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Reply to the reviewers

1. Point-by-point description of the revisions

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

In this study, the authors investigated the effect of nutritional stress (HSD and HFD) on cardiac function by assessing multiple parameters on adult flies. They next identified the adaptive transcriptomic changes in the heart in response to these nutritional stresses and screened for their roles under ND, HSD and HFD. They identified fit gene, encoding a satiety gene, expressed by cardiomyocytes and pericardial cells.

I think the characterisation is thorough; however, the conclusion is not well supported by the evidence. My main concern is that in many graphs, the difference between control and experiment is subtle, and, secondly, the authors showed some conflicting results (e.g. one RNAi showed a reduction of one parameter, however, the other independent RNAi did not. In this case, I believe the authors shouldn't conclude that the RNAi is functionally required, since the RNAis are meant to confirm each other.

First, we thank the reviewer for her/his constructive comments and suggestions. We obtained new results presented in the last version of the manuscript, which consistently support our conclusions and improve the study.

High-Sugar and High-Fat Diets modified cardiac performance

They assessed how HSD and HFD affect Adult fly heart performance. Instead of performing 3 weeks of dietary manipulation as has been done before by other groups, they put adult flies on HSD for 7 days and HFD for only 3 days.

We would like to clarify the nutritional challenge used. Cardiac function of flies was assessed at 10 days after emergence. Flies were put either in ND or HSD during these 10 days (ND and HSD conditions), or in ND for 7 days then transferred on HFD for 3days (HFD condition). Finally, all the females spent 10 days in a diet before being imaged or before hearts/brains dissection.

They found: HSD increases HP and SI, and reduces AI. The difference is too small and not consistent between different control lines. Also, when the difference is this small, p value does not tell much!

They probably intentionally induced a milder effect so that they could assess adaptive transcriptomic changes to this nutritional stress. In Fig. 1D SI is increased under HSD with control-KK, In Fig. S1C, SI is not changed under HSD with control-GD and control-GFP. Instead, DI is increased, which is also opposite to what they showed in Fig. 1 C. HFD increased ESD, EDD, SV, FS and CO.(Hypertrophy). This is not true with control-GD and control-GFP lines though! Comments: They have assessed many parameters in live animals with many different control lines, which is thorough. However, it is hard to draw any conclusions based on these conflicting results. Are these effect KK line specific?

Globally, we agree with the reviewer that the results, presented in the first version of the manuscript, for the control lines were difficult to understand due to the inconsistency of the phenotypes. In this revised version, we performed new results in Figure 1 and __S1 __regarding the effect of 10 days HSD and 3 days HFD exposure vs ND.

105 to 187 flies were imaged for the 3 control conditions, in the 3 diets concomitantly, to increase the power of our analysis. As mentioned in the main text (page 3, line 30-35; page 4, line 1-5), both diets deteriorate cardiac function with HFD leading to consistent phenotypes on heart diameters and rhythm and HSD milder effects. Indeed, the 3 control lines were uniformly affected by HFD after 3 days exposure, whereas 10 days in HSD was not sufficient to quantify a significant effect despite consistent the trends on several phenotypes (EDD, ESD, DI, AI and CO. These results revealed a different sensitivity of the cardiac performance when exposed to sugar and fat.

As described in the text, we were nevertheless confident that our approach would be good to investigate the early molecular dysregulations induced by sugar. This was the purpose of our analysis, presented in the follow-up of the manuscript.

Regarding the small differences measured in the phenotypes in HSD and HFD compared to ND, we would like to outline that the values presented are normalized values to control. Normalization is done for every independent experiment, performed at different dates, and permits the graphical representation of pooled values. Statistical analysis is performed using non-parametric Kruskal-Wallis test accordingly. Values are presented with the X axis cutting the Y axis at 0, this graphical representation also contributes to flattening the differences and p-values indicate their significance.

Analysis of the fly cardiac transcriptome upon nutritional stress

RNA seq to detect differentially expressed genes under HSD and HFD vs ND. Most DE genes are downregulated, which prompts them to assess how the downregulation of these genes adapts the animals to this nutritional stress.

High Sugar Diet downregulated 1c-metabolism and Leloir galactose pathways.

In this revised manuscript, we first present RT-qPCR validating the downregulation of Gnmt, Sardh and Galk expressions in the heart of 10days old HSD-fed females compared to ND-fed ones (Figure S3A).

We apologize for the confused explanations in the first version of the manuscript. We show new results in Figure 3 and __S3 __on the cardiac function of both Gnmt and Sardh, where following reviewer’s suggestion, both genes were knocked down in the heart in ND and Gnmt overexpressed in HSD. No available tools allowed us to test Sardh overexpression in HSD and we could not get some for Galk.

GNMT is downregulated under HSD and HFD.

In ND, GNMT knockdown increased ESD, EDD and CO. Sardh knockdown did the same? However, Sardh knockdown did not affect ESD significantly.

We reanalyze our first data and added new ones, comparing only knockdown or overexpression to the corresponding controls performed in concomitant experiments. Results are now shown in Figure 3C-E; S3C-H. Knocking down Gnmt in the heart increased HP, EDD, ESD and CO, Sardh knockdown in ND resulted in milder phenotypes but inducing significant hypertrophy in ND as Gnmt does. In both cases, FS was not impacted.

Both genes have been previously shown as beneficial to muscular function in time-restricted feeding context (Livelo et al., 2023, Nat.Comm.), illustrating that, even if both enzymes are involved in opposite reaction, their function has the same effect on organ/tissue function, as they did for heart diameters. The text corresponding to results and discussion were updated accordingly (pages 5, 11).

The conclusion here is: GNMT knockdown induces hypertrophy, similar to the effect of HFD.

In HSD, further knockdown of GNMT reduced (rescued) HP, suggesting downregulation of GNMT under HSD is adaptive. Should overexpress GNMT under HSD to see if this manipulation further increases HP, to claim GNMT downregulation is an adaptive change to high sugar stress.

We thank the reviewer for her/his suggestion. We now used UAS-GnmtWT (from FlyORF) to assess the role of Gnmt on cardiac function in HSD.

As shown in (Figure 3C-E; S3C,F), overexpressing Gnmt in the heart in HSD was sufficient to rescue some sugar induced phenotypes or to induce other dysfunctions, when compared to corresponding controls evaluated in the same experiments in ND and HSD. Notably, HP increase and CO decrease are rescued by Gnmt cardiac overexpression in HSD. Interestingly, the cardiac diastolic constriction induced by HSD is associated to increased FS and CO in this genotype in sugar diet. These new results strengthen the positive effect of Gnmt on cardiac function, improving it in HSD and preventing its deterioration in this diet.

Of note, Gnmt overexpression in ND did not trigger cardiac dysfunctions (data not shown).

The results and conclusions have been corrected.

Interestingly, HSD itself tends to decrease AI, a further knockdown of GNMT further decreases AI. This indicates GNMT downregulation under HSD contributes to AI reduction. Together, GNMT downregulation under HSD prevents HP from going higher, while its downregulation causes AI going down.

In the manscript, the authors claim that " Gnmt KD led reduced HP and AI, suggesting that it is able to counteract the effect of HSD observed in control flies on these phenotypes". This is not true according to the logic in Results section 1. As in section 1, the effect of HSD on AI is not significant, so the authors shouldn't say" HS tended to reduce AI".

Our reanalyzes and new results showed no Gnmt impact on AI, so these Figure panels were removed.

Why GNMT knockdown reduced FS under ND (Fig. S3C), while increasing FS under HSD (Fig. 3F)? If GNMT knockdown induces hypertrophy, I would expect it to increase FS.

Gnmt overexpression did not affect cardiac diameters in HSD, but it nevertheless led to an increased contractile efficacy compared to HSD controls (Figure S3F).

These new results strengthen the positive effect of Gnmt on cardiac function, preventing its deterioration in sugar diet. The text was modified accordingly.

High Fat Diet modulated CD36-scavenger receptor and Glut8 orthologues

In this revised manuscript, we present RT-qPCR validating the downregulation of Snmp1 expression and the slight upregulation of nebu’s in the heart of 10days old HFD-fed females compared to ND-fed ones (Figure S3B).

HFD: Snmp1 gene is downregulated, however, both overexpression and knockdown of Snmp1 in ND induced some phenotypes.

Indeed, as mentioned in the revised manuscript (page 6, lines 21-24), in heart of females fed ND, both Snmp1 knockdown (Snmp1KK) and overexpression (Snmp1WT) showed a reduction of EDD and ESD (Figure 3J; S3J) but FS is increased accordingly only in Snmp1KK.

As notified in the text, both downregulation and overexpression of Snmp1 led to side-phenotypes (page 6, lines 24-28): Snmp1KK exhibited abdominal fat increase (Figure S3K) and ostial cells seemed clearly malformed in Snmp1WT (Figure 3M). This may explain why the heart shows the same type of functional impairment in both genotypes.

We now discussed the hypothesis that these similar cardiac dysfunctions may result from Snmp1 being a regulator of organismal or cardiac lipid homeostasis. Indeed, increasing body fat content is deleterious as is increasing the import of fat in the cardiomyocytes. Finally, both affects cardiac cells’ health and functioning.

HFD: nebu has a role in regulating cardiac function under ND.

HSD and HFD revealed the secretory function of the heart

They identified diet-regulated secreted proteins that are required for cardiac dysfunction.

Cardiac Fit expression impacted Cardiac performance.

The author used Hand-G4 to knock down Fit using KK and GD lines, KK line showed a reduction in HP (Fig. 5A), but not GD line (Fig. S5D). How did the author conclude that Fit is required for cardiac function? Also, with the positive data, the difference is too subtle.

We apologize and agree that the contradictory or inconsistent results obtained with the two RNAi lines were confusing.

For this revised version, we first assess the effect of the two RNAi lines (KK and GD) on fit expression in the dissected hearts. RT-qPCR for KK line is presented in Figure S5A. GD line did not show a significant reduction of fit expression when expressed in the heart with Hand>, which can explain the former results presented (not shown but data are available). So, we removed all results obtained with the GD line in this revised version.

To confirm the KK effects, we used fit KO allele (fit81) and truncated version of fit, without its signal peptide (fitDeltaSP), which has a dominant negative effect, both previously published and validated (Sun et al. 2017, Nat. Comm.). These two mutants were used to investigate the cardiac function of fit in our analysis. Results presented in Figure 5 and S5 confirm the phenotypes already observed with the KK line when expressed with Hand> in the heart and with Lsp2> in the fat body.

Our results validate the effect of fit decrease on rhythmicity and contractility, the reverse effects being consistently observed in fit overexpression. In conclusion, we are confident in the requirement of Fit in the regulation of cardiac performance.

These new data are now included in the results section “Cardiac Fit expression impacted Cardiac performance” (pages 8-9)

**Referee cross-commenting**

i agree with the experiments proposed by reviewer 2.

Reviewer #1 (Significance (Required)):

The study aims to examine the effect of diet on cardiac function.

The strength is that a lot of characterisations were done.

the weakness is the functional data regarding fit could not be validated in two different RNAis, thus the evidence is not strong to support the conclusions.

We again would like to thank the reviewer for her/his remarks and suggestions. She/He highlights the weakness of the first analysis and this was an important and constructive feedbacks for us. We strengthened our results by increasing samples, reanalyzing data and performing mandatory new experiments that are now included in this revised version.

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

In this manuscript, Khamvongsa-Charbonnier et al. reported a RNA-seq analysis and RNA interference screening on high-fat and high-sugar-induced cardiomyopathy in Drosophila. The authors uncovered novel genes in 1C-metabolism, galactose metabolism, CD36-scavenger receptor and glucose transporter, as adaptative factors of cardiac function under high-fat and high-sugar treatment. The authors also identified a satiety hormone, Fit, as a cardiokine to control food intake and , expressed by dilp5 secretion. In summary, this study leverages the powerful genetic model Drosophila to uncover a number of new factors in regulating cardiac function under nutritional stresses and potentially offers new insights into molecular mechanisms underlying diet-related cardiac diseases. I have a few concerns, as listed below.

First, we would like to thank the reviewer for her/his comments and suggestions that deeply help us to improve the take-home messages of our manuscript. Following her/his recommendations and suggestions, we can now present a revised and stronger version of our manuscript.

1. Quantitative RT-PCR is required to validate the expression patterns of candidate genes identified from the RNAseq analysis.

RT-qPCR have been performed on hearts dissected from 10 days old females fed ND, HSD or HFD. Gnmt, Sardh and Galk validated downregulation are presented in Figure S3A, Snmp1 downregulation and nebu upregulation (trend but non-significant) in Figure S3B, fit downregulation in Figure S5A.

The authors state that the dysregulated gene expression patterns reflect acute adaptation to HSD and HFD stresses. Most of the candidate genes in this study were downregulated upon HSD and HFD. However, it is recommended that overexpression of these genes, rather than knockdown, is needed to confirm whether the downregulation of these candidate genes upon stresses is an adaptative response.

We agree with the reviewer and followed her/his recommendation when tools were accessible for our analysis.

For example, HSD feeding induces the heart period. Knocking down Gnmt, specifically in the heart, under the HSD feeding changes can reduce the heart period. This evidence is insufficient to suggest the protective role of Gnmt under the HSD diet. Gnmt has already been downregulated under the HSD. Further knockdown of Gnmt, instead of returning the Gnmt expression to normal levels, to protect cardiac contractile performance complicates the model.

We thank the reviewer for her/his suggestion. We used UAS-*GnmtWT * (from FlyORF) to perform these experiments.

As shown in (Figure 3C-E; S3C,F), knocking down Gnmt in the heart increased HP, EDD, ESD and CO. In the same Figure panels and in Figure S3F, we showed that overexpressing Gnmt with Hand> in HSD was sufficient to rescue some sugar induced phenotypes or to induce some, when compared to corresponding controls evaluated in the same experiments in ND and HSD. Gnmt overexpression in ND did not trigger cardiac dysfunctions (data not shown).

HP increase and CO decrease are rescued by Gnmt cardiac overexpression in HSD. Interestingly, the cardiac constriction induced by HSD is not rescued by Gnmt overexpression, but it is enough to increase FS and CO in sugar diet. These new results strengthen the positive effect of Gnmt on cardiac function, improving it in HSD and preventing its deterioration in this diet.

Sardh knockdown in ND, resulted in milder phenotypes but induced significant hypertrophy in ND as Gnmt does. No available tools allowed us to test its overexpression in HSD.

Nevertheless, as mentioned and discussed in the manuscript (page 5, line 27-30; page 11, lines 11-14), such protective role of muscular function and integrity has already been characterized in fly IFM in time-restricted feeding experiments for Gnmt and Sardh (Livelo et al., 2023, Nat.Comm.). Our experiments show that both genes encounter the same role in cardiac function upon nutritional stresses. The text was modified accordingly.

The authors suggest that the effect of nebu on heart contractility is not dependent on diet. However, based on the result from Figure 3O-P, the HFD treatment blocks the effect of nebu knockdown on heart contractility. The authors need to further explain these results and modify their conclusions accordingly.

We completely agree with the reviewer. We did not correctly analyze these results. We reanalyze our data, taking into account only the experiments of nebu knockdown that were performed in ND and in HFD concomitantly. Results are shown in Figure 3O-P; S3L-N.

As mentioned in the manuscript (page 7, lines 3-8), nebu knockdown led to identical HP decrease in both diets but its constrictive effect (reduction of heart diameters) in ND is abrogated by fat diet.

We modified the text accordingly in the results and discussion (page 7, lines 8-11; page 12, lines 7-12).

It is a bit confusing that knockdown of fit using Hand-Gal4 induced food intake, but knockdown of fit using tin-Gal4 or Dot-Gal4 did not significantly induce food intake (Fig 6A). The author did not provide any explanation of these results. What is even more confusing is that overexpressing fit using Dot-Gal4 decreased food intake, but overexpressing fit using Hand-Gal4 or tin-Gal4 did not significantly decrease food intake (Fig 6B). Why was the strong food intake phenotype not observed using Hand-Gal4 in both experiments? These confusing results lead to a question, which cell type is responsible for the production of cardiokine, Fit?

We apologize for the misleading results presented in the initial manuscript. We hope that our revised version will clarify Fit function regarding its remote impact.

Concerning the requirement of Fit function and the cell types that produces Fit, the results we obtained when evaluating cardiac performance strongly suggest that both cardiomyocytes and pericardial cells are important and recapitulate the effect of Hand> (Figure 5A-C; S5G-H).

In the case of food intake measurements, we now present results with newly performed food intake experiments for the Hand>fitWT (Figure 6D). They show a significant reduction of food intake in this condition, corroborating the results obtained with Dot>. We add a clarification in the manuscript for this point (page 10, lines 11-16).

When testing the role of cardiac Fit in Dilp5 secretion, the authors subjected flies to starvation stress. However, the main focus of the present study is on HSD and HFD. The RNAseq analysis showed that Fit expression was downregulated by both HSD and HFD. Can the authors show that Dilp5 secretion is reduced by both HSD and HFD? Most importantly, can the authors test whether overexpression of cardiac Fit blocks HSD- or HFD-reduced Dilp5 secretion?

We understand the point raised by the reviewer. First of all, we wanted to correlate the measured impact on food intake, when manipulating fit expression in the heart, to the level of Dilp release, as it has been used and validated in (Sun et al. 2017, Nat. Comm.). In this purpose, we used the same approach and protocol and results are shown in Figure 6 E-F.

As mentioned by the reviewer, fit expression is downregulated in both HSD and HFD (which we confirmed by RT-qPCR in Figure S5A). As suggested by the reviewer, we performed Dilp5 immunostaining on CNS from females that were fed HSD of HFD for 10 days. Our results, in Figure 6B (left panels) and corresponding quantifications in Figure 6C, show that both diets strongly induce a decrease in Dilp5 amount in the IPCs and that it was not due to an altered Dilp2 or Dilp5 expression in the CNS (Figure S6A). In this condition, overexpressing fit, which has a promoting effect on Dilp secretion (Figure 6B, right panels ND), may only have an additive effect. This is shown in Figure 6B-C.

Reviewer #2 (Significance (Required)):

In summary, this study leverages the powerful genetic model Drosophila to uncover a number of new factors in regulating cardiac function under nutritional stresses and potentially offers new insights into molecular mechanisms underlying diet-related cardiac diseases.

We again would like to thank the reviewer for her/his remarks and suggestions. Her/His important and constructive feedbacks helped us to improve and strengthen our study. Despite the weak points of the first version, she/he had supportive feedback and we deeply thank her/him. This revised version had improved results and analysis, thanks to the use of new genetic tools that strengthen this analysis.

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

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Referee #2

Evidence, reproducibility and clarity

In this manuscript, Khamvongsa-Charbonnier et al. reported a RNA-seq analysis and RNA interference screening on high-fat and high-sugar-induced cardiomyopathy in Drosophila. The authors uncovered novel genes in 1C-metabolism, galactose metabolism, CD36-scavenger receptor and glucose transporter, as adaptative factors of cardiac function under high-fat and high-sugar treatment. The authors also identified a satiety hormone, Fit, as a cardiokine to control food intake and , expressed by dilp5 secretion. In summary, this study leverages the powerful genetic model Drosophila to uncover a number of new factors in regulating cardiac function under nutritional stresses and potentially offers new insights into molecular mechanisms underlying diet-related cardiac diseases. I have a few concerns, as listed below.

1. Quantitative RT-PCR is required to validate the expression patterns of candidate genes identified from the RNAseq analysis.
2. The authors state that the dysregulated gene expression patterns reflect acute adaptation to HSD and HFD stresses. Most of the candidate genes in this study were downregulated upon HSD and HFD. However, it is recommended that overexpression of these genes, rather than knockdown, is needed to confirm whether the downregulation of these candidate genes upon stresses is an adaptative response. For example, HSD feeding induces the heart period. Knocking down Gnmt, specifically in the heart, under the HSD feeding changes can reduce the heart period. This evidence is insufficient to suggest the protective role of Gnmt under the HSD diet. Gnmt has already been downregulated under the HSD. Further knockdown of Gnmt, instead of returning the Gnmt expression to normal levels, to protect cardiac contractile performance complicates the model.
3. The authors suggest that the effect of nebu on heart contractility is not dependent on diet. However, based on the result from Figure 3O-P, the HFD treatment blocks the effect of nebu knockdown on heart contractility. The authors need to further explain these results and modify their conclusions accordingly.
4. It is a bit confusing that knockdown of fit using Hand-Gal4 induced food intake, but knockdown of fit using tin-Gal4 or Dot-Gal4 did not significantly induce food intake (Fig 6A). The author did not provide any explanation of these results.

What is even more confusing is that overexpressing fit using Dot-Gal4 decreased food intake, but overexpressing fit using Hand-Gal4 or tin-Gal4 did not significantly decrease food intake (Fig 6B). Why was the strong food intake phenotype not observed using Hand-Gal4 in both experiments?

These confusing results lead to a question, which cell type is responsible for the production of cardiokine, Fit? 5. When testing the role of cardiac Fit in Dilp5 secretion, the authors subjected flies to starvation stress. However, the main focus of the present study is on HSD and HFD. The RNAseq analysis showed that Fit expression was downregulated by both HSD and HFD. Can the authors show that Dilp5 secretion is reduced by both HSD and HFD? Most importantly, can the authors test whether overexpression of cardiac Fit blocks HSD- or HFD-reduced Dilp5 secretion?

Significance

In summary, this study leverages the powerful genetic model Drosophila to uncover a number of new factors in regulating cardiac function under nutritional stresses and potentially offers new insights into molecular mechanisms underlying diet-related cardiac diseases.

Reviewer #3 (Public review):

Summary:

Prudhomme et al report a detailed analysis of the role of vinculin in maintaining neuroepithelial integrity during cranial neurulation.

Strengths:

The authors use complementary experiments involving super-resolution microscopy, laser ablation, and live imaging of conditional knockout and ESC-derived embryos to demonstrate that loss of vinculin produces wide gaps between the adherens junctions of neuroepithelial cells at later stages of cranial neural fold elevation. The data presented are of extremely high quality, logically presented in a compelling story, and represent a very substantial contribution.

Weaknesses:

The authors are invited to consider the largely minor questions recommended below.

(1) The laser ablations reported are a correlate of cell border, or 'junctional' tension. Please avoid broad statements such as 'mechanical forces are upregulated' (abstract), which invoke gene-like regulation of tissue-level forces (in Newtons). Changes in junctional tension are likely to relate to changes in force generated, but their relationship is not simple: higher tensile stress withstood by the shorter length of junctions in cells with smaller apical surfaces does not necessarily translate into greater force being produced by that cell. The junctional tension readout measured is perfectly relevant to the paper, more so than tissue-level forces would have been.

(2) What is the mechanical mechanism by which loss of vinculin prevents neural fold elevation? The authors present exciting findings about the cellular consequences of losing Vcl at the late elevation stages when the tissue is quantifiably dysmorphic. A clear argument of how Vcl loss could lead to this dysmorphology would strengthen the paper, particularly given that junctional tension defects are excluded and apical non-constriction at the late stage is only mild.

(3) Can the authors comment on the likely impacts of Vcl deletion on the basal domain of the cell? For example, they could cite live-imaging of distinct behaviours in Williams et al Dev Cell 2014, and the NTD phenotypes of some integrin/focal adhesion mutant mice.

(4) The apparent uncoupling of apical area (larger in Vcl KO) from junctional tension (equivalent) in this model is noteworthy. Can the authors speculate on its potential basis?

(5) Live imaging in Figure 7C appears to show a marked reduction in apical area before cleavage furrow formation (T0-18min), suggesting a large apical constriction event (post-mitotic?), as previously reported (e.g., Ampartzidis et al Dev Biol 2023). Do junctional gaps appear during these constrictions?

(6) The live imaging setup used is clearly sufficient to identify differences between genotypes, so this is only a minor point. The gassing conditions listed in the methods specify 5% CO2, but E8.5 embryos also need low O2 to complete cranial closure. Was the O2 level controlled? Was tissue-level shape change observed to be consistent with ongoing neurulation during live-imaging?

(7) Neither the multi-cell laser ablations in the pre-print by De La O cited here, nor the narrower junctional ablations in Bocanegra-Moreno et al., Nat Phys, (2023), identified differences in recoil between developmental stages. Why might those results be different from the findings reported here (e.g., analysis region - not specified in the latter paper)? Limitations to interpreting junctional ablations between cells with different junction lengths include more of the recoil being dissipated by retraction of the longer ablated border.

(8) Is a truncated Vcl expressed in the ESC model, which could bind catenin without an F-actin anchor? The very high-contrast western shown is cropped so it is not clear whether the catenin-binding N-terminus is present. Does the antibody used recognise the head domain (this reviewer could not readily find the information)?

Rapport de Synthèse : Analyse du Traitement Judiciaire de l'Inceste en France

Résumé Exécutif

Ce document synthétise l'audition de Sihem Ghars, fondatrice du collectif Incesticide France, devant la commission d'enquête sur le traitement judiciaire des violences sexuelles incestueuses.

Le constat dressé est celui d'une défaillance systémique de l'institution judiciaire française, qualifiée de « torture institutionnelle » par le Comité contre la torture de l'ONU en 2025.

Le témoignage met en lumière une impunité quasi totale (moins de 1 % de condamnations) et un processus de « désenfantement » frappant les mères qui dénoncent les violences.

Au-delà des chiffres, le document expose les mécanismes de silenciation des victimes et propose des mesures d'urgence, affirmant que le cadre légal existant est suffisant mais que sa mise en œuvre est entravée par un déni institutionnel profond.

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1. État des Lieux et Données Chiffrées

Le collectif Incesticide France, créé en 2020, documente la situation de centaines de familles.

Les données présentées révèlent l'ampleur d'une crise qualifiée de « sanitaire » et de « pédophilique ».

• Représentativité du collectif : Plus de 600 familles suivies.

Une enquête citoyenne a recueilli 1 076 témoignages complets et 2 019 pièces justificatives en deux semaines.

• Impunité judiciaire :

  • Moins de 1 % de condamnations pour viols sur mineurs dans le cadre de l'inceste.

• Environ 3 % de condamnations pour les autres types de violences sexuelles.

• Parmi le 1 % de condamnés, la moitié bénéficie d'un sursis.

• Données médicales d'urgence : Les Unités Médico-Judiciaires (UMJ) ont recensé 614 bébés examinés pour viols en une seule année, soit environ 50 nourrissons par mois.

• Estimation de la menace : Sur la base d'études internationales, le témoignage estime à 2 millions le nombre d'auteurs de violences sexuelles sur mineurs en liberté en France.

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2. Défaillances Systémiques et « Torture Institutionnelle »

Le concept de « torture institutionnelle » s'appuie sur le rapport de mai 2025 du Comité contre la torture de l'ONU.

La France y est pointée pour de graves violations des droits fondamentaux.

Une chaîne d'acteurs en faillite

Le témoignage souligne l'échec successif de tous les intervenants de la chaîne de protection :

• Enquêteurs et police : Classements sans suite fréquents malgré des preuves matérielles (fissures anales constatées à l'hôpital, ADN, aveux sur PV).

• Experts psychiatres et psychologues : Manque de formation en psychotrauma.

Utilisation de rapports biaisés ou incompétents guidant les décisions des juges.

• Services sociaux (ASE) : Accusés de collusion ou d'incompétence.

Le terme « SS » est utilisé par certaines familles pour décrire des interventions perçues comme des « prises d'otages » d'enfants.

• Magistrats : Surcharge de travail empêchant une étude approfondie des dossiers (environ 10 dossiers par jour, soit des milliers de pages à traiter).

Le mensonge de l'État

La délégation française auprès de l'ONU aurait affirmé qu'aucune mère n'est poursuivie sans vérification préalable des signalements de viol.

Le témoignage dément formellement cette assertion, affirmant que les mères sont systématiquement poursuivies pour « non-représentation d'enfant » alors que l'enquête pénale est toujours en cours.

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3. La Situation des « Mères Empêchées » de Protéger

Le document rejette le terme de « mères protectrices », lui préférant celui de « mères empêchées » par une justice destructrice.

| Type de mesure | Conséquences pour la mère | | --- | --- | | Pénale | Poursuites pour non-représentation d'enfant, gardes à vue, incarcérations. | | Civile (JAF) | Astreintes financières massives (ex: 250 € à 600 € par jour de non-représentation). | | Éducationnelle | Accusations de « manipulation », de « syndrome d'aliénation parentale » (même si non reconnu) ou de « vengeance ». | | Droits parentaux | Retrait de l'autorité parentale, visites médiatisées (1h/mois sous surveillance) ou transfert de résidence chez l'agresseur présumé. |

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4. Les Stratégies des Agresseurs et la « Civilisation Parallèle »

Le témoignage décrit l'inceste non comme un acte isolé, mais comme une pratique ancrée dans une « civilisation parallèle » utilisant des mécanismes sophistiqués pour garantir l'impunité.

• Stratégies de silenciation :

  • Culpabilisation : Utilisation des réactions physiologiques de l'enfant (orgasme) pour induire une complicité.

• Initiation : Forcer l'enfant à agresser un tiers pour l'empêcher de dénoncer.

• Manipulation institutionnelle : Les agresseurs devancent souvent la mère en saisissant le Juge aux Affaires Familiales (JAF) pour instaurer un climat conflictuel avant que l'enfant ne parle.

• Profil des agresseurs : Des individus souvent insérés socialement (médecins, banquiers, magistrats) qui « hackent » l'enfance par une volonté de domination.

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5. Critiques des Dispositifs Existants

• Le FIGS (Fichier judiciaire des auteurs d'infractions sexuelles) : Qualifié de « grosse blague », car l'inscription nécessite généralement une condamnation à 5 ans de prison ferme, ce qui exclut la majorité des agresseurs bénéficiant de sursis ou de requalifications délictuelles (correctionnalisation).

• Certificat d'honorabilité : Inefficace tant qu'il repose sur un FIGS incomplet et tant que l'accès au fichier est interdit aux parents (contrairement aux États-Unis).

• Lois nouvelles : Le témoignage affirme qu'aucune nouvelle loi n'est nécessaire.

L'interdiction de violer un enfant est claire ; c'est le manque de volonté politique et judiciaire de l'appliquer qui fait défaut.

--------------------------------------------------------------------------------

6. Propositions de Mesures d'Urgence et Recommandations

Face à l'urgence, plusieurs axes d'action immédiate sont proposés :

• Utilisation des Ordonnances de Placement Provisoire (OPP) : Les procureurs devraient utiliser systématiquement leur pouvoir pour placer les enfants en sécurité dans la famille protectrice dès le signalement (mesure de 8 jours renouvelable).

• Recensement National : Création d'une cellule de crise pour répertorier et examiner les dossiers de toutes les familles ayant déposé plainte pour inceste et se sentant trahies par la justice.

• Amnistie/Immunité pour l'état de nécessité : Accorder l'immunité aux mères en exil (« en cavale ») ayant fui pour protéger leurs enfants.

• Priorité au JAF sur le Juge des Enfants (JE) : Centraliser les décisions de garde chez le JAF pour éviter les placements en foyer (ASE) souvent perçus comme des lieux de sur-victimisation.

• Ordonnance de « Levée du Doute » : En cas de classement pour « faits insuffisamment caractérisés », imposer un suivi psychiatrique obligatoire à l'agresseur présumé pour évaluation sur le long terme.

• Transparence des dossiers : Garantir aux parents un accès total et immédiat aux pièces de leur dossier pénal et aux rapports de l'ASE pour assurer le respect du contradictoire.

Citations Clés

« Le père violeur a un permis de violer son enfant par ordonnance de justice. »

« Ce n'est pas une injustice, ce sont des crimes, ce sont des viols par ordonnance de justice. »

« Le désespoir est l'ennemi de la justice. »

« On ne naît pas violeur d'enfants, on le devient, bien souvent en ayant été soi-même violé. C'est une impunité à vie qu'ils recherchent. »

I read this article about how numbers may seem clear and objective, even though they do not always reflect the real things they are supposed to measure. What stood out to me most was the part about sentiment ratings, IQ, and aggression scales. The article shows that even something as simple as 2+2=4 can become more complicated when the context changes. This made me think about social media too, because numbers like likes, views, and ratings often seem trustworthy even though they only show part of the picture.

This reminds of of 1984 by George Orwell, where the government brainwashed everyone into believing that 2+2=5 and that is the objective truth. But this article makes me think that 2+2=4 may be the false objective truth that we were all brain washed to believe and that we aren't opening our minds to the subjective truth of reality.

After re-reading the Introduction, I'm finding it fragmented and hard to follow. You start with definition, then quickly jump to multiple algorithms, introducing representations (grids, NavMesh, probabilistic maps) mid-flow. “Issues with pathfinding” comes late, even though it should frame the need for PathMaker. And details about PathMaker come after a long technical buildup. I suggest you organize this chapter as follows:

1 Introduction

1.1 Pathfinding: Context and Challenges

1.1.1 Common Pathfinding Approaches

1.1.2 Challenges in Pathfinding

1.2 PathMaker Overview and Contributions

1.3 Design Choices (Brief Overview)

1.4 Limitations of Existing Tools

1.5 Contributions Summary

1.6 Ethical Implications

**What is Pathfinding? ** Keep: Define pathfinding Enhance: Give 2–3 application examples (GPS, games, robotics) Briefly introduce grid-based focus (move this up earlier)

Move out / reduce: Detailed algorithm explanations (A*, Dijkstra) - shorten or defer emphasis Long discussion of representations - keep minimal here

A*, Dijkstra, Weighted Grids - combine & condense to provide minimal technical grounding

Keep very concise summaries (2–4 sentences each) Emphasize differences (optimality, cost handling, use cases) Avoid deep mechanics (no step-by-step descriptions)

Issues with Pathfinding - Move earlier

This is the main motivation Briefly connect to representations (grids vs NavMesh vs probabilistic) Keep probabilistic maps/NavMesh as examples, not deep dives

Project Overview - Move earlier

Refocus this section to explicitly answer:

What is PathMaker? What problem does it solve? Why is it different from existing tools?

Tighten: Avoid repeating motivation language Clearly list capabilities: map creation algorithm execution benchmarking metrics

Implementation Details - Keep (But de-emphasize in intro)

Keep short explanations of Rust + SDL2 Frame as: “lightweight, cross-platform, low-overhead” Avoid deep technical detail (belongs in methods)

Current State of the Art - need to connect to your project to show gaps Structure it as: Visualizers - lack benchmarking Game engines - too complex APIs - too low-level Benchmark libraries - lack usability/integration

End this section with a clear gap statement

Motivation - Connect gap -> need for your tool

Reduce repetition Focus on: - difficulty of evaluating algorithms in practice - need for controlled experimentation

Goals of the Project Convert into a clean list of features: Custom map creation Algorithm implementation support Automated benchmarking Visualization + analysis

Avoid repeating earlier explanations ** Ethical Implications**

Reviewer #1 (Public review):

Summary:

Using electron microscopy, the authors report discontinuities in the plasma membrane of C. elegans embryos. They associate these discontinuities with cell division and speculate that membrane rupture and subsequent resealing contribute to cytokinesis. They further discuss the proximity of these sites to vesicles and propose a role for vesicle-mediated membrane extension.

Weaknesses:

(1) The possibility that the membrane discontinuity is an artifact

Although the authors focus on discontinuities in the plasma membrane, similar discontinuities are also observed in mitochondria, the nuclear envelope, and yolk granules. This raises concerns about whether the electron micrographs presented are suitable for assessing membrane continuity.

Electron micrographs result from a lengthy sample preparation process, including high-pressure freezing, freeze substitution in acetone containing OsO4, gradual warming, uranyl acetate staining, resin embedding, and ultrathin sectioning. In general, lipids are soluble in acetone at temperatures above −30 {degree sign}C, and preservation of membrane structures relies heavily on efficient OsO4 fixation. Insufficient OsO4 treatment would be expected to reduce membrane contrast.

C. elegans embryos are encapsulated by an eggshell that forms at fertilization and gradually develops during the first few cell divisions. It is unclear how efficiently OsO4 in acetone penetrates the eggshell during freeze substitution, raising further concern about plasma membrane preservation under the conditions used.

(2) Lack of evidence linking membrane discontinuity to cell division

The reported plasma membrane discontinuities are not specific to mitotic cells. If this were a physiological process playing an important role in cytokinesis, it should occur in a temporally and spatially coordinated manner with nuclear division. However, it remains unclear at what stage of the cell cycle the membrane rupture occurs and where it is located relative to chromosomes and the mitotic spindle.

(3) Lack of evidence for extension of the separated membrane

Although the authors speculate that resealing of the ruptured membrane occurs via extension of the separated membrane, no direct evidence supporting this mechanism is presented. Proximity to vesicles alone does not demonstrate that membrane extension occurs through vesicle fusion. More direct evidence is required to support this claim.

(4) Inconsistency with published work

Numerous studies have examined cell division in developing C. elegans embryos using the GFP::PH(PLC1δ1) marker expressed from the ltIs38 transgene [pAA1; pie-1::GFP::PH(PLC1δ1) + unc-119(+)], generated by the Oegema lab (https://wormbase.org/species/c_elegans/transgene/WBTransgene00000911#01--10 ). To date, no study has reported membrane ruptures of the magnitude described here. The complexity of cell surface morphology from the 8- to 12-cell stages onward has been well documented, for example, by Fu et al. (2016) using light-sheet microscopy and 3D reconstruction (doi:10.1038/ncomms11088).

Supplementary Movies 5, 6, and 10 of this paper illustrate how single-plane images can easily produce apparent membrane discontinuities, for example, due to membrane orientations nearly parallel to the imaging plane.

The three single-plane images from only three embryos presented in Figure 6 are insufficient to support the authors' strong conclusions. Raw 3D data should be provided.

Reviewer #2 (Public review):

Summary:

Liang et al. explore an unusual observation of membrane discontinuities in dividing C. elegans embryonic cells. This report is the first to demonstrate that, instead of the classical invagination of membranes during cytokinesis, cells in the early embryos of C. elegans exhibit separation of sister membranes that extend independently. TEM images of high-pressure-frozen samples provide strong evidence for the presence of Membrane Openings (MOs) in cells at various stages of the cell cycle, predominantly during mitosis. High-resolution images (x 30,000) clearly show the wrinkled plasma membrane and smooth MOs.<br /> The electron microscopy data are supported by the live cell imaging of strains with fluorescently tagged membrane markers. This study opens up the possibility of tracking MOs at other stages of C. elegans development, and also asks if it might be a common phenomenon in other species that exhibit rapid embryonic growth and divisions.

