contacted by POA fibre terminals (line 168). By eye there are several green cells that don't have any red label on (or next to) them; additionally, even if there is a little bit of red signal next to a green cell: this is not definitive proof that this is a synaptic contact. It is therefore advisable to revisit the quantification and also revisit the interpretation/wording about synaptic contacts.

In relation to the above: Figure 2h suggests that all neurons are connected (the majority receiving inhibitory inputs), is this really the case, is there not a single neuron out of the 63 recorded pPVT neurons that does not receive direct synaptic input from the POA?

Thanks for the comments. For Figure 2-figure supplement 1, the baseline c-Fos activity in pPVT were indeed measured from mouse under room temperature. Observed activity may be attributed to the diverse functions that the pPVT is responsible for. Compared to the heat-exposed group, we observed significant increases in c-Fos signals, suggesting the effect of heat exposure.

For Figure 2-figure supplement 2, through targeted injection of AAV1-Cre into the POA, we achieved selective expression of Cre-dependent ChR2-mCherry in pPVT neurons receiving POA inputs. Following heat exposure, we observed substantial colocalization between heat-induced c-Fos expression (green signal) and ChR2-mCherry-labeled neurons (red signal) in the pPVT. This extensive overlap indicates that POA-recipient pPVT neurons are predominantly heat-responsive and likely mediate the behavioral alterations induced by chronic heat exposure. We have validated these signals and included updated quantification in our revised manuscript.

For Fig 2H, we specifically patched those neurons that were surrounded by red fluorescence under the microscope, ensuring that the patched neurons had a high likelihood of being innervated from POA. This is why all 63 recorded pPVT neurons were found to receive direct synaptic input from the POA.

(5) It would be nice to characterize the POA population that connects to the pPVT, it is possible/likely that not only warm-responsive POA neurons connect to that region but also others. The current POA-to-pPVT optogenetic fibre stimulations (Figure 4) are not selective for preoptic warm responsive neurons; since the POA subserves many different functions, this optogenetic strategy will likely activate other pathways. The referees acknowledge that molecular analysis of the POA population would be a major undertaking. Instead, this could be acknowledged in the discussion, for example in a section like "limitation of this study".

Thanks for the suggestion. We have supplemented this part in our revised manuscript.

(6) Figure 3a the strategy to express Gcamp in a Cre-dependent manner: it seems that the Gcamp8f signal would be polluted by EGFP (coming from the Cre virus injected into the POA): The excitation peak for both is close to 490nm and emission spectra/peaks of GCaMP8f (510-520 nm) and EGFP (507-510 nm) are also highly overlapping. We presume that the high background (EGFP) fluorescence signal would preclude sensitive calcium detection via Gcamp8f, how did the authors tackle this problem?

Thank you for pointing out this issue. We acknowledge that we included AAV1-EGFP when recording the GCaMP8F signal to assist in the post-verification of the accuracy of the injection site. But we also collected recording data from mice with AAV1-Cre without EGFP injected into POA and Cre-dependent GCaMP8F in pPVT, albert in a smaller number. We did not observe any obvious differences in the change in calcium signal between these two virus strategies, suggesting that the sensitivity of the GCaMP signals was not significantly affected by the increased baseline fluorescence due to EGFP.

(7) How did the authors perform the social interaction test (Figures 1F, G)? Was the intruder mouse male or female? If it was a male mouse would the interaction with the female mouse be a form of mating behavior? If so, the interpretation of the results (Figures 1F, G) could be "episodic heat exposure over the course of 3 weeks reduces mating behavior".

Thanks for the comment. For this female encounter test, we strictly followed the protocol by Ago Y, et al., (2015). During this test, both the strange male and female mice were placed into a wired cup (which is made up of mental wire entanglement and the size for each hole is 0.5 cm [L] x 0.5 cm [W]), which successfully prevented large body contact and the mating behavior but only innate sex-motivated moving around the cup. We have supplemented the details in the method part of our revised manuscript.

Ago Y, Hasebe S, Nishiyama S, Oka S, Onaka Y, Hashimoto H, Takuma K, Matsuda T (2015) The Female Encounter Test: A Novel Method for Evaluating Reward-Seeking Behavior or Motivation in Mice Int J Neuropsychopharmacol 18: pyv062.

Reviewer #3 (Public review):

In this study, Cao et al. explore the neural mechanisms by which chronic heat exposure induces negative valence and hyperarousal in mice, focusing on the role of the posterior paraventricular nucleus (pPVT) neurons that receive projections from the preoptic area (POA). The authors show that chronic heat exposure leads to heightened activity of the POA projection-receiving pPVT neurons, potentially contributing to behavioral changes such as increased anxiety level and reduced sociability, along with heightened startle responses. In addition, using electrophysiological methods, the authors suggest that increased membrane excitability of pPVT neurons may underlie these behavioral changes. The use of a variety of behavioral assays enhances the robustness of their claim. Moreover, while previous research on thermoregulation has predominantly focused on physiological responses to thermal stress, this study adds a unique and valuable perspective by exploring how thermal stress impacts affective states and behaviors, thereby broadening the field of thermoregulation. However, a few points warrant further consideration to enhance the clarity and impact of the findings.

(1) The authors claim that behavior changes induced by chronic heat exposure are mediated by the POA-pPVT circuit. However, it remains unclear whether these changes are unique to heat exposure or if this circuit represents a more general response to chronic stress. It would be valuable to include control experiments with other forms of chronic stress, such as chronic pain, social defeat, or restraint stress, to determine if the observed changes in the POA-pPVT circuit are indeed specific to thermal stress or indicative of a more universal stress response mechanism.

We also share similar considerations as the reviewer and indeed have conducted experiments to explore this possibility. Our findings suggest that the POA-pPVT pathway may also mediate behavioral changes induced by other chronic stress, e.g. chronic restraint stress. Nevertheless, given the well-known prominent role of POA neurons in heat perception, we do believe that the POA-pPVT has a specialized role in mediating chronic heat induced changes. The role of this pathway in other stress-related responses will need a more comprehensive study in the future.

(2) The authors use the term "negative emotion and hyperarousal" to interpret behavioral changes induced by chronic heat (consistently throughout the manuscript, including the title and lines 33-34). However, the term "emotion" is broad and inherently difficult to quantify, as it encompasses various factors, including both valence and arousal (Tye, 2018; Barrett, L. F. 1999; Schachter, S. 1962). Therefore, the reviewer suggests the authors use a more precise term to describe these behaviors, such as valence. Additionally, in lines 117 and 137-139, replacing "emotion" with "stress responses," a term that aligns more closely with the physiological observations, would provide greater specificity and clarity in interpreting the findings.

Thanks for the suggestion. We have modified the description of “emotion” to “emotional valence” in various places throughout the revised manuscript.

(3) Related to the role of POA input to pPVT,

a) The authors showed increased activity in pPVT neurons that receive projections from the POA (Figure 3), and these neurons are necessary for heat-induced behavioral changes (Figures 4N-W). However, is the POA input to the pPVT circuit truly critical? Since recipient pPVT neurons can receive inputs from various brain regions, the reviewer suggests that experiments directly inhibiting the POA-to-pPVT projection itself are needed to confirm the role of POA input. Alternatively, the authors could show that the increased activity of pPVT neurons due to chronic heat exposure is not observed when the POA is blocked. If these experiments are not feasible, the reviewer suggests that the authors consider toning down the emphasis on the role of the POA throughout the manuscript and discuss this as a limitation.<br /> b) In the electrophysiology experiments shown in Figures 6A-I, the authors conducted in vitro slice recordings on pPVT neurons. However, the interpretation of these results (e.g., "The increase in presynaptic excitability of the POA to pPVT excitatory pathway suggested plastic changes induced by the chronic heat treatment.", lines 349-350) appears to be an overclaim. It is difficult to conclude that the increased excitability of pPVT neurons due to heat exposure is specifically caused by inputs from the POA. To clarify this, the reviewer suggests the authors conduct experiments targeting recipient neurons in the pPVT, with anterograde labeling from the POA to validate the source of excitatory inputs.

For point (a), we acknowledge that pPVT neurons receiving POA inputs may also receive projections from other brain regions. While these additional inputs warrant investigation, they fall beyond the scope of our current study and represent promising directions for future research. Notably, compared to other well-characterized regions such as the amygdala and ventral hippocampus, the pPVT receives particularly robust projections from hypothalamic nuclei (Beas et al., 2018). Our optogenetic inhibition of POA-recipient pPVT neurons during chronic heat exposure effectively prevented the influence of POA excitatory projections on pPVT neurons. Furthermore, selective optogenetic activation of POA excitatory terminals within the pPVT was sufficient to induce similar behavioral abnormalities in mice, strongly supporting the causal role of POA inputs in mediating chronic heat exposure-induced behavioral alterations.

Beas BS, Wright BJ, Skirzewski M, Leng Y, Hyun JH, Koita O, Ringelberg N, Kwon HB, Buonanno A, Penzo MA (2018) The locus coeruleus drives disinhibition in the midline thalamus via a dopaminergic mechanism Nat Neurosci 21:963-973.

Regarding point (b), we acknowledge certain limitations in our in vitro patch-clamp recordings when attributing increased pPVT neuronal excitability to enhanced presynaptic POA inputs. Nevertheless, our brain slice recordings clearly demonstrated heightened excitability of pPVT neurons following chronic heat exposure. This finding was further corroborated by our in vivo fiber photometry recordings specifically targeting POA-recipient pPVT neurons, which confirmed that the increased pPVT neuronal activity was indeed modulated by POA inputs. The causal relationship was strengthened by our observation that optogenetic activation of POA excitatory terminals within the pPVT reproduced behavioral abnormalities similar to those observed in chronic heat-exposed mice. Additionally, our inability to induce circuit-specific LTP in the POA-pPVT pathway suggests that these synapses were already potentiated and saturated, reflecting enhanced excitatory inputs from the POA to pPVT. Collectively, these findings support our conclusion that increased excitatory projections from the POA to pPVT likely represent a key mechanism underlying chronic heat exposure-induced behavioral alterations in mice.

(4) The authors focus on the excitatory connection between the POA and pPVT (e.g., "Together, our results indicate that most of the pPVT-projecting POA neurons responded to heat treatment, which would then recruit their downstream neurons in the pPVT by exerting a net excitatory influence.", lines 169-171). However, are the POA neurons projecting to the pPVT indeed excitatory? This is surprising, considering i) the electrophysiological data shown in Figures 2E-K that inhibitory current was recorded in 52.4% of pPVT neurons by stimulation of POA terminal, and ii) POA projection neurons involved in modulating thermoregulatory responses to other brain regions are primarily GABAergic (Tan et al., 2016; Morrison and Nakamura, 2019). The reviewer suggests showing whether the heat-responsive POA neurons projecting to the pPVT are indeed excitatory (This could be achieved by retrogradely labeling POA neurons that project to the pPVT and conducting fluorescence in situ hybridization (FISH) assays against Slc32a1, Slc17a6, and Fos to label neurons activated by warmth). Alternatively, demonstrate, at least, that pPVT-projecting POA neurons are a distinct population from the GABAergic POA neurons that project to thermoregulatory regions such as DMH or rRPa. This would clarify how the POA-pPVT circuit integrates with the previously established thermoregulatory pathways.

Thanks for the comment and suggestion. We acknowledge that there are both excitatory and inhibitory projections from POA to pPVT. Although it is not our primary focus, we are aware of the substantial inhibitory inputs from POA to pPVT which suggests an important function. However, we do not think that this pathway, which would exert an opposite effect on POA-recipient pPVT neurons compared to the excitatory input, contributes to the long-term effect of chronic heat exposure. This is due to the increased, rather than decreased, excitability of the neurons. There is a possibility that this inhibitory input serves as a short-term inhibitory control for other purpose. Further work is needed to fully address this question.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

I have a number of suggested minor edits that would improve the readability and interpretation of figures for the reader. In many figures, there are places where it is unclear what is being tested, and making minor changes would make the manuscript flow more easily for the reader:

(1) The authors could add additional details about the behavior paradigms in the Figures, especially Figure 1. How long was the chronic heat exposure for? At what temperature? What is the length of time between the end of heat exposure and the start of behaviors? What was the schedule of testing for EPM and social behaviors? Was it all on the same day or on different days? These details will make it easier for the reader to understand the behavior tests.

We have revised our experimental scheme, especially Figure 1, and added more detailed descriptions in the method section. The modifications have also been applied to the other figures.

(2) In Figures 1J and 1K, it is a bit unclear what is being shown in the right panel, since there are no axes or labels to interpret what is being plotted.

We have added body kinetics (purple dot) in the left panel of Figure 1J and 1K to align with the right panels, and we have updated our descriptions in the figure legend.

(3) In general, Figure 1 would benefit from more headers/labels or schematics to demonstrate what is being tested (for example, it's unclear that forced swim, tail suspension, open field, aggression, sucrose preference, or acoustic startle are being studied unless the reader looks at the figure legend in depth. Simple schematics or titles for each panel would help.

We have added the abbreviated titles for each panel of Figure 1 to help readers to better understand what was being tested.

(4) Figure 2A would benefit from edits to the schematic so that it is clear that heat exposure is being done before the animal is sacrificed and cFos is stained.

We have revised the text to clarify that heat exposure occurred before the animal was sacrificed and c-Fos was stained.

(5) Figure 2D: would help if the quantification of overlap of cFos and rabies was shown in the figure in addition to reporting it in the text (84%).

We have added quantification in Figure 2D.

(6) The supplemental data in Figure 2 - Supplemental Figure 1 showing increased Fos in PVT and POA after heat exposure would actually help if it was in main Figure 2 so that the reader can more clearly see the rationale for choosing the POA-PVT circuit. But this is a matter of preference and up to the author where they want to show this data.

Thanks for the suggestion. But considering the layout and space, we will prefer to retain this part in Figure 2-supplemental figure 1.

(7) Figure 3 would benefit from a behavior schematic illustrating the time course of the experiment and what the heat exposure protocol is for each day (how many minutes heat 'on' vs 'off', the temperature of heat, etc). Also, what is different about day 22 that makes it chronic heat vs day 21? Currently, it is a bit hard to understand the protocol.

We have added the temperature and time of chronic heat exposure in the schematic of Figure 3. The “day 22” represented the time point after chronic heat exposure. And we measured the calcium activity of POA recipient pPVT neurons on day 22 to compare with day 1 to demonstrate that the activity changes of POA recipient pPVT neurons after chronic heat exposure.

(8) Figure 3D, it is unclear what the difference is between the Day 1 data on the left and Day 1 data on the right. Same with Figure 3H, unclear what the difference is between the left and the right.

The left panel and right panel reflect different parameters: frequency /min (left) and amplitude (△F/F) for Figure 3D-3H. By doing this, we want to reflect the dynamic activity changes of POA recipient pPVT neurons throughout chronic heat exposure process. Now, all figures in panel 3D to 3H have been revised to make them clearer in meaning.

(9) Figure 4A would benefit from schematics showing the stimulation protocol for chronic optogenetics (how many days? Frequency? Duration of time? Etc)

We have added detailed schematics in our Figure 4A.

Reviewer #2 (Recommendations for the authors)

(1) It is interesting that social behavior appears to be reduced upon long-term heat exposure but not after acute heat exposure. Interaction of animals, such as huddling, can be used by animals as a form of behavioral thermoregulation in cold environments and heat may drive animals apart to allow for better heat dissipation. The social interaction measured here is not huddling (because, I assume, the animals are separated by a divider?) but is this form of behavior measured here related to huddling/"social thermoregulation"? This could be discussed.

Our behavioral tests were performed at room temperature. Even though huddling is a type of social behavior, based on our observation, the tested mouse was actively revolving around the mental cap, suggesting this type of behavior is not related to huddling/social thermoregulation type of social behavior.

(2) Line 113: The statement "Chronic treatment did not change body temperature" should be clarified/rephrased because 90 minutes of 38 degrees centigrade exposure to heat will increase the body temperature of mice. It would be helpful if the authors made clear that they measure body temperature before the heat stimulus (and not during the heat stimulus), which is now only obvious if one digs into the methods section.

We have revised the text and clarified that body temperature was measured before the heat stimulus in the revised manuscript.

(3) Figure 1J and K: for the non-experts, these graphs are difficult to interpret, some more explanation is needed (what exactly is measured ?). We believe that the term "arousal" may not be justified in this context because the authors have not measured sleep patterns (EEG and EMG) to show that the mice arouse from a sleep (or sleep-like) stage; the authors may consider changing the terminology, e.g. something along the lines of "agitation" or "activity".

We have further elaborated the meaning of Figure 1J and K in our revised manuscript. The acoustic startle response is a well-recognized behavioral parameter reflecting arousal levels in rodent model. The more agitation in response to stimulus, the higher the arousal levels in mice. We have used the term “agitation” to describe mice’s performance in the acoustic startle response test.

Reviewer #3 (Recommendations for the authors):

(1) The authors suggest in the introduction of the manuscript that the HPA axis and other multifaceted factors may influence emotional changes caused by heat stress (lines 63-78). However, there are no experiments or discussions on how the POA-pPVT circuit interacts with these factors. In line with the study's proposed direction in the introduction section, it would be valuable to explore, or at least discuss, whether and how the POA-pPVT circuit interacts with the HPA axis or other neural circuits known to regulate emotional and stress responses. Alternatively, the reviewer suggests revising the content of the introduction to align with the focus of the study.

Although POA is known to possibly interact with the HPA axis via its connection with the paraventricular nucleus of the hypothalamus, there is hardly any evidence for the pPVT. Thus, we prefer not to speculate this question, which remains open, in our current manuscript.

(2) In Figure 5, the authors report that pPVT neurons that receive projections from the POA exhibited increased responses to stressful situations following chronic heat exposure. However, considering the long pre- and post-recording time gap of approximately three weeks, the additional expression of GCaMP protein over time could potentially account for the increased signal. Therefore, the reviewer recommends including a control group without heat exposure to rule out this possibility.

We have included Figure 3-figure supplement 1 in our manuscript to exclude the effect of expression of GCaMP protein over time on the recording of calcium signal.

(3) Related to Figure 2, a) Please include quantification data of the overlap between retrogradely labeled and c-Fos-expressing POA neurons, which can be presented as a bar graph in Figure 2. This would be beneficial for readers to estimate how many warm-activated POA neurons connected to the pPVT are actively engaged under these conditions.

In the revised manuscript, we have included the quantification analysis in Figure 2.

b) The images in Figure 2 - Figure Supplement 1 seem to degrade in quality when magnified, making it difficult to discern finer details. Higher-resolution images would greatly improve the clarity and help in accurately visualizing the c-Fos expression patterns in the POA and pPVT regions.

We have changed our images of Figure 2-figure supplement 1 to higher-resolution in the revised manuscript.

c) The c-Fos images in Figure 2D and Figure 2 - Figure Supplement 2C appear unusual in that the c-Fos signal seems to fill the entire cell, whereas c-Fos protein is localized to the nucleus. Could the authors clarify whether this image accurately represents c-Fos staining or if there might be an issue with the staining or imaging process?

We are confident that the green signals in both Figure 2D and Figure 2-figure supplement 2C, which did not occupy the whole cell body, have already accurately reflected the c-Fos and that they were nucleus staining. We have updated the amplified picture in Figure 2D.

d) In Supplemental Figure 2B, the square marking the region of interest should be clearly explained in the figure legend to ensure that readers can fully understand the context and focus of the image.

We have further modified our figure legend in Figure 2-figure supplement 1 in our revised manuscript.

It is crucial to understand your audience.

Burying the important information about the O-rings in the middle of the paragraph made the information seem inconsequential resulting in a big issue.

They did not present the negative piece of crucial information regarding the o ring safety the same as the other crucial point of the approving the launch. This mis read readers and led to major problems.

Bringing forward the O-ring data could have changed the outcome of the Challenger.

The crucial information about the O-ring seals should have been presented to the reader in a fashion that was clear and evident that this was a crucial problem.

在论文《信息美学：一次英雄实验》中提到的学者包括以下几位，他们对信息美学的形成和发展有着重要影响：

1. Max Bense（马克斯·本塞）

• 简介： Max Bense（1910-1990）是德国著名的哲学家、数学家和信息理论学者，通常被认为是信息美学的奠基人之一。他是斯图加特大学哲学与知识理论研究所的负责人。Bense的工作跨越了哲学、数学、物理学和美学的多个领域，尤其在信息理论与美学的结合方面具有开创性。

• 与信息美学的关系： Max Bense 提出了信息美学的基本理念，试图基于信息理论来创建一个客观的美学标准，排除了主观的审美感受。他认为，美学可以通过数学和信息理论的工具来量化，尤其是通过统计信息的方式，来定义美学对象的秩序与复杂度。他的理论为信息美学奠定了理论基础，尤其是在美学的客观性和定量分析方面的探索。

2. Abraham A. Moles（亚伯拉罕·莫莱斯）

• 简介： Abraham Moles（1920-1992）是法国的物理学家和心理学家，他的研究涉及信息学、符号学以及美学领域。Moles在信息美学中的贡献主要是通过美学信息的概念，将信息理论与美学结合。相比于Bense，Moles更为注重语义信息和美学信息之间的区分，强调艺术作品不仅仅是客观的信号集合，而是通过传递信息来影响观众的心理状态。

• 与信息美学的关系： Moles提出了美学信息的概念，认为艺术作品传递的美学信息与作品的物理信息是相对应的。他区分了语义信息和美学信息，并通过这一理论框架探讨艺术作品如何影响观众的情感和感知。Moles认为，美学信息不仅仅是符号系统中的信息，它还关乎如何通过作品的呈现方式影响观众的心理反应。他的思想推动了信息美学的进一步发展，尤其是在心理学与艺术交叉的领域。

3. George D. Birkhoff（乔治·D·比尔科夫）

• 简介： George D. Birkhoff（1884-1944）是美国著名的数学家，他的研究领域涵盖了数学美学，即如何将美学量化。Birkhoff提出了美学公式，该公式通过计算艺术作品的秩序与复杂度比率来衡量其美学价值，这一理论至今仍在美学和设计领域具有一定的影响力。

• 与信息美学的关系： Birkhoff的公式为信息美学提供了数学模型的基础。他的美学公式通过计算对象的秩序度和复杂度的比率（\(M = O / C\)）来衡量作品的美学价值。在信息美学中，Birkhoff的公式被转化为信息理论的术语，成为量化艺术作品美学的一部分。Birkhoff的工作为后来的信息美学奠定了数学基础，尤其是他通过量化秩序和复杂度为美学的客观性提供了理论支持。

4. Helmar Frank（赫尔玛·弗兰克）

• 简介： Helmar Frank 是德国的艺术学者，研究领域涉及信息理论和美学的结合。他提出了基于香农信息理论的美学信息测量方法，进一步发展了信息美学的理论框架。他的研究重点是美学信息如何通过统计方法来量化，并探讨了惊讶度和穿透度等概念。

• 与信息美学的关系： Frank在信息美学中扩展了信息测量的理论，提出了惊讶度和穿透度等度量指标，用以量化艺术作品中符号的突发性和频繁度。他利用香农的量化信息理论，通过计算作品中符号的信息内容和冗余度，为信息美学提供了更为细化的分析工具。他的贡献使得信息美学的量化更加丰富，并推动了美学信息理论的发展。

5. Rul Gunzenhäuser（鲁尔·冈岑豪泽）

• 简介： Rul Gunzenhäuser 是一位研究美学和信息理论的学者，他将Birkhoff的美学公式与信息理论结合，提出了通过信息理论量化艺术作品的复杂度和秩序的理论框架。

• 与信息美学的关系： Gunzenhäuser基于信息理论对Birkhoff的公式进行了解读，提出将复杂度与秩序转化为信息量和冗余度，并通过信息内容的最大化与冗余度最小化来衡量美学对象的美学价值。他的工作为信息美学提供了进一步的数学化手段，推动了信息美学理论的深入发展。

总结：

这些学者在信息美学中的贡献各具特色，但共同点是他们都试图将美学量化，通过数学模型和信息理论对艺术作品进行客观的评价。Max Bense 和 Abraham Moles 是信息美学的主要奠基人，他们的理论分别侧重于理性化的美学标准和美学信息的心理学层面；Birkhoff则为其提供了数学上的支持，通过秩序与复杂度的比率来量化美学；而Helmar Frank 和Rul Gunzenhäuser则进一步深化了信息美学的数学化，将信息理论的工具引入美学测量。

many sources used throughout. could show not much original research.

keywords are pointed out before the article, gives the reader an idea of what to expect to learn about. same with abstract

This work has been peer reviewed in GigaScience (https://doi.org/10.1093/gigascience/giaf007), which carries out open, named peer-review. These reviews are published under a CC-BY 4.0 license and were as follows:

Reviewer #2: Anuradha Wickramarachchi

Overall comments.

Authors of the manuscript have developed an iterative overlap graph construction algorithm to support genome assembly. This is both an interesting and a demanding area of research due to very recent advancements in sequencing technologies.

Although the text in the manuscript is interesting, grammar must be rechecked and revised. At some point it is difficult to keep track of the content and references to supplementary to make sense out of the content.

Specific comments

Page 1 Line 13: I believe the authors are talking about assembly sizes and not genome sizes. The sentences here could be a bit short to make them easy to understand.

Page 2 Line 19: Theoretical time complexity O(m2n2) is bit of an overstatement due to the heuristics employed by most assemblers. For example, mash distance, minimisers and k-mer bins are there to prevent this explosion of complexity. Either acknowledge such methods or provide a range for the time complexity. I would be interesting to know the time complexities of the methods expressed in sentence starting Line 15.

Page 5 Line 11: Was this performed with overlapping windows of 1gb? Otherwise, simulations may not have reads spanning across such regions.

Page 5 Line 14: It seems you are simulating 9 + 4 + 4 datasets. This is unclear, please make this into bullet points or separate paragraphs and explain clearly. Include simulator information in the table itself by may be making it landscape (in supplementary).

Fig 2: I believe authors should expand their analysis to more recent and popular assemblers. For example, wtdbg2 is designed for noisy reads and not specifically for more accurate R10/ HiFi reads. So please include, HiFi-asm, Flye where appropriate. Flye supports ONT out of the box and in my experience does produce good assemblies.

Although, you are evaluating read overlaps, it is hard to ignore assemblers themselves just because they do not produce intermediate overlaps graphs.

Page 5-9: In the benchmarks section, please include how True Positives and False Positives were labelled. Was this from simulation data?

Page 11: Use of xRead has been evaluated on genome assemblies. This is a very important and it is a bit unfortunate that existing assemblers are not very flexible in terms of plugging in new intermediate steps. It might be worth exploring into creating a new assembler using the wtpoa2 cli command of wtdbg2.

Page 16: What will happen if you only capture reads from a single chromosome due to longer length? I believe the objective is to gather longest reads capturing as much as possible covering the whole genome. Please comment on this.

Page 19: In the Github Readme the download URL was wrong. Please correct it to the latest release

Correct: https://github.com/tcKong47/xRead/releases/download/xRead-v1.0.0.1/xRead-v1.0.0.tar.gz Existing: https://github.com/tcKong47/xRead/releases/download/v1.0.0/xRead-v1.0.0.tar.gz

Make command failed with make: *** No rule to make target main.h', needed bymain.o'. Stop.

It seems the release does not have source code, but rather the compiled version. Please update github instructing how to compile code properly with a git clone.

EXTERNAL: ONCE AN ADULT ALWAYS ADULT LAWS STATE THAT IF A CHILD HAS A CRIMINAL RECORD IN AN ADULT COURT THEY MUST ALAYS SUBSEQUENTLY BE CHARGED IN ADULT COURTS. 34 STATES HAVE THESE LAWS

Connecting to god through night and other elements of nature.

This passage reflects deep themes of mourning, loss, and memory. The speaker is contemplating how to honor and remember someone they loved, considering what kind of art or images would be fitting for the "chamber walls" of the deceased's burial place. The "chamber walls" could be interpreted as the tomb or final resting place, and the speaker’s desire to adorn it with meaningful images indicates their wish to preserve the memory and significance of the person who has passed away. The question of what to "hang on the walls" suggests a search for meaningful symbols, reflections of the deceased’s life, or expressions of the love and grief felt by the speaker. These images are not merely for decoration but carry emotional weight, as they are intended to honor the deceased.

Qualifier

choice of legal systems: comparing the US and canada is easy (excerpt continues after this) WOOOOOOO

competência territorial absoluta, porque funcional

Art. 267. Extingue-se o processo, sem resolução de mérito:

Atual 485

O instrumento jurídico apto a constituir parceria da OSCIP com o Poder Público é o Termo de Parceria.

Frise que, nas OS, o instrumento jurídico é o Contrato de Gestão.

Para a constituição de um OS, deverá o Poder Executivo analisar a conveniência e oportunidade de se qualificar uma PJ sem fins lucrativos. Isto é, diferentemente das OSCIP, a qualificação desta lei é um ato discricionário e não vinculado.

.

Hypothes.is has a beta that would enable this asap on anything in your browser with in-context AI,

• RE 1151237
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. ALEXANDRE DE MORAES
• Julgamento: 03/10/2019
• Publicação: 12/11/2019

RECURSO EXTRAORDINÁRIO COM REPERCUSSÃO GERAL RECONHECIDA. COMPETÊNCIA PARA DENOMINAÇÃO DE PRÓPRIOS, VIAS E LOGRADOUROS PÚBLICOS E SUAS ALTERAÇÕES. COABITAÇÃO NORMATIVA ENTRE OS PODERES EXECUTIVO (DECRETO) E O LEGISLATIVO (LEI FORMAL), CADA QUAL NO ÂMBITO DE SUAS ATRIBUIÇÕES. 1. Tem-se, na origem, ação direta de inconstitucionalidade proposta perante o Tribunal de Justiça do Estado de São Paulo em face do art. 33, XII, da Lei Orgânica do Município de Sorocaba, que assim dispõe: “Art. 33. Cabe à Câmara Municipal, com a sanção do Prefeito, legislar sobre as matérias de competência do Município, especialmente no que se refere ao seguinte: (…) XII – denominação de próprios, vias e logradouros públicos e suas alterações”.

1. Na inicial da ação direta, a Procuradoria-Geral de Justiça do Estado de São Paulo sustenta que tal atribuição é privativa do Chefe do Poder Executivo.

2. O Tribunal de Justiça do Estado de São Paulo julgou procedente a ação no ponto, por considerar que a denominação de vias públicas compete tanto ao Poder Legislativo, quanto ao Executivo. Assim, reputou inconstitucional a norma, porque concede tal prerrogativa unicamente à Câmara Municipal.

3. A Constituição Federal consagrou o Município como entidade federativa indispensável ao nosso sistema federativo, integrando-o na organização político-administrativa e garantindo-lhe plena autonomia, como se nota na análise dos artigos 1º, 18, 29, 30 e 34, VII, c , todos da Constituição Federal.

4. As competências legislativas do município caracterizam-se pelo princípio da predominância do interesse local, que, apesar de difícil conceituação, refere-se àqueles interesses que disserem respeito mais diretamente às suas necessidades imediatas.

5. A atividade legislativa municipal submete-se à Lei Orgânica dos municípios, à qual cabe o importante papel de definir, mesmo que exemplificativamente, as matérias de competência legislativa da Câmara, uma vez que a Constituição Federal (artigos 30 e 31) não as exaure, pois usa a expressão interesse local como catalisador dos assuntos de competência municipal. Essa função legislativa é exercida pela Câmara dos Vereadores, que é o órgão legislativo do município, em colaboração com o prefeito, a quem cabe também o poder de iniciativa das leis, assim como o poder de sancioná-las e promulgá-las, nos termos propostos como modelo, pelo processo legislativo federal.

6. A Lei Orgânica do Município de Sorocaba, ao estabelecer, em seu artigo 33, inciso XII, como matéria de interesse local, e, consequentemente, de competência legislativa municipal, a disciplina de denominação de próprios, vias e logradouros públicos e suas alterações, representa legítimo exercício da competência legislativa municipal. Não há dúvida de que se trata de assunto predominantemente de interesse local (CF, art. 30, I).

7. Por outro lado, a norma em exame não incidiu em qualquer desrespeito à Separação de Poderes, pois a matéria referente à “denominação de próprios, vias e logradouros públicos e suas alterações” não pode ser limitada tão somente à questão de “atos de gestão do Executivo”, pois, no exercício dessa competência, o Poder Legislativo local poderá realizar homenagens cívicas, bem como colaborar na concretização da memorização da história e da proteção do patrimônio cultural imaterial do Município.