Strengths:

(1) Thorough verification of Membrane Openings (MO) by several methods:

(a) 4 independent sample batches.

(b) Examined historical collections.

(c) Analysed embryos at different stages of development. The absence of MOs in later stages (comma) serves as a negative control and gives confidence that MOs are genuine and not technical artifacts.

(2) Live cell imaging of strain with fluorescently labelled membranes provides real-time dynamics of membrane rupture.

(3) After observing the membrane rupture, the next obvious question is - what prevents the cytosol from leaking out? The EM images showing PBL and PEL - extracellular matrix serving as barriers for the cytosol are convincing.

Weakness:

(1) The association of membrane discontinuities with cell division is not convincing, as there are 159 cells out of 425 showing MOs, but it is not mentioned clearly how many of these are undergoing cell division. Also, it's not clear whether the 20 dividing cells analysed for MOs are a part of the 159 cells or a separate dataset. A graphical representation of the number of samples and observed frequencies would be helpful to understand the data collection workflow.

(2) In Figures 3A and 3B, the resolution of the images is not enough to verify 3A as classical membrane invagination and 3B as detached sister membranes.

(3) Figure 6 lacks controls. How does the classical invagination look in this strain? Also, adding nuclear dye would be informative, in order to correlate the nuclear division with membrane rupture, as claimed.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

Using electron microscopy, the authors report discontinuities in the plasma membrane of C. elegans embryos. They associate these discontinuities with cell division and speculate that membrane rupture and subsequent resealing contribute to cytokinesis. They further discuss the proximity of these sites to vesicles and propose a role for vesicle-mediated membrane extension. 

Weaknesses:

(1) The possibility that the membrane discontinuity is an artifact

Although the authors focus on discontinuities in the plasma membrane, similar discontinuities are also observed in mitochondria, the nuclear envelope, and yolk granules. This raises concerns about whether the electron micrographs presented are suitable for assessing membrane continuity.

Electron micrographs result from a lengthy sample preparation process, including high-pressure freezing, freeze substitution in acetone containing OsO4, gradual warming, uranyl acetate staining, resin embedding, and ultrathin sectioning. In general, lipids are soluble in acetone at temperatures above −30 {degree sign}C, and preservation of membrane structures relies heavily on efficient OsO4 fixation.

Insufficient OsO4 treatment would be expected to reduce membrane contrast.

C. elegans embryos are encapsulated by an eggshell that forms at fertilization and gradually develops during the first few cell divisions. It is unclear how efficiently OsO4 in acetone penetrates the eggshell during freeze substitution, raising further concern about plasma membrane preservation under the conditions used.

We thank the reviewer for raising this important technical concern. We have taken this question seriously since first observing membrane discontinuities six years ago, and we have since conducted extensive controls to rule out fixation artifacts. Below, we present multiple lines of evidence—ranging from technical reproducibility to orthogonal imaging approaches—that collectively demonstrate the biological reality of these structures.

(1) Technical expertise and standard protocols

Our laboratory has extensive experience with electron microscopy across diverse biological systems, including neurons, muscle cells, and hypodermis in C. elegans, as well as tissues from Drosophila, mouse, bacteria, and cultured cells (Chen et al., 2013; Ding et al., 2018; Guan et al., 2022; Y. Li et al., 2018; Miao et al., 2024; Qin et al., 2014; Wang et al., 2026; J. Xu et al., 2022; M. Xu et al., 2021; L. Yang et al., 2020; X. Yang et al., 2019; Zhu et al., 2022). Importantly, we did not introduce any novel or unconventional steps in our EM preparation; all protocols were standard and well-established. Thus, the observed membrane discontinuities are unlikely to stem from technical inexperience or idiosyncratic methods.

In addition to membrane discontinuities, we would like to emphasize that a large number of single plasma membranes separating adjacent cytoplasmic domains were also detected under EM (Figure 1, 3 and 4, for instance). This observation is particularly significant because the invagination model cannot generate single plasma membrane barriers between adjacent cytoplasmic domains. Instead, independent extension of detached sister membranes could explain the formation of cytoplasm-enclosed membranes. Furthermore, as the morphology and continuity of these single cytoplasm-immersed membrane structures are well preserved, this indicates successful EM processing and argues against inefficient fixation or other technical issues.

(2) Reproducibility across independent preparations and techniques

To test whether the discontinuities were preparation-specific, we examined four independent sample batches collected in the lab over the years. Membrane discontinuities, as well as cytoplasm-immersed membranes, on embryonic cells were consistently observed across all batches, indicating that the phenomenon is not dependent on a single preparation method. Furthermore, we validated our findings using two EM techniques: transmission electron microscopy (HPF-TEM) and dualbeam scanning electron microscopy (SEM). Membrane discontinuities were clearly identifiable with both techniques, further supporting their robustness.

(3) Validation using an independent public dataset

We examined the publicly available C. elegans embryo EM collection (WormAtlas). In several instances, particularly at the embryonic periphery where plasma membrane discontinuities are more readily visualized (https://www.wormimage.org/image.php?id=140265&page=1), we identified similar structures. The presence of these features in an independent dataset generated by different researchers confirms that they are not artifacts unique to our sample preparation.

(4) Developmental regulation of membrane discontinuities

We analyzed embryos across multiple developmental stages. Membrane discontinuities were observed in both intrauterine and laid embryos at early stages. However, as embryos reached the comma stage—a period marked by the onset of elongation and reduced cell proliferation—the incidence of discontinuities dropped dramatically (0/13, 0/17, and 0/30 cells examined). This developmental specificity argues strongly against a general fixation artifact, which would be expected to occur randomly across stages. Additionally, the eggshell is present throughout the embryonic stage of C. elegans; therefore, the dramatic reduction of membrane discontinuities in comma-stage of embryo argues against the possibility that the eggshell poses a fixation problem.

(5) Rigorous criteria for identifying membrane discontinuities

To ensure unbiased analysis, we systematically collected images from early embryonic cells using the following criteria:

(1) Random section selection: For each sample, we randomly selected one section containing the largest number of embryos or cells (Sup Figure 2) for initial analysis. We found membrane discontinuities in 159 cells distributed across 57 embryos, representing 95% of the total sampled embryos This portion of the data is summarized in Figure 1.

(2) Whole-membrane examination: Each putative membrane discontinuity was identified only after examining the entire plasma membrane of the cell on a given section. Importantly, aside from the discontinuity, the remainder of the plasma membrane remained intact. Moreover, in most cells, only a single discontinuity was present per section, arguing against random, widespread membrane tearing during preparation.

(3) Neighboring section verification: Because EM preparation yields serial sections, we verified nearly all membrane discontinuities by examining adjacent sections. Again, the same membrane discontinuity was confirmed only after inspecting the entire plasma membrane on those neighboring sections as well. We will include this verification protocol in the revised Methods and additional imaging of consecutive sections would be provided if needed.

(4) Serial section reconstruction: To further determine whether a dividing cell indeed contains one membrane rupture, we performed two serial reconstruction experiments.

First, we used HPF-TEM to analyze 105 consecutive sections of a metaphase cell, reconstructing the entire plasma membrane and chromosome configuration. We found that one membrane rupture largely encircled the chromosomal disc (Figure 2 and Video S1), spatially aligning with the future segregation zone. Second, we used AutoCUTS-SEM to collect approximately 600 sections covering ~95% of a telophase cell containing three nuclei sharing a common cytoplasm. This tri-nucleated cell was enclosed by three distinct plasma membranes, each harboring a single rupture site. These three ruptures converged to form a Y-shaped exposed cytoplasmic region spanning >351 sections (Figure 5). Collectively, these reconstructions demonstrate that each cell contains only one discontinuity from a 3D point of view, further supporting that the phenomenon is not due to random sample preparation damage.

(6) Orthogonal validation by live imaging: In addition to EM, we performed live imaging of plasma membrane dynamics. While live imaging provides important temporal context, we recognize its limitations in resolving membrane ultrastructure. The rapid kinetics of membrane extension (approximately 20–30 seconds for metaphase and less than 3 minutes for cytokinesis), combined with embryo motility, introduces spatiotemporal ambiguities. To capture dynamic membrane events, our live imaging using the GFP::PH membrane marker was performed at 4-second intervals, approaching the practical limit for single-section scanning of the embryo. With single-plane live imaging, nevertheless, both membrane ruptures and free-ended sister membrane structures could be detected (Figures 6), providing additional evidence that membrane rupture and independent extension of detached sister membranes underlie cytokinesis in C. elegans embryos. Notably, 3D membrane dynamics analysis using light-sheet microscopy (Fu et al. Imaging multicellular specimens with real-time optimized tiling light-sheet selective plane illumination microscopy. Nature Communications. 2016. DOI:10.1038/ncomms11088) revealed membrane ruptures in dividing early C. elegans embryonic cells, including during telophase or metaphase. Therefore, live imaging further validates the membrane rupture phenomena in dividing embryonic cells in C. elegans. 

While future advances in imaging technology may enable real-time visualization at near-EM resolution, our extensive, multi-year effort to test the artifact hypothesis has convinced us that these membrane discontinuities are genuine biological features of dividing C. elegans embryonic cells.

We are confident that the cumulative evidence presented here addresses the reviewer's concerns and demonstrates that the observed membrane discontinuities, as well as cytoplasm-immersed membranes, are not procedural artifacts but rather reflect a previously underappreciated aspect of plasma membrane dynamics during embryonic cell division.

(2) Lack of evidence linking membrane discontinuity to cell division 

The reported plasma membrane discontinuities are not specific to mitotic cells. If this were a physiological process playing an important role in cytokinesis, it should occur in a temporally and spatially coordinated manner with nuclear division. However, it remains unclear at what stage of the cell cycle the membrane rupture occurs and where it is located relative to chromosomes and the mitotic spindle.

Thank you for this insightful comment. We agree that establishing a direct link between plasma membrane discontinuities and mitotic progression is critical, and we appreciate the opportunity to clarify this point.

In C. elegans embryos, the early stages of development are characterized by rapid and extensive cell division. Within approximately 100 minutes, a two-cell embryo develops into an embryo containing nearly 30 cells. The majority of the electron microscopy analyses in our study were performed on embryos at stages with fewer than 30 cells, where most cells are actively dividing. Thus, it is reasonable to infer that the cells exhibiting membrane discontinuities are predominantly mitotic cells.

Supporting this notion, as embryos reached the comma stage—a period marked by the onset of elongation and reduced cell proliferation—the incidence of membrane discontinuities dropped dramatically (0/13, 0/17, and 0/30 cells examined). This developmental specificity strongly suggests that membrane discontinuities are tightly linked to cell division.

Importantly, mitotic features such as metaphase chromosomes aligned at the equatorial plane or two (or more) nuclei sharing common cytoplasm can be identified in EM images. In our single random EM section analysis, we captured membrane discontinuities in cells at metaphase, anaphase (characterized by fewer than 10 chromosomal clumps), and telophase (defined by two nuclei sharing cytoplasm). Hence, membrane discontinuities are indeed present on mitotic cells. In addition, a published work by Fu et al (Fu et al. Imaging multicellular specimens with real-time optimized tiling light-sheet selective plane illumination microscopy. Nature Communications. 2016. DOI:10.1038/ncomms11088) using light-sheet microscopy captured similar membrane discontinuities in cells displaying classical mitotic features, including anaphase or telophase.

To further investigate the spatial relationship between membrane ruptures and chromosome organization, we performed three-dimensional reconstructions on a metaphase cell. As shown in Figure 2 and Video S1, the membrane discontinuities largely encircled the condensed chromosome disc and were spatially aligned with the future segregation zone, further revealing the relative location of membrane discontinuities to chromosomes, at least at metaphase.

We further collected 3D information for a telophase cell containing three nuclei. This tri-nucleated cell was enclosed by three distinct plasma membranes, each harboring a single rupture site that merged to form a single rupture. The observation that membrane ruptures are present in a tri-nucleated cell is particularly informative. The tri-nucleated feature indicates that this cell underwent two rounds of cell division and that both divisions were at telophase. The presence of a single membrane rupture suggests that membrane discontinuities may persist throughout the cell cycle, as the second cell cycle began from a mother cell that still shared cytoplasm with its sister cell and already had one membrane rupture. Therefore, in addition to the mitotic phase, membrane discontinuities—at least in this context—also exist during the DNA synthesis stage.

(3) Lack of evidence for extension of the separated membrane 

Although the authors speculate that resealing of the ruptured membrane occurs via extension of the separated membrane, no direct evidence supporting this mechanism is presented. Proximity to vesicles alone does not demonstrate that membrane extension occurs through vesicle fusion. More direct evidence is required to support this claim.

Thank you for raising this important point. We appreciate the opportunity to clarify our conclusion.

In our study, EM analysis revealed the presence of cellular vesicles in close proximity to both free membrane edges and the already separated sister plasma membranes (Figure 4). However, we acknowledge that without advanced live-cell imaging, it is not possible to conclusively determine whether the extension of these separated sister membranes occurs through vesicle fusion.

We realize that a statement in the Discussion section—“The expansion of the plasma membrane is generally driven by vesicle fusion”(page 16)—may have inadvertently led the reviewer to interpret this as our own conclusion regarding the mechanism of membrane extension in this context. In fact, that statement was intended to reflect the current general understanding of membrane expansion, not to imply that we had demonstrated such a mechanism for the free-ended sister membranes. As we subsequently noted, “However, this remains speculative and requires further experimental validation.”

To avoid any misunderstanding, we will revise this section to clearly state that the mechanism by which the separated sister membranes extend remains unknown and that further investigation is needed to determine how existing models of membrane expansion may apply to or be adapted for this novel context.

We thank the reviewer again for their thoughtful comment, which has helped us improve the clarity of our manuscript

(4) Inconsistency with published work

Numerous studies have examined cell division in developing C. elegans embryos using the GFP::PH(PLC1δ1) marker expressed from the ltIs38 transgene [pAA1; pie-1::GFP::PH(PLC1δ1) + unc-119(+)], generated by the Oegema lab (https://wormbase.org/species/c_elegans/transgene/WBTransgene00000911#01--10 ). To date, no study has reported membrane ruptures of the magnitude described here. The complexity of cell surface morphology from the 8- to 12-cell stages onward has been well documented, for example, by Fu et al. (2016) using light-sheet microscopy and 3D reconstruction (doi:10.1038/ncomms11088).

Supplementary Movies 5, 6, and 10 of this paper illustrate how single-plane images can easily produce apparent membrane discontinuities, for example, due to membrane orientations nearly parallel to the imaging plane.

The three single-plane images from only three embryos presented in Figure 6 are insufficient to support the authors' strong conclusions. Raw 3D data should be provided.

Thank you for this important comment. We fully agree that the GFP::PH(PLC1δ1) marker, generated by the Oegema lab, has been widely and effectively used to study various aspects of C. elegans embryonic development. In fact, we also employed this same marker in our study to assess membrane integrity.

However, while live imaging provides invaluable temporal resolution, its limitations in resolving membrane ultrastructure are substantial. In C. elegans embryos, early development is marked by rapid and extensive cell divisions. Within approximately 100 minutes, a two-cell embryo develops into one containing nearly 30 cells. During this fast-dividing stage, the rapid kinetics of membrane extension—approximately 20–30 seconds during metaphase and less than 3 minutes during cytokinesis— combined with embryo motility, introduce considerable spatiotemporal ambiguities. Furthermore, the longstanding invagination model of cytokinesis has shaped interpretations in the field, which may have led to ambiguous structures such as free-ended extensions being dismissed as potential artifacts rather than recognized as alternative morphological features. Theoretical and computational models have largely been built upon invagination-centric assumptions, which may have further constrained conceptual frameworks. Therefore, fluorescence protein-based live imaging analysis alone could not serve as a convincing approach to challenge the current dogma of cell division, nor did we intend it to.

However, when reexamined in light of our findings, previous studies using this same GFP marker have in fact revealed membrane discontinuities that went unnoticed. For example, Fu et al (Fu et al. Imaging multicellular specimens with real-time optimized tiling light-sheet selective plane illumination microscopy. Nature Communications. 2016. DOI:10.1038/ncomms11088) using light-sheet microscopy and 3D reconstruction, captured membrane discontinuities in cells undergoing mitotic phases such as anaphase or telophase. Similarly, an earlier study by Harrell and Goldstein (Harrell and Goldstein. 2011. Internalization of multiple cells during C. elegans gastrulation depends on common cytoskeletal mechanisms but different cell polarity and cell fate regulators. Developmental Biology. DOI:10.1016/j.ydbio.2010.09.012) showed regions where the GFP::PH signal appeared fuzzy and discontinuous.

Nevertheless, given the inherent limitations of fluorescence microscopy in resolving membrane ultrastructure, high-resolution electron microscopy—supported by rigorous controls and serial section analysis—remains the gold standard for definitively identifying such membrane discontinuities.

We acknowledge that our findings are surprising. We did not set out to challenge the long-held view of membrane integrity during cell division. In fact, this study began when our dedicated EM technician, Jingjing Liang, first observed membrane discontinuity phenomena in control samples—wild-type embryos. Had she not come across this observation, we likely would never have pursued this line of inquiry.

We appreciate the opportunity to clarify these points and thank the reviewer for thoughtful engagement with our work.

Reviewer #2 (Public review):

Summary:

Liang et al. explore an unusual observation of membrane discontinuities in dividing C. elegans embryonic cells. This report is the first to demonstrate that, instead of the classical invagination of membranes during cytokinesis, cells in the early embryos of C. elegans exhibit separation of sister membranes that extend independently. TEM images of high-pressure-frozen samples provide strong evidence for the presence of Membrane Openings (MOs) in cells at various stages of the cell cycle, predominantly during mitosis. High-resolution images (x 30,000) clearly show the wrinkled plasma membrane and smooth MOs.

The electron microscopy data are supported by the live cell imaging of strains with fluorescently tagged membrane markers. This study opens up the possibility of tracking MOs at other stages of C. elegans development, and also asks if it might be a common phenomenon in other species that exhibit rapid embryonic growth and divisions. 

Strengths:

(1) Thorough verification of Membrane Openings (MO) by several methods: 

(a) 4 independent sample batches.

(b) Examined historical collections.

(c) Analysed embryos at different stages of development. The absence of MOs in later stages (comma) serves as a negative control and gives confidence that MOs are genuine and not technical artifacts. 

(2) Live cell imaging of strain with fluorescently labelled membranes provides realtime dynamics of membrane rupture.

(3) After observing the membrane rupture, the next obvious question is - what prevents the cytosol from leaking out? The EM images showing PBL and PEL - extracellular matrix serving as barriers for the cytosol are convincing.

Thanks to the reviewer for the encouragement. Highly appreciated.

Weakness:

(1) The association of membrane discontinuities with cell division is not convincing, as there are 159 cells out of 425 showing MOs, but it is not mentioned clearly how many of these are undergoing cell division. Also, it's not clear whether the 20 dividing cells analysed for MOs are a part of the 159 cells or a separate dataset. A graphical representation of the number of samples and observed frequencies would be helpful to understand the data collection workflow.

We sincerely thank the reviewer for raising this important question and appreciate the opportunity to clarify these points.

(1) Relationship between membrane discontinuities and cell division

In C. elegans embryos, early development is characterized by rapid and extensive cell division: within approximately 100 minutes, a two-cell embryo develops into one containing nearly 30 cells. Most of our electron microscopy (EM) analyses were performed on embryos at stages with fewer than 30 cells, in which the majority of cells are actively dividing. Therefore, it is reasonable to infer that the cells exhibiting membrane discontinuities (MOs) are predominantly mitotic. Supporting this, as embryos reached the comma stage—when cell proliferation declines and elongation begins—the incidence of MOs dropped sharply (0/13, 0/17, and 0/30 cells examined. This developmental specificity strongly links MOs to cell division.

Moreover, in single random EM sections, we observed MOs in cells displaying clear mitotic features, such as metaphase chromosomes aligned at the equatorial plate, or anaphase/telophase configurations (fewer than 10 chromosomal clumps or two nuclei sharing common cytoplasm). Thus, MOs are indeed present in mitotic cells.

From our 3D reconstruction (Figure 5), we identified a telophase cell containing three nuclei, each enclosed by its own plasma membrane, with each membrane harboring a single rupture that converged into a single opening. This tri-nucleated configuration indicates that the cell had undergone two rounds of division and was at telophase in both. The presence of a single membrane rupture in this context suggests that MOs can persist beyond mitosis, as the second cell cycle initiated from a mother cell that already shared cytoplasm with its sister and already contained a rupture. Thus, in this case, MOs were also present during DNA synthesis stage.

(2) Clarification of sample numbers and datasets

In Figure 1, we present results from a single EM section per embryonic cell, with sections randomly selected per embryo as detailed in Sup Figure 2. This initial dataset (425 cells) forms the basis of Figure 1.

From the same pool of 425 cells, we used additional EM sections—distinct from those shown in Sup Figure 2—to locate 20 dividing cells for analysis of membrane discontinuities. Thus, while these 20 cells originated from the same set of embryos, they were not derived from the sections used in Figure 1 or Sup Figure 2.

A graphical summary of sample numbers from the single-section analysis is already provided in Figure 1. Notably, cells with two clearly visible nuclei are more likely to be sectioned through or near their maximal diameter. In contrast, the randomly selected sections used for Figure 1 captured cells at variable planes, reducing the likelihood of observing MOs. Consistent with this, in the three embryos where no MOs were detected (one example is Sup Figure 2N), the sections likely passed through peripheral regions of the cells. Consequently, the frequency of MOs in randomly sectioned cells (Figure 1) is not directly comparable to that observed in the 20 dividing cells, which were analyzed using sections more likely to capture cells near their maximal diameter. These 20 dividing cells should therefore be considered a separate analysis. We will add detailed explanations in the Methods section to ensure this distinction is clearly understood.

We are grateful for the reviewer’s thoughtful feedback and believe these clarifications will improve the clarity and rigor of the manuscript.

(2) In Figures 3A and 3B, the resolution of the images is not enough to verify 3A as classical membrane invagination and 3B as detached sister membranes.

Thank you for your valuable comment. In the revised manuscript, we will provide additional images at higher magnification to better illustrate the classical membrane invagination in Figure 3A and the detached sister membranes in Figure 3B.

(3) Figure 6 lacks controls. How does the classical invagination look in this strain? Also, adding nuclear dye would be informative, in order to correlate the nuclear division with membrane rupture, as claimed. 

Thank you for this important comment. As we addressed how we correlated nuclear division with membrane rupture in response to weakness (1), below we will focus on how we may distinguish classical invagination from membrane rupture.

While live imaging provides invaluable temporal resolution, its limitations in resolving membrane ultrastructure are substantial. In C. elegans embryos, early development is marked by rapid and extensive cell divisions. Within approximately 100 minutes, a two-cell embryo develops into one containing nearly 30 cells. During this fast-dividing stage, the rapid kinetics of membrane extension—approximately 20–30 seconds during metaphase and less than 3 minutes during cytokinesis— combined with embryo motility, introduce considerable spatiotemporal ambiguities. Furthermore, the longstanding invagination model of cytokinesis has shaped interpretations in the field, which may have led to ambiguous structures such as free-ended extensions being dismissed as potential artifacts rather than recognized as alternative morphological features. Theoretical and computational models have largely been built upon invagination-centric assumptions, which may have further constrained conceptual frameworks. Therefore, fluorescence protein-based live imaging analysis alone could not serve as a convincing approach to challenge the current dogma of cell division, nor did we intend it to.

However, when reexamined in light of our findings, previous studies using GFP::PH or similar markers have in fact revealed membrane discontinuities that went unnoticed. For example, using light-sheet microscopy and 3D reconstruction, Fu et al captured membrane discontinuities in cells undergoing division such as anaphase or telophase (Fu et al. Imaging multicellular specimens with real-time optimized tiling light-sheet selective plane illumination microscopy. Nature Communications. 2016.DOI:10.1038/ncomms11088)

Similarly, an earlier study by Goldstein et al. (Harrell and Goldstein. 2011. Internalization of multiple cells during C. elegans gastrulation depends on common cytoskeletal mechanisms but different cell polarity and cell fate regulators. Developmental Biology. DOI:10.1016/j.ydbio.2010.09.012) showed regions where the GFP::PH signal appeared fuzzy and discontinuous.

Here, to capture dynamic membrane events, our live imaging using the GFP::PH membrane marker was performed at 4-second intervals, approaching the practical limit for single-section scanning of the embryo. With single-plane live imaging, both membrane ruptures and free-ended sister membrane structures (Figures 6) could be detected, providing additional evidence that membrane rupture and independent extension of detached sister membranes underlie cytokinesis in C. elegans embryos.

However, given the inherent limitations of fluorescence microscopy in resolving membrane ultrastructure, high-resolution electron microscopy—supported by rigorous controls and serial section analysis—remains the gold standard for definitively distinguishing invagination from membrane discontinuities.

While future advances in imaging technology may enable real-time visualization at near-EM resolution, our extensive, multi-year effort to test the artifact hypothesis has convinced us that these membrane discontinuities are genuine biological features of dividing C. elegans embryonic cells.

Reviewer #3 (Public review):

Summary:

In this manuscript, the authors challenge a dogma in cell biology, namely that cells are at any time point engulfed by a continuous plasma membrane. Liang et al. find that during C elegans embryogenesis, a high number of cells are not entirely surrounded by a plasma membrane but show membrane openings (MOs). These openings are enriched at the embryo's periphery, towards the eggshell. The authors propose that plasma membrane discontinuities emerge during metaphase of mitosis and that independent extension of "sister membranes" engulfs the daughter cells.

Strengths:

On the positive side, the authors find plasma membrane discontinuities not only by electron microscopy but also by fluorescence microscopy and provide information about the dynamics of membrane openings and their emergence. While this is assuring, the authors conclude that MOs emerge during metaphase. From what the authors show, this particular information cannot be deduced, as there is no dynamic capture of a membrane scission event together with a chromatin marker that would indicate mitosis. The authors could, however, attempt to find such events in live movies, given the high incidence of MOs reported from their EM data.

Thanks to the reviewer for the encouragement. Highly appreciated.

Weaknesses:

In order to convincingly demonstrate the absence of any plasma membrane in the respective regions of the embryonic periphery or between cells of the embryo, the authors would have to show consecutive serial TEM sections where MOs are detected over more z-planes, beyond the mere 3D reconstructions. Although the authors state in the methods section that continuous ultrathin sections were cut for the metaphase sample (page 21, line 472), consecutive sections are never shown in TEM. While we do see the 3D reconstructions, better documentation of the underlying TEM data is missing. It would be necessary to show a membrane opening in consecutive z sections. Alternatively, the authors could seek the possibility to convincingly back up their claims with volume imaging by focused ion beam scanning EM (FIBSEM), where cellular volumes can be sectioned in almost isotropic resolution

We Thank the reviewer for raising these important technical concerns. We have taken this question seriously since first observing membrane discontinuities six years ago, and we have since conducted extensive controls to rule out fixation artifacts.

First of all, in addition to membrane discontinuities, we would like to highlight that a large number of single plasma membranes separating adjacent cytoplasmic domains were detected by EM (Figure 1, 3 and 4). This observation is particularly significant because the invagination model cannot account for the formation of single plasma membrane barriers between adjacent cytoplasmic domains. Instead, independent extension of detached sister membranes offers a plausible explanation for the generation of cytoplasm-immersed membranes. Furthermore, the morphology and continuity of these single cytoplasm-immersed membrane structures are well preserved, indicating successful EM processing and arguing against potential issues such as inadequate fixation or other technical limitations.

Second, we applied rigorous criteria for identifying membrane discontinuities:

(1) To test whether the discontinuities were preparation specific, we examined four independent sample batches and validated our findings using two EM techniques: transmission electron microscopy (HPF-TEM) and dual-beam scanning electron microscopy (SEM).

(2) We analyzed embryos across multiple developmental stages. Membrane discontinuities were observed in both intrauterine and laid embryos at early stages. However, as embryos reached the comma stage—a period marked by the onset of elongation and reduced cell proliferation—the incidence of discontinuities dropped dramatically (0/13, 0/17, and 0/30 cells examined). This developmental specificity argues strongly against a general fixation artifact, which would be expected to occur randomly across stages. Additionally, the eggshell is present throughout the embryonic stage of C. elegans; therefore, the dramatic reduction of membrane discontinuities in comma-stage of embryo argues against the possibility that the eggshell poses a fixation problem.

(3) Each putative membrane discontinuity was identified only after examining the entire plasma membrane of the cell on a given section. Importantly, aside from the discontinuity, the remainder of the plasma membrane remained intact. Moreover, in most cells, only a single discontinuity was present per section, arguing against random, widespread membrane tearing during preparation. Because EM preparation yields serial sections, we verified nearly all membrane discontinuities by examining adjacent sections. Again, the same membrane discontinuity was confirmed only after inspecting the entire plasma membrane on those neighboring sections as well. We will include this verification protocol in the revised Methods and additional imaging of consecutive sections would be provided if needed.

To further determine whether a dividing cell indeed contains one membrane rupture, we performed two serial reconstruction experiments using consecutive sections, as the reviewer suggested. First, we used HPF-TEM to analyze 105 consecutive sections of a metaphase cell, reconstructing the entire plasma membrane and chromosome configuration. We found that one membrane rupture largely encircled the chromosomal disc (Figure 2 and Video S1), spatially aligning with the future segregation zone. Second, we used AutoCUTS-SEM to collect approximately 600 sections covering ~95% of a telophase cell containing three nuclei sharing a common cytoplasm. This tri-nucleated cell was enclosed by three distinct plasma membranes, each harboring a single rupture site. These three ruptures converged to form a Yshaped exposed cytoplasmic region spanning >351 sections (Figure 5). Collectively, these reconstructions demonstrate that each cell contains only one discontinuity from a 3D point of view, further supporting that the phenomenon is not due to random sample preparation damage.

(4) In addition to EM, we performed live imaging of plasma membrane dynamics. While live imaging provides important temporal context, we recognize its limitations in resolving membrane ultrastructure. The rapid kinetics of membrane extension (approximately 20–30 seconds for metaphase and less than 3 minutes for cytokinesis), combined with embryo motility, introduces spatiotemporal ambiguities. To capture dynamic membrane events, our live imaging using the GFP::PH membrane marker was performed at 4-second intervals, approaching the practical limit for single-section scanning of the embryo. With single-plane live imaging, nevertheless, both putative membrane ruptures (Figure 6A) and free-ended sister membrane structures could be detected (Figures 6B and 6C), providing additional evidence that membrane rupture and independent extension of detached sister membranes underlie cytokinesis in C. elegans embryos. Notably, 3D membrane dynamics analysis using light-sheet microscopy (Fu et al. Imaging multicellular specimens with real-time optimized tiling light-sheet selective plane illumination microscopy. Nature Communications. 2016. DOI:10.1038/ncomms11088). revealed membrane ruptures in dividing early C. elegans embryonic cells, including during telophase and metaphase. Therefore, live imaging further validates the membrane rupture phenomena in dividing embryonic cells in C. elegans. 

We are confident that the cumulative evidence presented here addresses the reviewer's concerns and demonstrates that the observed membrane discontinuities, as well as cytoplasm-immersed membranes, are not procedural artifacts but rather reflect a previously underappreciated aspect of plasma membrane dynamics during embryonic cell division.

Another critical issue concerns the detection of the membrane discontinuities in electron micrographs, which, in my opinion, is ambiguous. How do the authors reliably discriminate in their TEM images whether there is a plasma membrane or not? The absence - or weak appearance - of the stain of the electron dense material at membranes, which seems to be their criterion for MOs, is also apparent at other, intracellular membranes, like at the NE or at the ER (for example, see Figure 1C). Also, the plasma membrane itself appears unevenly stained in regions that the authors delineate as intact (for example, Figure 1C, 2B/1).

We thank the reviewer for raising this important concern.

First, our laboratory has extensive experience with electron microscopy across diverse biological systems, including neurons, muscle cells, and hypodermis in C. elegans, as well as tissues from Drosophila, mouse, bacteria, and cultured cells (Chen et al., 2013; Ding et al., 2018; Guan et al., 2022; Y. Li et al., 2018; Miao et al., 2024; Qin et al., 2014; Wang et al., 2026; J. Xu et al., 2022; M. Xu et al., 2021; L. Yang et al., 2020; X. Yang et al., 2019; Zhu et al., 2022). Importantly, we did not introduce any novel or unconventional steps in our EM preparation; all protocols were standard and well established. Thus, the observed membrane discontinuities are unlikely to result from technical inexperience or idiosyncratic methods.

Second, because EM preparation yields serial sections, we verified nearly all membrane discontinuities by examining adjacent sections. Specifically, a membrane discontinuity was confirmed only after inspecting the entirety of the plasma membrane in neighboring sections. We will include this verification protocol in the revised Methods section, and additional images of consecutive sections can be provided if needed.

Third, in addition to membrane discontinuities, a large number of single plasma membranes separating adjacent cytoplasmic domains were detected by EM (Figure 1, 3 and 4). This observation is particularly significant because the invagination model cannot account for the formation of single plasma membrane barriers between adjacent cytoplasmic domains. Instead, independent extension of detached sister membranes offers a plausible explanation for the generation of cytoplasm-immersed membranes. Furthermore, the morphology and continuity of these single cytoplasm-immersed membrane structures are well preserved, indicating successful EM processing and arguing against potential issues such as inadequate fixation or other technical limitations.

EM-related publications by Jingjing Liang:

Chen D, Jian Y, Liu X, Zhang Y, Liang J, Qi X, Du H, Zou W, Chen L, Chai Y, Ou G, Miao L, Wang Y, Yang C. 2013. Clathrin and AP2 Are Required for Phagocytic Receptor-Mediated Apoptotic Cell Clearance in Caenorhabditis elegans. PLoS Genetics 9:e1003517. DOI: https://doi.org/10.1371/journal.pgen.1003517

Ding L, Yang X, Tian H, Liang J, Zhang F, Wang G, Wang Y, Ding M, Shui G, Huang X. 2018. Seipin regulates lipid homeostasis by ensuring calcium‐dependent mitochondrial metabolism. The EMBO Journal 37:e97572. DOI: https://doi.org/10.15252/embj.201797572

Guan L, Yang Y, Liang J, Miao Y, Shang A, Wang B, Wang Y, Ding M. 2022. ERGIC2 and ERGIC3 regulate the ER‐to‐Golgi transport of gap junction proteins in metazoans. Traffic 23:140–157. DOI: https://doi.org/10.1111/tra.12830

Li Y, Zhang Y, Gan Q, Xu M, Ding X, Tang G, Liang J, Liu K, Liu X, Wang X, Guo L, Gao Z, Hao X, Yang C. 2018. C . elegans -based screen identifies lysosome-damaging alkaloids that induce STAT3-dependent lysosomal cell death. Protein & Cell 9:1013–1026. DOI: https://doi.org/10.1007/s13238-018-0520-0

Miao Y, Du Y, Wang B, Liang J, Liang Y, Dang S, Liu J, Li D, He K, Ding M. 2024. Spatiotemporal recruitment of the ubiquitin-specific protease USP8 directs endosome maturation. eLife 13:RP96353. DOI: https://doi.org/10.7554/eLife.96353

Qin J, Liang J, Ding M. 2014. Perlecan Antagonizes Collagen IV and ADAMTS9/GON-1 in Restricting the Growth of Presynaptic Boutons. Journal of Neuroscience 34:10311–10324. DOI: https://doi.org/10.1523/JNEUROSCI.5128-13.2014

Wang Z, Zhang L, Zhou B, Liang J, Tian Y, Jiang Z, Tao J, Yin C, Chen S, Zhang W, Zhang J, Wei W. 2026. A single MYB transcription factor GmMYB331 regulates seed oil accumulation and seed size/weight in soybean. Journal of Integrative Plant Biology 68:470– 485. DOI: https://doi.org/10.1111/jipb.70101

Xu J, Chen S, Wang W, Man Lam S, Xu Y, Zhang S, Pan H, Liang J, Huang Xiahe, Wang Yu, Li T, Jiang Y, Wang Yingchun, Ding M, Shui G, Yang H, Huang Xun. 2022. Hepatic CDP-diacylglycerol synthase 2 deficiency causes mitochondrial dysfunction and promotes rapid progression of NASH and fibrosis. Science Bulletin 67:299–314. DOI: https://doi.org/10.1016/j.scib.2021.10.014

Xu M, Ding L, Liang J, Yang X, Liu Y, Wang Y, Ding M, Huang X. 2021. NAD kinase sustains lipogenesis and mitochondrial metabolism through fatty acid synthesis. Cell Reports 37:110157. DOI: https://doi.org/10.1016/j.celrep.2021.110157

Yang L, Liang J, Lam SM, Yavuz A, Shui G, Ding M, Huang X. 2020. Neuronal lipolysis participates in PUFA‐mediated neural function and neurodegeneration. EMBO reports 21:e50214. DOI: https://doi.org/10.15252/embr.202050214

Yang X, Liang J, Ding L, Li X, Lam S-M, Shui G, Ding M, Huang X. 2019. Phosphatidylserine synthase regulates cellular homeostasis through distinct metabolic mechanisms. PLOS Genetics 15:e1008548. DOI: https://doi.org/10.1371/journal.pgen.1008548

Zhu J, Lam SM, Yang L, Liang J, Ding M, Shui G, Huang X. 2022. Reduced phosphatidylcholine synthesis suppresses the embryonic lethality of seipin deficiency. Life Metabolism 1:175–189. DOI: https://doi.org/10.1093/lifemeta/loac02

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

In their paper entitled "Alpha-Band Phase Modulates Perceptual Sensitivity by Changing Internal Noise and Sensory Tuning," Pilipenko et al. investigate how pre-stimulus alpha phase influences near-threshold visual perception. The authors aim to clarify whether alpha phase primarily shifts the criterion, multiplicatively amplifies signals, or changes the effective variance and tuning of sensory evidence. Six observers completed many thousands of trials in a double-pass Gabor-in-noise detection task while an EEG was recorded. The authors combine signal detection theory, phase-resolved analyses, and reverse correlation to test mechanistic predictions. The experimental design and analysis pipeline provide a clear conceptual scaffold, with SDT-based schematic models that make the empirical results accessible even for readers who are not specialists in classification-image methods.