8. Em nenhum momento, a Lei Orgânica Municipal afastou expressamente a iniciativa concorrente para propositura do projeto de lei sobre a matéria. Portanto, deve ser interpretada no sentido de não excluir a competência administrativa do Prefeito Municipal para a prática de atos de gestão referentes a matéria; mas, também, por estabelecer ao Poder Legislativo, no exercício de competência legislativa, baseada no princípio da predominância do interesse, a possibilidade de edição de leis para definir denominação de próprios, vias e logradouros públicos e suas alterações .

9. Recurso Extraordinário provido, para declarar a constitucionalidade do do art. 33, XII, da Lei Orgânica do Município de Sorocaba, concedendo-lhe interpretação conforme à Constituição Federal, no sentido da existência de uma coabitação normativa entre os Poderes Executivo (decreto) e o Legislativo (lei formal), para o exercício da competência destinada a “denominação de próprios, vias e logradouros públicos e suas alterações”, cada qual no âmbito de suas atribuições.

10. Fixada a seguinte tese de Repercussão Geral: "É comum aos poderes Executivo (decreto) e Legislativo (lei formal) a competência destinada a denominação de próprios, vias e logradouros públicos e suas alterações, cada qual no âmbito de suas atribuições".

Tema - 1070 - Competência para denominação de ruas, próprios, vias e logradouros públicos e suas alterações.

Tese - É comum aos poderes Executivo (decreto) e Legislativo (lei formal) a competência destinada a denominação de próprios, vias e logradouros públicos e suas alterações, cada qual no âmbito de suas atribuições.

Correction: P(F∩O)

so these are depression features established in a different study?

existence of contingent beings implies existence of non-contingent (necessary beings) we cannot postulate one w/o the other, as one implies the other

thus, any objectors would have to show wwhy contingency can exist w/o necesiity

Author response:

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

Recommendations for the authors:

Reviewing Editor Note:

The two reviewers have provided thoughtful and constructive feedback that we hope will be of use to the authors to improve their manuscript.

Reviewer #1 (Recommendations For The Authors):

The section on "Circuit evolution by duplication and divergence" (starting on line 622) should cite:

Chakraborty, Mukta, and Erich D. Jarvis. "Brain evolution by brain pathway duplication." Philosophical Transactions of the Royal Society B: Biological Sciences 370, no. 1684 (2015): 20150056.

and

Roberts, Ruairí JV, Sinziana Pop, and Lucia L. Prieto-Godino. "Evolution of central neural circuits: state of the art and perspectives." Nature Reviews Neuroscience 23, no. 12 (2022): 725-743.

It should also reference that the concept originated from genetics:

Ohno, Susumu. Evolution by gene duplication. Springer Science & Business Media, 1970

These papers have now been cited: “Duplication and divergence of circuits was also proposed as a possible mechanism for the evolution of brain pathways for vocal learning in song-learning birds, spoken language in humans [@chakraborty2015brain] and other circuits [@roberts2022evolution].”

and: Our reconstructions identified a potential case for circuit evolution by duplication and divergence [@tosches2017developmental; @roberts2022evolution], a concept that originated from genetics [@ohno1970evolution].

The terms outgoing and incoming synapses were confusing. The more common terminology is pre and postsynaptic elements. For example, in Fig 1, the label Sensory neuron outgoing and incoming was confusing because I mistakenly thought it was referring to the neurons and I could not figure out what an outgoing sensory neuron was.

We have now changed ‘incoming’ to ‘postsynaptic’ and ‘outgoing’ to ‘presynaptic’.

In L-O, there should be an indicator on the figures that they refer to the locations of synaptic sites, as it does in F.

We have now replaced the labels ‘incoming’ and ‘outgoing’ with ‘presyn’ and ‘postsyn’ for Figure 1 panels L-O to make it clear that these are synaptic sites.

Figure 2. - last panel of muscle motor - it would be helpful to have names of muscles instead of just having 5 'muscle motor' of different colors

Each muscle-motor module contains a large number and type of muscles and motor neurons. Labelling them by the name of individual muscle types is therefore not practical at this resolution. The three-day-old Platynereis larvae has 53 different muscle cell types. Their anatomy and classification, together with the details of motoneuron innervation have been described in detail elsewhere (Jasek et al 2022 https://doi.org/10.7554/eLife.71231).

Figure 3. D and E are hard to understand from the figure; The shading is the number of neurons; that scale should be shown somewhere.

We are not sure we understand the comment. These plots are histograms that show the distribution of the number of cells across categories. The y axis is the number of neuronal or non-neuronal cell types in each bin.

PageRank is an algorithm that Google uses. In Figure 4, it seems to be used to indicate centrality. A brief explanation in the text would be useful.

We have now added an explanation of the centrality measures used. “PageRank is an algorithm used by Google to rank webpages and scores the number and quality of the incoming links of a node [@page1999pagerank], betweenness centrality measures the number of shortest paths that pass through a node in a graph [@freeman1977set],  and authority measures the extent of inputs to a node by hubs in a network [@kleinberg1999authoritative].”

Figure 5. The labels on some images are not clear. They are on top of each other and elements of the figure

We have now moved the position of the labels to minimise overlap. We have also added an interactive html file with the network shown in Figure 5 panel A to help the exploration of the network. Added: “Figure 5—source data 1. Interactive html file with the network shown in panel A.”

There are differences in line thickness in several figures, such as Figure 9 (A and B) and Figure 12 (D and I and N) that presumably means numbers of synaptic contacts. It would be useful to know what the scale is.

We have now added labels of line thickness to the networks in Figure 4, Figure 5 – figure supplement 2, Figure 9, Figure 12, Figure 7 – figure supplement 1, Figure 15 and Figure 16.

Reviewer #2 (Recommendations For The Authors):

(1) Suggestions for improved or additional experiments, data, or analyses.

(2) Recommendations for improving the writing and presentation.

Perhaps we require a comprehensive inventory detailing all the innovations compared to previous, more limited publications, particularly in relation to the 2017 publication and 2020 preprint.

We have provided this detail in Supplementary table 1 that lists all cell types. We included the reference for previously published cell types in the ‘reference’ column except for those that were also described in the 2020 preprint. The current manuscript is a greatly revised and extended version of the original 2020 preprint. In addition, in the online connectome database (https://catmaid.jekelylab.ex.ac.uk), all cell types that were previously published are annotated with the notation ‘FirstAuthor_et_al_year’.

It is a bit frustrating given the huge amount of graphs, analyses, tables, and networks that are presented in the manuscript, we do not see much of the original EM pictures except for a few examples of cell type blow-ups. It would be useful for future workers in the field to have eventually a sort of compendium of how the authors actually recognized each cell type, without having to connect to the original CATMAID annotation.

Most neuronal cell types (with the exception of some characteristic sensory neurons such as photoreceptor cells and mechanosensory cells) were not classified based on ultrastructural features, but on features of neurite morphology, body position and synaptic connectivity. It would be therefore not possible to represent most of the cell types with a single layer of an original EM picture. However, in order to make the morphological skeleton characteristics more accessible to the reader, we have now added a comprehensive website ( https://jekelylab.github.io/Platynereis_connectome/)  including all cell types together with their interactive 3D rendering.

“Interactive 3D morphological renderings of each cell type together with their main annotations can also be explored on a webpage (https://jekelylab.github.io/Platynereis_celltype_compendium.html).”

The Platynereis 3-day larva is obviously only one transient stage in the developmental cycle of the animal, and it is a very specialized stage (called metatrochophore in annelid jargon), during which the animal does not yet feed, relying instead on its copious yolk. Moreover, it is a stage whose purpose is limited to dispersion, with no complex behavior or social interaction that later stages are going to display. While this work represents a substantial leap forward in understanding neural integration in a whole animal, it must be kept in mind that compared to an adult or growing juvenile, there are likely a considerable number of cells, cell types, and neural modules missing in this larva. This is clearly not a weakness of this study per se, but readers may find it interesting to be presented with this perspective and therefore more biological details about the Platynereis life cycle and associated behaviors.

Obviously, understanding how the constantly developing nervous system of a worm-like Platynereis gets reshuffled in time will be a great subject to investigate. The authors mention that the 3-day larva displays more than 4000 neuronal cells not yet differentiated. Readers may be interested in their location. Are there niches of neural stem cells? A description of what may be missing from the larva in terms of cell types compared to the adult may be useful.

We have now added further explanation into the Introduction about the early nectochaete larval stage: “The early nectochaete larva represents a transient dispersing stage in the life cycle of Platynereis. During this stage the larvae do not feed yet but rely on maternally provided yolk. Compared to the juvenile and adult stages it is expected that a considerable number of cell types will be only developing or completely missing at this stage. Three-day-old larvae do not yet have sensory palps and other sensory appendages (cirri), they do not crawl or feed and lack visceral muscles and an enteric nervous system.”

The location of developing neurons is shown in Figure 3—figure supplement 1 panel I.

Juvenile or adult cell types have not yet been described in any detail that is close to the level of detail we now provide for the nectochaete larva, therefore a meaningful comparison of cell-type complements across stages is not yet feasible.

(3) Minor corrections to the text and figures.

Figure 1: "outgoing" not "outgoung" in panels M, O, Q.

Corrected

Line 128: We may need a precise definition of "cable length".

We have included a definition of cable length in the Methods section under a new subheading ‘Quantitative analysis of neuron morphologies’.

In all Figures: information on the orientation of the worm's view is sometimes missing in figures, which could make interpretation difficult for the reader, especially for anterior views with no D/V indication. The authors should indicate the orientation for each panel or provide a general orientation in the figure if all panels are oriented the same.

We have now added D/V or A/P indication to all figures.

Figure 23: "right view, left side" is confusing.

We have changed this to “ Each panel shows a ventral (left panel) and a left-side view (right panel).”

Line 406 : the first mention of the Platynereis cryptic segment, as far as I know, is Saudemont et al, 2008.

Thank you for pointing this out. We added the citation.

Figure 45: descending and decussating, 2nd and 3rd line of the legend.

Corrected

The format of data source tables is not homogeneized with some files in Excel format and others in plain comma format.

We have homogeneized the file formats of the supplements and source data. We have .csv files or .rds (R data format) files for the more complex data, such as tibble graphs that cannot be represented in a simple .csv format.

Author response:

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

Reviewer #1 (Public Review):

Major concerns:

For studies investigating capsaicin binding to KEAP1, the authors used capsaicin concentrations that are toxic to cells (Figures S1D and 4F, G). In vivo studies were performed only in 3 rats per group. The T-test was used for the comparison of more than two groups. Given the well-known issues with the specificity of the NRF2 antibody, the authors should provide appropriate controls, especially for IF and IHC staining.

We sincerely appreciate your valuable comments. We repeated the experiments about CCK8 (Figure S1d) and Pull-down (Figure 4g), and then updated the results. In September 2022, GES-1 cells were more sensitive to capsaicin (CAP) because Gibco serum from North America was used. Later, in 2024, we changed the serum from Australia(Gibco: 10099-141), and we found that such GES-1 cells raised better, so we re-ran the test, and the IC50 was seen to be 304.8 μM, so concentrations used in this paper has no obvious toxicity to cells. What’s more, we repeated the Pull-down experiment with more reasonable concentrations of 32 μM and 100 μM, and the results were still in line with expectations. In summary, we concluded that the effect of CAP on GES-1 cells is closely related to the cell state, and that treatments of CAP from 32 to 100 μM can hinder the interaction between NRF2 and the Kelch domain of KEPA1. What’s more, at the cellular level, the experimental concentration of CAP was not more than 32 μM, which is a relatively safe concentration for cells.

Thank you very much for your comments. We also pay attention to using more repetitions to increase the reliability of the experimental results in animal experiments. Therefore, recently we supplemented the experiment of Nfe2l2Knockout mice in Figure 9 (6 mice per group). Additionally, thank you very much for your comments on the use of T-test analysis, we reviewed the statistics and changed them by one-way ANOVA.

Finally, thanks to your concern about the specificity of NRF2 antibody, we used commercialized NRF2 antibody which have been KO/KD validated (Cat No. 16396-1-AP, Proteintech) and can be used for IF and IHC staining. Each of our fluorescence result was equipped with Western Blotting in its active form at the size of 105-110 KDa for statistical analysis, the trend was consistent with the experimental results of IF and IHC, which fully proves the correctness of the results presented (Figure 2c and Figure S8j).

Reviewer #2 (Public Review):

Weaknesses:

One major weakness of the study is that plausibility is taken as proof for causality. The finding that capsaicin directly binds to Keap1 and releases Nrf2 from its fate of degradation (in vitro) is taken for granted as the sole explanation for the observed improved gastric health upon alcohol exposure (in vivo). There is no consideration or exclusion of any potential unrelated off-target effect of capsaicin, or proteins other than Nrf2 that are also controlled by Keap1. 

Another point that hampers full appreciation of the capsaicin effect in cells is that capsaicin is not investigated alone, but mostly in combination with alcohol only.

Thank you very much for this comment. In the introduction, we clarified as follows: “Currently, experiments conducted in rats have demonstrated that red pepper/capsaicin (CAP) had significant protective effects on ethanol-induced gastric mucosal damage, and the mechanism may be related to the promotion of vasodilation(6,7), increased mucus secretion(8) and the release of calcitonin gene-related peptide (CGRP)(9,10). However, it is noteworthy that whether the antioxidant activity of CAP works has not been fully investigated.” Therefore, we also recognize that CAP does not exert its effects through the KEAP1-NRF2 pathway alone. Your advice is very useful. We further explored the TRPV1 and DPP3 to detect the potential off-target effects of CAP respectively. Capsazepine (CAPZ), which is TRPV1 receptor antagonist did not affect the protection of CAP against GES-1 (Fig S4f and S4g), which may indicate that CAP activation of NRF2 does not have to depend on TRPV1. The binding of CAP with DPP3, containing an ETGE motif and can bind to KEPA1, was detected by BLI, and we found that the K_D between CAP and DPP3 was 1.653 mM(＞100 μM), which may indicate the potential off-target effect of CAP is low because CAP had a strong binding force with KEAP1 about 31.45 μM (Fig S4h and S4i).

Thank you very much for the comment of another point. Multiple experiments have shown that CAP significantly up-regulates NRF2 in the presence of additional stimuli such as EtOH (Figure 1i),  H₂O₂ (Figure 1l), PS-341(Figure 2e) and DTT (Figure 4d), which pattern is consistent with our understanding of allosteric regulation and as expected. Especially for the experiments of PS-341 and DTT, we had a group that only adds CAP, and it can be seen that the addition of CAP alone did not significantly up-regulate NRF2, which is completely different from traditional NRF2 activators (especially artificially designed covalent binding peptides which have serious side effects).  

Reviewer #3 (Public Review):

Weaknesses:

While the study provides valuable insights into the molecular mechanisms and in vivo effects of CAP, further clinical studies are needed to validate its efficacy and safety in human subjects. The study primarily focuses on the acute effects of CAP on ethanol-induced gastric mucosa damage. Long-term studies are necessary to assess the sustained therapeutic effects and potential side effects of CAP treatment.

Furthermore, the study primarily focuses on the interaction between CAP and the KEAP1-NRF2 axis in the context of ethanol-induced gastric mucosa damage. It may be beneficial to explore the broader effects of CAP on other pathways or conditions related to oxidative stress. CAP has been known for its interaction with the Transient Receptor Potential Vanilloid type 1 (TRPV1) channel and subsequent NRF2 signaling pathway activation. Those receptors are also expressed within the gastric mucosa and could potentially cross-react with CAP leading to the observed outcome. Including experiments to investigate this route of activation could strengthen the present study.

While the design of CAP nanoparticles is innovative, further research is needed to optimize the nanoparticle formulation for enhanced efficacy and targeted delivery to specific tissues.

Addressing these weaknesses through additional research and clinical trials can strengthen the validity and applicability of CAP as a therapeutic agent for oxidative stress-related conditions.

Thank you very much for these suggestions. We also believe that CAP is very valuable and promising for protecting EtOH induced gastric mucosal injury, and actively promote patent applications and if conditions permit, longer drug research for biosecurity is essential. Because of the inherently new discovery of the binding of CAP and KEAP1, and the important role of NRF2 in various oxidative stress-related diseases, we used Human umbilical cord mesenchymal stem cells (HUC-MSCs) and  H₂O₂ to explore the potential broader effects of CAP related to oxidative stress in cells (Figure 1l and 1m). At the same time, we also explored TRPV1 related experiments, and we were surprised to find that inhibiting TRPV1 did not affect the effect of CAP (Supplementary Figure 4f and 4g). We hope that more people can read this article and do more interesting research together.

Recommendations for the authors:

Reviewing Editor (Recommendations For The Authors):

Although this study has been conducted in rats, a direct proof that albumin-coated capsaicin nanoparticles act through activation of Nrf2 in protecting gastric mucosa against alcohol toxicity could be well conducted in commercially available Nrf2-deficient mice.

Thank you very much for your suggestion and the comment is very constructive for us to improve this paper. We purchased Nrf2-deficient mice (Cat. NO. NM-KO-190433) and performed experiments, and the results showed that knockout mice with Nrf2 were more sensitive to EtOH and the effects of CAP were partially eliminated (Figure 9), which further validated the role of Nrf2-related signaling pathway in EtOH-induced gastric mucosal injury and the therapeutic effect of CAP.

Reviewer #1 (Recommendations For The Authors):

Minor concerns include proofreading the paper. Actinomycin is not an inhibitor of translation.

Thank you for your comment. We have revised “Actinomycin” to “Cycloheximide”.

Reviewer #2 (Recommendations For The Authors):

- Please have a careful look at your conclusions: just because two effects happen at the same time and may be plausible explanations for each other, it does not mean that they are really in a causative relationship in your given test system (unless unambiguously proven by additional experiments).

Your suggestions are very constructive for us to improve this paper.

We further discussed the role of capsaicin with TRPV1, DPP3 and Nrf2deficient mice, hoping to make our conclusions more credible to some extent. 

- You may want to frankly discuss other targets of capsaicin (e.g. the TrpV1 receptor) that possibly could also account for your observations, and that binding to Keap1 not only releases Nrf2 from proteasomal degradation.

Thank you for your comment. As a result, we further explored the TRPV1 and DPP3 to detect the potential off-target effects of CAP respectively. Capsazepine (CAPZ), which is TRPV1 receptor antagonist does not affect the protection of CAP against GES-1 (Fig S4f and S4g). DPP3 with an ETGE motif was detected by BLI, and we found that the K_D between CAP and DPP3 was 1.653 mM, which may indicate the potential off-target effect of CAP is low (Fig S4h and S4i). At the same time, the activation of NRF2 by non-classical pathways such as CAP regulation of DPP3 or other proteins also deserves more discussion and experimental verification.

- For Figure 1G it does not become entirely clear what has been done (and thus deduction of conclusions is hampered).

Thank you for your comment. Network targets analysis (Figure 1g) was performed to obtain the potential mechanism of effects of CAP on ROS. Biological effect profile of CAP was predicted based our previous networkbased algorithm：drug CIPHER. Enrichment analysis was conducted based on R package ClusterProfiler v4.9.1 and pathways or biological processes enriched with significant P value less than 0.05 (Benjamini-Hochberg adjustment) were remained for further studies. Then pathways or biological processes related to ROS and significantly enriched were filtered and classified into three modules, including ROS, inflammation and immune expression. Network targets of CAP against ROS were constructed based on above analyses, and finally we combined proteomics to determine the research idea of this paper

-  Figure 1L: is there a reason/explanation why UC.MSC needs a comparably very high concentration of capsaicin.

Thank you for your comment. Because the experimental results of 8 μM and 32 μM on this cell were more stable, and the activation effect of NRF2 downstream was more obvious.

-  Figure 2C: it is surprising that naïve (unstressed /untreated cells) already show a rather high nuclear abundance of Nrf2 (shouldn´t Nrf2 be continuously tagged for degradation by Keap1).

Thank you for your comment. This is a real experimental result, and we have found in many experiments that the untreated group can also show NRF2 when immunoblotting. We think that this phenomenon may be related to the cell state at that time.

-  Figure 2E: the claim of synergy between CAP and the proteasome inhibitor is not justified with this single figure.

Thank you for your comment. Multiple experiments have shown that CAP significantly up-regulates NRF2 in the presence of additional stimuli such as EtOH (Figure 1i),  H₂O₂ (Figure 1l), PS-341 (Figure 2e) and DTT (Figure 4d), which pattern is consistent with our understanding of allosteric regulation and as expected. However, this synergy does warrant more research.

-  CHX is cycloheximide (in the main text it is referred to as actinomycin).

Thank you very much for your comment. We have revised “Actinomycin” to “Cycloheximide”.

-  Figures 2G-H: why switch to rather high concentrations? Is it due to the overexpression of Keap1?

Thank you for your comment. At the time of this part of the experiment, we had obtained in vitro data on the interaction of CAP and the Kelch domain of KEAP1 (about 32 μM). To keep the results uniform and valid, we chose a relatively higher concentration.

-  Figure 2I: in the pics of mitochondria the control mitochondria look way more punctuated (likely fissed) than the ones treated with EtOH or EtOH + CAP. Wouldn´t one expect that EtOH leads to mitochondrial fission and CAP can prevent it?

Thank you for your comment. MitoTracker® Red CMXRos (M9940, Solarbio, China) is a cell-permeable X-rosamine derivative containing weakly sulfhydryl reactive chloromethyl functional groups that label mitochondria. This product is an oxidized red fluorescent stain (Ex=579 nm, Em=599 nm) that simply incubates the cell and can be passively transported across the cell membrane and directly aggregated on the active mitochondria. Therefore, red does not represent broken mitochondria, but active mitochondria. Quantitative analysis of the mean branch length of mitochondria was calculated using MiNA software (https://github.com/ScienceToolkit/MiNA) developed by ImageJ.

-  Figure 3C: figure legend is somewhat poor.

Thank you for your comment. We have revised: “KEAP1-NRF2 interaction was detected with Surface plasmon resonance (SPR) in vitro.”

-  Figure 3E: given that CAP disrupts Nrf2/Keap1- PPI, why is there no Nrf2 stabilization seen in the fourth lane (input/lysate)?

Thank you for your comment. The fourth lane may promote the degradation of NRF2 due to overexpression of KEAP1.

-  Figure 3H: high basal Nrf2 levels in unstressed/untreated HEK WT cells, why?

Thank you for your comment. This is a real experimental result, and we have found in many experiments that the untreated group can also show NRF2 when immunoblotting in 293T cells. We think that this phenomenon may be related to the cell state at that time.

-  Figure 3G/I: this data suggests to me that the alcohol-mediated toxicity is Keap1-dependent (rather than the protection by CAP), doesn´t it?

Thank you for your comment. We can see that KEAP1-KO cells had a high expression of NRF2, which was also in line with our expectations, and EtOH-induced GES-1 damage may be closely related to oxidative stress.

-  Figure 4a: the inclusion of an additional Keap1 binding protein (one with an ETGE motif) would have been desirable (to get information on specificity/risks of off-target (unwanted) effects of CAP). 

Thank you for your comment. DPP3 with an ETGE motif was detected by BLI, and we found that the K_D between CAP and DPP3 was 1.653 mM, which may indicate the potential off-target effect of CAP is low (Fig S4h and S4i).

-  Figure 4D: why is there no stabilization of Nrf2 by CAP in lane 2 ? How can the DTT-mediated boost on Nrf2 levels be explained?

Thank you for your comment. Multiple experiments have shown that CAP significantly up-regulates NRF2 in the presence of additional stimuli such as EtOH (Figure 1i),  H₂O₂ (Figure 1l), PS-341 (Figure 2e) and DTT (Figure 4d), which pattern is consistent with our understanding of allosteric regulation and as expected. However, this synergy does warrant more research.

-  Figure 4f: 5% DMSO is a rather high solvent concentration, why so high (the solvent alone seems to have quite marked effects).

Thank you for your comment. Because our maximum concentration was set relatively high, we have also recognized relevant problems and resupplemented the more critical Pull-down experiment (Figure 4g). The current DMSO of 0.2% had no effect on the experimental results.

-  Figure 5: it should be described in the figure legend which mutant is used. Based on the previous data, I would expect an investigation of mutants carrying amino acid exchanges at the newly identified allosteric site.

Thank you for your comment. The mutated version involved substitutions at residues Y334A, R380A, N382A, N414A, R415A, Y572A, and S602A (the orthostatic site), which are residues reported to engage NRF2 and classic Keap1 inhibitors. The exploration of newly discovered allosteric sites is worthy of further study.

-  Figure 6/7: I am not expert enough to judge formulations and histology scores. However, the benefit of the encapsulated capsaicin does not become entirely clear to me, as CAP and IRHSA@CAP mostly do not significantly differ in their elicited response.

Thank you for your comment. On the one hand, nanomedicine improves the safety of administration: it helps to reduce the intense spicy irritation of CAP itself when administered in the stomach; On the other hand, the dosage of drugs is reduced to a certain extent to achieve better therapeutic effect.

-  Figure 7: rebamipide was introduced as positive control in the text with an activating effect on Nrf2, but there is no induction of hmox and nqo in Figure 7f, why?

Thank you for your comment. The effect of addition of positive control drug (Rebamipide) on NRF2 activation is not the focus of this paper. We speculate that the transcription and translation of related genes may not be completely synchronized when Rebamipide was taken at the same time.

-  Figure 8: the CAP effect on inflammation is visible, however, a clear causal connection between ROS/Nrf2/KEap1 is not given in the presented experiments.

Thank you for your comment. The simple mechanics of this paper are illustrated in the Graphic diagram. The activation of NRF2 exerts both antiinflammatory and antioxidant functions, which has been reported in many articles, but the causal relationship is still open to exploration.

Points related to presentation:  

-  The data with the encapsulated CAP appear a little as a sidearm that does not bolster your main message (maybe take out and elaborate on this topic more extensively in another manuscript).

-  Revise the introduction on the Nrf2 signaling pathway as it is written at the moment, someone outside the Nrf2 field might have trouble understanding it.

-  The use of language requires proofreading and revision.

Thank you for your comment. We rearranged and proofread it.

Reviewer #3 (Recommendations For The Authors):

Overall, the manuscript is well-written and the results are presented in a concise and comprehensible manner.

Some recommendations on the experimental evidence and further suggestions:

• The authors should state how they assessed the distribution of the data. Description of data with mean and standard deviation as well as comparisons between different groups with t-test assumes that the underlying data is normally distributed.

Your suggestions are very constructive for us to improve the paper.  The differences in the mean values between the two groups were analyzed using the student’s t-test, while the differences among multiple groups were analyzed using a one-way ANOVA test in the GraphPad Prism software.

Therefore, we checked and proofread the statistical analysis.

• Additional experiments further characterising and validating the activation of CAP via direct KELCH1-binding could include parallel experiments with similar agonists like dimethyl fumarate. It would be interesting to know how CAP activation compares to DMF activation.

Thank you very much for your comment. We believe that the activation of NRF2 by DMF has been widely reported and well-studied, so we did not purchase this drug for comparative study here. If it can be promoted clinically in the future, we may consider comparing with DMF.

• Also, the knock-down of NRF2 would be a suggested experiment to do because it rules out that the benefit of CAP is independent of KEAP1-NRF2 binding and activation.

Thank you very much for your suggestions. We purchased Nrf2-deficient mice and performed experiments, and the results showed that knockout mice with Nrf2 were more sensitive to ethanol and the effects of CAP were partially eliminated (Figure 9), which further validated the role of Nrf2-related signaling pathway in alcohol-induced gastric mucosal injury and the therapeutic effect of CAP.

Some corrections on text and figures:

• Figure 1b: incorrect spelling of DNA stain. Should be Hoechst33324.

Thank you very much for your comment. We have revised.

• Figure 1c: don't put the label inside the plot.

Thank you very much for your comment. We have revised.

• Figure 1d: choose less verbose axes titles (this also applies to other figures).

Thank you very much for your comment. We have revised.

• Figures 1e and 1f: please state the units.

Thank you very much for your comment. The enzyme activity of SOD and the content of MDA were compared with that of the control group.

• Heading 2.2: NRF2-ARE instead of NRF-ARE.

Thank you very much for your comment. We have revised.

• Line 118: missing expression after immune.

Thank you very much for your comment. We have revised.

• Figure 1g: names of proteins are not readable.

Thank you very much for your comment. We have revised.

• Line 120: You performed transcriptomic analyses to identify differentially expressed GENES not proteomic.

Thank you very much for your comment. This part of the work we do is proteomics.

• Line 122: Fold change should be stated in both directions, i.e. absolute FC like |FC| > 1. Or did you select only upregulated DEGs? Is it not log2 FC?

Thank you very much for your comment. We have revised.

• Figure 1h (and Supplementary Figure 1a): Missing heatmap legend for FC.

What do the colors show? Sample (column) description missing.

Thank you very much for your comment. We used red to indicate up-regulation, blue to indicate down-regulation, and the vertical coordinate on the right side were antioxidant genes such as GSS and SOD1, respectively, and the proportion between the treatment group and the model group (CAP + EtOH/EtOH) had been calculated and labeled.

• Line 145: A Western blot is not a proteomic analysis.

Thank you very much for your comment. We have revised: “Concurrently, the elevated expression levels of GSS and Trx proteins, which were also downstream targets of NRF2, further validated by western blotting (Figure 1j).”

• Supplementary Figure 2e-j: expression fold change is not the right quantity. The signal of the actual protein was quantified. And what are you comparing to with the statistics? The stars on one bar are not clear.

Thank you very much for your comment. The expression level of this part was normalized compared with that of the control group. The significance differentiation analysis is compared with the model group.

• What was the concentration of  H₂O₂ used?

Thank you very much for your comment. 200 μM  H₂O₂ was used.

• Figure 2d: use a more precise y-axis label.

Thank you very much for your comment. We do want to compare the amount of NRF2 entering the nucleus, so the relative expression is compared to the internal reference

• Figure 2g: missing molecular weight markers.

Thank you very much for your comment. Since the ubiquitination modification is a whole membrane, and only marking the size of HA and GAPDH is not beautiful enough here.

• Line 221: lactate is the endproduct of the anaerobic glycolytic pathway.

Thank you very much for your comment. We have revised.

• Supplementary Figure 3d: should it be PKM2 (instead of PKM) and LDHA (instead of LDH). Should fit with the text in the manuscript.

Thank you very much for your comment. We have revised.

• Supplementary Figures 3 e-f: brackets in y-axis labels are too bold.

Thank you very much for your comment. We have revised.

• Figures 3a and b. Brackets should only be used if two conditions are being compared statistically. Remove the one line with ns as it could imply that you have compared the first with the last condition only.

Thank you very much for your comment. We have revised.

• Consistent labeling of kDa in figures (no capital K in KDa).

Thank you very much for your comment. We have revised.

• Figure 4a. Move kDa on top of 70.

Thank you very much for your comment. We have revised.

• Figure 3 g-h: Why 2% EtOH. Used 5% previously?

Thank you very much for your comment. Because here we changed the 293T cell line, 5% EtOH concentration is too high on this cell.

• Supplementary Figure b-e: correct typo in y-axis label: expression.

Thank you very much for your comment. We have revised.

• Figure 4a: correct x-axis label for temperature unit. Too bold. Not readable.

Add a clear label and unit for y-axis.

Thank you very much for your comment. We have revised.

• Figure 4 b-c: should have a legend explaining colors.

Thank you very much for your comment. Our Figure legend already contains the meaning of colors: “(b) Computational docking of CAP molecule to KEAP1 surface pockets. The Keap1 protein is represented in gray, while the CAP molecule is shown in yellow. The seven key amino acids predicted to be crucial for the interaction are highlighted in blue. (c) Partial overlap of CAPbinding pocket with KEAP1-NRF2 interface. The KEAP1-NRF2 interaction interface is represented in purple.”

• Supplementary Figure 5a. Add axis units.

Thank you very much for your comment. We have revised.

• Figure 4e: Missing b ions value for number 19.

Thank you very much for your comment. This part is not missing, but corresponds to 19 of y ions.

• Figure 7f: adjust brackets - they are too bold.

Thank you very much for your comment. We have revised.

• Supplementary Figure 8b-i: labels not readable. c should be spleen.

Thank you very much for your comment. We have revised.

• Line 787: specify BH adjustment to Benjamini-Hochberg.

Thank you very much for your comment. We have revised.

• Check spelling of µl throughout the Methods section e.g. line 854 - shouldn't be "ul".

Thank you very much for your comment. We have revised.

• Line 974: correct spelling of species names: E. coli should be in italics.

Thank you very much for your comment. We have revised all of these corrections on text and figures. For me, the writing of papers will be more rigorous and careful in the future.