Strengths:

The study presents a coherent and well-executed investigation with several notable strengths. First, the main behavioral and EEG results in Figure 2 demonstrate robust pre-stimulus coupling between alpha phase and d′ across a substantial portion of the pre-stimulus interval, with little evidence that the criterion is modulated to a comparable extent. The inverse phasic relationship between hit and false-alarm rates maps clearly onto the variance-reduction account, and the response-consistency analysis offers an intuitive behavioral complement: when two identical stimuli are both presented at the participant's optimal phase, responses are more consistent than when one or both occur at suboptimal phases. The frontal-occipital phase-difference result suggests a coordinated rather than purely local phase mechanism, supporting the central claim that alpha phase is linked to changes in sensitivity that behave like changes in internal variability rather than simple gain or criterion shifts. Supplementary analyses showing that alpha power has only a limited relationship with d′ and confidence reassure readers that the main effects are genuinely phase-linked rather than a recasting of amplitude differences.

Second, the reverse-correlation results in Figure 3 extend this story in a satisfying way. The classification images and their Gaussian fits show that at the optimal phase, the weighting of stimulus energy is more sharply concentrated around target-relevant spatial frequencies and orientations, and the bootstrapped parameter distributions indicate that the suboptimal phase is best described by broader tuning and a modest change in gain rather than a pure criterion account. The authors' interpretation that optimal-phase perception reflects both reduced effective internal noise and sharpened sensory tuning is reasonable and well-supported. Overall, the data and figures largely achieve the stated aims, and the work is likely to have an impact both by clarifying the interpretation of alpha-phase effects and by illustrating a useful analytic framework that other groups can adopt.

Weaknesses:

The weaknesses are limited and relate primarily to framing and presentation rather than to the substance of the work. First, because contrast was titrated to maintain moderate performance (d′ between 1.2 and 1.8), the phase-linked changes in sensitivity appear modest in absolute terms, which could benefit from explicit contextualization. Second, a coding error resulted in unequal numbers of double-pass stimulus pairs across participants, which affects the interpretability of the response-consistency results. Third, several methodological details could be stated more explicitly to enhance transparency, including stimulus timing specifications, electrode selection criteria, and the purpose of phase alignment in group averaging. Finally, some mechanistic interpretations in the Discussion could be phrased more conservatively to clearly distinguish between measurement and inference, particularly regarding the relationship between reduced internal noise and sharpened tuning, and the physiological implementation of the frontal-occipital phase relationship.

We appreciate the reviewer’s thoughtful and constructive feedback, particularly regarding clarity and framing. In response, we have made several revisions to improve transparency and contextualization throughout the manuscript.

First, we now explicitly contextualize the relatively modest change in sensitivity by adding discussion of the contrast-titration procedure and its implications for effect size interpretation. Second, we address the coding error that led to unequal numbers of double-pass stimulus pairs across participants sooner in the manuscript by reporting the average number of pairs per participant in the Results (as well as the Methods), allowing for readers to interpret the results more appropriately. Third, we have provided additional detail, including precise stimulus timing parameters, electrode selection criteria, and a clearer explanation of the rationale for phase alignment in the Results (in addition to the Methods) section. Finally, we have revised portions of the Discussion to adopt more conservative language when interpreting our results, which more clearly distinguishes between empirical observations and mechanistic inferences, along with offering additional interpretations for the frontal-occipital phase relationship.

We believe these revisions substantially improve the clarity, transparency, and interpretability of the manuscript.

Reviewer #2 (Public review):

Summary:

The study of Pilipenko et al evaluated the role of alpha phase in a visual perception paradigm using the framework of signal detection theory and reverse correlation. Their findings suggest that phase-related modulations in perception are mediated by a reduction in internal noise and a moderate increase in tuning to relevant features of the stimuli in specific phases of the alpha cycle. Interestingly, the alpha phase did not affect the criterion. Criterion was related to modulations in alpha power, in agreement with previous research.

Strengths:

The experiment was carefully designed, and the analytical pipeline is original and suited to answer the research question. The authors frame the research question very well and propose several models that account for the possible mechanisms by which the alpha phase can modulate perception. This study can be very valuable for the ongoing discussion about the role of alpha activity in perception.

Weaknesses:

The sample size collected (N = 6) is, in my opinion, too small for the statistical approach adopted (group level). It is well known that small sample sizes result in an increased likelihood of false positives; even in the case of true positives, effect sizes are inflated (Button et al., 2013; Tamar and Orban de Xivry, 2019), negatively affecting the replicability of the effect.

Although the experimental design allows for an accurate characterization of the effects at the single-subject level, conclusions are drawn from group-level aggregated measures. With only six subjects, the estimation of between-subject variability is not reliable. The authors need to acknowledge that the sample size is too small; therefore, results should be interpreted with caution.

Conclusion:

This study addresses an important and timely question and proposes an original and well-thought-out analytical framework to investigate the role of alpha phase in visual perception. While the experimental design and theoretical motivation are strong, the very limited sample size substantially constrains the strength of the conclusions that can be drawn at the group level.

Bibliography:

Button, K., Ioannidis, J., Mokrysz, C. et al. Power failure: why small sample size undermines the reliability of neuroscience. Nat Rev Neurosci 14, 365-376 (2013). https://doi.org/10.1038/nrn3475

Tamar R Makin, Jean-Jacques Orban de Xivry (2019) Science Forum: Ten common statistical mistakes to watch out for when writing or reviewing a manuscript eLife 8:e48175 https://doi.org/10.7554/eLife.48175

We thank the reviewer for their supportive remarks on our design and analysis, and for raising this important statistical concern about our sample size (n=6). Our choice of a small sample size was driven by methodological considerations. Specifically, our reverse correlation analysis requires a large number of trials per participant, as it estimates perceptual tuning by regressing behavioral responses against fluctuations in the energy of stimulus features (orientation and spatial frequency). This approach, as well as the computation of signal detection theory (SDT) metrics such as d′ and criterion, depends on high trial counts to obtain reliable estimates, particularly given that our analysis further subdivides trials across eight phase bins. For this reason, we prioritized collecting a large number of trials per participant (∼5,000), which is consistent with established practices in psychophysical research.

Importantly, our approach means that our design is reliable on the individual level, which motivated us to include a new binomial probability testing in our revised paper. This binomial test helps address concerns about the generalizability of our results. Binomial testing considers each participant as an independent replication of the effect and then computes the p-value associated with the probability of having observed the given number of statistically significant participants by chance, with a false positive rate of 0.05. In our data, 3 out of 6 participants showed significant effects, which corresponds to a probability of 0.002 of having observed these effects by chance alone. We believe this converging evidence supports the replicability and generalizability of our results. To improve the transparency of the single-subject data, we have included single-participant results in the Supplemental Materials to allow readers to directly assess the consistency of effects across individuals and to better contextualize between-subject variability.

Thank you again for your suggestions, we believe that these additions have greatly improved our manuscript by demonstrating the robustness of our findings and increasing the transparency of our single-subject results.

Recommendations for the authors:

Reviewing Editor Comments:

The issue of generalizability arose during the review process, as your results are based on a small sample of participants who undertook a very large number of trials. In the revised version, it would be useful to discuss why this approach is valid, especially in the context of linking EEG with modeling (i.e., why it is more powerful than having many participants with fewer trials), and the extent to which your results can generalize to the population.

We sincerely appreciate all of the helpful comments provided by the reviewers and hope we can address the concerns of our experimental approach. In the introduction, we have emphasized the importance of our current small sample size design, which allows us to reliably compute our signal detection theory metrics across 8 phase bins in addition to including the reverse correlation analysis. In the methods section, we have added a description of the binomial probability statistical framework, which addresses the generalizability of our results. In this framework, each participant is viewed as an independent replication and the p-value reflects the probability of having observed the number of individually significant subjects from the total sample size by chance. In this regard, observing a significant effect in 3 out of 6 participants (as in our study) from chance alone has a 0.002 probability, which we believe is unlikely and instead reflects a true effect present in the general population.

Below I have copied our changes in the introduction and methods sections.

“... in a large number of trials (6,020 per observer, n = 6) across multiple EEG sessions. This approach ensures a sufficient number of trials in order to reliably compute signal detection theory (SDT) metrics across multiple alpha phase bins while also affording enough statistical power for reverse correlation analysis (Xue et al., 2024), making it preferred over having a larger sample size with fewer trials.”

“Additionally, we used a binomial probability testing framework that is designed for small sample sizes and treats each participant as an independent replication. As such, it computes the probability of having observed the number of statistically significant outcomes by chance given our sample size (Schwarzkopf & Huang, 2024).”

Reviewer #1 (Recommendations for the authors):

My suggestions are intended to be light-touch and focused on strengthening the clarity and durability of the Reviewed Preprint rather than on additional experimentation or major new analyses.

(1) Limitation statement for the double-pass coding error:

Add a short statement in the Methods or Results acknowledging that the coding error led to markedly fewer repeated stimulus pairs for the first three participants than for the last three. For the response-consistency result in Figure 2E, a simple acknowledgement that the available evidence is stronger for some participants than others will help readers calibrate their confidence without detracting from the main story.

Thank you for this suggestion, we have now added a statement to this effect in the Results section, in addition to the description already mentioned in the Methods section.

“To examine this, we implemented a double-pass stimulus presentation (~600 stimulus pairs for participants 1-3 and ~2,500 pairs for participants 4-6) and analyzed participant’s response consistency (Xue et al., 2024) to two identical stimuli.”

(2) Contextualizing the titrated performance level:

In the Discussion, explicitly note that contrast was titrated to keep d′ between approximately 1.2 and 1.8, which intentionally maintains moderate performance. This contextualization will help readers understand that while the phase-linked changes appear modest in absolute terms, they are mechanistically informative within this design.

Thank you, we have included a sentence to the Discussions speaking to this point.

“We also note that the observed modulation of d’ between optimal and suboptimal phases was relatively modest in absolute terms (0.21) in our study and could therefore require many trials per subject to detect. Two reasons for this modest effect size could be related to specific features of our task design. First, we titrated stimulus contrast to maintain consistent task performance. This titration could have reduced the magnitude of the phase effect on d’ that would otherwise be apparent if the stimulus intensity were kept constant. Additionally, the use of (relatively) high-contrast random noise likely means that trial-to-trial variability in perception is largely driven by random fluctuations in the noise properties and, to a lesser extent, internal brain state. Although both of these choices were necessary to perform SDT and reverse correlation analysis, they differ from many previous studies investigating alpha phase using only near-threshold detection in the absence of external noise and may contribute to an underestimation of the true effect size.”

(3) Methods clarifications:

(a) Replace placeholder text such as "{plus minus}" and "{degree sign}" with the appropriate symbols, and ensure that any equations implied in the reverse-correlation section are fully present.

Thank you for bringing this to our attention, these placeholder texts are an artifact of the conversion process and we will correct this.

(b) State explicitly that the 8 ms stimulus duration corresponds to a single frame on your 120 Hz display, which will clarify the timing in Figure 1A and the pre-stimulus windows in the phase analyses.

Thank you, we have added language to both the Method and Results sections explicitly indicating that the 8 ms stimulus choice corresponds to a single screen refresh. Additionally, we changed the text in Figure 1A to include inter-trial interval timing (as opposed to merely saying “Start Trial”):

“(A) Task design. Each trial contained a brief, filtered-noise stimulus (8 ms; one screen refresh) presented to the right or left of fixation with equal probability.”

“Each participant (n = 6) completed 5-6 EEG sessions of a Yes/No detection paradigm whereby participants reported the presence or absence of a brief (8 ms; one screen refresh) vertical Gabor target (2 cycles per degree) with concurrent confidence judgments (see Figure 1A), along with an additional imagination judgement (reported in the supplemental materials).”

(c) In the description of the post-stimulus taper, consider phrasing the rationale in terms of minimizing contamination from evoked responses rather than asserting that the taper ends before the earliest evoked response, which keeps the argument correct without committing to a precise latency boundary.

Thank you for this suggestion. We have changed our rationale for the taper to “minimizing”, rather than avoiding, the evoked response.

“This resulted in the post-stimulus data being flat after 70 ms, which is intended to minimize the evoked response in our data.”

(4) Analysis transparency:

(a) In the description of posterior electrode selection, explicitly note that channels were chosen solely on the basis of alpha power, independent of behavioural performance, and that the same electrodes were used for each participant across sessions.

We have gladly made this clarification to the methods.

“This was individually determined by rank-ordering 17 of the posterior channels (Pz, P3, P7, O1, Oz, O2, P4, P8, P1, P5, PO7, PO3, POz, PO4, PO8, P6, and P2) and algorithmically choosing the three with the highest power. This ensured that electrode selection was made independent of performance and instead was based upon maximizing alpha signal strength.”

(b) Describe the phase-alignment step used to center each participant's optimal bin before group averaging as a device for visualization and summary, and clarify that inferential statistics are based on the underlying, non-aligned data as appropriate. This will reassure readers who are cautious about circularity.

We agree that this should be made more explicit throughout the manuscript and have added statements clarifying this aspect in the Figure 2B caption, the Results, and Method sections.

“The data have been aligned across participants so that each individual's highest d’ was assigned to bin 8 (omitted from the plot), with the remaining data circularly shifted, and is averaged across -450 ms to stimulus onset. This graph is for visualization purposes only. Error bars represent ± 1 SEM. The pattern shows a clear phasic modulation of d’ across bins.”

“... requiring us to phase-align the performance data across participants in order to visualize the underlying phasic effects. To this end, we aligned all metrics (d’, c, HR, and FAR) by circularly shifting the data so that the bin with the highest d’ was assigned to bin 8, which was then omitted from further visualizations.”

“Bin 8 was then omitted from further visualizations. The shifted data were then averaged across all time points from -450 ms to 0 ms, based on significant effects at the group level, and averaged across participants. No statistics were conducted on these shifted variables and instead are for visualization purposes only.”

(c) Add a short note on the number of permutations and the cluster-forming threshold in the phase-coupling analyses, if not already stated in the Results or captions, to complete the description of your non-parametric testing procedure.

Thank you, we agree that reiterating this information in the Results section is helpful for the reader to clarify the analysis procedure.

“After smoothing the resultant vector length over time with a 50 ms moving average, we compared the observed vector lengths to a permuted threshold (95th percentile of 1,000 permutations) at each time point from –700 to 0 ms and performed cluster correction (95th percentile of the permuted cluster size) to account for multiple comparisons.”

(5) Discussion framing:

Make one or two small adjustments to your mechanistic phrasing so that the distinctions between measurement and interpretation are fully explicit:

(a) State that the combination of phase-d′ coupling, counterphased hit and false-alarm rates, response consistency, and phase-dependent classification images is "consistent with" a reduction in effective internal noise and sharper estimated tuning at optimal alpha phase, within the assumptions of your SDT and reverse-correlation framework.

Thank you for this suggestion. We have changed the language in the discussions to reflect this framing and interpretation of the results.

“Moreover, our data are consistent with a model in which the variability of internal responses changes systematically across the alpha cycle, as reflected in the inverse relationship between hit rate and false alarm rate.”

(b) Emphasize that reduced effective internal noise and sharpened sensory tuning are two complementary descriptions of a better match between sensory evidence and decision template rather than fully separable mechanisms.

Thank you, we have added this language for clarity of our interpretation.

“Together with decreases in the variance of sensory tuning during the optimal phase, our results suggest that alpha phase impacts sensitivity by shaping trial-to-trial variation in internal noise during perceptual decision making, leading to better matches between sensory evidence and decision templates as opposed to a change in the gain of internal sensory responses.”

(c) Note that the frontal-occipital phase relationship is consistent with a coordinated, possibly top-down component to the alpha-phase effect, while remaining agnostic about the precise physiological implementation.

Thank you for raising this additional interpretation. We have added this as a plausible alternative to the single-source account in the Discussion section.

“Moreover, our results suggest that prior literature reporting phasic effects in the alpha-band range from both frontal and occipital regions may plausibly be reporting the same effect from a single projected dipole source; however, these results are also consistent with two synchronized alpha sources which are anti-phase.”

Reviewer #2 (Recommendations for the authors):

Major issues:

Given that collecting more data may not be doable, the authors should take some actions to test the reliability of their results. For instance, simulations could be run to test the robustness of the results with such a small sample size (Zoefel, 2019). It would also be of interest to include in the report statistics and plots at the individual level, not only the aggregates. It is also important to report which electrodes were used in the analysis for each of the subjects, in the Methods section, it is clearly stated that these electrodes differed between subjects.

Thank you for these suggestions. To assess the reliability of our results at the single-subject level, we have included a new binomial probability test which is a framework suitable for small sample size experiments with large trail numbers (Schwarzkopf & Huang, 2024). Binomial testing views each individual as an independent replication and considers the probability of having observed the number of significant participants given the total number tested participants, and outputs the probability of having observed the results by chance. We believe this framework adequately addresses the reviewer’s concern of generalizability in addition to being well-suited to the design of our study.

To assess individual significance, we averaged the resultant vector length and permutations over the analysis window from -450 to 0 ms. If the resultant vector length exceeded the permutation for that participant, then they were considered to be a significant participant. In total, 3 out of 6 participants (participants 1, 4, and 5) showed significant d’ coupling. The binomial probability (equivalent to a p-value) of having observed this outcome as a result of three false positives at the individual-subject level is very small (p = 0.002), which is sufficiently low for psychological studies.

Below is the text which we have added to the Results and Methods sections.

“To interrogate the robustness of our findings at the single-subject level, we adopted a test of binomial probability, which is a statistical framework that treats each individual as an independent replication and is ideal for small sample size studies that utilize a large number of trials per observer (Schwarzkopf & Huang, 2024). For our data, we assessed individual significance by averaging the actual and permuted resultant vector lengths across time (-450 to 0ms) and comparing the real vector length to the 95% percentile of the permuted datasets. With this approach, 3 out of 6 participants showed significant d’-phase coupling which corresponds to a binomial probability of p = 0.002, indicating a very low probability that we observed these results by chance alone.”

“Additionally, we used a binomial probability testing framework that is designed for small sample sizes and treats each participant as an independent replication. As such, it computes the probability of having observed the number of statistically significant outcomes by chance given our sample size (Schwarzkopf & Huang, 2024). To assess significance at the participant level, we averaged the participant’s resultant vector length and permutations from -450 to 0 ms and obtained the 95th percentile of the time-averaged permutations. We then compared the averaged resultant vector lengths to the permutation thresholds for each subject, which revealed 3 out of 6 significant subjects. We then used the MATLAB function myBinomTest.m (Nelson, 2026) to compute the p-value associated with the probability of having observed 3 out of 6 significant subjects by chance (with a false-positive rate of 0.05).”

To address the reviewer's second request, we now include a supplemental figure which has each individual’s results for the main analysis (see Supplementary figure 3). These graphs, in addition to the methods, now provides the reader with each participant’s given set of analysis electrodes.

“Each participant had a different combination of electrodes which were used in the analyses; however, the same three channels were used across sessions within a participant (participant 1: POz, PO3, O1; participant 2: P7, PO7, PO4; participant 3: P2, P1, Pz; participant 4: O1, Oz, O2; participant 5: O2, PO8, PO4; participant 6: Oz, O2, O1).”

As an alternative approach, linear mixed models (LMM) could be used for statistics, as they are more suitable for small sample sizes (Wiley et al., 2019). LMM improve generalization by modelling subject-specific random effects. Although raw circular data is not suitable for LMM, the sine and cosine of the phases could have been used as predictors, for instance. Given that data were collected for 6 different sessions, sessions could be included as a factor in the model to improve statistical power.

We appreciate the suggestion but feel that LMMs would be a challenge in this case not only because the main predictor variables are circular, but because the main outcome variables are not defined on the single-trial level and require many trials to be computed (e.g., classification images, SDT measures, response consistency). As such, computing these measures within a session may also lead to noisier estimates than we had designed our experiment for. We therefore prefer the more straightforward approach we have taken in the paper, which has now been supplemented by a binomial test of individual-subject level significance.

Given that the number of subjects is quite small, I believe that individual data should be presented (either in the main text or supplementary materials) also for figures: 2A, B, C and D.

Thank you, we have included all of these results to the individual graphs in the Supplemental Materials (see Supplementary figure 3).

In plot 2B (HR and FAR) a p-value = 0.015 appears. However, in the text you write:

"Indeed, this showed that the difference between the HR and FAR vector angle was significantly clustered around a mean of 180{degree sign} (v = 3.78, p = 0.01), indicating that the phase angle associated with the greatest hits was counterphase to the phase angle associated with the greatest false alarms."

Which one is correct? Or do they refer to different tests?

We appreciate you catching this confusing discrepancy. The two values refer to the same test which has a p-value of 0.0145. In the figure, this value was rounded to the thousandths decimal place (i.e., 0.015), whereas in the text it was rounded to the hundredths value (0.01). We now consistently report p-values out to three decimal places throughout the manuscript.

Did you perform any statistical test for phasic modulation of dprime and criterion? I say that because in Figure 2B, you state that the data shows a "clear phasic modulation of d' across bins", but no statistic is mentioned. On the other hand, in Figure 2D, you state, "We did not & observe any significant phase-dependent relationship between phase and criterion." Is this sentence referring to both 2C and 2D panels or only to 2C?

Figure 2B and 2D show the phase-behavior relationship across bins after aligning the phase bins to each participant's “best” d’ bin. This bin is omitted from the plots because it is used for alignment, making the analysis circular. Accordingly, these panels were intended purely for visualization and were not used for statistical inference. Additional language has been added to the figure caption highlighting this aspect.

“The data have been aligned across participants so that each individual's highest d’ was assigned to bin 8 (omitted from the plot), with the remaining data circularly shifted, and is averaged across -450 ms to stimulus onset. This graph is for visualization purposes only.”

The primary statistical test for phase-behavior coupling was performed using permutation testing of the resultant vector length, which quantifies the magnitude of phase-dependent modulation. These results are shown in Figures 2A (for d′) and 2C (for criterion). In the original manuscript, we reported only the time points that survived cluster-based correction, but did not explicitly report the cluster p-values. We have now added these cluster p-values to the manuscript for completeness.

“The data revealed significant cluster-corrected coupling between alpha phase and d’ in the prestimulus window from -220 ms until stimulus onset (cluster p = 0.046),...”

Additionally, we have changed the caption of Figure 2 to be separate for C) and D).

“(C) No evidence for the coupling of criterion to pre-stimulus alpha-band phase. Graph C reveals the time course of the resultant vector lengths for alpha phase-criterion coupling, which shows no significant phase-dependent relationship between phase and criterion.

(D) The underlying shifted c across phase bins (shifted to participants’ optimal phase, as in graph B) did not visually demonstrate a phasic modulation pattern.”

Minor issues:

In general, the paper is very clear. I found a statement confusing in the Response consistency section:

"To quantify response consistency, we computed the proportion of trials in which participants provided the same response across the two identical trials. This procedure was done for each channel at each time point (from -450 to 0 ms) and then averaged."

Which makes no sense, as response consistency is independent of channel and time point. I believe here you refer to the phase, maybe by just changing the order (start with response consistency and then proceed to phase), the paragraph would be clearer.

We appreciate you catching this mistake. We have clarified the Methods section in the following way:

“To quantify response consistency, we computed the proportion of trials in which participants provided the same response across the two identical trials. Since the optimal phase changes over time, the set of trials were classified as either both having occurred during the optimal phase (or otherwise) for each time point (from -450 to 0 ms) and channel. The proportion of consistent responses was then averaged across channels and time.”

Could you include a plot of the power spectrum used for IAF estimation of all the subjects?

Thank you for the suggestion. In Supplemental Figure 3 we have included the power spectrum that was used to estimate IAF in addition to a topoplot of alpha power (IAF +/- 2 Hz) that has the analysis electrodes labelled.

Bibliography:

Wiley RW, Rapp B. Statistical analysis in Small-N Designs: using linear mixed-effects modeling for evaluating intervention effectiveness. Aphasiology. 2019;33(1):1-30. doi: 10.1080/02687038.2018.1454884.

Zoefel B, Davis MH, Valente G, Riecke L, How to test for phasic modulation of neural and behavioural responses, NeuroImage, Volume 202, 2019,116175, https://doi.org/10.1016/j.neuroimage.2019.116175.

Author Response:

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

Public Reviews:

Reviewer #1 (Public review):

Weaknesses:

Despite this compelling data regarding the protective role of HSF1 in the febrile response, what remains unexplained and complicates the authors' model is the observation that losing LvHSF1 at 'normal' temperatures of 25 ℃ is not detrimental to survival, even though viral loads increase and nSWD is likely still subject to LvHSF1 regulation. These observations suggest that WSSV infection may have other detrimental effects on the cell not reflected by viral load and that LvHSF1 may play additional roles in protecting the organism from these effects of WSSV infection, such as perhaps, perturbations to protein homeostasis. This is worth discussing, especially in light of the rather complicated roles of hormesis in protection from infection, the role of HSF1 in hormesis responses, and the findings from other groups that the authors discuss.

We are grateful for your unbiased advice by reviewer. And we have added the description about the role of HSF1 in hormesis responses in discussion in Lines 422-425 in the revised manuscript. Thank you.

Reviewer #2 (Public review):

Temperature is a critical factor affecting the progression of viral diseases in vertebrates and invertebrates. In the current study, the authors investigate mechanisms by which high temperatures promote anti-viral resistance in shrimp. They show that high temperatures induce HSF1 expression, which in turn upregulates AMPs. The AMPs target viral envelope proteins and inhibit viral infection/replication. The authors confirm this process in drosophila and suggest that there may be a conserved mechanism of high-temperature mediated anti-viral response in arthropods. These findings will enhance our understanding of how high temperature improves resistance to viral infection in animals.

The conclusions of this paper are mostly well supported by data, but some aspects of data analysis need to be clarified and extended. Further investigation on how WSSV infection is affected by AMP would have strengthened the study.

We are grateful for your unbiased advice by reviewer. We have provided additional experimental evidence and supplementary instructions in the revised manuscript. Thank you.

Reviewer #3 (Public review):

In the manuscript titled "Heat Shock Factor Regulation of Antimicrobial Peptides Expression Suggests a Conserved Defense Mechanism Induced by Febrile Temperature in Arthropods", the authors investigate the role of heat shock factor 1 (HSF1) in regulating antimicrobial peptides (AMPs) in response to viral infections, particularly focusing on febrile temperatures. Using shrimp (Litopenaeus vannamei) and Drosophila S2 cells as models, this study shows that HSF1 induces the expression of AMPs, which in turn inhibit viral replication, offering insights into how febrile temperatures enhance immune responses. The study demonstrates that HSF1 binds to heat shock elements (HSE) in AMPs, suggesting a conserved antiviral defense mechanism in arthropods. The findings are informative for understanding innate immunity against viral infections, particularly in aquaculture. However, the logical flow of the paper can be improved.

We are grateful for the positive comments and the unbiased advice by reviewer. We have improved the logical flow of the paper and added corresponding instructions in the revised manuscript. Thank you.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

(1) Figure 1: The analysis compares Group TW to Group W (not the other way around).

Thank you very much. To uncover the molecular mechanisms by which high temperature restricts WSSV infection, two shrimp groups, Group TW and Group W, were cultured at 25 °C. Group W comprised shrimp injected with WSSV and maintained at 25 °C continuously. In contrast, Group TW was subjected to a temperature increase to 32 °C at 24 hours post-injection (hpi). Gill samples were collected for analysis 12 hours post-temperature rise (hptr) and subjected to Illumina sequencing (Figure 1A). RNA-seq was used to identify genes responsive to high temperature, particularly those encoding potential transcriptional regulators. Thank you.

(2) The RNA-seq data in Figure 1 focus only on the TFs. The manuscript would benefit from showing all the RNA-seq data and the differentially expressed genes. In particular, are the AMPs upregulated at the same time point? This should not be the case if LvHSF1 were responsible for the transcription of the AMPs, given the time lag between transcription and translation.

Thank you for your suggestion. In Author response image 1, our previous study has revealed that classical heat shock proteins (such as HSP21, HSP70, HSP60, HSP83, HSP90, HSP27, HSP10, and Bip) were induced by RNA-seq between Group TW and Group W, suggesting heat shock proteins exert a crucial role in enhancing the resistance of shrimp to WSSV at elevated temperatures (32 ℃) and underscoring the reliability of our transcriptomic findings (Xiao et al., 2024).

Additionally, we also analyzed the AMPs expression between Group TW and Group W, and the results show that some antimicrobial peptides such as Lysozyme and C-type lectin are upregulated between Group TW and Group W. Notably, we did not detect upregulated expression of SWD between Group TW and Group W. We agree with the reviewer's point of view that there is a time lag between transcription and translation. Supplementary experimental evidences show that the expression level of LvHSF1 is strongly induced by WSSV stimulation, and then the expression level of SWD begins to increase. We have added a description in Lines 136-138 in the revised manuscript.

Author response image 1.

The Figure of the heat shock proteins in Group TW and Group W

Author response image 2.

Transcriptional expression levels of HSF1 and SWD after WSSV stimulation

Reference:

Xiao, B., Wang, Y., He, J., Li, C., 2024. Febrile Temperature Acts through HSP70-Toll4 Signaling to Improve Shrimp Resistance to White Spot Syndrome Virus. J Immunol 213, 1187-1201.

(3) The data showing the tissue distribution of LvHSF1 and nSWD is a rigorous approach and adds to the manuscript. A similar approach to understanding the time course of expression of AMPs in relationship to LvHSF1 expression levels would strengthen the authors' conclusions that LvHSF1 induction in response to high temperatures and viral infection, in turn, upregulates SWD and other antibacterial genes.

Thank you for your suggestion. As you good suggestion, we detected the transcriptional expression levels of HSF1 and SWD after WSSV stimulation for 0, 2, 4, 6, 8, 12, 16, 20, and 24 hours. The transcriptional expression level of SWD was set to 1.00 at 0 h, in the early stage of WSSV infection (0-12 h, except 6 h), the expression level of LvHSF1 is strongly induced, and then the expression level of SWD begins to increase. Theses results show that LvHSF1 induction in response to viral infection, in turn, upregulates SWD and other antibacterial genes. Thank you.

(4) The data (Figures 3 and 4) show that LvHSF1 is necessary to survive WSSV infection at high temperatures but does not affect survival at lower temperatures, even though LvHSF1 limits VP28 levels, and viral load at both temperatures is confusing. Does this suggest that LvHSF1 is not primarily important for protection against the virus but instead, for protection from the heat-induced damage caused by high temperatures, which would not be surprising? The manuscript would benefit if the authors could address this point. How do the authors envision the protection conferred by LvHSF1 only at high temperatures?

Thank you for your comment. Although no significant difference in shrimp survival rates was observed between LvHSF1-silenced shrimp and GFP-silenced shrimp at low temperature (25 °C), shrimp with silenced LvHSF1 exhibited increased viral loads in hemocytes and gills, suggesting that upregulation of HSF1 expression can protect shrimp from WSSV infection.

Notably, the tolerance temperature for L. vannamei growth ranges from 7.5 to 42 °C. When infected with WSSV, shrimp use behavioral fever to elevate their body temperature (~32 °C), thereby inhibiting WSSV infection (Rakhshaninejad et al., 2023; Xiao et al., 2024). And this temperature (~32 °C) will not cause heat-induced damage to the shrimp. Our results demonstrate that febrile temperatures induce HSF1, which in turn upregulates antimicrobial peptides (AMPs) that target viral envelope proteins and inhibit viral replication.

Only at high temperatures, we observed that knockdown of HSF1 did not affect shrimp survival rate (Figure 4A). Thank you again for your valuable feedback.

Reference:

Rakhshaninejad, M., Zheng, L., Nauwynck, H., 2023. Shrimp (Penaeus vannamei) survive white spot syndrome virus infection by behavioral fever. Sci Rep 13, 18034.

Xiao, B., Wang, Y., He, J., Li, C., 2024. Febrile Temperature Acts through HSP70-Toll4 Signaling to Improve Shrimp Resistance to White Spot Syndrome Virus. J Immunol 213, 1187-1201.

(5) Related to the previous comment, the authors do not clearly distinguish between basal effects of LvHSF1 or nSWD induction and heat-induced effects and the differences related to the requirement of LvHSF1 for protection. Simply increasing LvHSF1 levels can result in increased nSWD. SWD levels increase upon WSSV infection even at 25 ℃, and the knockdown experiments suggest that this could also occur through LvHSF1. It would be useful to explicitly differentiate between basal functions of HSF1 and induced functions.

Thank you for your suggestion. In previous responses, we have distinguished between basal effects of LvHSF1 or nSWD induction and heat-induced effects.

As your good suggestion, we injected GST or rHSF1 protein into shrimp, the results showed that recombinant protein HSF1 could significantly induced the expression level of SWD (Supplementary Fig. 5C). Further, after knockdown of SWD, shrimp were injection with rLvHSF1 mixed with WSSV. The results showed that the viral load was significantly lower than the control group 48 hours post WSSV infection (Supplementary Fig. 5D). We have added these results to the Supplementary Figure 5C&5D and added a description in Lines 253-255 and Lines 290-293 in the revised manuscript. Thank you for your constructive comments.

Reviewer #2 (Recommendations for the authors):

(1) Two temperatures are used in the experiments of shrimp. It seems that HSF1 is also upregulated by WSSV infection at 25 ℃. However, this upregulation seems not to be able to protect the animals. The authors compare the infection at 25 and 32 ℃ but did not discuss the findings.

Thank you for your comment. Although no significant difference in shrimp survival rates was observed between LvHSF1-silenced shrimp and GFP-silenced shrimp at low temperature (25 °C), shrimp with silenced LvHSF1 exhibited increased viral loads in hemocytes and gills, suggesting that upregulation of HSF1 expression can protect shrimp from WSSV infection. We have added a discussion of this finding in Lines 461-464 in the revised manuscript. Thank you.

(2) In the abstract the authors say that "These insights provide new avenues for managing viral infections in aquaculture and other settings by leveraging environmental temperature control." However, this point has not been discussed in the main text.

We appreciated your comments. We have added a discussion about the environmental temperature control in Lines 512-514 in the revised manuscript. Thank you.

(3) Line 142: "These results suggest that LvHSF1 may play a key role in enhancing shrimp resistance to WSSV at elevated temperatures." Although this type of conclusion has been made in many studies, I think it is impossible to see a "KEY role" based mainly on change in expression.

Thank you for your suggestion. We have revised this conclusion in the revised manuscript. Thank you.

(4) Section 2.1 Induction of Heat Shock Factor 1 in Response to WSSV at High Temperature

Figure 1. Identification of HSF1 as a key factor induced by high temperature.

The two titles are confusing. Whether the upregulation of HSF1 is a response to high temperature or WSSV infection? I think it is more likely a response to high temperature. Did the authors see the difference in HSF1 expression in shrimp with and without WSSV infection at high temperatures?

Thank you for your comment. We have modified the title of Section 2.1 in the revised manuscript. As your good suggestion, we have measured the expression of LvHSF1 after WSSV challenge at high temperatures (32 ℃) in revised Figure 2F-2H in Line 122 in the revised manuscript. The results demonstrate that the expression of LvHSF1 is strongly induced by WSSV stimulation at high temperatures (32 ℃) in the revised manuscript. Thank you.

(5) Figure 2. Upregulation of LvHSF1 in shrimp challenged by WSSV at both low and high temperatures. Results for WSSV challenge at high temperatures are not included in this figure.

Thank you for your suggestion. As your good suggestion, we have measured the expression of LvHSF1 after Poly (I: C) and WSSV challenge at high temperatures (32 ℃) in revised Figure 2C-2H. The results demonstrate that the expression of LvHSF1 is strongly induced by Poly (I: C) and WSSV stimulation at high temperatures (32 ℃). And we have added a description in Lines 168-179 in revised manuscript. Thank you.

(6) Section 2.2 Expression Profiles of LvHSF1 in Shrimp Under Varied Temperature Conditions and WSSV Challenge. Did the authors try poly IC and WSSV challenge at 32℃, and compare with the un-challenge group? Why were only low temperature was analyzed?

Thank you for your suggestion. As your good suggestion, we have measured the expression of LvHSF1 after Poly (I: C) and WSSV challenge at high temperatures (32 ℃) in revised Figure 2C-2H. And we have added a description about the expression of LvHSF1 after Poly (I: C) and WSSV challenge at high temperatures (32 ℃) in Lines 168-179 in revised manuscript. Thank you.

(7) Figure 2: Please indicate the temperature used in C-E and F-H in the figure legend. Statistical significance: compared with which group? Please provide information in the legend or show it in the bar chart.

Thank you for your suggestion. We have added the description of temperature used in revised Figures 2C-2E. The expression changes of HSF1 were compared with those of PBS control group at the corresponding time and we modified the comparison method of significance in revised Figures 2C-2E. Thank you.

(8) Figure 3H: There are two groups (dsGFP+PBS; dsHSF1+PBS) showing with the same symbol (dot line).

Thank you for your comment. The revised Figure 3H has used different symbols to distinguish the two groups. Thank you.

(9) Line 205: qPCR

Thank you for your careful checks. We have corrected this error in the revised manuscript. Thank you.

(10) Figure 5d and f: Please indicate the sample in each row.

Thank you for your suggestion. We have marked the samples in each row in the revised Figures 5d&5f.

(11) Figure 3 and Figure 4: Why different tissues were analyzed in the two experiments? Low temperature: gill and hemocytes. High temperature: gill and muscle? It is better to use the same tissues so that they can be compared. Please indicate the tissue analyzed in D and d.

Thank you for your suggestion. We have repeated the experiment to detect the copy number of WSSV in hemocyte at high temperature (32 °C) after LvHSF1 knockdown. The results showed that knockdown LvHSF1 showed increased viral loads in shrimp hemocyte (Figure 4C). We have supplemented the tissue information in Figure 4D&4d. Thank you.