DOI: 10.1016/j.celrep.2025.115264

Resource: (Aves Labs Cat# GFP-1020, RRID:AB_10000240)

Curator: @scibot

SciCrunch record: RRID:AB_10000240

What is this?

needed formulation to the argument from causal overdetermination against dualism

what are the implications of adding P4: For mental events to be causally relevant in the physical domain, mental events must cause physical events

this is shown to be necessaary bc w/o p4 above, it is shown that mental events can be causally relevant even if they arent causes

Further, we could disagree with her premise that double preventors or enabling events are not causes.

fasdasdfd

fadssefsdfsd

proto peirama

approach

paradeigma

Whitman uses natural imagery, particularly the lilac, the star, and the thrush, to symbolize grief, loss, and renewal. This deep connection between human emotion and the natural world is a hallmark of Romanticism.

Here it romantizes night. It shows the beauty of it's darkness and paints the dark and moody vibes of night.

Author response:

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

Public Reviews:

Reviewer #1 (Public Review):

Summary:

In this valuable study, the authors found that the macrolide drug rapamycin, which is an important pharmacological tool in the clinic and the research lab, is less specific than previously thought. They provide solid functional evidence that rapamycin activates TRPM8 and develop an NMR method to measure the specific binding of a ligand to a membrane protein.

Strengths:

The authors use a variety of complementary experimental techniques in several different systems, and their results support the conclusions drawn.

Weaknesses:

Controls are not shown in all cases, and a lack of unity across the figures makes the flow of the paper disjointed. The proposed location of the rapamycin binding pocket within the membrane means that molecular docking approaches designed for soluble proteins alone do not provide solid evidence for a rapamycin binding pocket location in TRPM8, but the authors are appropriately careful in stating that the model is consistent with their functional experiments.

Impact:

This work provides still more evidence for the polymodality of TRP channels, reminding both TRP channel researchers and those who use rapamycin in other contexts that the adjective "specific" is only meaningful in the context of what else has been explicitly tested.

Reviewer #2 (Public Review):

Summary:

Tóth and Bazeli et al. find rapamycin activates heterologously-expressed TRPM8 and dissociated sensory neurons in a TRPM8-dependent way with Ca2+-imaging. With electrophysiology and STTD-NMR, they confirmed the activation is through direct interaction with TRPM8. Using mutants and computational modeling, the authored localized the binding site to the groove between S4 and S5, different than the binding pocket of cooling agents such as menthol. The hydroxyl group on carbon 40 within the cyclohexane ring in rapamycin is indispensable for activation, while other rapalogs with its replacement, such as everolimus, still bind but cannot activate TRPM8. Overall, the findings provide new insights into TRPM8 functions and may indicate previously unknown physiological effects or therapeutic mechanisms of rapamycin.

Strengths:

The authors spent extensive effort on demonstrating that the interaction between TRPM8 and rapamycin is direct. The evidence is solid. In probing the binding site and the structural-function relationship, the authors combined computational simulation and functional experiments. It is very impressive to see that "within" a rapamycin molecule, the portion shared with everolimus is for "binding", while the hydroxyl group in the cyclohexane ring is for activation. Such detailed dissection represents a successful trial in the computational biology-facilitated, functional experiment-validated study of TRP channel structuralactivity relationship. The research draws the attention of scientists, including those outside the TRP channel field, to previously neglected effects of rapamycin, and therefore the manuscript deserves broad readership.

Weaknesses:

The significance of the research could be improved by showing or discussing whether a similar binding pocket is present in other TRP channels, and hence rapalogs might bind to or activate these TRP channels. Additionally, while the finding on TRPM8 is novel, it is worthwhile to perform more comprehensive pharmacological characterization, including single-channel recording and a few more mutant studies to offer further insight into the mechanism of rapamycin binding to S4~S5 pocket driving channel opening. It is also necessary to know if rapalogs have independent or synergistic effects on top of other activators, including cooling agents and lower temperature, and their dependence on regulators such as PIP2.

Additional discussion that might be helpful:

The authors did confirm that rapamycin does not activate TRPV1, TRPA1 and TRPM3. But other TRP channels, particularly other structurally similar TRPM channels, should be discussed or tested. Alignment of the amino acid sequences or structures at the predicted binding pocket might predict some possible outcomes. In particular, rapamycin is known to activate TRPML1 in a PI(3,5)P2-dependent manner, which should be highlighted in comparison among TRP channels (PMID: 35131932, 31112550).

Reviewer #3 (Public Review):

Summary:

Rapamycin is a macrolide of immunologic therapeutic importance, proposed as a ligand of mTOR. It is also employed as in essays to probe protein-protein interactions.

The authors serendipitously found that the drug rapamycin and some related compounds, potently activate the cationic channel TRPM8, which is the main mediator of cold sensation in mammals. The authors show that rapamycin might bind to a novel binding site that is different from the binding site for menthol, the prototypical activator of TRPM8. These solid results are important to a wide audience since rapamycin is a widely used drug and is also employed in essays to probe protein-protein interactions, which could be affected by potential specific interactions of rapamycin with other membrane proteins, as illustrated herein.

Strengths:

The authors employ several experimental approaches to convincingly show that rapamycin activates directly the TRPM8 cation channel and not an accessory protein or the surrounding membrane. In general, the electrophysiological, mutational and fluorescence imaging experiments are adequately carried out and cautiously interpreted, presenting a clear picture of the direct interaction with TRPM8. In particular, the authors convincingly show that the interactions of rapamycin with TRPM8 are distinct from interactions of menthol with the same ion channel.

Weaknesses:

The main weakness of the manuscript is the NMR method employed to show that rapamycin binds to TRPM8. The authors developed and deployed a novel signal processing approach based on subtraction of several independent NMR spectra to show that rapamycin binds to the TRPM8 protein and not to the surrounding membrane or other proteins. While interesting and potentially useful, the method is not well developed (several positive controls are missing) and is not presented in a clear manner, such that the quality of data can be assessed and the reliability and pertinence of the subtraction procedure evaluated.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

Major points

(1) Given the novelty of the STTD NMR approach, please provide more details and data for the reader.

• I would like to see all of the collected spectra so that readers can see and judge the effect sizes for themselves, perhaps as an additional supplementary figure.

We agree with the reviewer that the data transparency of the NMR measurements should be improved. We changed panel C of Figure 2 in the main text and provided all the STD and the computed STDD and STTD spectra recorded on one set of experiments. We carried out additional experimental replicas on new samples and addressed the variability of cell samples by rescaling the STD effects based on reference ¹H measurements. We provided supplementary spectra of the reference experiments without saturation (Figure S5) and the obtained STTD spectra from the three parallel NMR sessions (Figure S6).

• I appreciate the labels for STDD-1, STDD-2, and STTD on the lower two spectra of Figure 2C. Is the top spectrum from STD-1 or is it prior to saturation? In Figure 2C, what do the x1 and x2 notations on the right-hand side of the spectra indicate?

We showed the top spectrum as an overview and a demonstration of the spectral complexity of the samples. ¹H experiments were run before the STD measurements to assess the sample quality and stability. The demonstrated spectrum on sample 1 (TRPM8 with rapamycin in HEK cells) was recorded with more transients than the corresponding STDs, thus it is only visually comparable with the difference spectra after scaling (2x). Figure 2 was changed and all the spectra were replaced as mentioned before. All the recorded ¹H-experiments without saturation including the one removed are now available in the supplementary information (Figure S5).

• The STTD NMR results with WT TRPM8 are consistent with rapamycin binding directly to the channel. Testing whether rapamycin binding observed with STTD NMR is disrupted by one of the most compelling mutations (D796A, D802A, G805A, or Q861A) would be a further test of this direct interaction.

We thank the reviewer for the suggestion and agree that testing the most compelling mutants would be a promising next step. These mutations were generated in plasmid vectors and only transiently transfected into HEK cells. For NMR analysis we would need a high amount of cells stably overexpressing the mutant channels which were not available for experimentation.

• Given that this is not a methods paper, it is probably outside the scope to further validate the STTD NMR measurements by performing parallel ITC, SPR, MST, or radiolabeled ligand experiments. Nevertheless, I would be excited to see such a comparison since STTD NMR appears to have promise as an experimental technique for assessing ligand binding to membrane proteins that does not require large amounts of purified protein or radioactive isotopes.

We agree with the reviewer that additional independent biophysical measurements on the interactions are necessary to further validate the STTD methodology. This paper is a preliminary demonstration of the STTD concept and our group is currently working on the challenges of on-cell NMR (e.g., sample and spectral complexity) and the standardization of the proposed workflow.     

(2) Please clarify the methods used to model of rapamycin binding. Docking can be imprecise in TRP channels, even with a sophisticated docking scheme (Hughes et al., 2019, doi: https://doi.org/10.7554/eLife.49572.001).  

Thank you for mentioning this point and providing the reference. We have further clarified our methods and included the reference in our discussion, indicating the limitations of our approach.

• As a positive control, does the docking strategy accurately predict binding of known compounds (menthol, icilin, etc.) to TRPM8 consistent with cryo-EM structures?  

Yes, the binding site for menthol, based on a similar docking strategy as for rapamycin, is also presented, and matches with predictions from other publications. This is now clarified in the revised manuscript.

• Why was homology modeling to the human sequence used with the mouse structure but not the avian structure?  

At this onset of the project, only the avian structure was available, and it was used in the primary docking. Later, to get more precise docking relevant for human TRPM8 pharmacology, we did revert to the then available structure of the mouse ortholog.  

• How many rapamycin structural clusters were built, and how many structures were there in each cluster? How many were used? "most populated" is unspecific.  

Thank you for your comment. We have added the following highlighted information to the methods section to address your comment:

“Representative conformations of rapamycin were identified by clustering of the 1000-membered pools, having the macrocycle backbone atoms compared with 1.0 Å RMSD cut-off. Middle structures of the ten most populated clusters, accounting for more than 90% of the total conformational ensemble generated by simulated annealing, were used for further docking studies. To refine initial docking results and to identify plausible binding sites, the above selected rapamycin structures were docked again, following the same protocol as above, except for the grid spacing which was set to 0.375 Å in the second pass. The resultant rapamycin-TRPM8 complexes were, again, clustered and ranked according to the corresponding binding free energies. Selected binding poses were subjected to further refinement. The three most populated and plausible binding poses were further refined by a third pass of docking, where amino acid side chains of TRPM8, identified in the previous pass to be in close contact with rapamycin (< 4 Å), were kept flexible. Grid volumes were reduced to these putative binding sites including all flexible amino acid side chains (21.0-26.2 Å x 26.2-31.5 Å x 24.8-29.2 Å).”

However, it is important to clarify that the clusters are not built and their number is not specified by the user. The number of clusters found depends on how similar the structures are in the structural ensemble analyzed by clustering. A high number of clusters indicates a diverse, whereas a low number suggests a uniform structural ensemble. Furthermore, it is arbitrarily controlled by the similarity cutoff specified by the user. If the cutoff is selected well, then the number of structures is different in each cluster. There are some highly populated clusters and a few which only have one structure. The selection of how many cluster representatives are used is usually based on the decision of whether or not the sum of the population of selected clusters sufficiently covers the mapped conformational space.

• Additionally, the rapamycin poses were generated using a continuum solvent model that is unlikely to replicate the conditions existing in the lipid bilayer or in a lipid-exposed binding pocket as is predicted here. It is therefore possible that the rapamycin poses chosen for docking do not represent the physiological rapamycin binding pose, hampering the ability of the docking algorithm to find an appropriate docking pocket.  

• Furthermore, accurately docking that may bind to membrane-exposed pockets is a challenging problem, particularly because many scoring algorithms, including those employed by Autodock, do not distinguish between solvent-exposed and membrane-exposed faces of the protein. This affects the predicted binding energies.  

We appreciate the reviewer's insightful comments. We add a note in discussion part, mentioning these important limitations.  

• In Figure 4, it appears that the proposed rapamycin binding pocket is located at the interface between two subunits, but only one is shown. Is there any contact with residues in the neighboring subunit? Based on Figure S4, I assume not, but am unsure.

Based on the estimated distances, we do not think that there are any relevant interactions with residues from neighboring subunits. This is now indicated in the results section.

• Consider uploading the rapamycin-docked model to a public repository such as Zenodo for readers to examine and manipulate themselves  

As suggested, the model will be uploaded in a public repository. A link to the file on Zenodo is now included.

(3) Please discuss the spatial location of the proposed rapamycin binding pocket relative to the vanilloid binding pocket in TRPV1.

• The mutagenesis indicates that D745, D802, G805, and Q861 are most important for rapamycin sensitivity in TRPM8. Interestingly, the proposed rapamycin binding pocket appears to overlap spatially with the vanilloid binding pocket in TRPV1. Consistent with this, Q861 aligns with E570 in TRPV1, which is a critical residue for resiniferatoxin sensitivity. Indeed, similar to Q861's modeled proximity to the cyclohexyl ring, the hydroxyl group of the vanillyl moity of capsaicin (4DY in 7LR0, for example) is in proximity to E750 in TRPV1. Additionally, searching PubChem by structural similarity suggests that vanillyl head group of the TRP channel modulators capsaicin and eugenol are similar structurally to the trans-2Methoxycyclohexan-1-ol ring. Without overlaying the two structures myself, it is difficult to say more than that, but I encourage the authors to comment on any similarities and differences they observe.

• If the proposed rapamycin pocket is indeed similar to the location of the vanilloid binding site, the authors may wish to discuss other TRPM channel structures that show ligands and lipids bound to this pocket because this provides evidence that this pocket influences TRPM channel function. For example, how does the proposed rapamycin binding pocket compare to TRPM8 bound to agonist AITC (PDBID 8e4l), TRPM5 bound to inhibitor NDNA (7mbv), and TRPM2 bound to phosphatidylcholine (6co7)?

• Other TRP channel structures with ligands or lipids modeled in this region include TRPV1 bound to resiniferatoxin, capsaicin, or phosphatidylinositol (7l2j, 7l24, 7l2s, 7l2t, 7l2u, 7lp9, 7lpc, 7lqy, 7mz6, 7mz9, 7mza); TRPV3 bound to phosphatidylcholine (7mij, 7mik, 7mim, 7min, 7ugg); TRPV5 bound to econazole (6b5v) or ZINC9155 (6pbf); TRPV6 bound to piperazine (7d2k, 7k4b, 7k4c, 7k4d, 7k4e, 7k4f) or cholesterol hemisuccinate (7s8c); TRPC6 bound to BTDM (7dxf) or phosphatidylcholine (6uza); and TRP1 bound to PIP2 (6pw5).

We thank the reviewer for these valuable insights. We have included some additional discussion highlighting the similarities between the proposed rapamycin binding site and some of the other ligandchannel interactions in the TRP superfamily, in particular the well-known vanilloid binding site in TRPV1. However, to keep the discussion focused, we have not fully discussed all the indicated interactions, to best serve the clarity and scope of the manuscript.  

(4) I would like to see negative control calcium imaging and electrophysiology data with untransfected HEK cells to confirm that the observed activation is mediated by TRPM8 to parallel the TRPM8 KO sensory neuron experiments.  

This important information is now included in the revised manuscript (Figure S2).

(5) The DM-nitrophen Ca uncaging experiments are an interesting method to test Ca sensitivity of rapamycin, but the results make these experiments more complex to interpret. Ca has been shown to be an obligate cofactor for icilin sensitivity in TRPM8 under conditions where both the internal and external Ca concentrations are tightly controlled (Kuhn et al., 2009, doi: https://doi.org/10.1074/jbc.M806651200), which is necessary because TRPM8 allows Ca permeation through the pore when open. The large icilin-evoked currents in Figure 5A and 5B indicate that the effective intracellular calcium concentration is not zero prior to calcium uncaging, which may be high enough to mask any Ca-dependence of rapamycin that occurs at low Ca concentrations. Given this ambiguity, the inside-out patch clamp configuration would provide more control over the internal and external Ca concentration than is achieved in the Ca uncaging experiments. Because the authors have already demonstrated their ability to perform such experiments (Figure 2 panel B), it would be nice to see tests of Ca dependence using inside-out patch clamp.

As was already shown in Figure 2, Rapamycin activates TRPM8 in inside-out patches, and these experiments were performed using calcium-free cytosolic and extracellular solutions. Note that earlier studies have already shown that icilin activates outward TRPM8 currents in the full absence of calcium: see e.g. Janssens et al. eLife, 2016. Chuang et al. 2004. In the case of Icilin, increased calcium further potentiates the current, which is more prominent for the inward current.

In the Ca uncaging experiments, considering the Kd of DM-nitrophen of 5 nM, we expect that the intracellular calcium concentration before the UV flash would be approximately 15 nM. Taken together, both the inside-out experiments and the flash uncaging experiments confirm that rapamycin responses are not directly regulated by intracellular calcium, contrary to icilin.

(6) Sequence conservation within TRPM channels could be used in combination with the binding pocket model and mutagenesis to predict rapamycin selectivity for TRPM8 over other TRPMs. For example, some important residues, specifically G805 and Q861, are not conserved in TRPM3, which agrees with the lack of rapamycin sensitivity observed in TRPM3 (Figure S1). Further sequence comparison would provide testable hypotheses for future exploration of rapamycin sensitivity in other TRPMs that could validate the proposed binding pocket.

Thank you for the suggestion. We now indicate in the discussion that only some of the key residues are conserved and make suggestions for future studies.  

(7) Please unify the color scheme across the figures to improve clarity.

• The authors frequently use the colors blue, red, and green to represent menthol and rapamycin in the figures, but they are inconsistent in which one represents menthol and which represents rapamycin. It would be clearer for the audience if, for example, rapamycin is always represented with red and menthol is always represented with blue.  

Thank you for pointing this out. We have made the coloring schemes more uniform.

• In Figure 1, panel E, the coloring for Menthol and Pregnenolone Sulfate changes between the TRPM8+/+ and TRPM8-/- panels.  

Thank you for pointing this out. We have updated the coloring schemes to ensure consistency between the TRPM8+/+ and TRPM8-/- panels.

• Figure 3 B and E, perhaps color the plot background as a 3-color gradient (blue to white to red) rather than yellow and aqua. Center the white at the WT ratio, keeping the dashed line, with diverging gradients to, for example, blue for mutations that selectively affect menthol sensitivity and red for rapamycin.

Thank you for the suggestion – we have changed the figure accordingly.  

• Figure 4 panels A and B use the same color (green) to show two different things (menthol molecule and mutated residues that affect rapamycin sensitivity). It would be clearer for readers to change these colors to agree with a unified color scheme such that, for example, the menthol molecule is colored blue and the rapamycin-neighboring residues are colored red.

Thank you for the suggestion. We have updated the figure to use a unified color scheme, with the menthol molecule now colored green and the rapamycin-neighboring residues colored cyan, to enhance clarity for readers.

• I recommend adding a figure or panel that shows side chains for all mutations, colored by menthol/rapamycin selectivity, as indicated by the functional data in Figure 3B and 3E. This will highlight spatial patterns of the selective residues that are discussed in the text.

Thank you for your suggestion, we added all the side residues in Figure S10.

Minor points

(1) It would be nice to have one more concentration data point in the middle of the dose response curve shown in Figure 1 panel B. The response is not saturating at the top or foot of the curve in Figure 1 panel D, precluding a confident fit to a two-state Boltzmann function.

Instead of adding a single data point to this figure, we performed independent measurements on a plate reader system, comparing concentration responses at room temperature and 37 degrees. These data are now included as Figure S1.   

(2) The cartoon in Figure 2 panel B should be made more accurate. For example, only the transmembrane helices should be depicted embedded in the membrane, not the whole protein including the intracellular domain. Because the experiment was performed with cells, change the orientation of TRPM8 in the cartoon to show the intracellular domain of the protein facing away from the extracellular side of the membrane where the rapamycin is applied.

Thank you for this comment. We have corrected the cartoon accordingly

(3) Perhaps put the yellow circles under or around the carbon atoms to which the identified hydrogen atoms belong in Figure 2 panel E and Figure 4 panel C. I found it difficult to visualize and compare the STTD NMR results with the predicted binding pocket.

Thank you for the feedback. We have added yellow circles around the carbon atoms corresponding to the identified hydrogen atoms in Figure S9.  

(4) Regarding the sentence on p. 12 beginning "In agreement with this notion..."

• Include icilin, Cooling Agent-10, and WS-3 as other cooling agents whose sensitivity has been modulated by mutation of Y745

• Cryosim-3 responses were not tested in either of the two papers cited; please add citation to Yin et al., 2022, doi: https://doi.org/10.1126/science.add1268 .

• Other relevant papers include:

– Malkia et al., 2009, doi: https://doi.org/10.1186/1744-8069-5-62 which includes molecular docking showing the hydroxyl group of menthol interacting with Y745

– Beccari et al., 2017, doi: https://doi.org/10.1038/s41598-017-11194-0 Figure 5 shows disruption of icilin and Cooling Agent-10 sensitivity by Y745A

– Palchevskyi et al., 2023, doi: https://doi.org/10.1038/s42003-023-05425-6 Figure 3 shows disruption of icilin, cooling agent-10, WS-3, and menthol sensitivity by Y745A o Plaza-Cayon et al., 2022, https://doi.org/10.1002%2Fmed.21920 Review of TRPM8 mutations

• typo: Y754H should be Y745H

Thank you for these suggestions. We have added the above references to the text and corrected the typo.

(5) The authors use the competitive action of everolimus on rapamycin activation as evidence that the different macrolides are binding to the same binding pocket. In addition, prior work showed that Y745H and N799A mutations (which render TRPM8 insensitive to menthol and icilin, respectively) do not affect TRPM8 sensitivity to the structurally-related compound tacrolimus (Arcas et al., 2019). This is consistent with the docking and mutagenesis results presented here.

Thank you for this valuable suggestion. We discuss these data in the revised version.

(6) Rapamycin sensitivity has also been observed in TRPML1 (Zhang et al. 2019, doi: https://doi.org/10.1371/journal.pbio.3000252).

We added a short reference to this interesting finding in the discussion.

(7) The whole-cell currents are very large in several of the electrophysiology experiments (for example Figure 3 panel D and Figure S1), which could lead to artifacts of voltage errors as well as ion accumulation/depletion. However, because this paper is not relying on reversal potential measurements or trying to quantify V1/2, these errors are unlikely to affect the qualitative conclusions drawn.

This is a fair point, but indeed unlikely to affect our main conclusions. Note that we compensated between 70 and 90% of the series resistance, so we don’t expect voltage errors exceeding ~10 mV.

(8) Ligand sensitivity is frequently species-dependent in TRP channels, so it is interesting that multiple species were used here and that both human and mouse isoforms exhibit rapamycin sensitivity. It should be emphasized that human TRPM8 was used in the calcium imaging and electrophysiology experiments, as well as some docking models, while the mouse isoform was used in the sensory neuron experiments and a mutated avian isoform was used for some docking models.

This information is available in the Methods and we believe it is clear for the readers.

(9) Perhaps discuss the unclear mechanism of G805A action in icilin (but not menthol, cold, or praziquantel) sensitivity because it is not in direct contact with the ligand. For example, Yin et al., 2019 propose flexibility allowing Ca binding site and larger binding site for icilin.

Yin et al. (2019) suggests that the G805A mutation impacts icilin sensitivity by influencing the flexibility of the binding site and possibly affecting calcium binding. In our study, we found that G805A significantly reduces rapamycin sensitivity, likely due to its direct role in the rapamycin binding pocket rather than affecting calcium binding. This is now briefly mentioned in the results section.

(10) The Figure S1 legend indicates that n=5 for all panels, so please show normalized population IV curves rather than individual examples. Additionally, it would be interesting to see what happens when each agonist is co-applied with rapamycin. Does rapamycin potentiate or inhibit agonist activation in these channels and/or TRPM8?

We believe that normalized population IVs are not ideal for representing whole-cell currents, considering the substantial variation in current densities. We therefore prefer to show example traces in Figure S3 of the revised version but include mean values of current densities for all tested cells in the text.

While the effects of co-application of rapamycin with activating ligands could be of interest, we consider this somewhat outside the scope of the present manuscript. The combination of HEK293 cell experiments, along with results obtained in WT and TRPM8-deficient mice does, in our opinion, sufficiently describe the selectivity of rapamycin towards TRPM8 compared to other sensory TRP channels.

(11) Figure S1 panel A does not contain units for Rapamycin or AITC concentrations.

Thank you for pointing this out. The units were added to the figure.  

(12) It would be nice if the authors characterized the different mutations as predicted to contribute to site 1 (D796, H845, Q861, based on Figure S4), site 2 (D796, M801, F847, and R851), and/or site 3 (F847, V849, and R851).

The indicated mutants were all tested, as shown in Figure 3.

(13) The numbering scheme in Figure S4 does not appear to match the residue numbers in the rest of the paper for certain residues (HIS-844 rather than H845, PHE-846 rather than F847, VAL-848 rather than V849, ARG-850 rather than R851, and GLN-860 rather than Q861), and labels are often overlapping and difficult to see. I also find the transparent spheres very difficult to distinguish from the transparent background, which makes it difficult to appreciate the STTD NMR data overlay.

We apologize for the confusing numbering scheme. The lower numbers refer to the initial docking that was done using the avian TRPM8 ortholog. We have made a newer, clearer version of Figure S4 and inserted as Figure S9.  

(14) Please superpose the Ligplots in Figure S5 panels E and F as described in the LigPlus manual (https://www.ebi.ac.uk/thornton-srv/software/LigPlus/manual/manual.html) to facilitate easier comparison.

Thank you for the suggestion. We followed the suggestion to superpose the Ligplots as described but found that the result was visually cluttered and difficult to interpret. To avoid confusion, we instead decided to remove panels E and F from Figure S5, as we believe that the visualization in panels A-D is clear and informative.

(15) Some n values are missing in figure legends.

We checked all legends, and added n numbers were missing.

(16) There is an inconsistent specification of error bars as SEM in the figure legends, though it is specified in methods.

A question for my own edification: Here, you have looked at ligand interactions with the protein by saturating the protein resonances and observing transfer to the ligand. Would it be possible to instead saturate lipid or solute resonances and observe transfer to a ligand? I am curious whether this would be one way to measure equilibrium partitioning of ligand into a membrane and/or determine the effective concentration of a ligand in the membrane. Additionally, could one determine whether the compound is fully partitioned into the center of the membrane or just sitting on the surface?

The reviewer highlights an interesting aspect. The widely used WaterLOGSY NMR experiment (doi: 10.1023/a:1013302231549) saturates water molecules then the magnetization is transferred to the ligand of interest. Characteristic changes in ligand resonances are observed in the case of a binding event with proteins. On the other hand, the selective saturation of lipids is -while theoretically possible –technically challenging mainly because of the inherent low signal-dispersion of lipids and peak overlapping with ligand resonances. Additionally, lipid systems are more dynamic compared to proteins and ligand-lipid interactions could be weaker and less specific, significantly affecting the sensitivity of STD experiments.

Reviewer #2 (Recommendations For The Authors):

Major:

• Is it feasible to test rapamycin on TRPM8 with single-channel recording? This will allow us to better probe the mechanism of rapamycin activation and compare it with menthol, with parameters of singlechannel conductance and maximal open probability.

In our experience, it is very difficult to obtain single-channel recordings from TRPM8. The channel expresses at high densities, typically leading to patches contain multiple channels, making a proper analysis of mean open and closed times very difficult. Therefore, we have decided not to include such measurements in the manuscript.

• The authors classified rapamycin as a type I agonist, the type that stabilizes the open conformation, same as menthol but more prominent. Does that indicate that rapamycin work synergistically (rather than independently) with menthol, because co-application of them can allow them to add to each other in stabilizing the open conformation? I wonder if the authors agree that this could be tested with experiments as in Figure S3, by showing a much more prolonged deactivation with co-application of menthol and rapamycin than applying each alone.

Thank you for the insightful suggestion. We conducted co-application experiments, and our results show that the deactivation time is indeed significantly prolonged when both compounds are applied together compared to each alone. In fact, very little deactivation is seen when both compounds are co-applied, which made it virtually impossible to perform reliable fits to the deactivation time course for the Menthol+Rapamycin condition. Instead, we have now included summary results showing the percentage of deactivation after 100 ms. We included these findings in FigureS8.  

• It could be tested whether rapamycin activation of TRPM8 requires or overrides the requirement of PIP2 with inside-out patch by briefly exposing the patch to poly-lysine to sequester PIP2.

This is certainly a good suggestion for further follow-up studies. However, we considered that examination of the (potential) interaction between ligands and PIP2 was outside the scope of the current manuscript.

• Figure 1C suggests that the authors test rapamycin when there is a relatively high baseline TRPM8 activation (prior to rapamycin) activation. This raises the possibility that rapamycin is more a potentiator than an activator. I wonder if the following two experiments could address it: (1) perfuse rapamycin while holding at different membrane potentials, wash-off rapamycin in the solution and quickly (in a few seconds) test the activated current magnitude (before rapamycin dissociation), to compare whether a more depolarized membrane potential (high baseline open probability) allows rapamycin to potentiate more. (2) Perform the experiment at a higher temperature (low baseline open probability) and test whether rapamycin EC50 shifts to the right.

Thank you for the thoughtful suggestion. Overall, we are not really in favor of making a distinction between a potentiator and an activator since it is not really feasible to create a situation where TRPM8 activity is zero. As suggested, we performed the dose response experiment at a higher temperature (37 °C) and observed that rapamycin’s EC₅₀ shifts to the right FigureS2. This is similar to what has been observed for menthol on TRPM8 and for many other ligands on other temperature-sensitive TRP channels.

Minor:

(1) The author should report hill coefficient together with EC50 when showing dose-responses.

We have added Hill coefficients for all the fits.

(2) In Figure 1 (E, F), it might be clearer to use Venn-diagram to show whether there is overlapping among rapamycin-, menthol-, and cinnamaldehyde-responsive neurons. According to the authors' explanation, we can predict that rapamycin-insensitive, menthol-sensitive neurons should predominantly be cinnamaldehyde-responsive.

Thank you for your suggestion. In these experiments, we applied several agonists and the combination of them would result in a visually crowded Venn diagram difficult to interpret. However, we agree, with the reviewer’s suggestion, and discuss the percentage of the cinnamaldehyde+ neurons in the rapa- menthol+ population in Trpm8^-/- neurons.

(3) In Figure 3(C), since F847 does not respond to either menthol or rapamycin, it should be excluded from (B). Otherwise it is misleading.

Thank you for pointing this out. To clarify, we have included a calcium imaging trace for the F847 mutant, demonstrating a clear response to rapamycin in FigureS9. This additional data highlights that F847 does respond to rapamycin, albeit with a more modest response amplitude. This is now also clarified in the results section.  

(4) The word "potency" in pharmacology usually refers to a smaller EC50 number in dose-dependent experiments. In "Effect of rapamycin analogs on TRPM8" session, the authors use "potency" to refer to response to a single-dose experiment of different compounds. The experiment does not measure potency.

Thank you for pointing out this mistake. We have corrected the text and replaced “potency” with “efficacy”.

(5)  "2-methoxyl-" is misspelled in the text body.

We have corrected the typo.

(6) It will be nice to include "vehicle" in Figure 6B, or alternatively normalize all individual traces to vehicle. In Figure 6C and D, everolimus has almost no effect with compared to vehicle, and should not be shown as if it had ~8% in Figure 6B.

We have added the vehicle values to Figure 6B from the same experiments.

Reviewer #3 (Recommendations For The Authors):

(1) The NMR method presented here as novel and employed to identify a proposed molecule bound to a membrane protein (TRPM8 in this case) is not well explained and presented. Since several spectra need to be subtracted, the authors should present the raw data and the results of the subtractions step by step. Also, it seems that the height of the peaks in each spectra will be highly variable and thus a reliable criterion employed to scale spectra before subtraction. None of these problems are discussed of described.

The reviewer is right, that the data transparency should be improved and due to the high molecular complexity of the samples the size of the STD effects should be carefully scaled. We carried out additional experimental replicas on new samples and addressed the inherent sample/peak height variability by rescaling the STD effects based on reference ¹H measurements. We provided supplementary spectra of the reference experiments without saturation (Figure S5) and the computed STTD spectra from three parallel NMR sessions (Figure S6). We changed panel C of Figure 2 in the main text and provided all the STD and the computed STDD and STTD spectra recorded on one set of NMR experiments. We added the following paragraph to the main text: “To address the effect of the inherent variability of cellular samples on peak heights, STD effects were normalized based on the comparison of independent ¹H experiments (Figure S5). Three STTD replicates were computed, unambiguously confirming direct binding to TRPM8 in two datasets (Figure S6 A,B)”.