(12) Figure 2A The time for temperature treatment? hours or days?

Thank you for your comment. Transcriptional expression of LvHSF1 in different tissues of healthy shrimp subjected to low (25 °C) and high (32 °C) temperatures for 12 hours. We have supplemented this information in the legend of Figure 2A in Lines 840-841 in revised manuscript. Thank you.

(13) Line 249: purified by SDS-PAGE gel?

Thank you for your comment. We have modified this description in Lines 272-274 in current manuscript. Thank you.

(14) Line 258 "Next, to verify whether the anti-WSSV function of nSWD was mediated by LvHSF1 at high temperature". I think it is confusing to use "mediated" here. It seems that HSF1 is downstream of nSWD. Actually, HSF1 controls the expression of nSWD and thus regulates the anti-WSSV effect of shrimp at high temperatures.

We appreciated your comments. We have modified this description in Lines 282-283 in current manuscript. Thank you.

(15) Line 458 "The most probable anti-WSSV mechanism of nSWD is its direct interaction with WSSV envelope proteins VP24 and VP26, potentially inhibiting viral entry into target cells. I suggest the author analyze the entry of WSSV to see whether nSWD blocks this process.

Thank you for your comment. In general, the antimicrobial mechanism of action of AMPs is thought to involve direct membrane disruption, especially for enveloped virus (such as WSSV) (Wilson et al., 2013).

Thanks to the reviewers for their valuable comments. Our manuscript mainly focuses on the febrile temperature-inducible HSF in host antiviral immunity, and the role of HSF1 in regulating antimicrobial effectors (such as SWD). Due to the limitation of the manuscript's length, we will further investigate the functional mechanisms of SWD-specific anti-WSSV in future studies. Thank you.

Reference:

Wilson, S.S., Wiens, M.E., Smith, J.G., 2013. Antiviral Mechanisms of Human Defensins. Journal of Molecular Biology 425, 4965-4980.

(16) Line 435-456 The author discusses the difference between two shrimp species. Did the two studies measure the same immune parameters? I wonder whether the different observation is due to true differences or different methods they used to evaluate the response. If no immune response was promoted in the previous study, what's the possible anti-viral mechanism?

We appreciated your comments. Firstly, the shrimps in the two experimental groups have different adaptability to temperature. The optimal water temperature for M. japonicus growth ranges from 25 to 32 °C, and the tolerance temperature for L. vannamei growth ranges from 7.5 to 42 °C. Secondly, the experimental environmental factors are different in the two experimental groups. Ammonia is a key stress factor in aquatic environments that usually increases the risk of pathogenic diseases in aquatic animals, however, High temperatures (32°C) have been shown to inhibit the replication of WSSV and reduce mortality in WSSV-infected shrimp. Thirdly, the two studies tested different immune indicators. Ammonia-induced Hsf1 suppressed the production and function of MjVago-L, an arthropod interferon analog. In this study, our findings revealed the molecular mechanism through which the HSF-AMPs axis mediates host resistance to viruses induced by febrile temperature. Taken together, the benefits of HSF1 can be attributed to either the host or the pathogen, depending on the nature and context of the host-virus-environment interaction.

(17) Line 472 "directly bind to WSSV envelope proteins and inhibit WSSV proliferation"

I think it is confusing to use "proliferation" here. It seems that the binding of HSF affects the replication process. However, based on the authors' discussion, HSF may likely block viral entry.

Thank you for your suggestion. We have modified this description in Lines 505-507 in the current manuscript. Thank you.

Reviewer #3 (Recommendations for the authors):

In the manuscript titled "Heat Shock Factor Regulation of Antimicrobial Peptides Expression Suggests a Conserved Defense Mechanism Induced by Febrile Temperature in Arthropods", the authors investigate the role of heat shock factor 1 (HSF1) in regulating antimicrobial peptides (AMPs) in response to viral infections, particularly focusing on febrile temperatures. Using shrimp (Litopenaeus vannamei) and Drosophila S2 cells as models, this study shows that HSF1 induces the expression of AMPs, which in turn inhibit viral replication, offering insights into how febrile temperatures enhance immune responses. The study demonstrates that HSF1 binds to heat shock elements (HSE) in AMPs, suggesting a conserved antiviral defense mechanism in arthropods. The findings are informative for understanding innate immunity against viral infections, particularly in aquaculture. However, the logical flow of the paper can be improved. Following are my specific concerns.

Major comments

(1) The study design is pretty good, but the logical flow is not. The following should be improved.

(a) In Figure 1, the reason for selecting HSF1 as the focus of the study is not clearly explained.

Thank you for your comment. In a previous study, we have revealed that heat shock proteins exerted a significant role in enhancing the resistance of shrimp to WSSV at elevated temperature (32 ℃) (Xiao et al., 2024). GO functional enrichment analysis of DEGs between group TW and group W, indicating that most DEGs were involved in biological processes such as protein refolding, chaperone-mediated protein folding, and heat response. Therefore, special attention has been paid to heat shock factor 1 (HSF1), the master regulator of the heat shock response. We have added the description in Lines 136-138 in the revised manuscript. Thank you.

Reference:

Xiao, B., Wang, Y., He, J., Li, C., 2024. Febrile Temperature Acts through HSP70-Toll4 Signaling to Improve Shrimp Resistance to White Spot Syndrome Virus. J Immunol 213, 1187-1201.

(b) As the authors draw models in Figure 9, the established activation mechanism of HSF1 is via trimerization by the release of HSP90, which binds to misfolded proteins under stress conditions, such as heat shock. Therefore, the increase in the HSF1 mRNA level in Figure 1 is strange. The authors need to clarify this issue by explaining this established activation mechanism of HSF1 and also must provide the basis of upregulation of HSF1 by mRNA increase via citing papers in the Introduction.

We appreciated your comments. Under non-stress conditions, HSF monomers are retained in the cytoplasm in a complex with HSP90. During the stress response, such as high temperature, HSF dissociates from the complex, trimerizes, and converts into a DNA-binding conformation through regulatory upstream promoter elements known as heat shock elements (HSEs) (Andrasi et al., 2021). Previous studies have demonstrated that the expression of HSF1 was remarkably induced by stress response, such as high temperature (Ren et al., 2025), virus infection (Merkling et al., 2015), and ammonia stress (Wang et al., 2024). Our results also showed that the expression of LvHSF1 was significant induced by WSSV infection and high temperature (Figure 2). Therefore, this is not surprising that the increase in the HSF1 mRNA level in Figure 1.

In response, we have revised the proposed model to better reflect our experimental findings and the accompanying description. This revision ensures that the schematic is consistent with our data and accurately represents the proposed mechanism. We appreciate your careful review and constructive feedback.

Reference:

Andrasi, N., Pettko-Szandtner, A., Szabados, L., 2021. Diversity of plant heat shock factors: regulation, interactions, and functions. J Exp Bot 72, 1558-1575.

Ren, Q., Li, L., Liu, L., Li, J., Shi, C., Sun, Y., Yao, X., Hou, Z., Xiang, S., 2025. The molecular mechanism of temperature-dependent phase separation of heat shock factor 1. Nature Chemical Biology.

Merkling, S.H., Overheul, G.J., van Mierlo, J.T., Arends, D., Gilissen, C., van Rij, R.P., 2015. The heat shock response restricts virus infection in Drosophila. Sci Rep 5, 12758.

Wang, X.X., Zhang, H., Gao, J., Wang, X.W., 2024. Ammonia stress-induced heat shock factor 1 enhances white spot syndrome virus infection by targeting the interferon-like system in shrimp. mBio 15, e0313623.

(c) For RNA seq analysis in both in Figures 1 and 5, they need to provide changes in conventional HSF1 target chaperones (many HSPs) to validate their RNA seq data.

Thank you for your suggestion. In Authopr response image 1, our previous study has revealed that classical heat shock proteins (such as HSP21, HSP70, HSP60, HSP83, HSP90, HSP27, HSP10, and Bip) were induced by RNA-seq between Group TW and Group W, suggesting heat shock proteins exert a crucial role in enhancing the resistance of shrimp to WSSV at elevated temperatures (32 ℃) and underscoring the reliability of our transcriptomic findings (Xiao et al., 2024). We have added the description in Lines 136-138 in the revised manuscript.

In Figure 5, we have supplemented the heat shock proteins downregulated DEGs by transcriptome sequencing of dsGFP +WSSV (32 ℃) vs. dsLvHSF1 +WSSV (32 ℃) in Supplementary table 2. The results showed that the classical heat shock proteins were downregulated by the RNA-seq, underscoring the reliability of our transcriptomic findings. We have added the description in Lines 213-216 in the revised manuscript. Thank you.

Reference:

Xiao, B., Wang, Y., He, J., Li, C., 2024. Febrile Temperature Acts through HSP70-Toll4 Signaling to Improve Shrimp Resistance to White Spot Syndrome Virus. J Immunol 213, 1187-1201.

(d) In Figure 5, they did experiments by focusing on the changes by HSF1 knockdown at 32 ℃. However, the logical flow should be focusing on genes whose expression was increased by 32 ℃ compared with 25 ℃ (in figure 1), among them they need to characterize HSF1 target genes. Here as mentioned above, classical HSP genes must be included in addition to those AMP genes.

Thank you for your suggestion. As your good suggestion, we have supplemented the heat shock proteins downregulated DEGs by transcriptome sequencing of dsGFP +WSSV (32 ℃) vs. dsLvHSF1 +WSSV (32 ℃) in Supplementary table 2. The results showed that the classical heat shock proteins were downregulated by the RNA-seq, underscoring the reliability of our transcriptomic findings. We have added the description in Lines 213-216 in the revised manuscript. Thank you.

(e) What is the logical basis of just picking nSWD? It is another example of cherry-picking similar to picking HSF1 in Figure 1.

We appreciated your comments. To determine how temperature-induced LvHSF1 restricts WSSV infection, RNA-seq was performed to identify target genes regulated by HSF1. By analyzing the differentially expressed genes (DEGs), we screened eight candidate proteins for immunity-effector molecules, including SWD, CrustinⅠ, C-type lectin, Anti-lipopolysaccharide factor (ALF), and Vago. CrustinⅠ has been shown to play an important role in antiviral immunity (Li et al., 2020); C-type lectin (CTL1) can bind to the VP28, VP26, VP24, VP19, and VP14, thereby inhibiting the infection of WSSV (Zhao et al., 2009); Anti-lipopolysaccharide factor (ALF3) performs its anti-WSSV activity by binding to the envelope protein WSSV189 (Methatham et al., 2017); Vago can inhibit WSSV infection by activating the Jak/Stat pathway in shrimp (Gao et al., 2021). However, the detailed regulatory mechanism of SWD against WSSV was unclear, and particular attention was paid to the SWD. We have added the description in Lines 215-220 in the revised manuscript. Thank you for your valuable comments and the logic of the manuscript has been improved.

Reference:

Li, S., Lv, X., Yu, Y., Zhang, X., Li, F., 2020. Molecular and Functional Diversity of Crustin-Like Genes in the Shrimp Litopenaeus vannamei, Marine Drugs 18, 361.

Zhao, Z.Y., Yin, Z.X., Xu, X.P., Weng, S.P., Rao, X.Y., Dai, Z.X., Luo, Y.W., Yang, G., Li, Z.S., Guan, H.J., Li, S.D., Chan, S.M., Yu, X.Q., He, J.G., 2009. A novel C-type lectin from the shrimp Litopenaeus vannamei possesses anti-white spot syndrome virus activity. Journal of Virology 83, 347-356.

Methatham, T., Boonchuen, P., Jaree, P., Tassanakajon, A., Somboonwiwat, K., 2017. Antiviral action of the antimicrobial peptide ALFPm3 from Penaeus monodon against white spot syndrome virus. Dev Comp Immunol 69, 23-32.

Gao, J., Zhao, B.R., Zhang, H., You, Y.L., Li, F., Wang, X.W., 2021. Interferon functional analog activates antiviral Jak/Stat signaling through integrin in an arthropod. Cell Rep 36, 109761.

(f) Likewise, choosing Atta in S2 cells needs logic.

We appreciated your comments. Our manuscript revealed that febrile temperature inducible HSF1 confers virus resistance by regulating the expression of antimicrobial peptides (AMPs) in L. vannamei. Further, we want to know that whether HSF1 regulation of antimicrobial peptides is a conserved defense mechanism induced by elevated temperature in arthropods, and experiments were performed in an invertebrate model system (Drosophila S2 cells). Previous study showed that DmAMPs (such as Attacin A, Cecropins A, Defensin, Metchnikowin, and Drosomycin) exerted a significant role in the antiviral immunity in Drosophila (Zhu et al., 2013). Our results showed that the expression of Attacin A, Cecropins A and Defensin were remarkably induced by DmHSF, and the expression of Attacin A was the highest induced. Therefore, DmAtta was chosen as a representative to further demonstrate that DmHSF1 exerts its anti-DCV function by regulating DmAMPs. We have added the description in Lines 328-330 and Lines 361-364 in the revised manuscript. Thank you for your valuable comments and the logic of the manuscript has been improved.

Reference:

Zhu, F., Ding, H., Zhu, B., 2013. Transcriptional profiling of Drosophila S2 cells in early response to Drosophila C virus. Virol J 10, 210.

(2) From Figure 6I to 6K, the authors aimed to verify whether the anti-WSSV function of nSWD was mediated by LvHSF1 at high temperatures. However, what they showed was just showing that nSWD plays anti-WSSV function downstream of HSF1. The authors should show additional data for dsControl+rnSWD.

Thank you for your suggestion. As your suggestion, after knockdown of SWD, shrimp were injection with rLvHSF1 mixed with WSSV. The results showed that the viral load was significantly lower than the control group 48 hours post WSSV infection (Supplementary Fig. 5D). We have added these results to the Supplementary Figure 5C&5D and added a description in Lines 290-293 in the revised manuscript. Thank you for your constructive comments.

(3) For the physical interaction between nSWD and WSSV, it will be great if the authors perform Alphafold3 prediction analysis (Abramson et al PMID: 38718835).

Thank you for your suggestion. As you suggestion, we performed Alphafold3 prediction analysis on SWD and WSSV (VP24 and VP26). The predicted template modeling (pTM) score measures the accuracy of the entire structure. A pTM score above 0.5 means the overall predicted fold for the complex might be similar to the true structure. The Alphafold3 prediction results show that there is a possible interaction between SWD and WSSV. Notably, our manuscript demonstrated that rSWD could interact with VP24 and VP26 by pulldown assays and confocal analysis.

Author response image 3.

Alphafold3 prediction analysis of SWD&VP24 as follow (pTM = 0.64)

Author response image 4.

Alphafold3 prediction analysis of SWD&VP26 as follow (pTM = 0.53)

Minor comments

(1) In the Abstract and many other places, the authors need to specifically write "Drosophila S2 cells" instead of "Drosophila" because conventionally Drosophila implies fruit fly as an organism. We don't say cultured human cells as "human" or "Homo sapiens" in papers.

Thank you for your suggestion. We have modified the description of Drosophila in the revised manuscript. Thank you.

(2) Figure numbers can be reduced for better readability. I would combine Figures 1 and 2, and Figures 3 and 4. If the combined figures are too crowded, some can go to into supplementary figures.

Thank you for your suggestion. We have moved the Poly (I: C) data to Supplementary Figure 2 in the revised manuscript. However, we have added some experimental data to Figures 1, 2, 3, and 4. Therefore, we did not combine Figure 1 and Figure 2, and Figures 3 and 4. Thank you.

(3) One of the best-understood roles of HSF1 in physiology other than heat shock response is longevity, in particular with C. elegans. The authors need to mention this in the Discussion by citing the following recent review paper (Lee PMID: 36380728).

Thank you for your suggestion. We have supplemented the description of HSF1 regulating longevity and aging of organisms and cited the above reference in the revised manuscript (Lee and Lee, 2022). Thank you.

Reference:

Lee, H., Lee, S.V., 2022. Recent Progress in Regulation of Aging by Insulin/IGF-1 Signaling in Caenorhabditis elegans. Mol Cells 45, 763-770.

(4) Please make your own label for small letter panels or transfer small letter panels to supplementary figures.

Thank you for your suggestion. We have adjusted the relevant letter labels. The uppercase letters represent the main image of the Figure, and the small letter panels are the corresponding supplementary instructions in the revised manuscript. Thank you.

(5) In the introduction part, I recommend changing the references for HSFs and HSR with recent ones.

Thank you for your suggestion. We have added the latest references for HSFs and HSR in the Introduction part of the revised manuscript. Thank you.

(6) In Figure 1, it is not intuitive to understand the name groups W and TW.

We appreciated your comments. We have added the description of Group W and Group TW in revised Figure 1. Group W comprised shrimp injected with WSSV and maintained at 25 °C continuously. In contrast, Group TW was subjected to a temperature increase to 32 °C at 24 hours post-injection (hpi). Gill samples were collected for analysis 12 hours post-temperature rise (hptr) and subjected to Illumina sequencing. Thank you.

(7) Please add some kinds of sequence comparisons of SWD and nSWD for readers to understand the homology.

We appreciated your comments. We have added the multiple sequence alignment of SWD proteins in shrimp species in revised Supplementary Figure 3. Highly conserved amino acid residues and cysteine and residues are highlighted in red, indicating that LvSWD is a conserved antimicrobial peptide of the Crustin family. Thank you.

(8) Naming nSWD with "newly identified" is strange as it will not be new anymore as time goes by. Please change the name.

Thank you for your suggestion. We have modified the name of nSWD to SWD in the revised manuscript. Thank you.

(9) Please write the full name for Lv (Litopenaeus vannamei), Dm (Drosophila melanogaster), ds (double-stranded) before using LvHSF1, DmHSF1, and dsLvHSF1.

Thank you for your comments. We have added the full name of LvHSF1, DmHSF1, and dsLvHSF1 in the revised manuscript. Thank you.

(10) In Figure 2, it will be better to transfer poly I:C data to supplementary figures.

Thank you for your comments. We have moved the Poly (I: C) data to Supplementary Figure 2 in the revised manuscript. Thank you.

(11) The label for pGL3-nSWD-M12 is confusing. M1 and M2 are OK. Please change M12 with M1/2 or another one.

Thank you for your suggestion. We have changed pGL3-nSWD-M12 with pGL3-nSWD-M1/2 in the revised manuscript. Thank you.

Author Response:

The following is the authors’ response to the previous reviews

eLife Assessment

This article presents useful findings on how the timing of cooling affects the timing of autumn bud set in European beech saplings. The study leverages extensive experimental data and provides an interesting conceptual framework for the various ways in which warming can affect but set timing. The statistical analysis is compelling, but indicates some factors that may temper the authors' claims, while the designs of experiments offer incomplete support for the current claims as they rely on one population under extreme conditions for only one year each while a confounding effect (time in a chamber) sometimes lacks a control.

We thank the editor and reviewers for their consideration of our revised manuscript and for their constructive suggestions. In response to the editor’s guidance, we have ensured that: 1) the experimental design is clearly presented as physiological forcing, 2) the Solstice-as-Phenology-Switch concept is explicitly defined, limited, and framed as inferred, 3) conclusions are strictly aligned with the scope of the evidence, and limitations are acknowledged transparently.

We hope these revisions fully address the remaining concerns and clarify both the conceptual framework and the appropriate scope of inference.

Public Review:

Reviewer #1 (Public review):

The authors identified the summer solstice (June 21) as a phenological "switch point", but the flexibility of this switch point remains poorly understood. A more precise explanation of what "flexibility" means in this context is needed, along with a description of the specific experimental results that would demonstrate this flexibility.

We agree that the concept of “flexibility” required clearer definition and a more explicit link to the experimental results. In the Introduction, we now explicitly define flexibility as the capacity for the effective timing of the phenological switch to shift earlier or later depending on developmental progression, rather than occurring at a fixed calendar date. This switch occurs at the compensatory point between the antagonistic influences of early-season development [ESD effect] and late-season temperature [LST effect](L92-98). We have extended and clarified our explanation of the summer solstice’s role in this framework (L69-90). We propose that the solstice acts as an environmental switch that initiates the LST effect, as declining daylengths signal trees to become responsive to late-season cooling (L92-94). The compensatory point then occurs where the advancing ESD effect is balanced by the delaying LST effect. This point should therefore not be fixed to a calendar date but instead vary with developmental progression each year (L75-95).

In the Discussion, we clarify that flexibility is demonstrated experimentally by the observation that the magnitude of July cooling effects (LST effect) on autumn phenology depend on prior developmental rate (ESD effect) [3.4 times greater delay in late-leafing trees], indicating that the position of the compensatory point is development-dependent rather than fixed to June 21 (L398-410). We have made consistent edits throughout the Discussion, in particular in the ‘Support for the Solstice-as-Phenology-Switch Hypothesis’ subsection (L514-530).

The experiment did not directly measure the specific date of the phenological switch point. Instead, it was inferred by comparing temperature effects before and after the solstice. The manuscript should clearly state that this switch point remains an inferred conceptual node rather than a directly measured variable.

We fully agree and have clarified this in the revised manuscript. In the Discussion, we now clearly state that the compensatory point is a conceptual node inferred from responses to cooling before the solstice (June), directly after it (July), or later in the growing season (August) rather than a directly observed phenological event (L352-358 & L405-406).

In Experiment 1, the effect of bud type (terminal vs. lateral) was inconsistent across the overall model and the different leafing groups. The authors should provide a more thorough discussion of potential reasons for this inconsistency.

This inconsistency reflects biological complexity. In the Discussion, we now expand our interpretation to note that terminal and lateral buds may differ in developmental status, resource allocation and hormonal context. We emphasize that bud-type effects are therefore expected to be context-dependent and to interact with wholeplant developmental state, which plausibly explains why effects differ across leafing groups and models (L390-396).

In addition, the statistical model for Experiment 1 indicates that the measured variables (summer cooling and leaf emergence date) explain only 23.4% of the variation in bud formation timing. This leaves over 76% of the variation unexplained, suggesting that other important factors are involved. The discussion should address this limitation in greater depth, moving beyond a focus on the measured variables.

We now discuss the explained and unexplained variance in more detail. We also make it clear that our experiment was designed to test specific mechanistic pathways rather than to fully explain all phenological variability or maximise predictive power L417-419).

In the Discussion, we acknowledge that a substantial fraction of variation remains unexplained (L419-421). We discuss the possibility of other physiological mechanisms, such as photosynthetic assimilation, contributing to the unexplained variation (L421-427). However, large inter-individual variability is commonplace in autumn phenology. A low intra-class correlation coefficient (ICC = 0.26; see L276-280 for methods) suggests much of the remaining variation is attributable to individual-level differences rather than missing explanatory variables (L429-431). In line with the literature, we suggest that genetic and epigenetic differences likely contributed significantly to inter-individual variation, even within a single provenance population (L431-434). In this context of high individual variability, leaf-out timing (ESD effect) and summer cooling treatment (LST effect) together explaining 23.4% of variation in bud set timing is biologically meaningful and demonstrates the mechanistic importance of these processes (L438-441). For completeness, we also briefly discuss alternate sources of within-treatment variability (L434-437).

Reviewer #2 (Public review):

I think the experiments are interesting, but I found the exact methods of them somewhat extreme compared to how the authors present them.

We appreciate this concern and have substantially revised the manuscript to clarify the experimental logic. In the Introduction, we now state explicitly that the study uses temperature regimes that were designed as strong physiological forcing treatments, intended to deeply constrain development and isolate mechanisms rather than to simulate natural or future climatic conditions (L113-115).

In the Methods, we have enhanced our description of the non-linear effects of temperatures below 10°C on physiological processes (L154-158).

At the start of the Discussion, we have added a dedicated paragraph clarifying the scope of inference: the experiment tests causality and constraint (i.e. whether specific physiological processes can drive phenological shifts), not quantitative responses under realistic climate scenarios (L346-363). Throughout the Discussion, we have revised language that could be read as scenario-based interpretation, replacing it with mechanistic phrasing.

Further, given that much of the experiment happened outside, I am not sure how much we can generalize from one year for each experiment, especially when conducted on one population of one species.

Given the large individual variation expected in phenological experiments, we used single experimental populations of single provenance beech saplings to minimise uncontrolled for variation arising from genetic differences (L358-360). This allowed us to elucidate mechanisms despite noisy biological heterogeneity associated with phenology.

In the last round of revision, we toned down statements of generalisation. In the Discussion, we now go further to clarify what mechanistic understanding can be gleamed directly from our findings and then cautiously make suggestions how these mechanisms may play out in natural systems. We repeatedly state the intention of the study as mechanistic inference rather than predictive power, e.g. “However, extrapolations to more complex natural ecosystems should be made with caution as our experimental design prioritised mechanistic inference over generalisability and predictive power.” (L417-419). Alongside our previous calls for tests on other species, we now additionally call for tests on other provenances of beech (L511-512).

I was also very concerned by the revisions.

If this concern stems from the confusion regarding line-numbers and the two submitted versions of the manuscript (with tracked changes and without tracked changes; as required by eLife), then we hope that situation is now clarified. Otherwise, the authors do not understand why our previous revisions would be perceived as being concerning. Regardless, we have made every attempt to address the remaining comments comprehensively.

Further, I am at a loss about their hypothesis, when they write in their letter: "Importantly, the Solstice-asPhenology-Switch hypothesis does not assume that the reversal is fixed to June 21." Why on earth reference the solstice if the authors do not mean to exactly reference the solstice?

We appreciate this important conceptual point. The Solstice-as-Phenology-Switch hypothesis is central to our conceptual model and therefore requires clear explanation. In concert with our changes in response to Reviewer 1’s comment regarding flexibility, we have substantially revised and improved our description of this hypothesis (L69-108).

Whilst the summer solstice is fixed to a calendar date (June 21), the timing of when trees change their autumn phenological responses to temperature is not (L88-90 & L515-517). This occurs when the compensatory point of two antagonistic effects is crossed. Higher early-season development rates (which are driven by temperature) have an advancing (negative) effect on autumn phenology, which we now refer to as the ESD effect (L71-78). Warmer late-season temperatures have a delaying (positive) effect because trees become phenologically susceptible to cooling, i.e. overwintering responses are induced in response to cooling, which we now refer to as the LST effect (L78-82). The point in time when these two effects balance each other out, i.e. the net effect = 0, is the compensatory point (L95-97 & L523-525). The reason this point occurs after the solstice, is because the LST effect only becomes active when days begin to shorten (L92-94 & L522-523). The solstice acts as an environmental switch, initiating trees’ susceptibility to cooling. Therefore, the solstice is referenced in the hypothesis because it forms a daylength barrier. In this framework, the compensatory point cannot occur earlier than the solstice because day lengths are still increasing (L517-519).

In the Introduction and Discussion, we clarify that the solstice is referenced as a biologically meaningful photoperiodic cue, not as a fixed threshold date. We now emphasise that the hypothesis concerns a seasonal reversal in responses to temperature structured around photoperiod, whose effective timing depends on developmental state, rather than a reversal occurring precisely on June 21. To avoid confusion, we have reworded phrases such as “summer solstice effect reversal” to “reversal of phenological responses to temperature after the summer solstice” (L371). In accordance, we have also changed the title to “Developmental constraints mediate the reversal of temperature effects on the autumn phenology of European beech after the summer solstice”.

The following comments stem from the first round of review. We have previously revised the manuscript in accordance with these comments. For most of these points we do not see further cause for changes except for any overlap with comments above. We therefore predominantly copy our previous responses in quotes for clarity, the exception being the comment regarding the framing of our results in relation to natural systems.

The comments below relate to my original review with many of them still applying.

Methods: As I read the Results I was surprised the authors did not give more info on the methods here. For example, they refer to the 'effect of July cooling' but never say what the cooling was. Once I read the methods I feared they were burying this as the methods feel quite extreme given the framing of the paper.

“We understand the concern regarding the structure of the manuscript and note that the methods section was moved to the end of the paper in accordance with eLife’s recommended formatting. We have now moved the methods section before the results to ensure that readers are familiar with the treatments before encountering the outcomes.

Regarding presentation, treatment details are now described in both the Methods and the relevant figure legends. Given this structure, we have chosen not to restate the full treatment conditions in the main Results text to avoid repetition.”

The paper is framed as explaining observational results of natural systems, but the treatments are not natural for any system in Europe of which I have worked in. For example a low of 2 deg C at night and 7 deg C during the day through end of May and then 7/13 deg C in July is extreme. I think these methods need to be clearly laid out for the reader so they can judge what to make of the experiment before they see the results.

We appreciate the reviewer’s concern regarding the use of relatively extreme temperature treatments and the need to ensure that our conclusions are consistent with the motivation for using them. The manuscript was also revised in this regard in the previous round, and we copy the relevant responses at the bottom of this response. Despite this, we agree that further explanation of how our experimental treatments suited the aims of our study was still required.

The aim of these treatments was not to reproduce typical ambient conditions, but to act as a mechanistic probe. Such mechanisms are not readily identifiable from observations or mild manipulations, because the expected effects are small relative to natural variability; stronger perturbations are therefore required to generate a diagnostic contrast. By strongly constraining development in the early-season, and by providing a robust cooling signal in the late-season, we sought to reveal the causal structure underlying the observed solstice-related reversal in temperature effects on autumn phenology.

Temperatures below 10°C intensively slow down cell division and mitotic rates, these rates then rapidly and non-linearly approach 0 as temperatures drop towards 0°C (Körner, 2021). As reflected in L152-158 of the revised manuscript, we selected a spring cooling regime of 2–7 °C to strongly slow developmental processes while maintaining a clear thermal safety margin that eliminates the risk of frost damage. Although a milder cooling regime (e.g. 5–10 °C) would be less extreme, it would also be expected to produce only a comparatively small reduction in developmental rates, thereby substantially reducing our ability to generate distinct early- and late-developing individuals and to detect carry-over effects on autumn phenology. Applying strong cooling therefore increases signal-to-noise and allows us to detect the underlying mechanism, which would not be possible with temperature treatments that represent average contemporary climatic variation.

The use of conditions out with the norm is a standard practice to elucidate mechanisms in ecology, where organisms are often pushed to their physiological limits or transplanted into environments fundamentally different to those which they are adapted (Somero, 2010; Berend et al., 2019). Experiments targeting autumn phenology have utilised a broad range of environmental conditions from moderate to extreme manipulations (Tanino et al., 2010). For example, to test the controls of growth cessation and dormancy induction in Prunus species, one study applied a range of treatments including constant 9°C temperature and 24 hour photoperiod between April and July (Heide, 2008).

Our experimental design aimed to reduce rates of development, cell division and maturation. In the Methods, we describe this aim and clearly state that the experimental design was not intended to mimic natural climatic variation (L154-156 & L181-186). Importantly, our conclusions are framed at the level of direction, timing, and interaction of effects, rather than the magnitude expected under contemporary or future field conditions (L360-363).

This framing intends to reflect the primary inference of this study, which concerns when and why temperature effects reverse around the solstice, and how this timing depends on developmental state and diel temperature exposure, rather than making quantitative predictions for present-day or future climates. This aligns our conclusions with the experimental design. We have further revised the Discussion to explain these aims and conclusions more clearly, including the addition of a subsection at the beginning titled “Experimental forcing and scope of inference” (L346-363). We have also set up this expectation in the Introduction (L113-115).

Additionally, we have improved the Discussion in a number of related aspects.

We explicitly separate mechanistic conclusions and any relation to natural systems, remaining cautious to not overgeneralise or overstate our findings (L417-419).

We now include a dedicated paragraph explaining that, although these specific conditions are not likely to be found in beech’s range, analogous developmental constraints can arise during cold springs, late cold spells following budburst, or at high-elevation and continental sites where temperatures remain low despite increasing photoperiod (L540-545, L583-588). We further explain that because developmental progression integrates temperature cumulatively over time, even short episodes of strong cooling can exert lasting carry-over effects on seasonal timing, thereby linking the forced experimental responses to processes relevant under natural, fluctuating conditions (L545-550).

We explicitly state that the decoupling of day and night temperatures was not intended to represent realistic meteorological states (L458-460). We explain that this design was used diagnostically to isolate inherently diel physiological processes (e.g. nocturnal growth, cell division and expansion versus daytime carbon assimilation), and that the observed responses demonstrate the importance of diel timing of temperature exposure rather than the realism of the imposed cycles (L460-468).

Previous response:

We recognise that our temperature treatments were severe and do not mimic real world scenarios. They were deliberately designed to create large contrasts in developmental rates, thereby maximising our ability to detect the mechanisms underpinning the solstice switch. For example, the severe cooling between 4 April and 24 May was specifically designed to slow spring development as much as possible without damaging the plants. We have added text in the Methods to clarify this aim.

I also think the control is confounded with growth chamber experience in Experiment 1. That is, the control plants never experience any time in a chamber, but all the treatments include significant time in a chamber. The authors mention how detrimental chamber time can be to saplings (indeed, they mention an aphid problem in experiment 2) so I think they need to be more upfront about this. The study is still very valuable, but -- again -- we may need to be more cautious in how much we infer from the results.

We appreciate the reviewer’s concern about the potential confounding effect of chamber exposure in experiment 1. We have now discussed this limitation more explicitly, adding further explanation to the Methods and Discussion.

Note that chamber-related problems (e.g. aphid infestations) primarily occurred under warm chamber conditions, whereas our experiment 1 cooling treatments maintained low temperatures that suppressed such issues. This means that an equivalent “warm chamber control” could have been associated with its own artefacts, as trees kept under warm chamber conditions would have been exposed to additional stressors that were not present under natural growing conditions. To address this point, we included a chamber control in experiment 2. While aphid abundance was indeed higher in the warm chamber controls, chamber exposure itself had no detectable effect on autumn phenology. This suggests that the main findings of experiment 1 are unlikely to be artefacts of chamber conditions.

Nevertheless, we agree that chamber exposure remains a potential limitation of experiment 1, which requires clear acknowledgement. We now state this more explicitly in the manuscript while also emphasising that our results are supported by experiment 2 and by converging lines of external evidence.

Also, I suggest the authors add a figure to explain their experiments as they are very hard to follow. Perhaps this could be added to Figure 1?

We have now added figures to the methods section to depict the experimental timelines and settings more clearly (Figs. 2 and 3).

Finally, given how much the authors extrapolate to carbon and forests, I would have liked to see some metrics related to carbon assimilation, versus just information on timing.

We agree that carbon assimilation is an important component of forest carbon dynamics. However, the primary aim of this study was to identify how developmental state and diel cycles mediate temperature effects on autumn phenology, rather than to quantify carbon assimilation per se. Assessing photosynthetic controls on autumn phenology would require a substantially different experimental design and is therefore beyond the scope of the present study.

That said, we were able to include measurements of photosynthetic assimilation during pre-solstice cooling (now presented as Fig. S12 for all treatments). These data show that cooling strongly reduced assimilation across all treatments, despite their markedly different phenological outcomes. This supports our interpretation that variation in assimilation alone cannot explain the observed phenological responses, consistent with previous manipulative and observational studies reporting a weak role of late-season assimilation in controlling autumn phenology.

Fagus sylvatica: Fagus sylvatica is an extremely important tree to European forests, but it also has outlier responses to photoperiod and other cues (and leafs out very late) so using just this species to then state 'our results likely are generalisable across temperate tree species' seems questionable at best.

We agree that Fagus sylvatica has a stronger photoperiod dependence than many other European tree species. As we note in our response to Reviewer 1, our findings align with previous research across temperate northern forests. Within our framework, interspecific variation in leaf-out timing would not alter the overall response pattern, though it could shift the specific timing of effect reversals. For example, earlier-leafing species may approach completion of development sooner and thus show sensitivity to late-season cooling earlier than F. sylvatica. Nevertheless, we acknowledge the importance of not overstating generality. We have therefore revised the manuscript to phrase conclusions more cautiously and highlight the need for further research across species.

And the referenced response to Reviewer one:

We agree that extrapolation from our experiments on Fagus sylvatica to other species and natural forests requires caution. However, it is precisely the controlled nature of our design that allowed us to isolate the precise mechanisms that appear to underpin the solstice switch, highlighting the role of diel and seasonal temperature variation. In natural systems, additional variables such as competition, precipitation, and soil heterogeneity can strongly influence phenology, but they also make it difficult to disentangle causal mechanisms. By minimising these confounding factors, our experiment provided a clear test of how temperature before and after the solstice regulates growth cessation.

To acknowledge the limitation, we have toned down statements about generalisation (e.g. “likely generalisable” to “other temperate tree species may display similarities”) and explicitly call for follow-up studies across species and forest contexts. At the same time, we highlight that our findings align with independent evidence from manipulative experiments, satellite observations, flux measurements, and groundbased phenology, which suggests the mechanisms we report may extend beyond the specific populations studied here.”

As described in responses above, we have further clarified what can be directly concluded from our study, avoiding overgeneralisation.

Measuring end of season (EOS): It's well known that different parts of plants shut down at different times and each metric of end of season -- budset, end of radial expansion, leaf coloring etc. -- relate to different things. Thus I was surprised that the authors ignore all this complexity and seem to equate leaf coloring with budset (which can happen MONTHS before leaf coloring often) and with other metrics. The paper needs a much better connection to the physiology of end of season and a better explanation for the focus on budset. Relatedly, I was surprised the authors cite almost none of the literature on budset, which generally suggests is it is heavily controlled by photoperiod and population-level differences in photoperiod cues, meaning results may different with a different population of plants. 

We thank the reviewer for pointing out that our discussion of the responses of different EOS metrics needs more clarity. We agree with much of this perspective, and we have added an additional analysis of leaf chlorophyll content data to use leaf discolouration as an alternative EOS marker. On this we would like to make two important points:

Firstly, we agree that bud set often occurs before leaf discolouration, although this can depend on which definition of leaf discolouration is used. In experiment 1, budset occurred on average on day-of-year (DOY) 262 and leaf senescence (50% loss of leaf chlorophyll) occurred on DOY 320. However, we do not necessarily agree that this excludes the combined discussion of bud set and leaf senescence timing. Whilst environmental drivers can affect parts of plants differently, often responses from different end-of-season indicators (e.g. bud set and loss of leaf chlorophyll) are similar, even if only directionally. Figure S11 shows how, across both experiments, treatment effects were tightly conserved (R² = 0.49) amongst the two phenometrics. In accordance with these revisions, we have updated the manuscript title to “Developmental constraints mediate the summer solstice reversal of climate effects on the autumn phenology of European beech”.