Importantly since this signal subtraction method is proposed as a new development, control experiments employing well-established pairs of ligand and membrane protein receptor should be performed to demonstrate the reliability of the method.

We agree with the reviewer, that the STTD experiment as a new development needs further validation, however, this paper is a preliminary demonstration of a new strategy building on the well-established STD and STDD NMR methodologies. Our group is actively engaged in studying additional biological samples to enhance our understanding of the applicability of STTD NMR. These efforts also aim to address challenges such as sample and spectral complexity by refining and standardizing the proposed workflow.

(2) The tail currents shown in supplementary figure 3 are clearly not monoexponential. The fit to a single exponential can be seen to be inadequate and thus the comparison of kinetics of control, rapamycin and menthol is incorrect. At least two exponentials should be fitted and their values compared.

We agree that the decay in the (combined) presence of agonists deviates from a simple monoexponential behavior. While we agree that fitting with two (or more) exponentials would provide a better fit, this also comes with greater variations/uncertainties in the fit parameters. This is particularly the case when inactivation is very slow and incomplete, or when the difference between slow and fast exponential time constants is <5, as seen with rapamycin and rapamycin +menthol. Therefore, we decided to provide monoexponential time constants as a proxy to describe the clear slowing down of activation and deactivation time courses in the presence of Type I agonists.   

Also related to this aspect, recordings of TRPM8 currents can not be leak subtracted with a p/n protocol, thus a large fraction of the initial tail current must be the capacitive transient. There is no indication in the methods of how was this dealt with for the fitting of tail currents.

As explained in the methods, capacitive transients and series resistance were maximally compensated. Therefore, we do not agree that a large fraction of the initial tail current must be capacitive. This can also be clearly seen in experiment such as Figure 1C, where the inward tail current is fully abolished in the presence of a TRPM8 antagonist. Likewise, very small and rapidly inactivating tail currents can be seen during voltage steps under control conditions (e.g. Figure S7  and S8 in the revised version).  

(3) The docking procedure employed, as the authors show, is not appropriate for membrane proteins since it does not include a lipid membrane. It is not clear in the methods section if the MD minimization described applies only to the rapamycin molecule or to rapamycin bound to TRPM8.  

It is also not clear if the important residue Q861 (and other residues that are identified as interacting with rapamycin) were identified from dockings or proposed based on other evidence.

(4) Identifying amino acid residues that diminish the response to a ligand, does not uniquely imply that they form a binding site or even interact with said ligand. It is entirely possible that they can be involved in the allosteric networks involved in the activating conformational change. This caveat should be clearly posited by the authors when discussing their results.

In our study, we identified several residues that significantly reduce the response to rapamycin when mutated, while retaining robust responses to menthol, which indicates that these mutations do not affect crucial conformational changes leading to channel gating. While our cumulative data suggest that these residues may be involved in direct interaction with rapamycin, we recognize the alternative possibility that they allosterically affect rapamycin-induced channel gating. This is now clearly stated in the first paragraph of the discussion.

Ulteriore conferma dell'importanza di tutti gli autori antichi o moderni si rileva anche nella scelta di essi che hanno scritto in qualsiasi lingua o che hanno adoperato dei testi di riferimento in qualsiasi lingua poichè è sempre patrimonio bibliotecario culturale, senza quindi dover presentare una preferenza linguistica a tal riguardo

L'autore un questione sta sottolineando l'importanza e la necessità di disporre qualsiasi natura argomentativa del libro e di qualsiasi epoca giacchè, è di estrema rilevanza possedere testi sia moderni che antichi in quanto la cultura propriamente detta non deve essere trascurata in nessun'epoca

Reviewer #1 (Public review):

Summary:

This manuscript by Guo and Uusisaari describes a series of experiments that employ a novel approach to address long-standing questions on the inferior olive in general and the role of the nucleo-olivary projection specifically. For the first time, they optimized the ventral approach to the inferior olive to facilitate imaging in this area that is notoriously difficult to reach. Using this approach, they are able to compare activity in two olivary regions, the PO and DAO, during different types of stimulation. They demonstrate the difference between the two regions, linked to Aldoc-identities of downstream Purkinje cells, and that there is co-activation resulting in larger events when they are clustered. Periocular stimulation also drives larger events, related to co-activation. Using optogenetic stimulation they activate the nucleo-olivary (N-O) tract and observe a wide range of responses, from excitation to inhibition. Zooming in on inhibition they test the assumption that N-O activation can be responsible for suppression of sensory-evoked events. Instead, they suggest that the N-O input can function to suppress background activity while preserving the sensory-driven responses.

Strengths:

This is an important study, tackling the long-standing issue of the impossibility to do imaging in the inferior olive and using that novel method to address the most relevant questions. The experiments are technically very challenging, the results are presented clearly and the analysis is quite rigorous. There is quite a lot of room for interpretation, see weaknesses, but the authors make an effort to cover many options.

Weaknesses:

The heavy anesthesia that is required during the experiment could severely impact the findings. Because of the anesthesia, the firing rate of IO neurons is found to be ~0.1 Hz, significantly lower than the 1 Hz found in non-anesthetized mice. This is mentioned and discussed, but what the consequences could be cannot be understated and should be addressed more. Although the methods and results are described in sufficient detail, there are a few points that, when addressed, would improve the manuscript.

Author response:

Reviewer #1 (Public review):

Summary:

This manuscript by Guo and Uusisaari describes a series of experiments that employ a novel approach to address long-standing questions on the inferior olive in general and the role of the nucleoolivary projection specifically. For the first time, they optimized the ventral approach to the inferior olive to facilitate imaging in this area that is notoriously difficult to reach. Using this approach, they are able to compare activity in two olivary regions, the PO and DAO, during different types of stimulation. They demonstrate the difference between the two regions, linked to Aldoc-identities of downstream Purkinje cells, and that there is co-activation resulting in larger events when they are clustered. Periocular stimulation also drives larger events, related to co-activation. Using optogenetic stimulation they activate the nucleoolivary (N-O) tract and observe a wide range of responses, from excitation to inhibition. Zooming in on inhibition they test the assumption that N-O activation can be responsible for suppression of sensoryevoked events. Instead, they suggest that the N-O input can function to suppress background activity while preserving the sensory-driven responses.

Strengths:

This is an important study, tackling the long-standing issue of the impossibility to do imaging in the inferior olive and using that novel method to address the most relevant questions. The experiments are technically very challenging, the results are presented clearly and the analysis is quite rigorous. There is quite a lot of room for interpretation, see weaknesses, but the authors make an effort to cover many options.

Weaknesses:

The heavy anesthesia that is required during the experiment could severely impact the findings. Because of the anesthesia, the firing rate of IO neurons is found to be 0.1 Hz, significantly lower than the 1 Hz found in non-anesthetized mice. This is mentioned and discussed, but what the consequences could be cannot be understated and should be addressed more. Although the methods and results are described in sufficient detail, there are a few points that, when addressed, would improve the manuscript.

We sincerely thank the reviewer for their encouraging comments and recognition of our study’s significance. We fully acknowledge the confounding effects of the deep anesthesia used in our experiments, which was necessary to ensure the animals’ welfare while establishing this technically demanding methodology. We elaborate on these effects below and will further clarify them in the revised manuscript.

Ultimately, the full resolution of this issue will require recordings in awake animals, as we consider our approach an advancement from acute slice preparations but not yet a complete representation of in vivo IO function. However, key findings from our study—such as amplitude modulation with co-activation and the potential role of IO refractoriness in complex spike generation—could be further explored in existing cerebellar cortical recordings from awake, behaving animals. We hope our work will motivate re-examination of such datasets to assess whether these mechanisms contribute to overall cerebellar function.

Reviewer #1 (Recommendations for the authors):

On page 10 the authors indicate that 2084 events were included for DAO and 1176 for PO. Is that the total number of events? What was the average and the range per neuron and the average recording duration?

Thank you for pointing out lack of clarity. The sentence should say "in total, 2084 and 1176 detected events from DAO and PO were included in the study". We will add the averages and ranges of events detected per neuron in different categories, as well as the durations of the recordings (ranging from 120s to 270s) to the tables.

On page 10 it is also stated that: "events in PO reached larger values than those in DAO even though the average values did not differ". Please clarify that statement. Which parameter + p-value in the table indicates this difference?

Apologies for omission. Currently the observation is only visible in the longer tail to the right in the PO data in Figure 2B2. We will add the range of values (3.0-75.2 vs 3.1-39.6 for PO and DAO amplitudes, respectively) in text and the tables in the revision.

Abbreviating airpuff to AP is confusing, I would suggest not abbreviating it.

Understood. We will change AP to airpuff in the text. In figure labels, at least in some panels, the abbreviation will be necessary due to space constraints.

What type of pulse was used to drive ChrimsonR? Could it be that the pulse caused a rebound-like phenomenon with the pulse duration that drove the excitation?

As described on line 229 and in the Methods, we used 5-second trains of 5-ms LED light pulses. Importantly, these stimulation parameters were informed by our extensive in vitro examination of various stimulation patterns (Lefler et al., 2014), which consistently produced stable postsynaptic responses without inducing depolarization or rebound effects. Additionally, Loyola et al. (2024) reported no evidence of rebound activity in IO cells following optogenetic activation of N-O axons in the absence of direct neuronal depolarization. We will incorporate these considerations into the discussion, while also acknowledging that unequivocal confirmation of “direct” rebound excitation would require intracellular recordings, such as patch clamp experiments.

The authors indicate that the excitatory activity was indistinguishable in shape from other calcium activity, but can anything be said about the timing (the scale bar in Figure 4A2 has no value, is it the same 2s pulse)?

Apologies for oversight in labeling the scale bar in Figure 4A2 (it is 2s). While we deliberately refrain from making strong claims regarding the origin of the NO-evoked spikes, their timing can be examined in more detail in Figure 4 - Supplement 1, panels C and D. We will make sure this is clearly stated in the revised text.

Did the authors check for accidental sparse transfection with ChrimsonR of olivary neurons in the post-mortem analysis?

Good point! However, we have never seen this AAV9-based viral construct to drive trans-synaptic expression in the IO, nor is this version of AAV known to have the capacity for transsynaptic expression in general.

No sign of retrograde labeling (via the CF collaterals in the cerebellar nuclei) was seen either. Notably, the hSyn promoter used to drive ChrimsonR expression is extremely ineffective in the IO. Thus, we doubt that such accidental labeling could underlie the excitatory events seen upon N-O stimulation. We will add these mentions with relevant references to the discussion of the revised manuscript.

On page 18 the authors state that: "The lower SS rate was attributed to intrinsic factors of PNs, while the reduced frequency of CSs was speculated to result from increased inhibition of the IO via the nucleo-olivary (N-O) pathway targeting the same microzone." I think I understand what you mean to say, but this is a bit confusing.

Agreed. We will rephrase this sentence to clarify that a lower SS rate in a given microzone may lead to increased activation of inhibitory N-O axons that target the region of IO that sends CF to the same microzone.

Is airpuff stimulation not more likely to activate PO dan DAO because of the related modalities (more face vs. more trunk/limbs?), and thereby also more likely to drive event co-activation (as it is stated in the abstract).

We agree that the specific innervation patterns of different IO regions likely explain the discrepancy between previous reports of airpuff-evoked complex spikes in cerebellar cortical regions targeted by DAO and the absence of airpuff responses in the particular region of DAO accessible via our surgical approach. As in the present dataset virtually no airpuff-evoked events were seen in DAO regions, we are unable to directly compare airpuff-evoked event co-activation between PO and DAO. The higher co-activation for PO was observed for "spontaneous" activity.

The Discussion addresses the question of why N-O pathway activation does not remove the airpuff response.

Given the potentially profound effect, I would propose to expand the discussion on the role of aneasthesia, including longer refractory periods but also potential disruption of normal network interactions (even though individually the stimulations work). Briefly indicating what is known about alpha-chloralose would help interpret the results as well.

We fully agree that the anesthetic state introduces confounding factors that must be considered when interpreting our results. We will expand the discussion to address how anesthesia, particularly alphachloralose as well as tissue cooling, may contribute to prolonged refractory periods and potential disruptions in normal network interactions. However, we recognize that certain aspects cannot be fully resolved without recordings in awake animals. For this reason, we characterize our preparation as an "upgraded" in vitro approach rather than a fully representative in vivo model.

Please clearly indicate that the age range of P35-45 is for the moment of virus injection and specify the age range for the imaging experiment.

Apologies for the oversight. We will indicate these age ranges in the results (as they are currently only specified in Methods). The P35-45 range refers to moment of virus injection.

The methods indicate that a low-pass filter of 1Hz was used. I am sure this helps with smoothing, but does it not remove a lot of potentially interesting information. How would a higher low-pass filter affect the analysis and results?

We acknowledge that applying a 1 Hz low-pass filter inevitably removes high-frequency components, including potential IO oscillations and fine details such as spike "doublets." However, given the temporal resolution constraints of our recording approach, we prioritized capturing robust, interpretable events over attempting to extract finer features that might be obscured by both the indicator kinetics and imaging speed.

While a higher cut-off frequency could, in principle, allow more precise measurement of rise times and peak timings, it would also amplify high-frequency noise, complicating automated event detection and reducing confidence in distinguishing genuine neural signals from artifacts. Given these trade-offs, we opted for a conservative filtering approach to ensure stable event detection. Future work, particularly with faster imaging rates and improved sensors (GCaMP8s) will be used to explore the finer temporal structure of IO activity. We will deliberate on these matters more extensively in the revised discussion.

Reviewer #2 (Public review):

The authors developed a strategy to image inferior olive somata via viral GCaMP6s expression, an implanted GRIN lens, and a one-photon head-mounted microscope, providing the first in vivo somatic recordings from these neurons. The main new findings relate to the activation of the nucleoolivary pathway, specifically that: this manipulation does not produce a spiking rebound in the IO; it exerts a larger effect on spontaneous IO spiking than stimulus (airpuff)-evoked spiking. In addition, several findings previously demonstrated in vivo in Purkinje cell complex spikes or inferior olivary axons are confirmed here in olivary somata: differences in event sizes from single cells versus co-activated cells; reduced coactivation when activating the NO pathway; more coactivation within a single zebrin compartment.

The study presents some interesting findings, and for the most part, the analyses are appropriate. My two principal critiques are that the study does not acknowledge major technical limitations and their impact on the claims; and the study does not accurately represent prior work with respect to the current findings.

We thank the reviewer for recognising the value of the findings in our "reduced" in vivo preparation, and apologize for omissions in the work that led to critique. We will elaborate on these matters below and prepare a revised manuscript.

The authors use GCaMP6s, which has a tau1/2 of >1 s for a normal spike, and probably closer to 2 s (10.1038/nature12354) for the unique and long type of olivary spikes that give rise to axonal bursts (10.1016/j.neuron.2009.03.023). Indeed, the authors demonstrate as much (Fig. 2B1). This affects at least several claims:

a. The authors report spontaneous spike rates of 0.1 Hz. They attribute this to anesthesia, yet other studies under anesthesia recording Purkinje complex spikes via either imaging or electrophysiology report spike rates as high as 1.5 Hz (10.1523/JNEUROSCI.2525-10.2011). This discrepancy is not acknowledged and a plausible explanation is not given. Citations are not provided that demonstrate such low anesthetized spike rates, nor are citations provided for the claim that spike rates drop increasingly with increasing levels of anesthesia when compared to awake resting conditions.

We fully acknowledge that anesthesia is a major confounding factor in our study. Given the unusually invasive nature of our surgical preparation, we prioritized deep anesthesia to ensure the animals’ welfare. This, along with potential cooling effects from tissue removal and GRIN lens contact, likely contributed to the observed suppression of IO activity.

We recognize that reported complex spike rates under anesthesia vary considerably across studies, and we will expand our discussion to provide a more comprehensive comparison with prior literature. Notably, different anesthetic protocols, levels of anesthesia, and recording methodologies can lead to widely different estimates of firing rates. While we cannot resolve this issue without recordings in awake animals, we will clarify that our observed rates likely reflect both the effects of anesthesia and specific methodological constraints. We will also incorporate additional references to studies examining cerebellar activity under different anesthetic conditions.

More likely, this discrepancy reflects spikes that are missed due to a combination of the indicator kinetics and low imaging sensitivity (see (2)), neither of which are presented as possible plausible alternative explanations.