Secondly, shifts in bud set timing remain the primary focus of the manuscript as these shifts are of direct physiological relevance to plant development and dormancy induction, whereas leaf discolouration may simply follow bud set as a symptom of developmental completion. This is supported by our results, which show stronger responses of bud set than leaf senescence (Figs. 4 & 5 vs. Figs. S9 & S10).

Following the reviewer’s suggestion, we have included more references on the topic of bud set and its environmental controls. The reviewer rightly stresses that photoperiod is considered the most important factor. Photoperiod is therefore key in our conceptual model. However, the responses we observed in F. sylvatica cannot be explained by photoperiod alone. For example, in experiment 1, July cooling delayed the autumn phenology of late-leafing trees but had negligible impact on early-leafing trees, even though both experienced the exact same photoperiod. Moreover, in experiment 2, day, night and full-day cooling showed substantial variations in their effects despite equal photoperiod across the climate regimes. This is why we suggest that the annual progression of photoperiod modulates the responses to temperature variations instead of eliciting complete control.

Following the addition of an analysis of leaf senescence data, we also revised the terminology in places (including the title) from “primary growth cessation/bud set” to the broader term “autumn phenology.” This term is intended to encompass two distinct but related physiological processes—bud set and leaf senescence—both of which are commonly used as markers of autumn phenology and the end of the growing season.

Somewhat minor comments:

(1) How can a bud type -- which is apical or lateral -- be a random effect? The model needs to try to estimate a variance for each random effect so doing this for n=2 is quite odd to me. I think the authors should also report the results with bud type as fixed, or report the bud types separately.

We have revised the analysis to include bud type as a fixed effect. There are only very minor numerical adjustments (e.g. rounding to 4.8 days instead of 4.9) and inferences are not altered. We also report the bud type effects for experiment 1 and experiment 2.

(2) I didn't fully see how the authors results support the Solstice as Switch hypothesis, since what timing mattered seemed to depend on the timing of treatment and was not clearly related to solstice. Could it be that these results suggest the Solstice as Switch hypothesis is actually not well supported (e.g., line 135) and instead suggest that the pattern of climate in the summer months affects end of season timing?

Our responses to the main comments in this new round of revision have comprehensively covered this topic.

References

Berend K, Haynes K, MacKenzie CM. 2019. Common garden experiments as a dynamic tool for ecological studies of alpine plants and communities in northeastern North America. Rhodora 121: 174.

Heide OM. 2008. Interaction of photoperiod and temperature in the control of growth and dormancy of Prunus species. Scientia Horticulturae 115: 309–314.

Körner C. 2021. Alpine Plant Life: Functional Plant Ecology of High Mountain Ecosystems. Cham: Springer International Publishing.

Somero GN. 2010. The physiology of climate change: how potentials for acclimatization and genetic adaptation will determine ‘winners’ and ‘losers’. Journal of Experimental Biology 213: 912–920.

Tanino KK, Kalcsits L, Silim S, Kendall E, Gray GR. 2010. Temperature-driven plasticity in growth cessation and dormancy development in deciduous woody plants: a working hypothesis suggesting how molecular and cellular function is affected by temperature during dormancy induction. Plant Molecular Biology 73: 49–65.

Reviewer #1 (Public review):

Summary:

The authors use a gambling task with momentary mood ratings from Rutledge et al. and compare computational models of choice and mood to identify markers of decisional and affective impairments underlying risk-prone behavior in adolescents with suicidal thoughts and behaviors (STB). The results show that adolescents with STB show enhanced gambling behavior (choosing the gamble rather than the sure amount), and this is driven by a bias towards the largest possible win rather than insensitivity to possible losses. Moreover, this group shows a diminished effect of receiving a certain reward (in the non-gambling trials) on mood. The results were replicated in a general online sample where participants were divided into groups with or without STB based on their self-report of suicidal ideation on one question in the Beck Depression Inventory self-report instrument. The authors suggest, therefore, that adolescents diagnosed with depression or anxiety with decreased sensitivity to certain rewards may need to be monitored more closely for STB due to their increased propensity to take risky decisions aimed at (expected) gains (such as relief from an unbearable situation through suicide) regardless of the potential losses. However, such a result was only found in the clinical sample and cannot be generalized more broadly based on the current findings.

Strengths:

(1) The study uses a previously validated task design and replicates previously found results through well-explained model-free and model-based analyses.

(2) Sampling of adolescents at high risk can help target early preventative diagnoses and treatments for suicide.

(3) Replication of the results in an online cohort increases confidence in the findings.

(4) The models considered for comparison are thorough and well-motivated. The chosen models allow for teasing apart which decision and mood sensitivity parameters relate to risky decision-making across groups based on their hypotheses.

(5) Novel finding of mood (in)sensitivity to non-risky rewards and its relationship with risk behavior in STB.

Weaknesses:

(1) Sample size of 25 for S- group is low-powered, which is explicitly mentioned as a study limitation.

(2) Modeling in the mediation analysis focused on predicting risk behavior in this task from the model-derived bias for gains and suicidal symptom scores. Thus, the implications of this work are more relevant to a basic-science understanding of the etiology of suicidal behavior than they are useful as a predictor of suicidal behavior, and it is not clear that a psychiatrist or psychologist could use this task to potentially determine who is at higher risk of attempting suicide and must be more closely monitored. Indeed, relationships between task parameters and behavior and suicidal behavior was limited to the clinical sample with a diagnosis of depression or anxiety disorder, and did not extend to the online sample. Therefore, the claim that these findings provide "computational markers for general suicidal tendency among adolescents" is unwarranted.

Reviewer #2 (Public review):

Summary:

This article addresses a very pertinent question - what are the computational mechanisms underlying risky behaviour in patients having attempted suicide. In particular, it is impressive how the authors find a broad behavioral effect whose mechanisms they can then explain and refine through computational modeling. This work is important because currently, beyond previous suicide attempts, there has been a lack of predictive measures. This study is the first step towards that: understanding the cognition on a group level. Before then being able to include it in future predictive studies (based on the cross-sectional data, this study by itself cannot assess the predictive validity of the measure).

Strengths:

- Large sample size<br /> - Replication of their own findings<br /> - Well-controlled task with measures of behaviour and mood + precise and well-validated computational modeling

Questions, based on revised manuscript and replies to other reviewers:

(1) Replies to reviewers in general: Bayes Factors have been added, it would be good to also use common verbal terms to describe them (e.g. 'anecdotal', 'moderate' etc). For example, my reading of table S8 would be that for gambling rate there is only anecdotal evidence that it does not relate to PSWQ, BDI, and moderate evidence it does not relate to TAI.

(2) Reply to reviewer 1 Q2 (Predicting STB):<br /> For the regression predicting suicidal ideation, it seems to me that what you did was a regression STB ~ gambling behaviour + approach + mood? Could you clarify? I had expected as a test of whether the task can predict STB risk something slightly different - a cross-validation (LOO or maybe 5-fold in the large sample): STB ~ gambling behaviour + approach [parameter from model] + mood [parameter from model]; and then computing in the left out participants: predicted STB. Then checking correlation between STB and predicted STB. This would allow testing whether the diverse task measures together predict STB (with the caveat, that it's cross-validated, rather than hold-out sample, unless you could train on one sample (in lab) and test on the other (online).

(3) Reply to reviewer 2 Q1 (parameter recovery): I'm looking at S3, it seems to still show only the scatter plots and not the correlation matrices, which are now added as text notes. Can you actually show these matrices? An off-diagonal correlation of 0.63 appears quite high. I think it needs to be discussed exactly which parameters those are, and whether that impacts the interpretation of the results.

(4) Reply to reviewer 3 Q3 (mood model): I would have imagined that the response would involve changing the mood equations (equation 8 main text) to include a term for whether the participant gambled or not, independent of the gamble value.

Author response:

The following is the authors’ response to the original reviews

eLife Assessment

This valuable study combined careful computational modeling, a large patient sample, and replication in an independent general population sample to provide a computational account of a difference in risk-taking between people who have attempted suicide and those who have not. It is proposed that this difference reflects a general change in the approach to risky (high-reward) options and a lower emotional response to certain rewards. Evidence for the specificity of the effect to suicide, however, is incomplete, which would require additional analyses.

We thank the editors and reviewers for this important assessment. Based on clinical interviews, we included patients with and without suicidality (S⁺ and S^- groups). However, in line with suicidal-related literature (e.g., Tsypes et al., 2024), two groups also differed substantially in the severity of symptoms (see Table 1). To address the request for evidence on specificity to suicidality beyond general symptom severity, we performed separate linear regressions to explain in gambling behaviour, value-insensitive approach parameter (β_gain), and mood sensitivity to certain rewards (β_CR) with group as a predictor (1 for S⁺ group and 0 for S^- group) and scores for anxiety and depression as covariates. Results remained significant after controlling anxiety and depression (ps < 0.027; Table S8). Given high correlations among anxiety and depression questionnaires (rs > 0.753, ps < 0.001), we performed Principal Components Analysis (PCA) on the clinical questionnaire to extract the orthogonal components, where each component explained 86.95%, 7.09%, 3.27%, and 2.68% variance, respectively. We then performed linear regressions using these components as covariates to control for anxiety and depression. Our main results remained significant (ps < 0.027; Table S9). We believe that these analyses provide evidence that the main effects on gambling and on mood were specific to suicide.

Moreover, as Reviewer 3 pointed out, these “absence of evidence” cannot provide insights of “evidence of absence”. Although we median-split patients by the scores of general symptoms (e.g., depression and anxiety-related questionnaires) and verified no significant differences in these severities (Figure S11), we additionally conducted Bayesian statistics in gambling behavior, value-insensitive approach parameter, and mood sensitivity to certain rewards. BF₀₁ is a Bayes factor comparing the null model (M₀) to the alternative model (M₁), where M₀ assumes no group difference. BF₀₁ > 1 indicates that evidence favors M₀. As can be seen in Table S7, most results supported null hypothesis, suggesting that general symptoms of anxiety and depression overall did not influence our main results. Overall, we believe that these analyses provide compelling evidence for the specificity of the effect to suicide, above and beyond depression and anxiety.

Beyond these specific findings, this work highlights the broader utility of computational modelling and mood to better understand behavioral effect, showing how to use both mood and choice data to better comprehend a psychiatric issue. 

Public Reviews:

Reviewer #1 (Public review):

Summary:

The authors use a gambling task with momentary mood ratings from Rutledge et al. and compare computational models of choice and mood to identify markers of decisional and affective impairments underlying risk-prone behavior in adolescents with suicidal thoughts and behaviors (STB). The results show that adolescents with STB show enhanced gambling behavior (choosing the gamble rather than the sure amount), and this is driven by a bias towards the largest possible win rather than insensitivity to possible losses. Moreover, this group shows a diminished effect of receiving a certain reward (in the non-gambling trials) on mood. The results were replicated in an undifferentiated online sample where participants were divided into groups with or without STB based on their self-report of suicidal ideation on one question in the Beck Depression Inventory self-report instrument. The authors suggest, therefore, that adolescents with decreased sensitivity to certain rewards may need to be monitored more closely for STB due to their increased propensity to take risky decisions aimed at (expected) gains (such as relief from an unbearable situation through suicide), regardless of the potential losses.

Strengths:

(1) The study uses a previously validated task design and replicates previously found results through well-explained model-free and model-based analyses.

(2) Sampling choice is optimal, with adolescents at high risk; an ideal cohort to target early preventative diagnoses and treatments for suicide.

(3) Replication of the results in an online cohort increases confidence in the findings.

(4) The models considered for comparison are thorough and well-motivated. The chosen models allow for teasing apart which decision and mood sensitivity parameters relate to risky decision-making across groups based on their hypotheses.

(5) Novel finding of mood (in)sensitivity to non-risky rewards and its relationship with risk behavior in STB.

Weaknesses:

(1) The sample size of 25 for the S- group was justified based on previous studies (lines 181-183); however, all three papers cited mention that their sample was low powered as a study limitation.

We thank the Reviewer for rising this concern. We agree that the sample size for S^- group (n=25) is modest, and the prior studies we cited also acknowledged limited power. We wanted to point out that we obtained a comparable sample size to a prior study. In the revision, we therefore updated the section to justify this sample size in which we acknowledge the limited power of our study in the limitation section. Please see our clarification below:

Page 32:

“Third, despite replicating our main results in an independent dataset (n=747), the modest S^- subgroup size (n=25) has a limited statistical power.”

(2) Modeling in the mediation analysis focused on predicting risk behavior in this task from the model-derived bias for gains and suicidal symptom scores. However, the prediction of clinical interest is of suicidal behaviors from task parameters/behavior - as a psychiatrist or psychologist, I would want to use this task to potentially determine who is at higher risk of attempting suicide and therefore needs to be more closely watched rather than the other way around (predicting behavior in the task from their symptom profile). Unfortunately, the analyses presented do not show that this prediction can be made using the current task. I was left wondering: is there a correlation between beta_gain and STB? It is also important to test for the same relationships between task parameters and behavior in the healthy control group, or to clarify that the recommendations for potential clinical relevance of these findings apply exclusively to people with a diagnosis of depression or anxiety disorder. Indeed, in line 672, the authors claim their results provide "computational markers for general suicidal tendency among adolescents", but this was not shown here, as there were no models predicting STB within patient groups or across patients and healthy controls.

Thank you for these thoughtful comments. Our study focuses on why adolescent patients with suicidality have increased risk behavior, aiming to provide a mechanism-based target for suicide prevention. Therefore, our dependent variable in the mediation model was gambling behavior. We also agree that the clinically relevant question is whether suicidality can be predicted from task-derived behavior/parameters. We thus used risky behavior and the potential mental parameters to predict STB. Linear regressions showed that gambling behavior, as well as the value-insensitive approach parameter, can predict suicidal symptom scores among patients (former: β = 9.189, t = 2.004, p = 0.048; latter: β = 5.587, t = 2.890, p = 0.005). In healthy controls, these predictions failed (gambling behavior: β = 1.471, t = 0.825, p = 0.411; approach: β = 0.874, t = 1.178, p = 0.241). These results suggest that clinical relevance of these findings apply exclusively to people with a diagnosis of depression or anxiety disorder. We found same patterns for the mood parameter (mood sensitivity to certain rewards: patients: β = -28.706, t = -2.801, p = 0.006; healthy controls: β = -2.204, t = -0.528, p = 0.599). In sum, we believe that our statement of “computational markers for general suicidal tendency among adolescents” is reasonable now. Please see our revisions below:

Page 17:

“Furthermore, linear regression showed that gambling rate can predict the current suicidal ideation score (BSI-C, β = 9.189, t = 2.004, p = 0.048) among patients, but not among HC (β = 1.471, t = 0.825, p = 0.411), suggesting that gambling behavior has patient-specific predictive utility for suicidal symptoms.”

Page 19:

“Furthermore, linear regression showed that approach parameter can predict the current suicidal ideation score (β = 5.587, t = 2.890, p = 0.005) among patients, but not among HC (β = 0.874, t = 1.178, p = 0.241), suggesting that value-insensitive approach parameter has patient-specific predictive utility for suicidal symptoms.”

Page 21:

“Furthermore, linear regression showed that mood sensitivity to CR can predict the current suicidal ideation score (β = -28.706, t = -2.801, p = 0.006) among patients, but not among HC (β = -2.204, t = 0.528, p = 0.599), suggesting that mood sensitivity to CR has patient-specific predictive utility for suicidal symptoms.”

(3) The FDR correction for multiple comparisons mentioned briefly in lines 536-538 was not clear. Which analyses were included in the FDR correction? In particular, did the correlations between gambling rate and BSI-C/BSI-W survive such correction? Were there other correlations tested here (e.g., with the TAI score or ERQ-R and ERQ-S) that should be corrected for? Did the mediation model survive FDR correction? Was there a correction for other mediation models (e.g., with BSI-W as a predictor), or was this specific model hypothesized and pre-registered, and therefore no other models were considered? Did the differences in beta_gain across groups survive FDR when including comparisons of all other parameters across groups? Because the results were replicated in the online dataset, it is ok if they did not survive FDR in the patient dataset, but it is important to be clear about this in presenting the findings in the patient dataset.

Thank you for raising the important issue of multiple testing and for asking us to clarify exactly which tests were covered by the FDR procedure. In the clinical dataset we conducted a large number of inferential tests (χ², t-tests, ANOVAs, regressions) spanning: (i) group differences in demographic/clinical characteristics; (ii) sanity checks (e.g., anxiety/depression questionnaires); (iii) primary hypotheses (e.g., group differences in risky behavior); (iv) model-based analyses (parameter checks and between-group contrasts); and (v) control/sensitivity analyses. Post-hoc t-tests were performed only when the three-group ANOVA was significant. This yielded >150 p-values. FDR was applied using all these p-values. Please see our clarification below:

Supplementary Page 4:

“Supplementary Note 8: Clarification for FDR correction.

In the clinical dataset we conducted a large number of inferential tests (χ<sup2\</sup>, t-tests, ANOVAs, regressions) spanning: (i) group differences in demographic/clinical characteristics; (ii) sanity checks (e.g., anxiety/depression questionnaires); (iii) primary hypotheses (e.g., group differences in risky behavior); (iv) model-based analyses (parameter checks and between-group contrasts); and (v) control/sensitivity analyses. Post-hoc t-tests were performed only when the three-group ANOVA was significant. This yielded >150 p-values. FDR was applied using all these p-values.”

(4) There is a lack of explicit mention when replication analyses differ from the analyses in the patient sample. For instance, the mediation model is different in the two samples: in the patient sample, it is only tested in S+ and S- groups, but not in healthy controls, and the model relates a dimensional measure of suicidal symptoms to gambling in the task, whereas in the online sample, the model includes all participants (including those who are presumably equivalent to healthy controls) and the predictor is a binary measure of S+ versus S- rather than the response to item 9 in the BDI. Indeed, some results did not replicate at all and this needs to be emphasized more as the lack of replication can be interpreted not only as "the link between mood sensitivity to CR and gambling behavior may be specifically observable in suicidal patients" (lines 582-585) - it may also be that this link is not truly there, and without a replication it needs to be interpreted with caution.

Thank you for these important comments. This study focused on cognitive and affective computational mechanisms underlying increased risky behavior in STB. Accordingly, we compared patients with STB (S⁺) with patients without STB (S^-) and healthy controls (HC) to examine the effects of STB on risky behavior. Therefore, group comparison, instead of dimensional measure of suicidal symptoms by Beck Scale for Suicidal Ideation, can answer our research questions directly.

To enhance consistency between the clinical and replication datasets, we included all participants in each dataset when performing the mediation analysis. Given that S^- and HC did not differ in gambling behavior or the approach parameter in the clinical dataset, we merged these two groups. In the replication dataset, to mirror the S⁺ vs. S^- contrast used clinically, we categorized the general sample into S+ and S^- based on BDI item 9. The mediation results remained significant in both datasets (the clinical dataset: a×b = 0.321, 95% CI = [0.070, 0.549], p = 0.016; the replication dataset: a×b = 0.143, 95% CI = [0.016, 0.288], p = 0.031), suggesting that STB is associated with increased risk behavior via stronger approach motivation.

We also acknowledge the non-replication of the correlation between gambling behavior and mood sensitivity to certain rewards in the online sample. While this pattern might indicate that the link is specific to suicidal patients, it may also reflect sample-specific or unstable effects; thus, we now state this explicitly and interpret the finding with caution. Please see our revisions below:

Page 15:

“We next verified our results in an independent dataset, including the same task and BDI questionnaire in 747 general participants (500 females; age: 20.90±2.41) (46). One item in BDI involves the measurement of STB. In item 9 of BDI, participants chose one option that describes them best: Option 1, “I don't have any thoughts of killing myself.”; Option 2, “I have thoughts of killing myself, but I would not carry them out.”; Option 3, “I would like to kill myself.”; Option 4, “I would kill myself if I had the chance.”. In line with the current definition of S⁺/S^- in the clinical dataset, we identified S⁺ group as choosing Option 2, 3, or 4, while participants selecting Option 1 were categorized as S^- group.”

Page 19:

“Given significant correlations between group, approach parameter, and gambling rate for gain trials (ps < 0.017), we further conducted a mediation analysis with the assumption of the mediating effect of approach motivation of suicidality on the risk behavior. Given that we aimed to test the effect of STB, with S^- and HC as controls, and given that S^- and HC did not differ in gambling behavior or in the approach parameter, we merged these two groups for the mediation analysis. Results supported our hypothesis (a×b = 0.321, 95% CI = [0.070, 0.549], p = 0.016; Figure 2C), confirming that suicidal thoughts and behavior increase risk behavior through stronger approach motivation.”

Page 26:

“However, we did not observe any significant correlation between mood sensitivity to CR and gambling behavior (ps > 0.389), which suggests that the link between mood sensitivity to CR and gambling behavior may be specifically observable in suicidal patients. Alternatively, this non-replicated result may also reflect sample-specific or unstable effects, which needs to be interpreted with caution.”

(5) In interpreting their results, the authors use terms such as "motivation" (line 594) or "risk attitude" (line 606) that are not clear. In particular, how was risk attitude operationalized in this task? Is a bias for risky rewards not indicative of risk attitude? I ask because the claim is that "we did not observe a difference in risk attitude per se between STB and controls". However, it seems that participants with STB chose the risky option more often, so why is there no difference in risk attitude between the groups?

Thank you for pointing out the ambiguity. In our manuscript, “motivation” and “risk attitude” are defined at the computational level. Following prior work with this task Rutledge et al., (2015, 2016), we decompose observed gambling into (i) value-dependent valuation parameters that capture risk attitude (e.g., risk aversion and loss aversion, which scale the subjective value of outcomes), and (ii) value-insensitive, valence-dependent biases that capture approach/avoidance motivation. Accordingly, a higher gambling rate does not imply a change in risk attitude per se: it can arise from an increased value-insensitive approach bias even when risk-attitude parameters are comparable between groups—which is what we observe for S⁺ vs. controls. We have clarified this point in the computational modeling section.

Pages 12-13:

“Please note that a higher gambling rate does not imply a change in risk attitude per se: it can arise from an increased value-insensitive approach bias even when risk-attitude parameters are comparable between groups. Risk attitude is indeed conceptualized in economics as the curvature of the utility function (i.e., the subjective value) of the objective outcomes, with concave curves associated with risk aversion, and convex curves associated with risk seeking (54,56). By contrast, the approach or avoidance bias apply to all the value. A possible interpretation of the approach bias is that participant approach the option with the highest possible gain (the lottery) in the gain frame; the avoidance bias would then reflect a tendency to systematically avoid the highest potential losses (the lottery) in the loss frame.”

Reviewer #2 (Public review):

Summary:

This article addresses a very pertinent question: what are the computational mechanisms underlying risky behaviour in patients who have attempted suicide? In particular, it is impressive how the authors find a broad behavioural effect whose mechanisms they can then explain and refine through computational modeling. This work is important because, currently, beyond previous suicide attempts, there has been a lack of predictive measures. This study is the first step towards that: understanding the cognition on a group level. This is before being able to include it in future predictive studies (based on the cross-sectional data, this study by itself cannot assess the predictive validity of the measure).

Strengths:

(1) Large sample size.

(2) Replication of their own findings.

(3) Well-controlled task with measures of behaviour and mood + precise and well-validated computational modeling.

Weaknesses:

I can't really see any major weakness, but I have a few questions:

(1) I can see from the parameter recovery that the parameters are very well identified. Is it surprising that this is the case, given how many parameters there are for 90 trials? Could the authors show cross-correlations? I.e., make a correlation matrix with all real parameters and all fitted parameters to show that not only the diagonal (i.e., same data is the scatter plots in S3) are high, but that the off-diagonals are low.

Thank you for raising these thoughtful concerns. The current task consisted of 90 choices and 36 mood ratings. There were 5 choice parameters and 4 mood parameters. The apparently strong identifiability is not unexpected, as 90 choice trials and 36 mood ratings are comparable to those in prior computational modeling literature (Blain & Rutledge, 2022).

As suggested, we computed cross-correlations between all generating (“true”) and recovered (“fitted”) parameters. The resulting matrix showed high diagonal (choice winning model: rs > 0.91; mood winning model: rs > 0.90) and low off-diagonal (choice winning model: abs(rs) < 0.63; mood winning model: abs(rs) > 0.40) correlations, further supporting parameter recovery. Please see our clarifications below:

Supplementary Pages 2-3:

“Parameter recovery: Figure S3 shows good parameter recovery for both choice and mood winning model (choice: rs > 0.91, ps < 0.001; intraclass coefficients > 0.78; mood: rs > 0.90, ps < 0.001; intraclass coefficients > 0.86). Moreover, we computed cross-correlations between all generating (“true”) and recovered (“fitted”) parameters. The resulting matrix showed high diagonal (choice winning model: rs > 0.91; mood winning model: rs > 0.90) and low off-diagonal (choice winning model: abs(rs) < 0.63; mood winning model: abs(rs) > 0.40) correlations, further supporting parameter recovery.”

Page 10：

“The numbers of choice trials and mood ratings were comparable to those in prior computational modeling studies (34,35).”

(2) Could the authors clarify the result in Figure 2B of a correlation between gambling rate and suicidal ideation score, is that a different result than they had before with the group main effect? I.e., is your analysis like this: gambling rate ~ suicide ideation + group assignment? (or a partial correlation)? I'm asking because BSI-C is also different between the groups. [same comment for later analyses, e.g. on approach parameter].

Thank you for pointing out the lack of clarity. We performed group difference analysis and correlation of suicidal ideation analysis, separately. We first performed group difference analysis to test our hypothesis of STB effects. We then conducted correlational analysis to further specify our findings.

(3) The authors correlate the impact of certain rewards on mood with the % gambling variable. Could there not be a more direct analysis by including mood directly in the choice model?

Thank you for this insightful suggestion. As suggested, we tried to integrate mood into choice models by adding mood bias component(s) in line with previous literature (Vinckier et al., 2018). The first model (mcM1) assumes that mood biases choice, building on cM3 (the winning choice model). cmM2 further separated the mood bias parameter into two components according to participants’ choices.

However, model comparison using BIC supported cM3 (Table S6), that is, without consideration of mood in choice modeling. This can be due to the lack of block design in our experimental design unlike e.g., Vinckier et al., (2018) and Eldar & Niv, (2015). Please see our clarifications below:

Supplementary Pages 3-4:

“Supplementary Note 6: integration of mood into choice models

Although we modeled choice and mood separately to examine cognitive and affective mechanisms underlying increased risk behavior in adolescent suicidal patients, one interesting question was whether mood responses influence subsequent gambling choices and how to model them. First, we median-split mood responses (except the final rating) to compare gambling rate. Results showed a trend for less gambling rate in higher mood (t = -1.971, p = 0.050). However, there was no significant group difference (F = 0.680, p = 0.507). Second, with the assumption that mood biases choice, we constructed mcM1 based on cM3 (the winning choice model).

Based on our finding of the negative correlation between mood sensitivity to certain rewards and gambling rate in S⁺, we separated β_Mood parameter into β_Mood-CR and β_Mood-GR (cmM2).

Model comparison using BIC supported cM3 (Table S6), that is, without consideration of mood in choice modeling. The mood bias parameters in neither cM2 nor cM3 reached significance (ps > 0.091), which may be due to the absence of a blocked design in our experiment, unlike in Vinckier et al. (2018) and Eldar and Niv (2015).”

(4) In the large online sample, you split all participants into S+ and S-. I would have imagined that instead, you would do analyses that control for other clinical traits. Or, for example, you have in the S- group only participants who also have high depression scores, but low suicide items.

Thank you for this insightful suggestion. Following prior suicide-related literature (Tsypes et al., 2024), we controlled for depression by including them as covariates. Note that depression scores were derived from our established bifactor model (Wang et al., 2025), which decomposed depression from the anxiety. These results remained largely significant (ps ≤ 0.050), except a marginally significant effect of group on gambling behavior (p = 0.059). Despite a trend, this effect with covariates of depression-related questionnaires is strong in our clinical cohort (p = 0.024; Table S8). This suggests that the link between suicidality and risky behavior persists above and beyond general depressive symptoms.

Please see our clarifications below:

Page 26:

“After controlling for depression severity using our established bifactor model (see ref 60 for details), these results remained significant (ps ≤ 0.050), except a marginally significant effect of group on gambling behavior (p = 0.059). Despite a trend, this effect with covariates of depression-related questionnaires is strong in our clinical cohort (p = 0.024; Table S8). This suggests that the link between suicidality and risky behavior persists above and beyond general depressive symptoms.”

Reviewer #3 (Public review):

This manuscript investigates computational mechanisms underlying increased risk-taking behavior in adolescent patients with suicidal thoughts and behaviors. Using a well-established gambling task that incorporates momentary mood ratings and previously established computational modeling approaches, the authors identify particular aspects of choice behavior (which they term approach bias) and mood responsivity (to certain rewards) that differ as a function of suicidality. The authors replicate their findings on both clinical and large-scale non-clinical samples.

(1) The main problem, however, is that the results do not seem to support a specific conclusion with regard to suicidality. The S+ and S- groups differ substantially in the severity of symptoms, as can be seen by all symptom questionnaires and the baseline and mean mood, where S- is closer to HC than it is to S+. The main analyses control for illness duration and medication but not for symptom severity. The supplementary analysis in Figure S11 is insufficient as it mistakes the absence of evidence (i.e., p > 0.05) for evidence of absence. Therefore, the results do not adequately deconfound suicidality from general symptom severity.

Thank you for this important comment. Based on clinical interviews, we included patients with and without suicidality (S⁺ and S^- groups). However, in line with suicidal-related literature (e.g., Tsypes et al., 2024), two groups also differed substantially in the severity of symptoms (see Table 1). To address the request for evidence on specificity to suicidality beyond general symptom severity, we performed separate linear regressions to explain in gambling behaviour, value-insensitive approach parameter (β_gain), and mood sensitivity to certain rewards (β_CR) with group as a predictor (1 for S⁺ group and 0 for S^- group) and scores for anxiety and depression as covariates. Results remained significant after controlling anxiety and depression (ps < 0.027; Table S8). Given high correlations among anxiety and depression questionnaires (rs > 0.753, ps < 0.001), we performed Principal Components Analysis (PCA) on the clinical questionnaire to extract the orthogonal components, where each component explained 86.95%, 7.09%, 3.27%, and 2.68% variance, respectively. We then performed linear regressions using these components as covariates to control for anxiety and depression. Our main results remained significant (ps < 0.027; Table S9). We believe that these analyses provide evidence that the main effects on gambling and on mood were specific to suicide.

As pointed out, these “absence of evidence” cannot provide insights of “evidence of absence”. Although we median-split patients by the scores of general symptoms (e.g., depression and anxiety-related questionnaires) and verified no significant differences in these severities (Figure S11), we additionally conducted Bayesian statistics in gambling behavior, value-insensitive approach parameter, and mood sensitivity to certain rewards. BF₀₁ is a Bayes factor comparing the null model (M₀) to the alternative model (M₁), where M₀ assumes no group difference. BF₀₁ > 1 indicates that evidence favors M₀. As can be seen in Table S7, most results supported null hypothesis, suggesting that general symptoms of anxiety and depression overall did not influence our main results. Overall, we believe that these analyses provide compelling evidence for the specificity of the effect to suicide, above and beyond depression and anxiety.

Please see our revisions below:

Page 17:

“Within patients, this group effect on gambling rate remained significant after controlling for sex, illness duration, family history, diagnosis, and various medications use (ps < 0.05), as well as general symptoms (e.g., depression and anxiety; p = 0.024; also see Figure S11, Table S7 and Table S8). Given high correlations among anxiety and depression questionnaires (rs > 0.753, ps < 0.001), we performed Principal Components Analysis (PCA) to extract main components, where each component explained 86.95%, 7.09%, 3.27%, and 2.68% variance, respectively. To further control for anxiety and depression, linear regression using these components as covariates revealed that the group effect on gambling rate remained significant (p = 0.024; Table S9).”

Pages 18-19:

“Within patients, this group effect on the approach parameter remained significant after controlling for sex, illness duration, family history, diagnosis, and various medications use (ps < 0.05), as well as general symptoms (e.g., depression and anxiety; p = 0.027; also see Figure S11, Table S7 and Table S8). Linear regression using PCA components as covariates revealed that the group effect on approach parameter remained significant (p = 0.027; Table S9).”

Page 21:

“Within patients, this group effect on βCR remained significant after controlling for gambling rate, earnings, mood-related outcome effect, mood drift effect, sex, illness duration, family history, diagnosis, and various medications use (ps < 0.032), as well as general symptoms (e.g., depression and anxiety; p = 0.001; also see Figure S11, Table S7 and Table S8). Linear regression using PCA components as covariates revealed that the group effect on this mood parameter remained significant (p = 0.001; Table S9).”

(2) The second main issue is that the relationship between an increased approach bias and decreased mood response to CR is conceptually unclear. In this respect, it would be natural to test whether mood responses influence subsequent gambling choices. This could be done either within the model by having mood moderate the approach bias or outside the model using model-agnostic analyses.

Thank you for this important suggestion. As suggested, one interesting question was whether mood responses influence subsequent gambling choices and how to model them. First, we median-split mood responses (except the final rating) to compare gambling rate. Results showed a trend for less gambling rate in higher mood (t = -1.971, p = 0.050). However, there was no significant group difference (F = 0.680, p = 0.507). Second, with the assumption that mood biases choice, we constructed mcM1 based on cM3 (the winning choice model). Based on our finding of the negative correlation between mood sensitivity to certain rewards and gambling rate in S⁺, we separated β_Mood parameter into β_Mood-CR and β_Mood-GR (cmM2). Model comparison using BIC supported cM3 (Table S6), that is, without consideration of mood in choice modeling. This can be due to the lack of block design in our experimental design unlike e.g., Vinckier et al., (2018) and Eldar & Niv, (2015). Please see Supplementary Pages 3-4:

(3) Additionally, there is a conceptual inconsistency between the choice and mood findings that partly results from the analytic strategy. The approach bias is implemented in choice as a categorical value-independent effect, whereas the mood responses always scale linearly with the magnitude of outcomes. One way to make the models more conceptually related would be to include a categorical value-independent mood response to choosing to gamble/not to gamble.

We apologise for the unclear statement. The approach bias is implemented in choice as a continuous value-independent effect, ranging from -1 to 1.

It was true that the mood responses always scale with the magnitude of outcomes, since mood ratings were request after the outcomes. Therefore, mood parameters and the approach bias were both continuous.

We also attempted to integrate mood into choice modelling. See Response 2 for Reviewer 3 for details.

(4) The manuscript requires editing to improve clarity and precision. The use of terms such as "mood" and "approach motivation" is often inaccurate or not sufficiently specific. There are also many grammatical errors throughout the text.

Thank you for this important suggestion. We have now explained motivation and mood in the Introduction section and the computational modeling section. Please see our clarifications below:

Pages 3-4:

“A growing literature indeed shows that risky behavior can be far better explained after adding value-insensitive approach and avoidance components to prospect theory(18,19), that is by including a decision bias in favor of the highest gain (approach) and another decision bias against the lowest loss (avoidance), above and beyond options value difference. This class of models highlights the important role of value-insensitive motivational components in decision making in addition to risk attitude-driven valuation (e.g., loss/risk aversion)(20).”

Page 5:

“Although mood is thought to persist for hours, days, or even weeks(30-33), momentary mood, measured over the timescale in the laboratory setting, represents the accumulation of the impact of multiple events at the scale of minutes(30,32,34-38). Momentary mood external validity is demonstrated e.g., through its association with depression symptoms(37). Mood is different from emotions, which reflect immediate affective reactivity and is more transient (e.g., from surprise to fear)(31-33,39).”

We have corrected grammatical errors throughout the manuscript.

5) Claims of clinical relevance should be toned down, given that the findings are based on noisy parameter estimates whose clinical utility for the treatment of an individual patient is doubtful at best.

Thank you for this comment. We agree that we did not evaluate the noise in our estimate e.g., by assessing the test-retest reliability on the task parameters, which is outside the scope of the study, and it is indeed possible that parameter estimate is somehow noisy. Therefore, we tone down the clinical relevance of our results. Please see our revision below:

Page 32:

“Next, we did not evaluate the noise in our estimate e.g., by assessing the test-retest reliability on the task parameters and it is indeed possible that parameter estimate is somehow noisy.”

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

(1) Title: I believe "aberrant mood dynamics" is both too general and overstating the results of this study, which did not measure mood dynamics longitudinally. "Aberrant" is also overly pathologizing. I would suggest sticking more directly to the results, for instance, "Insensitivity of momentary mood to non-risky rewards in adolescent suicidal patients".

Thank you for this suggestion. We have now corrected it.

(2) Abstract: in line 61, "Our study uncovers the cognitive and affective mechanisms" suggests that these are the only ones, and you uncovered them. Of course, there could be more mechanisms contributing to risk behavior in STB, so I would suggest removing the word "the" or adding "one of the".

Thank you for this suggestion. We have now corrected it.

(3) One major weakness of this study is that suicidal thoughts and behaviors were not assessed via a clinical instrument such as the Columbia Suicide Severity Rating Scale - this should be mentioned upfront.

Thank you for this comment. According to medical records and information from family and friends by the researcher and psychiatrists, patients with suicidal thoughts and behaviors were categorized as suicidal group (S⁺), while patients without suicidal thoughts and behaviors were identified as control group (S^-). Note that medical records and information were recorded from clinical interviews where the psychiatrists were vigilant for signs of suicidal ideation and inquired about suicidal-related thoughts and behaviors from both the patients and their families. Therefore, the current group operation was possibly comparable to Columbia Suicide Severity Rating Scale.

(4) Table 1: female/male are sex, not gender (gender is man/woman/transgender/non-binary).

Thank you for this suggestion. We have now corrected it.

(5) Equation 1: It would be good to clarify what happens in gain-only or loss-only trials (the other value is then 0, but this can be clarified as it is not technically a loss or a gain).

Thank you for this suggestion. We have now corrected it. Please see below for our revision:

Page 12:

“Please note that V_gain is 0 in gain trials and V_loss is 0 in loss trials.”

(6) Figure 1E: The model prediction is not informative here. Given the linear regression model, there is no other option except that the mean prediction would overlap with the mean empirical measurement (unless the model was specified incorrectly). The same is true in Figure 2A.

Thank you for this suggestion. We have now removed plots for model prediction.

(7) Figure 1G: There was no analysis of the differences between groups in terms of earnings, given that the ANOVA was not significant. Still, if the claim is that risky behavior is sometimes suboptimal in this task, it would be good to show that there is a correlation between, say, symptoms of STB across groups and 1) risky behavior and 2) earnings.

Thank you for this insightful comment. In the patient cohort, risky behavior (gambling rate)—but not earnings—predicted the current suicidal ideation score (BSI-C, β = 9.189, t = 2.004, p = 0.048; earnings, β = 0.001, t = 0.582, p = 0.562). The lack of association for earnings is consistent with the task design, in which there is no stable optimal policy and payouts are only a coarse proxy for decision quality. Future work in learning paradigms, where optimality is well defined, may be better suited to test earnings-based links to STB. We have clarified this point below:

Page 32:

“Second, although we assumed that increased risky behavior in STB was suboptimal, the current task was not suited to test this, given the task design of random feedback for gambling option. Future work in learning paradigms, where optimality is well defined, may be better suited to test earnings-based links to STB.”

(8) Line 290: "beta_gain: -1-1" is unclear. I believe you meant beta_gain \in [-1,1].

Thank you for this suggestion. We have now corrected it to make it clear.

(9) The gain and loss biases are modeled as minimum and maximum probabilities for choosing the gamble. This is a legitimate choice for value-agnostic biases, but it is not the traditional choice (as far as I know). I wonder if the same results would hold with the more traditional formulation of the bias as an added constant to the utility of the gamble, i.e., p(gamble) = 1/(1+ exp(-mu(U_gamble + beta_gain - U_certain)). I believe in this case, you would also not have to specify different equations for positive or negative biases, or to limit the bias to the range of [-1,1] (indeed, the bias would be in reward-equivalent units).

Thank you for this suggestion. The winning choice model we used here was consistent with previous literature (Rutledge et al., 2015 & 2016), which decomposed the decision process into risk-attitude-driven valuation (e.g., loss and risk aversion) and value-insensitive motivational components. These approach/avoidance parameters are a decision bias in favor of the highest gain (approach) and another decision bias against the lowest loss (avoidance), above and beyond options value difference.

As suggested, we also compared the traditional bias choice model. Model comparison did not support this. Please see our revision below:

Supplementary Page 4:

“We also considered the traditional bias parameter (cM4), rather than approach/avoidance parameters. We limited the bias to the range of [-100, 100], which was in reward-equivalent units.

However, model comparison did not support cM4 (Table S6).”

(10) Also, for equations 5-8, it seems that 5-6 are identical to 7-8 except for the use of beta_gain versus beta_loss. You might want to consider simplifying by putting beta in the equations and specifying in the text that, depending on the trial type (loss or gain), the relevant beta is used.

Thank you for this suggestion. We have now simplified it. Please see response to Reviewer 2, point 3.

(11) It is not clear what equations are applied to mixed trials in cM3.

Sorry for the confusion. We have now clarified this point.

Page 12:

“Approach/avoidance parameters are not applied to in mixed trials.”

(12) Model comparison: the mood models are nested within each other (e.g., mM3 can be derived from mM1 by setting beta_EV = beta_RPE). In this case, model comparison can use the likelihood ratio test instead of BIC, which can be too conservative (and therefore does not support the extra beta parameter for RPE, different from previous results in the literature). I wonder if a likelihood ratio test would lead to results more in line with previous findings with this task?

Thanks for this suggestion. We agree that mM1 (CR+EV+RPE) and mM3 (CR+GR) are nested. However, our model space also included unnested models, such as mM5 (CR+GR_better+GR_worse). Therefore, it was not reasonable in our model space to use likelihood ratio tests.

(13) Line 346: The replication sample is described as "healthy participants," however, their health (or mental health) status was not assessed, and they may as well have mental health concerns. I would suggest calling this a general sample or an undifferentiated sample - but not a healthy sample.

Sorry for the confusion. We have now corrected this phrase.

(14) Line 363: "in addition to the replication of previous findings in the validation dataset" is unclear. Are those tests not two-tailed?

Sorry for the unclear statement. In the replication analyses, we used one-tailed t-tests because the direction of the effect was revealed on the clinical dataset. Please see our clarification below:

Page 15:

“For the replication of previous findings in the validation dataset, we used one-tailed tests in line with our clinically motivated directional hypothesis.”

(15) Line 372: "validating our group manipulation" - the presented work does not have a manipulation. Maybe you meant "validating our grouping of participants"?

Thank you for this suggestion. We have now corrected it to make it clear.

(16) Figure 2B: It is not clear how the data were binned for illustration purposes only, and why this binning is necessary (I have not seen it in other papers) - presenting the data from each subject and the correlation line with error margins (as is done here) should be sufficient.

Thank you for flagging this. For illustration only, we binned the data proportional to group sizes: in the patient sample (S^- n = 25; S⁺ n = 58; ≈1:2), we displayed 3 bins for S^- and 6 bins for S⁺. We agree that binning is not necessary; all statistics were computed on raw, unbinned data. The binned panel was included solely for visualization, consistent with our prior work (Blain et al., 2023).

(17) Table 2: delta BIC should be presented per subject (that is, divided by the number of subjects in each group), as the groups are of different sizes, so as presented now, the columns are not comparable across groups.

Thank you for the helpful suggestion. Our goal in Table 2 is not to compare ΔBIC magnitudes across groups, but to identify the winning model within each group. The ΔBICs are aggregated at the group level solely to rank models for that group. Dividing by the number of participants would rescale each group’s column by a constant and would therefore not affect the within-group ranking or the conclusion that cM3 is the best model in all groups. For this reason, we retain the current presentation and interpret each column within group rather than across groups.

(18) Line 640 - the effect of expectations and prediction errors on mood was not only shown in healthy people, but also in people with depression (Rutledge et al., 2007, https://pubmed.ncbi.nlm.nih.gov/28678984/)

Thank you for this comment. Indeed, Rutledge et al., (2017) showed evidence for CR+EV+RPE mood model in adult people with depression. However, our study recruited adolescents with depression or anxiety, given that adolescent period might provide a developmental window for opportunities for early intervention of suicidality. Therefore, it is also possible that the current winning model was specific to adolescents. Please see our clarifications below:

Page 28:

“It is also possible that the current winning model was specific to adolescents. Given that Rutledge et al., (2017) supported the “CR-EV-RPE model” in adults with depression, our study with adolescent populations may suggest a developmental change for mood sensitivities.”

(19) Supplemental material: Is the R2 section about R-squared? Perhaps you can use superscript on the 2 to make that clearer? For Figure S2, how was model recovery determined? Should I interpret the confusion matrix as suggesting that the winning model for each and every simulated subject was the generating model, or was the winning model determined for the whole simulated population in each of the 100 simulations? Traditionally, confusion matrices use the former measure, but the results of 100% recoverability make me suspect the latter was used here. In Figure S3, should we not be looking at simulated parameters and recovered parameters? What are "real parameters" here?

Thank you for these important comments. We now consistently denote the coefficient of determination as R² (with a superscript 2) throughout the manuscript and Supplementary Materials.

For the model recovery analysis in Figure S2, we have clarified that the confusion matrix is computed at the population level. Specifically, for each of the 100 simulations we generated a full dataset under each candidate model, fit all models to that dataset, and selected the winning model based on group-level model evidence (BIC). Each cell in the confusion matrix therefore reflects the proportion of simulations in which model j was selected as the best-fitting model when the data were generated by model i. This operation was reasonable because the decision of the winning model is made on the population-level dataset rather than on individual subjects.

In Figure S3, the term “real parameters” referred to the parameters used to generate the simulated data. To avoid confusion, we now relabel these as “simulated (generating) parameters” and explicitly describe the figure as showing the relationship between simulated (generating) parameters and recovered parameters. Please see our revisions below:

Supplementary Pages 2-3:

“Model recovery: We generated 100 simulated datasets for each model (3 choice models and 8 mood models) using the fitted parameters of each model as the ground truth. Each dataset contained 201 trials and included 3 (or 8) sets of simulated data corresponding to the respective models. For each simulated dataset, we then fit all models and determined the winning model at the population level based on group-level BIC, yielding a confusion matrix in which each entry represents the proportion of simulations in which model j was selected as the best-fitting model when the data were generated by model i. As shown in Figure S2, all models are highly identifiable, indicating excellent recovery performance for both the choice and mood models.”

“Parameter recovery: Figure S3 shows good parameter recovery for both choice and mood winning model (choice: rs > 0.91, ps < 0.001; intraclass coefficients > 0.78; mood: rs > 0.90, ps < 0.001; intraclass coefficients > 0.86). Moreover, we computed cross-correlations between all generating (“generating”) and recovered (“fitted”) parameters. The resulting matrix showed high diagonal (choice winning model: rs > 0.91; mood winning model: rs > 0.90) and low off-diagonal (choice winning model: abs(rs) < 0.63; mood winning model: abs(rs) > 0.40) correlations, further supporting parameter recovery.”

Typos:

(1) Line 90: original → originate

(2) Line 596-598 - the same phrase is repeated twice.

(3) Line 616: on the other word → hand.

Sorry for the mistakes. We have now corrected them throughout the manuscript.

Reviewer #2 (Recommendations for the authors):

For people unfamiliar with interpersonal theory or motivational-volitional model, or three-step theory (lines 105-106), could you briefly explain the key idea of mood and suicide before going to the decision-making tasks? And from this, maybe motivate the predictions in your task? In particular, in the abstract and introduction, the phrasing could be a bit more concise and simpler. In the abstract, sentences were sometimes quite long. In the introduction, some paragraphs are somewhat repetitive. In the discussion, there were some typos.

Thank you for these suggestions. We have now explained the key idea of mood and suicide before going to the decision-making tasks in the introduction, which can be seen below:

Pages 4-5:

“Contemporary theories of suicide converge on the idea that STB is initially caused by low mood experience. The interpersonal theory of suicide proposes that suicidal desire arises when people simultaneously feel socially disconnected (“thwarted belongingness”) and like a burden on others (“perceived burdensomeness”), experiences that are tightly linked to chronically low mood(25). The motivational–volitional model(26) and the three-step theory(27,28) similarly emphasize that when negative mood and feelings of defeat or entrapment are experienced as inescapable, they can give rise to suicidal ideation, and that the progression from ideation to suicide attempts depends on additional factors such as reduced fear of death, increased pain tolerance, and a tendency to act impulsively under intense affect. Some official organizations, e.g., National Institute of Mental Health, have also listed mood problems as warning signals(8). Interestingly, within the framework of decision making under uncertainty, gambling on lotteries with a revealed outcome has been found to induce high mood variance(29), providing an opportunity to assess the relationship between deficient mood and increased gambling decisions in STB.”

We have also refined the wording and corrected typos throughout the manuscript.

Reviewer #3 (Recommendations for the authors):

(1) Since many readers might only read the abstract, it is important that it is both informative and accurate. I have two suggestions in this respect. First, for the abstract to be more informative, it may be helpful to indicate already there that these are value-insensitive approach-avoidance parameters, in the sense that they favor/disfavor the gamble regardless of the potential outcomes' magnitude or probability. This issue is also present throughout the text, where the phrases "approach and avoidance motivation" are referred to as if they have established and precise computational definitions. In my view, these terms could just as easily be interpreted as parameters that multiply the value of potential gains or losses, which is not what the authors mean. It would be helpful to clarify this terminology.

Thank you for these suggestions. In line with previous literature (Rutledge et al., 2015 & 2016), approach and avoidance motivation are indeed defined at the computational level, referring to a decision bias in favor of the highest gain (approach) and another decision bias against the lowest loss (avoidance), above and beyond options value difference. We have cited these papers in the manuscript. We also make it clear to further clarify approach and avoidance parameters in the abstract and introduction. Please see our revisions below:

Page 2 (Abstract):

“Using a prospect theory model enhanced with value-insensitive approach-avoidance parameters revealed that this rise in risky behavior resulted only from a heightened approach parameter in S⁺.Altogether, model-based choice data analysis indicated dysfunction in the approach system in S⁺, leading to greater propensity for gambling in the gain domain regardless of the lottery expected value.”

Page 3 (Introduction):

“A growing literature indeed shows that risky behavior can be far better explained after adding value-insensitive approach and avoidance components to prospect theory(18,19), that is by including a decision bias in favor of the highest gain (approach) and another decision bias against the lowest loss (avoidance), above and beyond options value difference. This class of models highlights the important role of value-insensitive motivational components in decision making in addition to risk attitude-driven valuation (e.g., loss/risk aversion)(20).”

(2) The statement "our study uncovers the cognitive and affective mechanisms contributing to increased risk behavior in STB" is overstating the findings, as the study may have uncovered some contributing mechanisms, but likely not all of them. Removing the word "the" would fix this issue.

Thank you for this suggestion. We have now corrected it.

(3) Since mood is typically defined as lasting hours, it's inappropriate to refer to ratings that only reflect the last few trials as self-reports of mood. To be sure, I view the distinction between emotions and moods as quantitative, not qualitative, so I do not think there is a problem studying the former to understand the latter, but to avoid confusion, the terminology should follow common usage.

Thank you for this suggestion. We follow previous work and operational definitions regarding mood (Rutledge et al., 2014, Eldar & Niv, 2015, Vinckier et al., 2018). Emotion is usually a very brief response to a specific stimulus (Emanuel & Eldar, 2023), e.g., leading to rapid changes like surprise then fear. In contrast, mood is defined as a diffuse state that is not specific to one stimulus. Here, we operationally and computationally define mood as an affective state reflecting the recent history of safe and gamble outcomes. We now clarify that point in the main text. Please see our revision below:

Page 5:

“Although mood is thought to persist for hours, days, or even weeks(30-33), momentary mood, measured over the timescale in the laboratory setting, represents the accumulation of the impact of multiple events at the scale of minutes(30,32,34-38). Momentary mood external validity is demonstrated e.g., through its association with depression symptoms(37). Mood is different from emotions, which reflect immediate affective reactivity and is more transient (e.g. from surprise to fear)(31-33,39).”

(4) Line 78: The phrases "increase in risk attitude", "decrease in loss attitude", and "decrease in value-independent choice biases" are unclear to me in terms of their directionality. An attitude might be avoidant or embracing. If it is the former then increasing it would decrease risk-taking.

Thank you for pointing out the ambiguity. We have now corrected them throughout the manuscript. Please see our revision below:

Page 4:

“We therefore hypothesized that heightened approach motivation, or weakened avoidance motivation, would account for increased risk behavior in STB.”

(5) Line 125: I was not sure why one would expect the mood response to gamble-related quantities (EV and RPE) to be lower in STB and not higher.

Sorry for the typo. We hypothesized that mood would respond more strongly to gambling-related quantities—expected value (EV) and reward prediction error (RPE)—in adolescents with STB than in controls, given prior evidence that STB is associated with greater risk-taking.

(6) The text could use proofreading, as there are many typos. These are from the first 100 lines alone:

a) Abstract: regardless the lotteries -> regardless of the lotteries'.

b) Line 78: it remains whether.

c) Line 80: can each -> each can.

d) Line 90: may original from.

Sorry for the mistakes. We have now corrected them throughout the manuscript.

(7) The rationale for focusing on the S+ group for mood model comparison is incorrect. The purpose is to identify parameters that vary as a function of suicidality, and for that, the S- group is just as important.

Thank you for this comment. We agree that the S^- group is as important as the S⁺ group. A direct comparison was complicated because the winning mood models differed (S⁺: mM3; S^-: mM5; Table 3). To ensure comparability, we checked results from both model specifications (mM3 and mM5). The conclusions were convergent: mood sensitivity to certain rewards (CR) was lower in S⁺ than in S^- (see Fig. 3 for mM3 and Fig. S8 for mM5).

(8) There appears to be a contradiction between the inclusion criteria, which include having experienced suicidal thoughts and behaviors, and the definition of the S- group as not having suicidality.

Thank you for pointing out this mistake. The corrected version of inclusion criteria can be seen on Page 7:

“Patients were included if they met the following criteria: 1) both the researcher and psychiatrists agreed on their group classification; 2) they had a current diagnosis of major depressive disorder (MDD; unipolar depression), generalized anxiety disorder (GAD), or bipolar disorder with depressive episodes (BD), confirmed by two experienced psychiatrists using the Structured Clinical Interview for DSM-IV-TR-Patient Edition (SCID-P, 2/2001 revision; see Supplementary Note 1 for details); 3) they were between 10 and 19 years of age; 4) they had no organic brain disorders, intellectual disability, or head trauma; 5) they had no history of substance abuse; 6) they had no experience of electroconvulsive therapy.”

(9) It would be helpful to specify whether mood modeling was based on objective or subjective values, and why.

Thank you for this helpful suggestion. We have now clarified whether mood modeling was based on objective or subjective values, and why. Specifically, we constructed two model families: one in which mood was driven by objective monetary outcomes (objective values) and one in which mood was driven by subjective values derived from each participant’s fitted choice model (subjective values). We then used the VBA_groupBMC function in the VBA toolbox to perform family-wise model comparison, with 8 candidate mood models within each family. Consistent with previous literature, the objective-value family provided a clearly superior fit to the data (exceedance probability, EP = 1.000). Based on this result and for parsimony, we report and interpret the mood modeling results from the objective-value family in the main text. We have clarified this point below:

Supplement Pages 4-5:

“Supplementary Note 9: Mood model comparison using subjective values.

To identify whether mood modeling was based on objective or subjective values, we constructed two model families: one in which mood was driven by objective monetary outcomes (objective values) and one in which mood was driven by subjective values derived from each participant’s fitted choice model (subjective values). We then used the VBA_groupBMC function in the VBA toolbox (Daunizeau et al., 2014) to perform family-wise model comparison, with 8 candidate mood models within each family. Consistent with previous literature, the objective-value family provided a clearly superior fit to the data (exceedance probability, EP = 1.000).”

Reviewer #1 (Public review):

Summary

From transcriptomic comparisons of adult mouse cochlear and vestibular hair cells, Xu et al. provide a broad and well-organized overview of differences across 4 established hair cell types (2 cochlear and 2 vestibular). They go on to demonstrate the power of such analyses to provide functional insights by focusing on the differentiated expression of ciliary genes, building to the hypothesis that kinociliary motility occurs in adult vestibular hair cells.

Background

Cilia are prominent in sensory receptors, including vertebrate photoreceptors, olfactory neurons and mechanosensitive hair cells of the inner ear and lateral line. Cilia can be motile or nonmotile depending on their axonemal structure: motile cilia require dynein and the inner 2 singlet microtubules of the 9+2 array. Primary cilia, present early in development, are considered to have sensory functions and to be nonmotile (Mill et al., Nature Rev Gen 2023).

In hair cells, the kinocilium anchors and polarizes the mechanosensitive hair bundle of specialized microvilli. The kinocilium matures from the primary cilium of a newborn hair cell; behind it the bundle of mechanosensory microvilli rises in a descending staircase of rows. During maturation of the mammalian cochlea, all hair cells lose the kinocilium, though not the associated basal body. The consensus for many years has been that most vertebrate kinocilia, and especially mammalian kinocilia, are nonmotile, based largely on the lack of spontaneous motility in excised mammalian vestibular organs, but also on the impression that the rare examples of spontaneous beating motility even in non-mammalian hair cells are associated with deterioration of the preparation (Rüsch & Thurm 1990).

Strengths

In comparing RNA expression across the 4 major types of mouse hair cells - 2 cochlear and 2 vestibular - Xu et al. provide rich data sets for exploration of structure-function differences between these highly specialized cell types. The revised paper significantly improves the organization, interpretation and readability of the presentation of overall findings. smFISH and immuno-staining back up key RNA data, and comparisons are made with published data.

The ciliary motility focus of the rest of the paper is creative and highly interesting. The authors curated the ciliary genes into types associated with different aspects of beating motility, and also investigated the expression of genes typical of primary cilia, which are considered to have sensory and cell signaling functions and to be nonmotile. Their data justify suggesting a role for kinociliary motility (or force generation) in adult mammalian vestibular hair cells, in opposition to a long-held assumption. The results should stimulate investigation of the implications for mechanosensitivity.

Weaknesses

Data

Functional data on kinocilia motility: The technical difficulty in making such measurements in small mouse hair bundles led the authors to work with bullfrog crista bundles. Though not extensively studied here, the ciliary motility shown is convincing. Mouse hair bundle motions are also shown but the evidence connecting the data to kinociliary motion are more suggestive than convincing. But the authors are not dogmatic about these data, and it is reasonable to show them.

Interpretation

The authors take the view that kinociliary motility is likely to be normally present but is rare in their observations because conditions are not right. But while others have described some (rare) kinociliary motility in fish organs (Rusch & Thurm 1990), they interpreted its occurrence as a sign of pathology. Indeed, in this paper, it is not clear what role kinociliary motility would play in mature hair bundles. The authors have added a discussion of this question in the revision.

An underlying rationale for the hypothesis that ciliary motility manifests in mammalian vestibular hair cells seems to rest on the presence of the necessary mRNA and its contrasting absence in cochlear hair cells. Another way to look at this difference could be that evolution acted on cochlear hair cells to shed kinocilia as one of many changes to improve mechanosensitivity at much higher sound frequencies. In vestibular hair cells, kinociliary motion might be useful to enhance mechanostimulation in the developing vestibule (as suggested in this revision) and not so active in maturity. Nevertheless, with their scholarly analysis of the expression of ciliary genes, the authors make a significant argument for further investigation of when and why hair cell kinocilia show active motility.

Reviewer #2 (Public review):

Summary:

In this study the authors compared the transcriptomes of the various different types of hair cells contained in the sensory epithelia of the cochlea and vestibular organs of the mouse inner ear. The analysis of their transcriptomic data lead to novel insights into the potential function of the kinocilium.

Strengths:

The novel findings for the kinocilium gene expression along with the demonstration that some kinocilia demonstrate rhythmic beating as would be seen for known motile cilia is fascinating. It is possible that perhaps the kinocilium known to play a very important role in the orientation of the stereocilia, may have a gene expression pattern that is more like a primary cilium early in development and later in mature hair cells more like a motile cilium. Since the kinocilium is retained in vestibular hair cells it makes sense that it is playing a different role in these mature cells than its role in the cochlea.

Another major strength of this study which cannot be overstated is that for the transcriptome analysis they are using mature mice. To date there is a lot of data from many labs for embryonic and neonatal hair cells but very little transcriptomic data on the mature hair cells. They do a nice job in presenting the differences in marker gene expression between the 4 hair cell types. This information is very useful to those labs studying regeneration or generation of hair cells from ES cell cultures. One of the biggest questions these labs confront is what type of hair cell develop in these systems. The more markers available the better. These data will also allow researchers in the field to compare developing hair cells with mature hair cell to see what genes are only required during development and not in later functioning hair cells.

Comments on revision:

I am satisfied with the revision, the authors made an effort to incorporate the changes requested.

Author Response:

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

Public Reviews:

Reviewer #1 (Public review):

Weaknesses:

(1) Data:

(a) The main weakness in the data is the lack of functional and anatomical data from mouse hair bundles. While the authors compensate in part for this difficulty with bullfrog crista bundles, those data are also fragmentary - one TEM and 2 exemplar videos. Much of the novelty of the EM depends on the different appearance of stretches of a single kinocilium - can we be sure of the absence of the central microtubule singlets at the ends?

Our single-cell RNA-seq findings show that genes related to motile cilia are specifically expressed in vestibular hair cells. This has not been demonstrated before. We have also provided supporting evidence using electrophysiology and imaging from bullfrogs and mice. Although no ultrastructural images of mouse vestibular kinocilia were provided in our study, transmission electron micrograph of mouse vestibular kinocilia has been published (O’Donnell and Zheng, 2022). The mouse vestibular kinocilia have a “9+2” microtubule configuration with nine doublet microtubules surrounding two central singlet microtubules. This finding contrasts with a previous study, which demonstrated that the vestibular kinocilia from guinea pigs lack central singlet microtubules and inner dynein arms, whereas outer dynein arms and radial spokes are present (Kikuchi et al., 1989). The central pair of microtubules is absent at the end of the bullfrog saccular kinocilium (Fig. 7A). We would like to point out that the dual identity of primary and motile cilia is not just based on the TEM images. The kinocilium has long been considered a specialized cilium, and its role as a primary cilium during development has been demonstrated before (Moon et al., 2020; Shi et al., 2022).

In most motile cilia, the central pair complex (CPC) does not originate directly from the basal body; instead, it begins a short distance above the transition zone, a feature that already illustrates variation in CPC assembly across systems (Lechtreck et al., 2013). The CPC can also show variation in its spatial extent: for example, in mammalian sperm axonemes, it can terminate before reaching the distal end of the axoneme (Fawcett and Ito, 1965). In addition, CPC orientation differs across organisms: in metazoans and Trypanosoma, the CPC is fixed relative to the outer doublets, whereas in Chlamydomonas and ciliates it twists within the axoneme (Lechtreck et al., 2013). Such variation has been described in multiple motile cilia and flagella and is therefore not unique to vestibular kinocilia. What appears more unusual in our data is the organization at the distal tip, where a distinct distal head is present, similar to cilia tip morphologies recently described in human islet cells (Polino et al., 2023). Although this feature is intriguing, we interpret it primarily as a structural signature rather than as evidence for a specialized motile adaptation, and we have moderated our interpretation accordingly in the revision.

(b) While it was a good idea to compare ciliary motility expression in published P2 datasets for mouse cochlear and vestibular hair cells for comparison with the authors' adult hair cell data, the presentation is too superficial to assess (Figure 6C-E; text from line 336) - it is hard to see the basis for concluding that motility genes are specifically lower in P2 cochlear hair cells than vestibular hair cells. Visually, it is striking that CHCs have much darker bands for about 10 motility-related genes.

While these genes (e.g., Dynll1, Dynll2, Dynlrb1, Cetn2, and Mdh1) appear more highly expressed in P2 cochlear hair cells, they are not uniquely associated with the axoneme. For example, Dynll1/2 and Dynlrb1 are components of the cytoplasmic dynein-1 complex (Pfister et al., 2006), Cetn2 has multiple basic cellular functions beyond cilia (e.g., centrosome organization, DNA repair), and Mdh1 encodes a cytosolic malate dehydrogenase involved in central metabolic pathways such as the citric acid cycle and malate–aspartate shuttle. This contrasts with axonemal dyneins, which are uniquely required for cilia motility. To avoid ambiguity, we have marked such cytoplasmic or multifunctional genes with red asterisks in both Fig. 5G and Fig. 6D in the revised manuscript.

Our comparison showed that key genes for motile machinery are not detected in cochlear hair cells. For example, Dnah6 and Dnah5 are not expressed in the P2 cochlear hair cells. Dnah6 and Dnah5 encode axonemal dynein and are part of inner and outer dynein arms. Importantly, we did not detect the expression of CCDC39 and CCDC40 in kinocilia of P2 cochlear hair cells. Furthermore, axonemal CCDC39 and CCDC40, the molecular rulers that organize the axonemal structure in the 96-nm repeating interactome were not detected in cochlear hair cells. We have revised the text to emphasize key differences.

(2) Interpretation:

The authors take the view that kinociliary motility is likely to be normally present but is rare in their observations because the conditions are not right. But while others have described some (rare) kinociliary motility in fish organs (Rusch & Thurm 1990), they interpreted its occurrence as a sign of pathology. Indeed, in this paper, it is not clear, or even discussed, how kinociliary motility would help with mechanosensitivity in mature hair bundles. Rather, the presence of an autonomous rhythm would actively interfere with generating temporally faithful representations of the head motions that drive vestibular hair cells.

Spontaneous flagella-like rhythmic beating of kinocilia in vestibular HCs in frogs and eels (Flock et al., 1977; Rüsch and Thurm, 1990) and in zebrafish early otic vesicle (Stooke-Vaughan et al., 2012; Wu et al., 2011) has been reported previously. Based on Rüsch and Thurm (1990), spontaneous kinocilia motility occurred under non-physiological conditions and was interpreted as a sign of cellular deterioration rather than a normal feature. We speculate that deterioration under non-physiological conditions may lead to the disruption of lateral links between the kinocilium and the stereociliary bundle, effectively unloading the kinocilium and allowing it to move more freely. Additionally, fluctuations in intracellular ATP levels may contribute, as ciliary motility is highly ATP-dependent; when ATP is depleted, beating ceases. Similar phenomena have been documented in respiratory epithelia, where ciliary activity can temporarily pause. Nevertheless, the fact that kinocilia can exhibit spontaneous motility under these conditions indicates that they possess the motile machinery necessary for such beating. Irrespective of the condition, cilia without the molecular machinery required for motility will not be able to move.

We agree with the reviewer that, based on the present data, it is difficult to know the functional role of kinocilia and whether the presence of such autonomous rhythm would interfere with temporal fidelity. Spontaneous bundle motion, driven by the active process associated with mechanotransduction, was observed in bullfrog saccular hair cells (Benser et al., 1996; Martin et al., 2003). We have revised the discussion to clarify this important point of the reviewer. Specifically, we will emphasize that our observations of ciliary beating in the ex vivo conditions may not reflect its properties in the mature in vivo context, but rather a byproduct of motile machinery clearly present in the kinocilia. We speculate that this machinery in mature hair cells could operate in a more subtle mode—modulating the rigor state of dynein arms or related axonemal structures to influence kinociliary mechanics and, in turn, bundle stiffness in response to stimuli or signaling cues. Such a mechanism could either enhance sensitivity or introduce filtering properties, thereby contributing to the fine control of mechanosensory function without compromising temporal fidelity. Future studies using loss-of-function approach will be needed to reveal the unexplored role(s) of kinocilia for vestibular hair cells in vertebrates.

We note that spontaneous activity exits throughout nervous system. It allows the nervous system to maintain baseline activity and interpret signals. Retinal cells are spontaneously active even in the dark and spiral ganglion neurons also fire spontaneously. Spontaneous hair bundle motion driven by mechanotransduction-related mechanism has been observed in bullfrog saccular hair cells. So, it is unlikely that spontaneous kinocilia beating would interfere with generating temporally faithful representations.

Could kinociliary beating play other roles, possibly during development - for example, by interacting with forming accessory structures (but see Whitfield 2020) or by activating mechanosensitivity cell-autonomously, before mature stimulation mechanisms are in place? Then a latent capacity to beat in mature vestibular hair cells might be activated by stressful conditions, as speculated regarding persistent Piezo channels that are normally silent in mature cochlear hair cells but may reappear when TMC channel gating is broken (Beurg and Fettiplace 2017). While these are highly speculative thoughts, there is a need in the paper for more nuanced consideration of whether the observed motility is normal and what good it would do.

We thank the reviewer for these excellent suggestions. We agree that kinociliary motility could plausibly serve roles during development, for example by guiding hair bundle formation or by contributing to early mechanosensitivity and spontaneous neural activity before mature stimulation mechanisms are established. It is also possible that the motility machinery represents a latent capacity in mature vestibular hair cells that could be reactivated under stress or pathological conditions. We have revised the Discussion to address these possibilities and to provide a more nuanced consideration of whether the observed motility is normal and what potential functions it might serve.

Reviewer #2 (Public review):

Summary:

In this study, the authors compared the transcriptomes of the various types of hair cells contained in the sensory epithelia of the cochlea and vestibular organs of the mouse inner ear. The analysis of their transcriptomic data led to novel insights into the potential function of the kinocilium.

Strengths:

The novel findings for the kinocilium gene expression, along with the demonstration that some kinocilia demonstrate rhythmic beating as would be seen for known motile cilia, are fascinating. It is possible that perhaps the kinocilium, known to play a very important role in the orientation of the stereocilia, may have a gene expression pattern that is more like a primary cilium early in development and later in mature hair cells, more like a motile cilium. Since the kinocilium is retained in vestibular hair cells, it makes sense that it is playing a different role in these mature cells than its role in the cochlea.

Another major strength of this study, which cannot be overstated, is that for the transcriptome analysis, they are using mature mice. To date, there is a lot of data from many labs for embryonic and neonatal hair cells, but very little transcriptomic data on the mature hair cells. They do a nice job in presenting the differences in marker gene expression between the 4 hair cell types. This information is very useful to those labs studying regeneration or generation of hair cells from ES cell cultures. One of the biggest questions these labs confront is what type of hair cells develop in these systems. The more markers available, the better. These data will also allow researchers in the field to compare developing hair cells with mature hair cells to see what genes are only required during development and not in later functioning hair cells.

We would like to thank reviewer 2 for his/her comments and hope that the datasets provided in this manuscript will be a useful resource for researchers in the auditory and vestibular neuroscience community.

Joint Recommendations for the authors:

(1) Figure 1 - Explain how hair cell types are recognized after dissociation. Figure 1 will not be clear in this regard for non-aficionados. Some of the dissociated cells shown appear quite distorted and even unhealthy - e.g., the bottom right crista type II hair cell; the second from left crista type I hair cell; can you address why this doesn't matter for the purposes of this study?

HC types in Fig. 1C were identified based on their morphological features: Type I HCs are flask-shaped with a narrow neck while type II HCs are cylindrical and short. We have replaced those cells with new images. In our study, HCs were identified based on their marker genes. Although some HCs such as those shown in Fig. 3C were impossible to avoid during preparation of single cells for library (most people did not examine their morphology), quality of mRNA and sequencing was high, better than those datasets published in previous studies.

(2) Line 98 - Explain accessory cells (as opposed to supporting cells).

We changed accessory cells to other cell types.

(3) Line 246 - The primary cilium is...

Changed.

(4) Figure 6D - The scale bar is missing. Please use arrows to point to the genes you call out in the text. Also, the genes called out in the text as differently expressed (line 342) are quite faint bands in both cell types. It would be a service to the reader to point them out in the panel.

A scale bar has been added. We also marked those genes as suggested and edited the text accordingly.

(5) Figure 7 - mixes frog crista and mouse middle ear images with waveforms and FFTs from frog crista, mouse middle ear, and mouse crista. Related to these still images are 2 videos of frog kinocilium beating (2 hair cells). The mouse images must be underwhelming, or we would have been shown those, yet they were considered adequate to analyze.

Yes, the spontaneous kinocilia motion of mouse crista HCs is very small. The peak motion is about 40 nm, which is very close to the resolution of our camera. That is why we used photodiode technique to detect its motion. Photodiode is more sensitive, and this technique allows us to observe dynamic response waveform.

(6) I recommend labeling each figure panel with the tissue of origin to avoid confusion.

Labeled as suggested.

(7) I suggest dropping the mouse middle ear data, as they are not directly adequate as a positive control (or no more so than the more beautiful frog data).

We keep the waveforms of middle ear cilia movement in Fig. 7. The main reason is that we would like to show the magnitude difference between airway cilia and kinocilia. The kinocilia movement was at least an order of magnitude less than the movement of airway cilia. This has led to our effort to generate a model to predict the 96-nm modular repeat and explain why kinocilia movement in mice is much smaller than airway cilia and bullfrog kinocilia.

(8) Focus on the hair bundle motions:

(a) Show the waveforms for the frog crista hair cells and their FFTs.

These images were captured many years ago using camera. The kinocilia motion is between 5 and 10 Hz. We did not present any waveforms of kinocilia motion since we no longer have access to bullfrogs. However, although we did not present response waveforms, the videos are very powerful for visualization of kinocilia beat of bullfrog saccular HCs.

(b) Find some way to show us how you measured the mouse hair bundle beating.

Photodiode technique was used to measure spontaneous kinocilia motion in mice. More details are now included in the text.

(c) Does EGTA break links between kinocilium and stereocilia? (Could that contribute to the higher beat frequency?) Just applying the same treatment and viewing from above could clarify whether kinocilia dissociate from stereocilia rows. This would likely be more straightforward with an otolith organ.

All these links (tip links, side links) are vulnerable to Ca concentration and Ca-free medium is often used to break these links as shown in many previous studies. Breaking the kinocilia links leads to reduced load to the kinocilia, which may result in larger motion of the kinocilia. The frequency is inherent to motile machinery and subject to temperature and intracellular ATP concentration. When facing upward, the hair bundles in otolith organ do not have a good contrast against HCs in the background. This makes measurement of their motion difficult, especially when the motion is small and random and can’t be averaged to improve signal to noise ratio. Besides, unlike cochlear HCs whose hair bundles are short and can easily be oriented in parallel with light path, the long hair bundle of vestibular HCs is more difficult to orient and image. For these reasons, we chose to use crista hair bundles for our measurements since they can be oriented in perpendicular to the light path without interference from background HCs. The lateral motion of the entire bundle is also relatively easy to measure in this preparation.

(6) Is there no reason to cite McInturff et al. (2018), given that they compared type I and II VHC transcriptomes at P12 and P100? This database is also available on gEAR.

Their studies are now cited. We also compared their datasets with ours.

(7) Line 374 - Eatock et al., 1998 citation does not work for this purpose. Eatock & Songer (2011) would be better, or Li, Xue, Peterson (2008): mouse utricle anatomy; significant discussion of relative heights of kinocilia and tallest stereocilia.

Changed and cited.

(8) In Figure 3, 2 of the 18 panels in B are missing labels.

The bar, applied to all panels, was there at the bottom of Fig. 3B. The bar is bigger and more visible in the revision.

(9) Line 187 should "Sppl1" be Spp1?

Corrected.

(10) Define BBSome on line 244.

Added.

(11) Looking at Figure 5, it seems that all the motile genes are expressed in the vestibular hair cells and not the cochlear hair cells. It is surprising that there are any cilia-related genes expressed in these adult cochlear hair cells, given that they do not retain their cilia into adulthood. Could the authors make a comment on this finding in the discussion? Also, are there any ciliopathies that show a vestibular defect but normal hearing in mice or humans? Have you compared the cilia-related gene expression in neonatal/embryonic vestibular hair cells to your dataset?