We acknowledge that the combination of slow indicator kinetics and limited optical power in our miniature microscope setup constrains the temporal resolution of our recordings. However, we are confident that we can reliably detect events occurring at intervals of 1 second or longer. This confidence is based on data from another preparation using the same viral vector and optical system, where we observed spike rates an order of magnitude higher.

That said, we do not make claims regarding the presence or absence of somatic events occurring at very short intervals (e.g., 100-ms "doublets," as described by Titley et al., 2019), as these would likely fall below our temporal resolution. We will clarify this limitation in the revised manuscript to ensure that the constraints of our approach are fully acknowledged.

While GCaMP6s is not as sensitive as more recent variants (Zhang et al., 2023, PMID 36922596), our previous work (Dorgans et al., 2022) demonstrated that its dynamic range and sensitivity are sufficient to detect both spikes and subthreshold activity in vitro. Although the experimental conditions differ in the current miniscope experiments, we took measures to optimize signal quality, including excluding recordings with a low signal-to-noise ratio (see Methods). This need for high signal fidelity also informed our decision to limit the sampling rate to 20 fps. In future work, we plan to adopt newer GCaMP variants that were not available at the start of this project, which should further improve sensitivity and temporal resolution.

Many claims are made throughout about co-activation ("clustering"), but with the GCaMP6s rise time to peak (0.5 s), there is little technical possibility to resolve co-activation. This limitation is not acknowledged as a caveat and the implications for the claims are not engaged with in the text.

As noted in the manuscript (L492-), "interpreting fluorescence signals relative to underlying voltage changes is challenging, particularly in IO neurons with unusual calcium dynamics." We acknowledge that the slow rise time of GCaMP6s ( 0.5 s) limits our ability to precisely resolve the timing of co-activation at very short intervals. However, given the relatively slow timescales of IO event clustering and the inherent synchrony in olivary network dynamics, we believe that the observed co-activation patterns remain meaningful, even if finer temporal details cannot be fully resolved.

To ensure clarity, we will expand this section to explicitly acknowledge the temporal resolution limitations of our approach and discuss their implications for interpreting co-activation. While the precise timing of individual spikes within a cluster may not be resolvable, the observed increase in event magnitude with coarse co-activation suggests that clustering effects remain functionally relevant even when exact spike synchrony is not detectable at millisecond resolution.

This finding is consistent with the idea that co-activation enhances calcium influx, leading to larger amplitude events — a relationship that does not require perfect temporal resolution to be observed. The fact that this effect persists across a broad range of clustering windows (as shown in Figure 2 Supplement 2) further supports its robustness. While we cannot make strong claims about precise spike timing within these clusters nor about the mechanism underlying enhanced calcium signal, our results demonstrate that co-activation may influence IO activity in a quantifiable way. We will clarify these points in the revised manuscript to ensure that our findings are appropriately framed given the temporal constraints of our imaging approach.

The study reports an ultralong "refractory period" (L422-etc) in the IO, but this again must be tempered by the possibility that spikes are simply being missed due to very slow indicator kinetics and limited sensitivity. Indeed, the headline numeric estimate of 1.5 s (L445) is suspiciously close to the underlying indicator kinetic limitation of 1-2 s.

Our findings suggest a potential refractory period limiting the frequency of events in the inferior olive under our recording conditions. This interpretation is supported by the observed inter-event interval distribution, the inability of N-O stimulation to suppress airpuff-evoked events, and lower bounds reported in earlier literature on complex spike intervals recorded in awake animals under various behavioral contexts. Taking into account the likely cooling of tissue, a refractory period of 1.5s is not unreasonable. Of course, we recognize that the slow decay kinetics of GCaMP6s may cause overlapping fluorescence signals, potentially obscuring closely spaced events. This is in line with data presented in the Chen et al 2013 manuscript describing GCaMp6s (PMID: 36922596; Figure 3b showing events detected with intervals less than 500 ms).

The consideration of refractoriness only arose late in the project while we were investigating the explanations for lack of inhibition of airpuff-evoked spikes. Future experiments, particularly in awake animals, will be instrumental in validating this interpretation. To ensure that the refractory period is understood as one possible mechanism rather than a definitive explanation, we will rephrase the discussion to clarify that while our data are compatible with a refractory period, they do not establish it conclusively.

The study uses endoscopic one-photon miniaturized microscope imaging. Realistically, this is expected to permit an axial point spread function (z-PSF) on the order of 40um, which must substantially reduce resolution and sensitivity. This means that if there *is* local coactivation, the data in this study will very likely have individual ROIs that integrate signals from multiple neighboring cells. The study reports relationships between event magnitude and clustering, etc; but a fluorescence signal that contains photons contributed by multiple neighboring neurons will be larger than a single neuron, regardless of the underlying physiology - the text does not acknowledge this possibility or limitation.

We acknowledge that the use of one-photon endoscopic imaging imposes limitations on axial resolution, potentially leading to signal contributions from neighboring neurons. To mitigate this, we applied CNMFe processing, which allows for the deconvolution of overlapping signals and the differentiation of multiple neuronal sources within shared pixels. However, as the reviewer points out, if two neurons are perfectly overlapping in space, they may be treated as a single unit.

To clarify this limitation, we will expand the discussion to explicitly acknowledge the impact of one-photon imaging on signal separation and to emphasize that, while CNMFe helps resolve some overlaps, perfect separation is not always possible. As already noted in the manuscript (L495-), "the absence of optical sectioning in the whole-field imaging method can lead to confounding artifacts in densely labeled structures such as the IO’s tortuous neuropil." We will further elaborate on how this factor was considered in our analysis and interpretation.

Second, the text makes several claims for the first multicellular in vivo olivary recordings. (L11; L324, etc).

I am aware of at least two studies that have recorded populations of single olivary axons using two-photon Ca2+ imaging up to 6 years ago (10.1016/j.neuron.2019.03.010; 10.7554/eLife.61593). This technique is not acknowledged or discussed, and one of these studies is not cited. No argument is presented for why axonal imaging should not "count" as multicellular in vivo olivary recording: axonal Ca2+ reflects somatic spiking.

We appreciate the reviewer’s point and acknowledge the important prior work using two-photon imaging to record olivary axonal activity in the cerebellar cortex. However, while axonal calcium signals do reflect somatic spiking, these recordings inherently lack information about the local network interactions within the inferior olive itself.

A key motivation for our study was to observe neuronal activity within the IO at the level of its gap-junctioncoupled local circuits, rather than at the level of its divergent axonal outputs. The fan-like spread of climbing fibers across rostrocaudal microzones in the cerebellar cortex makes them relatively easy to record in vivo, but it also means that individual imaging fields contain axons from neurons that may be distributed across different IO microdomains. As a result, while previous work has provided valuable insight into olivary output patterns, it has not allowed for the examination of coordinated somatic activity within localized IO neuron clusters.

With apologies, we recognize that this distinction was not sufficiently emphasized in our introduction. We will clarify this key point and ensure that the important climbing fiber imaging studies are properly cited and contextualized in the revised manuscript.

Reviewer #2 (Recommendations for the authors):

The authors state: "we found no reports that examined coactivation levels between Z+ and Z- microzones in cerebellar complex spike recordings" (L359). Multiple papers (that are not cited) using AldolaceC-tdTomato mice with two photon Purkinje dendritic calcium imaging showed synchronization (at similar levels) within but not across z+/z- bands. (2015 10.1523/JNEUROSCI.2170-14.2015, 2023 https://doi.org/10.7554/eLife.86340).

We apologize for the misleading phrasing. We will rephrase this statement to: "While complex spike coactivation within individual zebrin zones has been extensively studied (references), we found no reports directly comparing the levels of intra-zone co-activation between Z+ and Z microzones."

Additionally, we will ensure that the relevant studies demonstrating synchronization within zebrin zones, as well as (lack of) interactions between neighboring zones, are properly cited and discussed in the revised manuscript.

The figures could use more proofreading, and several decisions should be reconsidered:

Normalizing the amplitude to maximum is not a good strategy, as it can overemphasize noise or extremely small-magnitude signals, and should instead follow standard convention and present in fixed units (3A2, 4B2, and even 2C).

As noted earlier, we have excluded recordings and cells with high noise or a low signal-to-noise ratio for event amplitudes, ensuring that such data do not influence the color-coded panels. Importantly, all quantitative analyses and traces presented in the manuscript are normalized to baseline noise level, not to maximal amplitude, ensuring that noise or low-magnitude signals do not skew the analysis.

The decision to use max-amplitude normalization in color-coded panels was made specifically to aid visualization of temporal structure across recordings. This approach allows for clearer comparisons without the distraction of inter-cell variability in absolute signal strength. However, we recognize the potential for confusion and will revise the Results text to explicitly clarify that the color-coded visualizations use a different scaling method than the quantitative analyses.

x axes with no units: Figures 2B2, 2E1, 3B2, 3C2, 5B2, 5C2, 5D2.

No colorbar units: 5A3 (and should be shown in real not normalized units).

No y axis units: 5D1.

No x axis label or units: 5E1.

5E3 says "stim/baseline" for the y-axis units and then the first-panel title says "absolute frequencies" meaning it’s *not* normalized and needs a separate (accurate) y-axis with units.

Illegibly tiny fonts: 2E1, 3E1, etc.

We will correct all these in the revised manuscript. Thank you for careful reading.

descrições dos capítulos

En la acepción de Wenger, práctica, aprendizaje e identidad van de la mano. Es difícil, sino imposible, diferenciar lo uno de lo otro y son más bien dimensiones de mirada. Pareciera que el párrafo quiere diferenciar dos comunidades distintas: de práctica y de aprendizaje en lugar de dos maneras distintas de ver a la comunidad. Sugeriría un redacción que haga más claras esas facetas de las comunidades en lugar de su aparente disyuntiva.

lỗ chẩm - the largest foramen of the skull (Lỗ lớn nhất của hộp sọ) - kết nối thuỳ chẩm (occipital lobe) và cột sống (spinal cord) => Ở cả hai bên lỗ chẩm là lồi cầu chẩm (occipital condyle). Các lồi cầu này tạo thành khớp với đốt sống cổ thứ nhất. These condyles form joints with the first cervical vertebra. khả năng là form an information passage to exercise movement decisions since spinal cord nối với hệ thần kinh vận động?

DOI: 10.3389/fendo.2025.1514264

Resource: None

Curator: @scibot

SciCrunch record: RRID:AB_3661729

What is this?

El feedback es de gran ayuda para motivar y alentar a las y los estudiantes a interesarse en su propio aprendizaje.

Es interesante como diferentes plataformas cuentan con la facilidad de retroalimentación, algo que en la presencialidad se ha visto olvidado.

Muchos de los prejuicios que se tienen de la Educación en línea, justo es el acompañamiento de docentes, pero nos podemos dar cuenta que existen multiples medios para interactuar y guíar a las y los estudiantes.

Considero que estas capas se relacionan directamente al Diseño curricular o bien al Diseño Instruccional, desde la perspectiva pedagógica.

El proceso para crear una evaluación auténtica puede ser complicado, ya que muchas veces no sabemos que actividades si pueden ser evaluables y cuales no, esto dependiendo de los objetivos de aprendizaje, de la interacción en el grupo y la modalidad de los cursos.

Es importante que en un entorno digital y no presencial, se fomente más interacción y un aprendizaje donde los estudiantes participen la mayor parte del tiempo

Subrraya el uso de herramientas electrónicas para estructurar o medir el proceso de enseñanza-aprendizaje.

Cuando das o recibes un feedback debes comprender que no es un ataque hacia tu persona, son una serie de recomendaciones y mejoras hacia tu trabajo.

El feedback también tiene una serie de procesos, mismos que no deberían quedarse en los comentarios y correcciones que te dan o que realizas. Para que un feedback sea eficaz debe llevarse a la práctica.

Feed Back es ecencial, y es tambien una opción altamente recomendable para proporcionar retorno.

Feedback esta presente en todo el proceso de aprendizaje y no solo es hacer correciones, o palabras de aliento, sino retroalimentaciones

Evaluar no es sólo calificar, aunque sí es una de sus dimenciones no es la única, pese a estar en la Licenciatura en Pedagogía hay colegas que continúan replicando la medición del aprendizaje de las y los estudiantes cuando podemos proponer y aplicar otras manera de evaluar, incluso que las y los mismos estudiantes evalúen el curso o el taller en pro de retroalimentar y mejorar el quehacer pedagógico en los procesos de enseñanza y aprendizaje.

Cuántas y cuántos de nosotros hemos escuchado o hablado, a lo largo de nuestra formación educativa, la preocupación sobre la acreditación u obtención de notas altas en vez de centrarnos en aprender. Diferenciando a la memorización, apelando a una educación bancaria en vez de formativa que propicie el reconocimiento de los saberes propios y adquiridos, además de la transformación del entorno de las y los educandos.

Entre a membrana e a parede celular, logo não entra na célular

• Informativo nº 840
• 18 de fevereiro de 2025.
• Processo
• Processo em segredo de justiça, Rel. Ministra Nancy Andrighi, Segunda Seção, por unanimidade, julgado em 6/2/2025, DJEN 13/2/2025.

Ramo do Direito DIREITO CIVIL, DIREITO PROCESSUAL CIVIL

TemaPaz, Justiça e Instituições Eficazes <br /> Penhora de imóvel. Bem de família. Impenhorabilidade legal. Fraude à execução. Ineficácia da doação. Proteção da impenhorabilidade mantida. Imóvel qualificado como bem de família antes da doação. Situação inalterada pela alienação apontada como fraudulenta.

Destaque - É possível o reconhecimento da manutenção da proteção do bem de família que, apesar de ter sido doado em fraude à execução aos seus filhos, ainda é utilizado pela família como moradia.

Informações do Inteiro Teor - Cinge-se a controvérsia em definir se se a doação ou a alienação gratuita de bem de família impenhorável pode configurar fraude à execução e afastar a proteção legal da impenhorabilidade.

• Na Segunda Seção do Superior Tribunal de Justiça, prevalece o entendimento de que o reconhecimento da ocorrência de fraude à execução e sua influência na disciplina do bem de família deve ser aferida casuisticamente, de modo a evitar a perpetração de injustiças - deixando famílias ao desabrigo - ou a chancelar a conduta ardilosa do executado em desfavor do legítimo direito do credor, observados os parâmetros do art. 792 do Código de Processo Civil e da Lei n. 8.009/1990.

• De acordo com o entendimento mais recente do STJ, "o parâmetro crucial para discernir se há ou não fraude contra credores ou à execução é verificar a ocorrência de alteração na destinação primitiva do imóvel - qual seja, a morada da família - ou de desvio do proveito econômico da alienação (se existente) em prejuízo do credor. Inexistentes tais requisitos, não há alienação fraudulenta" (REsp 1.227.366/RS, Quarta Turma, DJe 17/11/2014).

• Considerando que a consequência da fraude à execução é apenas a ineficácia da alienação em relação ao exequente (art. 792, § 1º, do CPC), para aferir a incidência ou não da regra da impenhorabilidade do bem de família, é necessário analisar, primeiro, a situação do imóvel anterior à alienação, para verificar se houve ou não alteração na sua destinação primitiva.

• Assim, havendo alegação de alienação em fraude à execução envolvendo bem de família impenhorável, será necessário analisar: i) se, antes da alienação, o imóvel já se qualificava como um bem de família, não incidindo nenhuma exceção legal, como aquelas previstas no art. 3º da Lei n. 8.009/1990; e ii) se, após a alienação, o imóvel manteve a qualidade de bem de família, ou seja, se continuou a servir de moradia à entidade familiar.

• Em sendo positivas as respostas, conclui-se pela incidência da proteção legal da impenhorabilidade do bem de família, tendo em vista que não houve alteração na situação fática do imóvel, a despeito da alienação.

• Por conseguinte, não haverá interesse na declaração de fraude e ineficácia da alienação em relação ao exequente, diante da ausência de consequência sobre o imóvel que continuaria sendo bem de família e, portanto, impenhorável.

Art. 79. São imunes ao IBS e à CBS as exportações de bens e de serviços para o exterior, nos termos do art. 8º desta Lei Complementar, asseguradas ao exportador a apropriação e a utilização dos créditos relativos às operações nas quais seja adquirente de bem ou de serviço, observadas as vedações ao creditamento previstas nos arts. 49 e 51, as demais disposições dos arts. 47 e 52 a 57 desta Lei Complementar e o disposto neste Capítulo.

ICMS. está previsto pro ICMS não entrar na BC do IBS e CBS. Porém não há previsão para que o IBS/CBS não entre na BC do ICMS.

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