There are many kinocilia related genes still expressing adult cochlear HCs. It is not surprising to see many kinocilia related genes in cochlear HCs. Most of these genes are related to primary cilia structure including the basal body and transporters in cilia. The basal body is still present in cochlear HCs. Many other primary cilia-related proteins are also expressed in soma, especially those related to signal transduction, microtubule cytoskeleton, actin cytoskeleton, vesicle transport, metabolic enzyme, protein folding, translation, nuclear transport, ubiquitination, RNA binding, mitochondrial proteins and transcription factors. Of course, some of them are vestigial. We added discussion of this in the text. Comparison between neonatal cochlear and vestibular was presented in Fig. 6D. We compared those genes related to the axonemal repeat (96 nm repeat complex). Due to quality of mRNA, the total genes and genes related to kinocilia detected in previous developmental studies were much less than our datasets. While we detected 112 out of 128 genes related to axonemal repeat, only 90 genes were detected in previous studies (Burns et al., 2015; McInturff et al., 2018). Therefore, we only compared neonatal cochlear and vestibular HCs using their datasets. As far as we know, no ciliopathies with vestibular defects but normal hearing have been reported in mice or humans. But we plan to use a Ccdc39 mutant mouse model to examine how loss of function of a key motile cilia signature gene would affect kinocilia motility and vestibular function.

(12) How is "expression level" in the violin plots being calculated? Is this a measure of read count? The normalization is cursorily explained in the methods. Is this value comparable across genes? Did the authors switch to z-score by Figure 6?

We dissected the auditory and vestibular sensory epithelia from the same groups of mice and prepared libraries and sequenced them at the same time. All parameters are the same. The violin Plots are based on values presented in Supplementary Table 1. Each dot in the plot reflects an aggregated number of reads across all cells for each gene. They are all normalized across different HC types and biological repeats. The details for normalization are now provided.

(13) The authors comment on the 16/128 motile cilia axonemal repeat genes that are not expressed in the vestibular hair cells. Listing these somewhere may be helpful to the readers.

We thank the reviewer for this helpful suggestion. Most of the 128 motile cilia axonemal repeat genes were listed in Figs 8C and S5, along with known loss-of-function mutations and ciliopathy associations identified in human diseases or observed in animal models. To improve clarity, we have now included Table S2, which provides the complete list of all 128 motile cilia axonemal repeat genes, including those not expressed in vestibular HCs.

(14) Figure 5D needs some refinement. While the authors used databases, including CiliaCarta, SYSCILIA gold standard, and CilioGenics, to identify the primary cilia-related genes, they have included many genes that are not highly specific to primary cilia function (e.g., HSP90, HSPA8, DNAJA4, GNAS...). Perhaps the authors would be able to do a better job of specifically querying primary cilia function by using genes that are common to these three databases.

We presented comparison and analysis based on three major cilia databases, which are generated from proteomics of cilia from different tissues/organisms. In addition, we have provided more comprehensive list of primary cilia-related genes in Fig. S2. While majority of cilia-related genes/proteins are highly conserved, some genes/proteins are tissue-/organism-specific. Majority of the genes presented in Fig. 5D of our manuscript are shared among all three databases. The cilium is a complex structure, composed of proteins for microtubule cytoskeleton, actin cytoskeleton, vesicle transport, metabolic enzyme, signaling, and protein folding. It also contains proteins for translation, nuclear transport, ubiquitination, RNA binding as well as mitochondrial proteins and transcription factors (https://ciliogenics.com/?page=Home). Proteins such as HSP90 and HSPA8 are important for protein folding. HSPA8 also functions as an ATPase in the disassembly of clathrin-coated vesicles during transport of membrane components through the cell. GNAS is part of a G protein complex that transmits signals. DNAJA4 is one of the high-confidence cilia proteins (mean score of 1.26, expression rank is 938). These proteins are detected in cilia according to CilioGenics (https://ciliogenics.com/?page=Home). These proteins are not highly specific to cilia and are expressed in soma as well. Most of these proteins for signaling such as WNT (Supplementary Fig. 2) are detected in both cilia and soma.

(15) The authors state, "Furthermore, we observed robust spontaneous kinocilia motility in bullfrog crista HCs and small spontaneous bundle motion in mouse crista HCs." This statement should be moderated by acknowledging that this motility was observed in only some cells. The authors favor the hypothesis that the lack of motility in some crista HCs is due to depolarization or damage to the sample. The authors should also acknowledge the possibility that there may be cell-to-cell variability in the motility of the kinocilia.

We address these issues in public review section. We modified the statement as suggested.

(16) The first few pages of the Results section include many lists of genes. Readability may be improved if this is curtailed modestly.

Changed as suggested. We removed comparison among different types of HCs and replotted Fig. 2B. This has reduced the number of genes mentioned in the text.

Author Response:

The following is the authors’ response to the previous reviews

Public Reviews:

Reviewer #1 (Public review):

In this revised manuscript, Qin and colleagues aim to delineate a neural mechanism that is engaged specifically in the sated flies to suppress the intake of sugar solution (the "brake" mechanism for sugar consumption). They identified a three-step neuropeptidergic system that downregulates the sensitivity of sweet-sensing gustatory sensory neurons in sated flies. First, neurons that release a neuropeptide Hugin (which is an insect homolog of vertebrate Neuromedin U (NMU)) are in active state when the concentration of glucose is high. This activation depends on the cell-autonomous function of Hugin-releasing neurons that sense hemolymph glucose levels directly. Next, the Hugin neuropeptides activate Allatostatin A (AstA)-releasing neurons via one of Hugin receptors, PK2-R1. Finally, the released AstA neuropeptide suppresses sugar response in sugar-sensing Gr5a-expressing gustatory sensory neurons through AstA-R1 receptor. Suppression of sugar response in Gr5a-expressing neurons reduces fly's sugar intake motivation. They also found that NMU-expressing neurons in the ventromedial hypothalamus (VMH) of mice (which project to the rostal nucleus of the solitary tract (rNST)) are also activated by high concentrations of glucose independent of synaptic transmission, and that injection of NMU reduces the glucose-induced activity in the downstream of NMU-expressing neurons in rNST. These data suggest that the function of Hugin neuropeptide in the fly is analogous to the function of NMU in the mouse.

The shift of the narrative, which focuses specifically on the hugin-AstA axis as the "brake" on the satiety signal and feeding behavior, clarified the central message of the presented work. The authors have provided multiple lines of compelling evidence generated through rigorous experiments. The parallel study in mice adds a unique comparative perspective that makes the paper interesting to a wide range of readers.

While I deeply appreciate the authors' efforts to substantially restructure the manuscript, I have a few suggestions for further improvements. First, there remains room for discussion whether the "brake" function of the hugin-AstA axis is truly satiety state-dependent. The fact that neural activation (Fig. Supp. 8), peptide injection (Fig. 3A, 4A), receptor knockdown (Fig. 3C,G, 4E), and receptor mutants (Fig. Supp. 10, 12) all robustly modulate PER irrespective of the feeding status suggests that the hugin-AstA axis influences feeding behaviors both in sated and hungry flies. Additionally, their new data (Fig. Supp. 13B, C) now shows that synaptic transmission from hugin-releasing neurons is necessary for completely suppressing feeding even in sated flies. If the hugin-AstA axis engages specifically in sated (high glucose) state, disruption of this neuromodulatory system is expected to have relatively little effect in starved flies (in which the "brake" is already disengaged).

We thank the reviewer for pointing out this inconsistency. We have corrected this interpretation. Specifically:

(1) We removed statements suggesting that the circuit is fully disengaged during starvation.

(2) We now state that endogenous hugin activity is reduced during starvation, but the circuit retains modulatory capacity when experimentally perturbed.

(3) The Discussion now emphasizes that the system operates as a state-modulated inhibitory tone rather than a strictly fed-state switch.

We believe this revised framing resolves the discrepancy.

In this context, it is intriguing that the knockdown of PK2-R2 hugin receptor modestly but consistently decreases proboscis extension reflex specifically in starved flies (Fig. 3D, H). The manuscript does not discuss this interesting phenotype at all. Given the heterogeneity of hugin-releasing neurons (Fig. Supp. 7), there remains a possibility that a subset of hugin-releasing neurons and/or downstream neurons can provide a complementary (or even opposing) effect on the feeding behavior.

We agree that this is an important observation. Although the effect size is modest, it is reproducible and suggests that hugin signaling may not operate as a strictly linear pathway.

To address this:

(1) We added a paragraph in the Results acknowledging the PK2-R2-dependent phenotype.

(2) We included a discussion noting the potential functional heterogeneity of hugin neurons.

(3) The schematic model (now Figure Supplementary 17, previously Figure Supplementary 16) includes a dashed line indicating a possible parallel PK2-R2-dependent branch.

Given these intriguing yet unresolved issues, it is important to acknowledge that whether this system is "selectively engaged in fed states to dampen sweet sensation (in Discussion)" requires further functional investigations. Consistent effects of manipulation of the hugin-AstA system across multiple experimental approaches underscores the importance of this molecular circuitry axis for controlling feeding behaviors. Moderation of conclusions to accommodate alternative interpretation of data will be beneficial for field to determine the precise mechanism that controls feeding behaviors in future studies.

We fully agree with the reviewer. Our original description of the circuit as a “satiety brake” implied exclusive engagement in fed states, which is not strictly supported by the behavioral data. Although endogenous hugin activity is elevated under fed conditions (as shown by CaMPARI), experimental manipulations demonstrate that the circuit retains functional capacity to modulate feeding behavior across feeding states.

To address this concern, we have:

(1) Removed the term “satiety-specific brake” throughout the manuscript.

(2) Reframed the circuit as a glucose-responsive, state-modulated inhibitory module.

(3) Revised the Discussion to explicitly state that the hugin–AstA pathway biases sweet sensitivity according to circulating glucose levels rather than functioning as an on/off switch.

(4) Substantially revised Supplementary Figure 17 to reflect graded modulation across metabolic states rather than binary state engagement.

These changes better align our conclusions with the experimental observations.

Reviewer #2 (Public review):

Summary:

The question of how caloric and taste information interact and consolidate remains both active and highly relevant to human health and cognition. The authors of this work sought to understand how nutrient sensing of glucose modulates sweet sensation. They found that glucose intake activates hugin signaling to AstA neurons to suppress feeding, which contributes to our mechanistic understanding of nutrient sensation. They did this by leveraging the genetic tools of Drosophila to carry out nuanced experimental manipulations, and confirmed the conservation of their main mechanism in a mammalian model. This work builds on previous studies examining sugar taste and caloric sensing, enhancing the resolution of our understanding.

Strengths:

Fully discovering neural circuits that connect body state with perception remains central to understanding homeostasis and behavior. This study expands our understanding of sugar sensing, providing mechanistic evidence for a hugin/AstA circuit that is responsive to sugar intake and suppresses feeding. In addition to effectively leveraging the genetic tools of Drosophila, this study further extends their findings into a mammalian model with the discovery that NMU neural signaling is also responsive to sugar intake.

Weaknesses:

The effect of Glut1 knockdown on PER in hugin neurons is modest in both fed and starved flies, suggesting that glucose intake through Glut1 may only be part of the mechanism.

We agree that the modest PER phenotype suggests that Glut1-mediated glucose uptake represents one component of glucose sensing in hugin neurons. We have clarified this in the Discussion and now explicitly state that additional glucose-sensing mechanisms may contribute to hugin activation.

Additionally, many of the manipulations testing the "brake" circuitry throughout the study show similar effects in both fed and starved flies. This suggests that the focus of the discussion and Supplemental Figure 16 on a satiety-specific "brake" mechanism may not be fully supported by the data.

We fully agree that the previous framing overstated state specificity.

As described above, we have:

(1) Removed “satiety-specific brake” terminology.

(2) Reframed the circuit as a glucose-responsive inhibitory module.

(3) Revised the Discussion to explicitly acknowledge modulation across feeding states.

(4) Updated the schematic model (Figure Supplementary 17, formerly Figure Supplementary 16) accordingly.

Recommendations for the authors:

Reviewing Editor (Recommendations for the authors):

Both the reviewers and I agree that the conclusion about a "satiety-dependent" brake needs to be modified to discuss the phenotypes that are also observed under starved conditions. Reviewer 1 would further like to emphasize that the authors are not required to follow through with the specific recommendations suggested by them. Modifying the conclusion and Supplementary Figure 16 should suffice.

We sincerely thank the Reviewing Editor for the clear guidance. We fully agree that our previous framing of the hugin–AstA circuit as a strictly “satiety-dependent” brake may have overstated the state specificity of the system.

In response to this recommendation, we have:

(1) Revised the Abstract, Results, and Discussion to moderate the conclusion and explicitly acknowledge the phenotypes observed under starved conditions.

(2) Reframed the circuit as a glucose-responsive, state-modulated inhibitory module, rather than a satiety-exclusive brake.

(3) Supplementary Figure 17 (formerly Figure Supplementary 16) has been substantially revised to illustrate graded modulation across metabolic states rather than binary engagement.

We appreciate the clarification that no additional experiments were required and are grateful for the opportunity to improve the conceptual framing of our work.

Please include full statistical reporting in the main manuscript (e.g., figure legends or results).

We have revised all figure legends to include full statistical reporting.

Reviewer #1 (Recommendations for the authors):

By re-framing their finding as the "brake" mechanism on satiety-induced suppression of feeding behavior and sensitivity to sweet taste, the authors substantially improved the clarity of their findings and their significance. The additional data (Fig. Supp. 13B, C) allows "apple-to-apple" comparisons of behavioral data. I support the publication of this manuscript with no further experiments, although I have several suggestions for the text.

As I write in the public review, I have a reservation on the authors' argument that hugin-AstA system is the "'satiety brake' - that is selectively engaged in fed states to dampen sweet sensation (lines 392-394)". Manipulation of both hugin system (Fig. 2C, Fig. 3A, C, D, G, Fig. Supp. 8A, C, Fig. Supp. 10A-C, Fig. Supp. 13B, C) and AstA system (Fig. 4A, E, Fig. Supp., 8C, D, Fig. Supp. 12A-C, Fig. Supp. 13D) all indicate that hugin-AstA system suppresses feeding regardless of the satiety state. Specifically, Fig. Supp. 13B shows that synaptic blockade does further increases PER, causing contradictions to authors' statements ("silencing hugin+ neurons led to enhanced sweet-driven feeding behavior (line 299-300)" and "...further silencing has little additional effect (line 402)"). The CaMPARI data (Fig. 1J) provides the link between the activity levels of hugin-releasing neurons and satiety state. However, the fact that eliminating hugin-AstA signal can promote further PER in starved flies suggests that this brake is not completely satiety-dependent. I ask authors to at least discuss this perceived discrepancy between their data and conclusions.

Also, the authors' finding that PK2-R2 reduction actually suppresses PER specifically among starved flies (Fig. 3D, H), albeit with relatively small effect size, suggests that hugin-AstA axis is not a singular, linear pathway as authors suggest in Fig. Supp. 16. While delineating the PK2-R2-dependent pathway is beyond the scope of this study, at least a line of discussion would be helpful.

Minor comments:

(1) Fig. Supp. 8 (dTRPA1 activation of hugin and AstA neurons), and Fig. Supp. 13B-D (inhibition of hugin and AstA neurons) should be in the main figure given its relevance to the narrative of this manuscript.

We agree with the reviewer regarding their importance. The key behavioral panels from these figures have now been moved to the main figures to strengthen the narrative flow.

(2) Fig. Supp. 11 (PER and imaging using decapitated heads only), despite its creativity, leaves me wonder how PER of fly heads looks like. It is a highly artificial and invasive experiment. Supplementary movies would be helpful.

We apologize for the lack of clarity in our description. In this experiment, flies were not decapitated. Instead, we surgically severed the connection between the brain and the ventral nerve cord (VNC), while keeping the body and proboscis musculature intact. Thus, the flies remained physically intact, and PER was measured using the same behavioral protocol as in intact animals.

We have revised the figure legend to clarify this point and avoid confusion. Because the behavioral procedure was identical to standard PER assays and the flies retained normal proboscis motor function, we did not include supplementary videos.

(3) Expression patterns of PK2-R1 and AstA-R2 in proboscis are mentioned in text but with no data (lines 229 and 279). I strongly encourage authors to show images.

We have now included the relevant expression images in the revised manuscript.

(4) A citation for the "previous study (line 486)" describing PER method is required.

The appropriate citation has been added.

Author Response:

The following is the authors’ response to the previous reviews

Public Review:

We thank the editor and reviewers for their thoughtful and constructive feedback, which has enabled us to greatly strengthen the manuscript. We apologize for the delay in resubmitting this as we were dealing with a large turnover in the lab due to trainee graduations which has We have carefully revised the text, figures, and supplementary materials in response to these comments. Below, we summarize the key revisions made followed by a point-by-point response to the reviewers’ critiques.

(1) Performed CUTS analyses in human neuronal system: In the revised manuscript, we included new data demonstrating that the CUTS system can be applied to additional cellular models, specifically neuronal cells (Figure 5, Figure S4). To address whether CUTS functions effectively in neuronal contexts, we generated stable CUTS-expressing lines in differentiated BE(2)-C and ReN VM–derived differentiated neurons (Figure 5A-D, Figure S4 A-C). To ensure this was neuronal expression, we developed a new Tet-On3G system construct where the Tet-On3G transactivating protein is driven by the SYN1 promoter to ensure neuron-specific inducible expression for these experiments.

(2) Define the relationship between CUTS and endogenous/physiological cryptic exons inclusion: To evaluate how well the CUTS system reflects physiological cryptic exon regulation, we performed RT-PCR analysis of several cryptic exons previously reported by us and evaluated CUTS activation at the RNA level in parallel (Figure S2E) . CUTS is sensitive to low-mild reductions in TDP-43 levels, whereas the tested endogenous cryptic exons exhibit variable responses to TDP-43 knockdown.

(3) Defining stress-induced TDP-43 loss of function: We included new data demonstrating that the CUTS system can detect TDP-43 loss of function induced by acute sodium arsenite (NaAsO₂) treatment in HEK cells (Figure 3D–I). We have also tested additional stressor as part of a separate ongoing study where this work will be expanded upon (Xie et al., 2025). We selected this paradigm since TDP-43 loss of function in response to acute NaAsO₂ treatment is also supported by work from other labs(Huang et al., 2024).

(4) Implications of using a TDP-43 Loss-of-Function sensor for therapeutic applications: In the revised manuscript, we clarify that CUTS-TDP43 is auto-regulated and we highlight two potential therapeutic applications: i) TDP-43 Knockdown-and-replacement: CUTS-TDP43 provides a strategy for simultaneous depletion of pathological TDP-43 species while enabling autoregulated re-expression of wild-type TDP-43. This design mitigates the risk of supraphysiologic overexpression, a known liability in conventional replacement approaches, by restoring TDP-43 within a self-limiting regulatory network that maintains homeostatic control. ii) Aggregation-independent correction: Because CUTS is autoregulatory, it can be repurposed to regulate alternative downstream effectors, including splicing modifiers or TDP-43 functional interactors, without expressing TDP-43 itself. This approach provides a potential aggregation-independent strategy to compensate for TDP-43 loss-of-function (LOF) by restoring downstream splicing. We are evaluating this work in a follow up study (Xie et al., 2025). In these ongoing studies, we show that CUTS-regulated expression of splicing proteins in response to TDP-43 loss restored subsets of cryptic exon events (24/28 events evaluated). These findings suggest CUTS as a versatile tool for both autoregulated TDP-43 replacement and trans-regulatory therapeutic correction. We expanded on this concept in the discussion section of this revised manuscript. We also note that autoregulatory TDP-43 biosensor strategies have been proposed in related systems, including TDP-Reg, underscoring broader interest in self-regulated TDP-43 systems (Wilkins et al., 2024).

(5) Clarified mechanism of TDP-43 5FL causing strong loss of function: The TDP-43 5FL exhibits reduced RNA binding capacity, and we previously showed that the lack of RNA binding promotes aberrant homotypic phase separation of TDP-43 (Mann et al., 2019). Expression of RNA-deficient TDP-43 variant forms nuclear “anisomes” (Yu et al., 2021), which evidence suggests sequesters endogenous TDP-43 protein into insoluble structures. We expanded on this in our results section in this revised manuscript.

(6) Improved figure clarity and data presentation: To enhance clarity and organization, we maintained the main structure of the manuscript while reorganizing figures and improved data visualization. Some examples include:

Figure 1: We revised the schematic layout for greater clarity and simplicity. The figure now focuses more specifically on the CUTS data, with additional data on the UNC13A-TS and CFTR-TS moved to Figure S1. To improve readability, titles were added to all schematic panels. Visual consistency was also improved by refining the color labelling for each sensor in Figures 1C and 1D and adjusting the corresponding bar graphs accordingly.

Figure 2: We reorganized the figure to clearly distinguish between protein and mRNA analyses for greater clarity. In the revised layout, western blot quantifications of TDP-43 and CUTS (GFP) signals are shown in Figures 2D and 2E, respectively, while the corresponding qPCR analyses are presented in Figures 2H and 2I. Minor edits include removing the percentage knockdown and fold-change annotations from the graphs and incorporating these values into a mini-table in Figure S2E.

The original Figure 2D and 2G were reincorportated as reference panels in Figure S2A–B, while new graphs showing CUTS protein-level changes as a function of TDP-43 knockdown were added (Figure S2C–D). We also incorporated new data showing the behavior of endogenous cryptic exons under low siTDP-43 treatment (Figure S2E).

Figure 3: We added new data demonstrating that the application of the CUTS system in detecting TDP-43 loss of function induced by stress conditions. Specifically, we show that sodium arsenite (NaAsO₂) treatment leads to TDP-43 functional impairment detectable by CUTS and supported with endogenous cryptic exon via RT-PCR (Figure 3D-I).

Figure 5 and Figure S4: We introduced a new figure that demonstrates the effective application of the CUTS system in differentiated neuronal systems, thereby extending its usability to disease-relevant cell types.

Figures 2SA and 4B were edited to include the corresponding labels on the sides of each image for clarity. Sup Figure 2A was moved to Sup Figure 3A, while Figure 4B remains in its original configuration.

We thank the reviewers again for their insightful critiques and helpful suggestions, which have enabled us to substantially improve the manuscript. Please find our detailed response to each review below:

Reviewer #1 (Public review):

Summary:

The authors create an elegant sensor for TDP -43 loss of function based on cryptic splicing of CFTR and UNC13A. The usefulness of this sensor primarily lies in its use in eventual high throughput screening and eventual in vivo models. The TDP-43 loss of function sensor was also used to express TDP-43 upon reduction of its levels.

Strengths:

The validation is convincing, the sensor was tested in models of TDP-43 loss of function, knockdown and models of TDP-43 mislocalization and aggregation. The sensor is susceptible to a minimal decrease of TDP-43 and can be used at the protein level unlike most of the tests currently employed,

Weaknesses:

Although the LOF sensor described in this study may be a primary readout for high-throughput screens, ALS/TDP-43 models typically employ primary readouts such as protein aggregation or mislocalization. The information in the two following points would assist users in making informed choices.

(1) Testing the sensor in other cell lines

We thank the reviewer for raising this important point. In agreement with this suggestion, we generated ReN VM cell lines and used a neuroblastoma cell line model (BE(2)-C) expressing the TetOn3G CUTS system under a human synapsin I (hSYN1) promoter. In this construct the transactivator protein is under the control of a neuronal specific hSYN1 promoter whereas the classical TetOn3G system uses a CMV-like promoter. Several studies have reported reduced activity or silencing of CMV and PGK-driven transgenes in neurons. Therefore, we for our neuronal experiments, we removed this promoter to generate a new version of a doxycycline-inducible CUTS system in which Tet-On 3G transactivator is now driven by the hSYN1 promoter which will express CUTS in response to doxycycline treatment. In this improved construct, we also replaced mCherry with mScarlet to enhance the fluorescent signal.

To test this neuronal-adapted system, we established stable CUTS expression in undifferentiated BE(2)-C cells, a subclone of the SK-N-BE(2) neuroblastoma line that has been used to study TDP-43–dependent splicing function(Brown et al., 2022). This model can be differentiated into neuron-like cells within 10 days, as shown in Supplementary Figure 4A. Using this model, we confirmed that TDP-43 knockdown leads to robust activation of the CUTS system (Figure 5B-E). We additionally tested this in in a stable polyclonal ReN VM cells following differentiation into cortical-like neurons (Figure 5D, Figure S4B-C).

(2) Establishing a correlation between the sensor's readout and the loss of function (LOF) in the physiological genes would be useful given that the LOF sensor is a hybrid structure and doesn't represent any physiological gene. It would be beneficial to determine if a minor decrease (e.g., 2%) in TDP-43 levels is physiologically significant for a subset of exons whose splicing is controlled by TDP43.

We agree with the reviewer that correlating the sensor’s readout with physiological TDP-43 splicing targets is essential to validate its biological relevance. To this end, we complemented our sensor expression profile with endogenous cryptic exons (CEs) sensitive to TDP-43 depletion. We tested a panel of five physiological cryptic exons regulated by TDP-43 (LRP8, EPB41L4A, ARHGAP32, HDGFL2, and ACBD3). To address the reviewer’s concerned, we performed RT-PCR on samples from the low-dose siTDP-43 experiment shown in Figure S2E.

The endogenous CEs used in the panel were selected based on our own and others’ preliminary observations. Among these, HDGFL2 showed a particularly robust increase in cryptic exon inclusion at very low siTDP-43 concentrations (38 pM), while untreated samples showed almost no CE inclusion. This finding strongly supports a direct mechanism linking mild TDP-43 reduction to loss of physiological splicing control.

(3) Considering that most TDP-LOF pathologically occurs due to aggregation and or mislocalization, and in most cases the endogenous TDP-43 gene is functional but the protein becomes non-functional, the use of the loss of function sensor as a switch to produce TDP-43 and its eventual use as gene therapy would have to contend with the fact that the protein produced may also become nonfunctional. This would eventually be easy to test in one of the aggregation modes that were used to test the sensor.. However, as the authors suggest, this is a very interesting system to deliver other genetic modifiers of TDP-43 proteinopathy in a regulated fashion and timely fashion.

We thank the reviewer for this thoughtful point and agree that in the disease-relevant context where endogenous TDP-43 is intact but TDP-43 function is lost due to mislocalization and/or aggregation, a re-supply of TDP-43 risks sequestration and loss of activity. In our manuscript, the CUTS-TDP43 module was presented as a control circuit proof-of-concept rather than a stand-alone approach: it demonstrates that CUTS can (i) sense LOF with high dynamic range and proportionality, and (ii) drive a payload under negative feedback such that total TDP-43 remains near baseline while partially rescuing a splicing readout (CFTR minigene) under knockdown conditions.

Importantly, we evaluated CUTS in aggregation/mislocalization-prone contexts: ΔNLS, 5FL, and ΔNLS+5FL variants trigger CUTS activation (ref), allowing us to quantify LOF arising from these aggregation modes. This confirms that CUTS can operate precisely in the very settings where sequestration is likely to occur.

To directly address the reviewer’s suggestion, in the revision we (i) clarify in the Discussion that CUTS-TDP43 is a circuit demonstration and not our proposed monotherapy in aggregation-dominant disease; and (ii) expand our therapeutic framing into two approaches:

Knockdown-and-replacement: concurrently deplete aggregation-prone/endogenous pathologic TDP-43 species (i.e., mutant TDP-43) while using CUTS to re-deliver wild-type TDP-43 under autoregulation. Aggregation-independent correction: use of CUTS to deliver modifiers that bypass TDP-43 sequestration (e.g., downstream effectors or splicing correctors that restore LOF consequences without expressing TDP-43 itself).

(4) I don't think the quantity of siRNA is directly proportional to the degree of TDP-43 knockdown/extent of TDP-43 loss. Therefore, to enhance the utility of the dose-response curves, I'd suggest using TDP-43 levels as the variable on the x-axis, rather than the amount of siRNA administered or even just adding a plot alongside the current plots would enable readers to quickly evaluate LOF response levels concerning the protein. While I understand that the sensitivity of Western blots for quantification might be why the authors have not created the graphs in this manner, having this information would be useful.

We appreciate the reviewer’s insightful comment. As noted, in the original version of the graph, we incorporated the percentage of TDP-43 knockdown corresponding to each siTDP-43 concentration (indicated in red text). However, we agree that this format was not easy to interpret, given the amount of information presented. To address this, we generated two new plots in which the x-axis represents TDP-43 levels (percentage of remaining protein or mRNA), and the y-axis shows the fold change in CUTS signal measured by (i) TDP-43 protein pixel intensity and (ii) TDP-43 mRNA levels, respectively. These new plots are now included as Supplementary Figures 2C–D, which allow a clearer visualization of CUTS readout in relation to actual TDP-43 levels rather than siRNA dose. As the reviewer anticipated, the reason we did not originally present the data in this format was that at low siTDP-43 concentrations, the fold change is minimal and more difficult to quantify by Western blot. Nevertheless, we have now incorporated the revised plots to strengthen the interpretation of the dose–response relationship. Additionally, we experience batch effects across siRNA lots. We believe this revised format should enhance the clarity of the result.

(5) p3 line 74: one of the reasons cited as a pitfall of using the endogenous cryptic exons exhibit variable responses to TDP-43 loss and may be cell type-specific. has the sensor been used in different cell lines?

We tested the CUTS system in differentiated neuronal models using two differentiated neuronal cell types, BE(2)C and ReN VM cells. The results are presented in Figure 5 and Figure S4 of the revised manuscript.

(6) The order of the text describing 1A and 1B is confusing. The text starts describing the TS cassettes referring to 1A using the CUTS cassettes which haven't been introduced yet as an example. I'd suggest reorganising this section. The graph, always in 1A showing readout proportional to GFP should be taken out or highlighted in the figure legend that it is theoretical.

We agree with the reviewer’s point. In the original schematic (Figure 1A), we included the CUTS system as an example to introduce the TS cassette design, since it contains the three possible sensor configurations. However, we recognize that this could be confusing. Therefore, we have removed the CUTS cassette from Figure 1A, along with the theoretical graph showing GFP readout proportional to the degree of TDP-43 LOF. In agreement with this change, we also restructured Figure 1. As the focus is the CUTS system, we have moved the Western blot and quantification of UNC13A-TS and CFTR-TS to Supplementary Figure 1.

Reviewer #2 (Public review):

Summary:

The authors goal is to develop a more accurate system that reports TDP-43 activity as a splicing regulator. Prior to this, most methods employed western blotting or QPCR-based assays to determine whether targets of TDP-43 were up or down-regulated. The problem with that is the sensitivity. This approach uses an ectopic delivered construct containing splicing elements from CFTR and UNC13A (two known splicing targets) fused to a GFP reporter. Not only does it report TDP-43 function well, but it operates at extremely sensitive TDP-43 levels, requiring only picomolar TDP-43 knockdown for detection. This reporter should supersede the use of current TDP-43 activity assays, it's cost-effective, rapid and reliable.

Strengths:

In general, the experiments are convincing and well designed. The rigor, number of samples and statistics, and gradient of TDP-43 knockdown were all viewed as strengths. In addition, the use of multiple assays to confirm the splicing changes were viewed as complimentary (ie PCR and GFPfluorescence) adding additional rigor. The final major strength I'll add is the very clever approach to tether TDP-43 to the loss of function cassette such that when TDP-43 is inactive it would autoregulate and induce wild-type TDP-43. This has many implications for the use of other genes, not just TDP-43, but also other protective factors that may need to be re-established upon TDP-43 loss of function.

Weaknesses:

(1) Admittedly, one needs to initially characterize the sensor and the use of cell lines is an obvious advantage, but it begs the question of whether this will work in neurons. Additional future experiments in primary neurons will be needed.

We thank the reviewer for highlighting the importance of validating the sensor in neuronal models, given the central role of TDP-43 dysfunction in ALS/FTD and related neurodegenerative disorders. While initial characterization in established cell lines provides experimental control and scalability, we agree that demonstrating functionality in neuronal systems is essential. To address this, we adapted the CUTS platform for neuronal application by incorporating the human synapsin-1 (hSYN1) promoter into the Tet-On 3G system to enable inducible, neuronal specific expression. We validated this configuration in differentiated BE(2)-C cells (Figures 5A-C, S4A-C), where CUTS retained robust responsiveness to TDP-43 perturbation. In parallel, we generated stable CUTS-expressing ReN VM neural progenitor cells and differentiated them for three weeks prior to functional assessment (Figures 5A-C, S4A-C). In both neuronal models, CUTS was functional and responsive to TDP-43 siRNA. We are currently optimizing promoter selection and expression paradigms for fully differentiated iPSC-derived neuronal models and will be the subject of future studies.

(2) The bulk analysis of GFP-positive cells is a bit crude. As mentioned in the manuscript, flow sorting would be an easy and obvious approach to get more accurate homogenous data. This is especially relevant since the GFP signal is quite heterogeneous in the image panels, for example, Figure 1C, meaning the siRNA is not fully penetrant. Therefore, stating that 1% TDP-43 knockdown achieves the desired sensor regulation might be misleading. Flow sorting would provide a much more accurate quantification of how subtle changes in TDP-43 protein levels track with GFP fluorescence.

We thank the reviewer for this thoughtful suggestion. We agree that flow cytometry and sorting of GFP-positive populations would provide a higher-resolution, single-cell–level relationship between TDP-43 abundance and sensor output. Such an approach would reduce heterogeneity arising from incomplete siRNA penetrance and allow more precise quantification of how incremental changes in TDP-43 protein levels track with GFP fluorescence. In the present study, our goal was to establish proof-of-principle functionality of the CUTS circuit and to demonstrate that graded TDP-43 depletion produces a proportional sensor response at the population level. While GFP signal heterogeneity is visible in imaging panels, we hypothesize that this variability likely reflects known differences in siRNA uptake and transfection efficiency rather than instability of the circuit itself. Importantly, bulk measurements consistently demonstrated dose-dependent sensor regulation across independent experiments, supporting the robustness of the system despite cellular heterogeneity. Furthermore, we were able to quantify CUTS activation in HeLa TARDBP^-/- cells. We also note that CUTS was developed as a practical tool for rapid assessment of TDP-43 LOF in standard laboratory settings. Although flow cytometry increases resolution, the ability to detect functional perturbation using bulk fluorescence measurements supports the utility of the system for routine and high-throughput applications.

We agree that flow cytometry would provide a more refined analysis of the dynamic range and sensitivity of CUTS, particularly for defining thresholds such as minimal TDP-43 knockdown required for measurable activation. We plan to include this work in future studies. Specifically, we have implemented FACs sorting of CUTS-expressing cells in a parallel study in which we are conducting a CRISPR knockout screen to identify modifiers of TDP-43 splicing function. For this, we incorporate TDP-43 knockdown followed by FACs to stratify cells based on CUTS activation. This strategy enables direct evaluation of the relationship between the extent of TDP-43 LOF and CUTS sensor activation. These analyses are ongoing and provide a more quantitative analyses linking TDP-43 depletion to CUTS activation and address the reviewer’s concern regarding heterogeneity in bulk measurements. We plan to include this in a future study.

(3) Some panels in the manuscript would benefit from additional clarity to make the data easier to visualize. For example, Figure 2D and 2G could be presented in a more clear manner, possibly split into additional graphs since there are too many outputs.

We thank the reviewer for this suggestion. In response, we have split the graphs previously shown in Figures 2D and 2G to improve clarity, as we agree that these panels contained an extensive amount of data. We Specifically split Figure 2D into two separate graphs showing TDP-43 and GFP pixel intensity from Western blots on the Y-axis, plotted against low siTDP-43 treatment on the X-axis. Please see this data as Figure 2 D and Figure 2E in the new manuscript.

Furthermore, for Figure 2G we also split into graphs showing the fold change of mRNA for TDP-43 and the CUTS cryptic exon plotted against low siTDP-43 treatment on the X-axis. Please see this data as Figure 2 H and Figure 2I in the new manuscript. We have maintained the previous graphs in Supplementary Figure 2 to preserve the full dataset for reference.

(4) Sup Figure 2A image panels would benefit from being labeled, its difficult to tell what antibodies or fluorophores were used. Same with Figure 4B.

We appreciate the reviewer’s careful observation. In both figures, we are showing mCherry and GFP signals. In the revised version, we have added the corresponding labels to the side of each image for clarity. Therefore, Sup Figure 2A has been moved and is now Sup Figure 3A, while Figure 4B remains in its original configuration.

(5) Figure 3 is an important addition to this manuscript and in general is convincing showing that TDP43 loss of function mutants can alter the sensor. However, there is still wild-type endogenous TDP-43 in these cells, and it's unclear whether the 5FL mutant is acting as a dominant negative to deplete the total TDP-43 pool, which is what the data would suggest. This could have been clarified.

The TDP-43 5FL variant exhibits reduced RNA-binding capacity, and we previously demonstrated that impaired RNA binding promotes aberrant homotypic phase separation of TDP-43. Consistent with this mechanism, expression of RNA-binding–deficient TDP-43 variants induces the formation of nuclear “anisomes” which have been shown to sequester endogenous TDP-43 into insoluble fractions via dominant-negative mechanisms (Cohen et al., 2015; Keating et al., 2023; Mann et al., 2019; Yu et al., 2021). These findings support a model in which disruption of RNA engagement alters TDP-43 biophysical behavior and promotes functional depletion through self-association. We have expanded this mechanistic explanation in the Results section of the revised manuscript to better contextualize the behavior of the 5FL construct and its impact on endogenous TDP-43.

(6) Additional treatment with stressors that inactivate TDP-43 could be tested in future studies.

We appreciate this suggestion and agree with this important point. Due to the lack of methods to directly induce endogenous TDP-43 aggregation and loss of function, the use of stressors has become a partial solution to address this issue. In line with this, our group has tested several stressors in follow-up research, including sodium arsenite (NaAsO₂), puromycin, KCl, MG132, sorbitol, and tunicamycin, using HEK cells expressing the CUTS system(Xie et al., 2025). We were able to show a dose-response relationship in relative GFP intensity under these conditions, with sodium arsenite showing the strongest effect, consistent with previous reports(Huang et al., 2024). To provide additional relevant findings in the current manuscript, we expanded this analysis by testing sodium arsenite in the CUTS system while also including endogenous cryptic exons. We therefore added a new figure showing the effect of sodium arsenite on the CUTS system, including GFP intensity measurements, qPCR using CUTS cryptic exon primers, and three endogenous cryptic exon reporters (ATG4B, GPSM2, and KCNQ2).

Overall, the authors definitely achieved their goals by developing a very sensitive readout for TDP-43 function. The results are convincing, rigorous, and support their main conclusions. There are some minor weaknesses listed above, chief of which is the use of flow sorting to improve the data analysis. But regardless, this study will have an immediate impact for those who need a rapid, reliable, and sensitive assessment of TDP-43 activity, and it will be particularly impactful once this reporter can be used in isolated primary cells (ie neurons) and in vivo in animal models. Since TDP-43 loss of function is thought to be a dominant pathological mechanism in ALS/FTD and likely many other disorders, having these types of sensors is a major boost to the field and will change our ability to see sub-threshold changes in TDP-43 function that might otherwise not be possible with current approaches.

(7) Regarding the methods, they seem a bit sparse and would benefit from additional detail. For example, I do not see a section in the methods where microscopy images were quantified (%GFP positive cells for example). This information is important and is lacking in the current form.

We thank the reviewers, and we add the following information in the method section: For live imaging quantification, we measured the mean GFP signal intensity for each group. The values were averaged, and the fold change was calculated and plotted. For immunofluorescent imaging, we first created maximum intensity projection images. We then applied masks to the GFP, mCherry, and Hoechst signals. By overlapping the GFP and mCherry signals, we identified the number of GFP-positive cells. Similarly, by overlapping the mCherry signal with the Hoechst mask, we identified the CUTS-expressing cells. We then calculated the ratio of GFPpositive cells to CUTS-expressing cells and plotted it as a percentage of GFP-positive cells. All analyses were performed using the Nikon NIS software. This information is included in the methods of the revised manuscript.

Reviewer #3 (Public review):

The DNA and RNA binding protein TDP-43 has been pathologically implicated in a number of neurodegenerative diseases including ALS, FTD, and AD. Normally residing in the nucleus, in TDP-43 proteinopathies, TDP-43 mislocalizes to the cytoplasm where it is found in cytoplasmic aggregates. It is thought that both loss of nuclear function and cytoplasmic gain of toxic function are contributors to disease pathogenesis in TDP-43 proteinopathies. Recent studies have demonstrated that depletion of nuclear TDP-43 leads to loss of its nuclear function characterized by changes in gene expression and splicing of target mRNAs. However, to date, most readouts of TDP-43 loss of function events are dependent upon PCR-based assays for single mRNA targets. Thus, reliable and robust assays for detection of global changes in TDP-43 splicing events are lacking. In this manuscript, Xie, Merjane, Bergmann and colleagues describe a biosensor that reports on TDP-43 splicing function in real time. Overall, this is a well described unique resource that would be of high interest and utility to a number of researchers. Nonetheless, a couple of points should be addressed by the authors to enhance the overall utility and applicability of this biosensor.

(1) While the rationale for selecting UNC13A CE as the reporting CE species is understood given the relevance to disease, could the authors please comment on whether other CE sequences would behave similarly or as robustly? This is particularly critical given the multitude of different splicing changes that can occur as a result of TDP-43 loss of function (ie cryptic exons of differing sensitivity, skiptic exons, premature polyadenylation).

We thank the reviewer for this question regarding generalizability beyond the UNC13A CE. While UNC13A was selected due to its strong disease relevance and well-characterized sensitivity to TDP-43 loss-of-function (LOF), our platform is not intrinsically restricted to this sequence. In the manuscript, we directly compared three architectures: UNC13A-TS, CFTR-TS, and the combined CUTS sensor incorporating additional UG motif optimization. Under matched conditions in stable HEK293 lines, CUTS demonstrated superior specificity and sensitivity, exhibiting near-zero baseline activity and a proportional, log-linear response across low-dose siTDP43 (38–1200 pM) (Figures 1–2). Importantly, this head-to-head comparison demonstrates that sensor performance can be engineered and optimized beyond a single CE species.

TDP-43 LOF is known to induce a spectrum of RNA processing defects, including cryptic exons with differing sensitivities and cell-type dependence, premature polyadenylation events (e.g., STMN2), and, under conditions of excess nuclear TDP-43, exon skipping (“skiptic exons”). This diversity supports the concept in which alternative CE elements, or other TDP-43 regulated RNAs, can be incorporated into the same sensor backbone and tuned for specific biological scenarios (cell type, specific stress responses, etc...). Consistent with this, the recently described TDP-REG system (Wilkins et al., 2024) designed and AI-generated de novo CE sequences to express reporters or gene payloads, and screened multiple candidates to identify the appropriate RNA elements required for this response. These findings demonstrate that CE sequences beyond UNC13A can serve as robust TDP-43 sensing elements when optimized. Our results complement this work by demonstrating that CUTS achieves tight baseline control and a steep dynamic range (>110,000-fold induction over baseline in HEK293 cells), while maintaining compatibility across both non-neuronal and neuronal model systems, as shown in the revised manuscript.

In the revised manuscript, we show direct comparisons indicating that CUTS outperforms single-CE sensors such as UNC13A-TS and CFTR-TS under identical conditions. This supports independent work from other groups that alternative CE sequences can be engineered into effective sensors, depending on their paradigm and model systems. We have clarified this in the revised Discussion and now note that CUTS is adaptable to alternative CE inserts.

(3) Could the authors provide evidence of the utility of their biosensor in disease relevant systems that do not rely on TDP-43 KD? For example, does this biosensor report on TDP-43 loss of function in C9orf72 iPSNs in a time-dependent manner? Alternatively, groups have modeled TDP-43 proteinopathy in wildtype iPSNs via MG132 treatment.

We thank the reviewer for this important suggestion. We agree that demonstrating CUTS responsiveness in disease-relevant models independent of artificial TDP-43 knockdown would further strengthen its translational relevance. In the current study, our primary objective was to establish the sensitivity, dynamic range, and autoregulatory properties of the CUTS circuit under controlled perturbation of TDP-43 levels. siRNA-mediated depletion provides a reliable approach to establish the relationship between graded TDP-43 LOF and the CUTS sensor sensitivity/specificity. That said, CUTS is designed to detect functional TDP-43 loss irrespective of the upstream cause. As the reviewer notes, disease-relevant systems, such as C9orf72 iPSC-derived neurons and proteotoxic stress paradigms (e.g., MG132-induced impairment of TDP-43 nuclear function), are important for future studies. We are currently evaluating CUTS in iPSC-derived neuronal models of TDP-43 proteinopathy, but are optimizing the induction system, promoters, and timing. It should be noted that C9orf72 iPSC neurons do not exhibit TDP-43 LOF using standard differentiation protocols. Regarding pharmacological stress, we have shown that acute sodium arsenite treatment can activate CUTS (Figure 3). In a concurrent study under revision, we show that MG132 similarly causes TDP-43 LOF and CUTS activation (Xie et al., 2025). Notably, none of these induce complete nuclear loss of TDP-43; instead, they show nuclear TDP-43 retention or modest mislocalization. This suggests that TDP-43 LOF may also result from nuclear redistribution and dysfunction under these stress conditions, rather than from complete nuclear loss. We look forward to presenting these ongoing studies in the future.

References

Brown A-L, Wilkins OG, Keuss MJ, Kargbo-Hill SE, Zanovello M, Lee WC, Bampton A, Lee FCY, Masino L, Qi YA, Bryce-Smith S, Gatt A, Hallegger M, Fagegaltier D, Phatnani H, NYGC ALS Consortium, Newcombe J, Gustavsson EK, Seddighi S, Reyes JF, Coon SL, Ramos D, Schiavo G, Fisher EMC, Raj T, Secrier M, Lashley T, Ule J, Buratti E, Humphrey J, Ward ME, Fratta P. 2022. TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A. Nature 603:131–137. doi:10.1038/s41586-022-04436-3

Cohen TJ, Hwang AW, Restrepo CR, Yuan C-X, Trojanowski JQ, Lee VMY. 2015. An acetylation switch controls TDP-43 function and aggregation propensity. Nat Commun 6:5845. doi:10.1038/ncomms6845

Huang W-P, Ellis BCS, Hodgson RE, Sanchez Avila A, Kumar V, Rayment J, Moll T, Shelkovnikova TA. 2024. Stress-induced TDP-43 nuclear condensation causes splicing loss of function and STMN2 depletion. Cell Rep 43:114421. doi:10.1016/j.celrep.2024.114421

Keating SS, Bademosi AT, San Gil R, Walker AK. 2023. Aggregation-prone TDP-43 sequesters and drives pathological transitions of free nuclear TDP-43. Cell Mol Life Sci 80:95. doi:10.1007/s00018-023-04739-2

Mann JR, Gleixner AM, Mauna JC, Gomes E, DeChellis-Marks MR, Needham PG, Copley KE, Hurtle B, Portz B, Pyles NJ, Guo L, Calder CB, Wills ZP, Pandey UB, Kofler JK, Brodsky JL, Thathiah A, Shorter J, Donnelly CJ. 2019. RNA Binding Antagonizes Neurotoxic Phase Transitions of TDP-43. Neuron 102:321-338.e8. doi:10.1016/j.neuron.2019.01.048

Wilkins OG, Chien MZYJ, Wlaschin JJ, Barattucci S, Harley P, Mattedi F, Mehta PR, Pisliakova M, Ryadnov E, Keuss MJ, Thompson D, Digby H, Knez L, Simkin RL, Diaz JA, Zanovello M, Brown A-L, Darbey A, Karda R, Fisher EMC, Cunningham TJ, Le Pichon CE, Ule J, Fratta P. 2024. Creation of de novo cryptic splicing for ALS and FTD precision medicine. Science 386:61–69. doi:10.1126/science.adk2539

Xie L, Zhu Y, Hurtle BT, Wright M, Robinson JL, Mauna JC, Brown EE, Ngo M, Bergmann CA, Xu J, Merjane J, Gleixner AM, Grigorean G, Liu F, Rossoll W, Lee EB, Kiskinis E, Chikina M, Donnelly CJ. 2025. Contextdependent Interactors Regulate TDP-43 Dysfunction in ALS/FTLD. BioRxiv. doi:10.1101/2025.04.07.646890

Yu H, Lu S, Gasior K, Singh D, Vazquez-Sanchez S, Tapia O, Toprani D, Beccari MS, Yates JR, Da Cruz S, Newby JM, Lafarga M, Gladfelter AS, Villa E, Cleveland DW. 2021. HSP70 chaperones RNA-free TDP-43 into anisotropic intranuclear liquid spherical shells. Science 371. doi:10.1126/science.abb4309.

Author Response:

The following is the authors’ response to the previous reviews

Public Review:

Reviewer #1 (Public review):

The weaknesses are in the clarity and resolution of the data that forms the basis of the model. In addition to general whole embryo morphology that is used as evidence for CE defects, two forms of data are presented, co-expression and IP, as well as a strong reliance on IF of exogenously expressed proteins. Thus, it is critical that both forms of evidence be very strong and clear, and this is where there are deficiencies; 1) For vast majority of experiments general morphology and LWR was used as evidence of effects on convergent extension movements rather than keller explants or actual cell movements in the embryo. 2) the microscopy would benefit from super resolution microscopy since in many cases the differences in protein localization are not very pronounced. 3) the IP and Western analysis data often shows very subtle differences, and some cases not apparent.

Major points.

(1) Assessment of CE movement

The authors conducted an analysis of the subcellular localization of PCP core proteins, including Vangl2, Pk, Fz, and Dvl, within animal cap explants (ectodermal explants). The authors primarily used the length-to-width ratio (LWR) to evaluate CE movement as a basis for their model. However, LWR can be influenced by multiple factors and is not sufficient to directly and clearly represent CE defects. While the author showed that Prickle knockdown suppresses animal cap elongation mediated by Activin treatment, they did not test their model using standard assays such as animal cap elongation or dorsal marginal zone (DMZ) Keller explants. Furthermore, although various imaging analyses were performed in Wnt11-overexpressing animal caps and DMZ explants, the Wnt11-overexpressing animal caps did not undergo CE movement. Given that this study focuses on the molecular mechanisms of Vangl2 and Ror2 regulation of Dvl2 during CE, the model should be validated in more appropriate tissues, such as DMZ explants.

(2) Overexpression conditions

Another concern is that most analyses were performed with overexpression conditions. PCP core proteins (Vangl2, Pk, Dvl, and Fz receptors) are known to display polarized subcellular localization in both the neural epithelium and DMZ explants (Ref: PCP and Septins govern the polarized organization of the actin cytoskeleton during convergent extension, Current Biology, 2024). However, in this study, overexpressed PCP core proteins failed to show polarized localization. Previous studies, such as those from the Wallingford lab, typically used 10-30 pg of RNA for PCP core proteins, whereas this study injected 100-500 pg, which is likely excessive and may have created artificial conditions that confound the imaging results.

(3) Subtle and insufficient effects

Several of the reported results show quite modest changes in imaging and immunoprecipitation analyses, which are not sufficient to strongly support the proposed molecular model. For example, most Dvl2 remained localized with Fz7 even under Vangl2 and Pk overexpression (Fig. 4). Similarly, Wnt11 overexpression only slightly reduced the association between Vangl2 and Dvl2 (Sup. Fig. 8), and the Ror2-related experiments also produced only subtle effects (Fig. 8, Sup. Fig. 15).

We thank reviewer 1 for careful reading of our revised manuscript, and additional constructive criticisms. Since the two reviewers had divergent opinions towards our revised manuscript, we think that it might be more productive to request a Version of Record at this point, and have our proposed model debated/ tested by others in the field. We will keep the reviewer’s suggestions in mind while design ongoing studies. We would like to address the criticisms collectively below:

(1) The primary goal of our current manuscript is to build a mechanistic model for non-canonical Wnt signaling through elucidating the functional relationships between Dvl, Vangl, PK and Ror during CE. They each have been studied extensively in prior literature using DMZ injected embryos, and DMZ, Keller and animal cap explants, so there is little doubt that the reduced LWR following their over-expression or knockdown in DMZ is due to disruption of CE. In the context of our study in the current manuscript, we primarily performed their co-injections in different combinations to differentiate synergistic vs. antagonistic relationship, and in the majority cases we relied on epistatsis to draw conclusions (e.g. Fig. 1; Fig. 2h, I; Suppl. Fig. 6; Suppl. Fig. 14). Nevertheless, we did follow the reviewer’s suggestion and used animal cap elongation as an additional assay to confirm that Pk and Vangl2 did synergize to disrupt CE, and their synergy could be blocked by Dvl2 co-overexpression; the new data is added to Fig. 1 (Fig. 1h, h’). Therefore, given the prior literature, our new animal cap explant data, and the specific scope of our current study, we feel that the LWR measurement is a reasonable assay to determine CE phenotype in this manuscript. We fully agree with the reviewer that our model will need to be tested at the cellular level through live imaging of DMZ explants; it is indeed the direction of our future study, but is beyond the scope of the current manuscript.

(2) A salient feature of non-canonical Wnt signaling is that loss or over-expression of any components can often cause identical CE defects at the tissue/ embryo level. We used many co-injection experiments to demonstrate that this is due, at least in part, to a counterbalance between Dvl/Ror and Vangl/PK (e.g. Fig. 1; Fig. 2h, I; Suppl. Fig. 6; Suppl. Fig. 14). It is in this context that we planned the imaging and biochemical experiments to determine the possible molecular mechanisms underlying their functional interaction, and we feel that the moderate over-expression used is reasonable in this case for us to build the first integrated model. We do plan to test our model using lower expression in the future. To acknowledge the limitation of our study, we also added the following sentences in the Discussion:

“We acknowledge, however, that our model explains primarily the potential molecular actions underlying the regulation of CE at the tissue level. Whether and how our model may explain the cellular behavior during CE, such as polarized remodeling of cell junction or extension of cell protrusions, will require further study.”

(3) The Wnt11 induced reduction of Dvl2-Vangl2 co-IP (Suppl. Fig. 8, 15) may be moderate, but is statistically significant and reproducible, and we have reported similar findings in two other publications (DOI: 10.1093/hmg/ddx095; DOI: 10.1038/s41467-025-57658-0). Given the limitation of co-IP, we had to rely on high level over-expression to make the experiments feasible. We are building proximity based assays such as NanoBRET, and plan to verify the result with lower level expression in the future.

Reviewer #2 (Public review):

We thank the reviewer for the encouraging comments, and the suggestion to clarify the description related to Suppl. Fig. 15. We made revision according to the reviewer’s suggestion, and added Suppl. Fig. 16 to further examine the effect of Ror2 knockdown on the steady state interaction between Dvl2 and Vangl2 using imaging approach.

image

Reviewer #1 (Public review):

Summary:

This manuscript presents an end-to-end pipeline, intended to accelerate EM-based connectomics by combining low-resolution imaging for large volumes with synapse-level imaging only in selected regions of interest. In principle, this strategy can substantially reduce imaging time, computational demands, analysis time, and overall cost.

General note:

Overall, I found the manuscript interesting and valuable, particularly as a description of how one laboratory has assembled and applied a practical workflow to reconstruct and analyze the central complex across multiple insect species. In that sense, the work is compelling as an account of a real, functioning strategy for comparative connectomics, and I appreciated reading it. My main reservation is not about the relevance of the biological problem or the utility of the pipeline in the authors' own hands, but about whether the manuscript, in its current form, fully meets the expectations of a paper that is focused on tools and resources. The expectation would be that this paper would be a venue for sharing new techniques, software tools, datasets, and other resources intended to be usable by the community. Here, because much of the pipeline appears to build on existing methods and software, the key value added should be a particularly clear demonstration of how these components were adapted, integrated, validated, and documented for this specific use case in a way that others could realistically reproduce and adopt. At present, that translational and reproducibility-oriented component does not yet seem sufficiently developed, despite the clear promise of the overall approach.

Major comments:

(1) The work is valuable as a practical integration and application of multiple existing tools into a coherent pipeline, together with a new multi-resolution imaging strategy. However, the manuscript at times reads as though it introduces an entirely novel workflow. I would encourage the authors to clarify the contribution more explicitly: which components are genuinely new (for example, the acquisition strategy and the end-to-end integration/validation), and which are adaptations of already established methods or software. This would make the scope and novelty of the paper easier to assess.

(2) The most distinctive element is the multi-resolution acquisition strategy. However, as described, the selection of high-resolution regions seems to be decided a priori based on anatomy (guided by xCT localization of the CX), rather than being determined automatically from the data (i.e., ROI placement is anatomy-driven rather than data-driven). A more data-driven or machine learning-guided ROI strategy would strengthen the methodological contribution and the adaptability to new scenarios, along the lines of approaches such as SmartEM [1].

(3) The manuscript emphasizes open-source availability and reduced barriers to entry, but the current software release, as referenced, does not yet appear to support straightforward external reuse. Since much of the pipeline builds on existing methods, the main added value lies in how these technologies were adapted, combined, and validated for the present problem. A clear and complete explanation of this adaptation is therefore essential, but is currently missing. I would suggest the following concrete improvements:<br /> a) Provide a single landing page or umbrella repository that links each pipeline step in the paper to the corresponding codebase, including version tags/commits and expected inputs/outputs for each step.<br /> b) Include step-by-step tutorials for each component.<br /> c) Provide an example dataset together with a full reproduction walkthrough in a controlled environment.<br /> d) Clearly explain the required parameters and configuration for each step, including how they should be adjusted for other datasets or scenarios.<br /> e) Follow packaging and distribution best practices (for example, PyPI/conda releases, Docker containers, and version pinning).

(4) In my own attempt to set up and run parts of the released code, I encountered issues that currently limit reproducibility. For example, when creating an environment for EMalign (https://github.com/Heinze-lab/EMalign), the required Python version is not specified, and installation did not succeed under Python 3.12 due to dependency constraints. Additionally, synful_312 (https://github.com/Heinze-lab/synful_312) and SegToPCG (https://github.com/Heinze-lab/SegToPCG) appear to be empty despite being referenced in the manuscript. These are fixable issues, but addressing them is important if the paper is to deliver on its "low entry cost" claim.

(5) Table 1 reports acquisition times, which is helpful. However, the multi-resolution approach adds essential processing steps that appear due to the strategy followed (e.g., "XY alignment high-res" and "high-res to low-res alignment"). Please include registration/alignment (and other major post-processing) runtimes and resource requirements, such as storage, in a comparable table so readers can assess true end-to-end cost.

References:

[1] Meirovitch, Y., et al. "SmartEM: machine learning-guided electron microscopy." Nature Methods (2025).

Reviewer #2 (Public review):

Summary:

The paper proposes a workflow to accelerate EM connectomics by combining multi-scale imaging with image processing and analysis (image alignment, registration, neuron tracing, automated segmentation and synapse prediction, proof-reading) to derive a brain region connectome. The paper argues and (partially) demonstrates that this approach facilitates comparative connectomics.

The data acquisition pipeline uses a well-established sample preparation protocol, uCT guided acquisition, and SBEM imaging at cellular and synaptic resolution.

Data processing and analysis combine existing state-of-the-art components and focus on the alignment and complementary analysis of the two SBEM resolution levels. The paper applies the workflow to the central complex of six different insects and performs some preliminary analysis based on this (which is acceptable for a resource/tool).

Disclaimer for the rest of the review: I am an expert in image analysis and segmentation, so I have mainly focused on these aspects as I am not qualified to analyze the details of image acquisition.

Strengths:

The paper addresses an important problem and promises an acceleration and democratization of comparable connectomics. The time savings of the imaging approach are well-motivated and derived. The methods used for image alignment, segmentation, synapse detection, and proofreading are state-of-the-art.

Weaknesses:

I see two major weaknesses in the paper:

(1) The paper introduces the (approximate) equivalence of the projectome and connectome in the insect brain very prominently in the introduction and uses this as a central motivation for the multi-resolution image acquisition protocol. But - to me - it is unclear how this principle is really used in the analysis presented in the last results and if this assumption is evaluated at all. Specifically, Figure 4 a shows the anatomical neuron reconstructions (from cellular resolution SBEM), d-g show connectome-level analysis from the synaptic resolution data. The only link I can see between the two is that the neural processes in the synapse-resolution data can be mapped to the neurons from the cellular resolution data, thanks to the image alignment. This is certainly important, BUT it is only tangentially related to the projectome vs. connectome claim from the introduction. This claim implies that a tentative connectome is derived from projectome-level data (e.g. by assuming a uniform probability of synapse-formation given surface or distance between projections) that is then validated by the "true" connectome data from synaptic resolution. Instead, what is actually solved - to my understanding - is mapping the local connectome to the projectome. While related, these are different things and the current framing of the paper and the quite brief description of the section on comparative connectomics (also no corresponding Methods section) make this claim inadequately supported.

(2) Reporting on segmentation and proofreading is purely qualitative. Given that this is claimed as a core contribution of the paper (e.g. statement in line 497 and following), I would expect substantially more reporting and evaluation of this claim:<br /> a) Report the actual time needed for proofreading the segmentations in CAVE. I could not find any numbers on this.<br /> b) Report the initial segmentation quality of the model: How many errors does it make? Note: There is a brief mention of VoI-based quantification in Methods (around line 1060), but the results are not reported.

What should be done: Report the error rates (with an accurate measure such as skeleton VoI) independently for all 6 volumes. Given that the authors have the proofread versions, this is feasible. Only then can the claims be made here be evaluated. Note that the F1-score of synapse prediction is quantified. This is a good starting point, but could also be extended to further species in order to assess the actual transferability. Furthermore, none of the data from the study seems to be available. The training data of the network has to be made available. If possible, high-resolution data should be proofread too.

Further points:

(1) Why isn't reconstruction at the cellular level addressed with ML? This is surely possible and should be easier than the full connectome analysis. Similar to before, the actual times needed for tracing with CATMAID are not reported; the manuscript only states that this can be done in minutes for a neuron, but it's unclear if this is the best or average case. It would help to have quantitative numbers to assess whether automation would bring any benefits.

(2) Finally, regarding the underlying software. I did not try this myself due to time constraints, but did check the repositories. They seem to be in an ok state with some documentation in a README. However, given the central role of the software contribution, I would expect a centralized doc page that explains how to use the different parts of the software, including a full example with sample data. Without this, application by other labs - a central claim - will be difficult.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors test the hypothesis that whole-brain functional magnetic resonance imaging in behaving mice, coupled with reinforcement-learning modeling, can dissociate neural substrates of initial cue-reward acquisition versus contingency reversal, and potentially reveal underappreciated contributors to cognitive flexibility. Using a head-fixed go/no-go odor discrimination task with subsequent rule reversal in a subset of mice, they model trial-by-trial state-action values with a model-free Q-learning algorithm (hierarchical Bayesian fit) and use the model-derived decision variable as a parametric regressor in whole-brain analyses. They report that acquisition-related signals prominently involve ventral and dorsal striatal regions, whereas reversal learning additionally recruits the periaqueductal gray (negative correlation with the decision variable) and shows an apparent double dissociation between nucleus accumbens and periaqueductal gray responses for hit versus correct-rejection outcomes during reversal.

Strengths:

(1) The reversal manipulation is implemented without explicit punishment, targeting suppression of previously rewarded actions under reward omission - an underexplored regime for midbrain contributions beyond canonical threat/pain framing.

(2) The manuscript provides a credible MR-compatible olfactory/licking platform with synchronized sniff/lick/valve/reward timing and high-field imaging, supporting feasibility and broader utility for mesoscale systems neuroscience in rodents.

(3) Trial-by-trial value estimates from a Q-learning variant are fit via hierarchical Bayesian inference and explicitly integrated into subject-level general linear models with a mouse hemodynamic response function, which is appropriate for leveraging within-subject dynamics in small-N rodent fMRI.

(4) The decision-variable maps during acquisition recover expected basal ganglia involvement (including nucleus accumbens and dorsal striatum), providing face validity; the reversal-stage map yields an interpretable set of cortical/striatal/pallidal regions plus periaqueductal gray/hippocampus.

(5) The finite impulse response analysis stratified by behavioral outcomes (hit, false alarm, correct rejection, miss) adds interpretability beyond the model regressor alone, and the reported crossover interaction between nucleus accumbens and periaqueductal gray is potentially impactful if robust.

Weaknesses:

(1) The core claim regarding selective periaqueductal gray engagement rests on a subset of n = 6 mice for reversal. With permutation-based whole-brain inference and very small cluster sizes, the robustness of the periaqueductal gray effect to reasonable analytic perturbations is not yet convincing. I would suggest providing leave-one-animal-out analyses for the periaqueductal gray cluster/ROI effects and reporting how often the key findings survive.

(2) The authors note that due to temporal resolution and hemodynamics, they cannot separate stimulus, choice, and feedback and therefore model "whole trials." This limitation creates ambiguity about whether periaqueductal gray signals reflect value updating, action inhibition (no-lick), reward omission, autonomic arousal, or motor preparation/withholding, especially given the strong hit versus correct-rejection opponency. I would suggest adding targeted analyses that disambiguate "withholding" from "reversal-related updating".

(3) ROIs are defined from the whole-brain decision-variable maps and then interrogated by outcome types; the manuscript acknowledges non-independence. This can inflate apparent dissociations. It would be better if the authors define ROIs independently (anatomical periaqueductal gray/nucleus accumbens masks, or split-half ROI definition with held-out data) and repeat the key ROI conclusions.

(4) The reversal group is a subset of the acquisition cohort and also experiences a different task phase structure and additional sessions; the paper attempts to address exposure differences descriptively. I would suggest that the authors formally test whether periaqueductal gray effects are explained by session count, time-in-scanner, or learning rate differences (e.g., include these as covariates, or match sessions more strictly).

(5) The platform records sniffing and licking, but the imaging models described include motion, global, and ventricle regressors and do not clearly include trialwise lick/sniff covariates. Given the periaqueductal gray's known autonomic and defensive coordination roles, physiological state confounding is a major concern. Could the authors incorporate sniff and lick metrics (and their derivatives) as nuisance regressors and show whether the periaqueductal gray effects persist?

Reviewer #1 (Public review):

Summary:

In their manuscript, Zhou and colleagues present a detailed look at how the JSP functions differently in the various cells of a breast tumor. The authors have effectively shown that the JSP acts as a double-edged sword, as it helps T cells fight cancer but also allows tumor cells to grow and avoid ferroptosis. These findings are important because they identify a useful biomarker to predict how TNBC patients might respond to PD-1 inhibitors.

Strengths:

This work is important because it provides a clear explanation for the conflicting roles of the JSP in the tumor environment. The evidence is solid, as it combines data from thousands of patients with single-cell analysis and lab experiments to confirm the role of STAT4 in cancer progression and immunity.

Weaknesses:

However, there are areas for improvement in the scope of the review, the depth of analysis, and the potential for broader clinical implications. The authors are encouraged to address these issues to enhance the scientific and clinical impact of the study.

Major Issues:

(1) The authors demonstrate that STAT4 upregulates SLC47A1, but this is currently supported only by expression correlation and western blot data. To confirm a direct link, the authors are encouraged to perform ChIP-qPCR or luciferase reporter assays to show that STAT4 binds directly to the SLC47A1 promoter.

(2) The conclusion that the MIF-CD74 axis drives immunosuppression is based on computational inference. To support this, the authors could consider mining publicly available breast cancer spatial transcriptomics data to show the co-localization of MIF and CD74. Alternatively, performing simple dual-color immunofluorescence staining on a few clinical sections would effectively demonstrate the physical proximity of these cells.

(3) TNBC is highly heterogeneous and includes subtypes like mesenchymal and immunomodulatory groups. The authors should analyze whether the JSP score or STAT4 levels vary significantly between these subtypes, as this could further refine the selection of patients for JAK1 inhibitors.

(4) While the JSP score works well in the current datasets, the authors should consider validating its predictive accuracy in additional independent immunotherapy cohorts, such as the TONIC trial, to ensure the biomarker is robust across different treatment settings.

Minor Issue:

The manuscript mentions a U-shaped trajectory of JSP activity during tumor transition. A more detailed biological explanation of why the pathway activity initially drops and then rises would add depth to the discussion.

.

It is so important to find picture books that use translanguaging. These books can serve as models. Students can see how authors use multiple languages and apply that to their own writing. But these books also serve a deeper purpose. They show students that their languages belong in school. Many students are used to thinking they should only write in English. Seeing these books challenges that idea. It almost gives them permission to use all their language resources and write in a way that feels natural to them. It tells them, “these authors are doing it and you can do it too.”

This is why text choice is so important. The books we use can send powerful messages to students. Books that include translanguaging show students that their language practices are important and something we want to see in their own writing. This is why the students’ “faces seemed to open up like a thank you.”

Talia Zoref: I agree, translanguaging is beneficial for students because they are not limited by one language. They may want to say something in a certain way but can only do so in their native language. It also makes them feel more connected as they may talk another language at home. These students are not put into a box when they have this flexibility. Translanguaging promotes student identity and culture through cultural names, places, and phrases.

I find this description of translanguaging to be a very good one because translanguaging does not limit or isolate language and rather it sees it as a collection of linguistic abilities that can be used in unison to express meaning and feeling. Being worded as an "active negotiation of language" is such an interesting way to put it because as somebody who translanguages every single day, it sounds like a good way to express the idea that the two languages aren't competing, but are instead flowing in our brains and bargaining depending on the situation or circumstance that we find ourselves in.

On 2026-01-19 13:00:58, user Gene C Koh wrote:

Gene Ching Chiek Koh, Serena Nik-Zainal

Department of Genomic Medicine, University of Cambridge, CB2 0QQ, UK.

We commend Kanwal et al. for their timely evaluation of the in vivo mutagenic potential of CX-5461. This follows our report that CX-5461 induces substantial mutagenesis in cultured mammalian cells1. The authors analysed samples from four patients treated with CX-5461, including marrow aspirates, trephine biopsies, PBMCs, and skin lesions collected at early treatment timepoints (baseline; days 1, 2, or 9; and end-of-treatment of a 21-/28-day cycle), and used error-corrected duplex sequencing to detect low-frequency mutations. They concluded that CX-5461 exposure did not increase single-/ double-base substitution or indel burdens, nor reproduced the mutational signatures reported in our in vitro study. While we welcome their contribution, several methodological and interpretive shortcomings limit the conclusions that can be drawn.

1. Data presentation<br /> Figures 1–3 present absolute mutation counts instead of frequencies normalized to total informative duplex bases per sample. In duplex sequencing, normalization is a basic requirement to account for variability in sequencing depth and library complexity; without it, true mutation accrual or fold-change differences versus controls (if any) cannot be assessed reliably.

2. Experimental controls, assay sensitivity, and performance<br /> The study lacks essential positive and negative controls making it impossible to evaluate whether the sequencing and analytical processes used by the authors have worked. Clinical samples with known mutational signatures detectable through this approach should have been included to confirm assay sensitivity and substantiate a true negative finding. This is fundamental. Samples from patients unexposed to CX-5461 were also required as negative controls to establish background variability, affording confidence intervals and statistical robustness.<br /> Moreover, the authors have not shown awareness of the assay’s limit of detection (LOD). What is the smallest measurable fold-change at the reported sequencing depth? Without this, one cannot determine the smallest mutational differences that could have been missed. The authors have not disclosed quality-control metrics required to understand whether sufficient data quality was achieved for detecting differential mutagenesis. P/S: TwinStrand kit has an error rate ~0.5e-7 to 1e-7 depending on the protocols, and this can be considerably higher if DNA quality is low or from fixed biopsies.

3. Lack of curation, comparisons to literature<br /> The reported mutation counts did not make sense (baseline values exceeding treated samples, patient samples sometimes lower than kit control). The authors should perform some ‘sanity check’ comparisons with published mutation frequencies of respective normal adult tissues from other duplex-sequencing studies2,3. Analytical rigour would include, for example, examining whether detected variants represent driver mutations from clonal haematopoiesis or occurred in genes under post-treatment selection. Such analyses would have demonstrated critical evaluation of data quality and biological relevance.

4. Cell-type considerations, sampling window<br /> Most analysed compartments—PBMCs, MACS-sorted marrow fractions—are dominated by mature, non-dividing cells that rarely fix new mutations. A more relevant population for assessing mutagenicity is the haematopoietic stem and progenitor cells (HSPCs), typically <0.5% of marrow cells. A null result in the analysed compartments could just mean no widespread mutation fixation in mature immune cells; it does not exclude the possibility of mutagenesis in progenitors below the detection threshold of the current assay.<br /> In addition, samples were taken at very early timepoints (days 1, 2, 9, or EOT) of the first treatment cycle. At such intervals, mutagenic events are unlikely to have become fixed, as mutagen-induced DNA damage will need time to become embedded through DNA repair and replication. Exposure in terminally-differentiated cells might yield no detectable mutations. If exposure occurs on dividing cells, mutational footprints may only become detectable months or years after exposure. The current dataset lacks the temporal window necessary to assess cumulative in vivo mutagenicity.

5. Expected evidence of prior treatments <br /> All four patients reportedly had “measurable, relapsed, or refractory advanced haematologic malignancies without any standard therapeutic options available”4. Although treatment histories were not provided, these patients likely received multiple prior therapies (e.g., doxorubicin, cyclophosphamide, etc) that could induce characteristic mutational signatures in normal haematopoietic cells5. Were signatures of prior therapy detected by the authors? Their absence raises concerns regarding the overall assay sensitivity and/or suggests that sampling strategy was suboptimal for detecting mutagenic exposures.

6. Interpretation of model data<br /> While critical of our findings in cultured human cells as “not adequately representative of physiological human tissue” – a limitation we explicitly acknowledged in our manuscript’s title and discussion – the authors cited a C. elegans study6 in support of their argument of “low non-selective mutagenic potential of CX-5461”. This interpretation is incorrect: the worm study reported high copy-number aberrations, high SNV burdens, and a distinct A>T/T>A-rich signature after CX-5461 exposure, with survival requiring multiple repair pathways (homology-directed repair, microhomology-mediated end joining, nucleotide excision repair, and translesion synthesis). If anything, these cross-species findings reinforce rather than contradict our observations that CX-5461 is highly mutagenic. The concentrations used in that study were chosen to promote viability in the worms, not to minimise mutagenicity. Selective viability does not equate to selective mutagenicity.

7. Clinical mutagenicity testing<br /> We agree that clinical safety assessments must be rigorous and physiologically relevant. The authors dismissed our experiments as not rivalling the “GLP-compliant, non-mutagenic” results of the CX-5461 drug development pathway. However, those mutagenicity data are not available in the public domain and have neither been shared by the authors nor the company that distributes CX-5461.

We urge the authors to reconsider and not simply dismiss our findings. First, the primary clinical quality mutagenicity assay (required by agencies such as the US Food and Drug Administration (FDA), European Medicines Agency, and UK Medicines and Healthcare Regulatory Agency (MHRA)) referred to by the authors comprises the Ames test – a reverse gene mutation test performed in prokaryotes (e.g., E.coli, Salmonella).

Second, according to the FDA’s ICH S2(R1) guidance for a standard battery of mutagenicity assays (Safety Implementation Working Group of the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use), additional genotoxic assays should be performed in mammalian cells in vitro (where some of the more common assays include metaphase chromosome aberration assays, the micronucleus assay, and the mouse lymphoma L5178Y cell Tk (thymidine kinase) gene mutation assay (MLA)) or in in vivo studies as necessary.

Third, the FDA guidance acknowledges that “no single test is capable of detecting all genotoxic mechanisms relevant in tumorigenesis” and that the standard battery serves primarily for hazard identification rather than comprehensive assessment of mutagenic potential. For negative in vivo results, the ICH S2(R1) guidance requires evidence of adequate target-tissue exposure (e.g., toxicity in the tissue, TK/PK data, or direct tissue concentrations) to validate interpretability. Without such data, negative findings have limited meaning, especially where in vitro systems demonstrate strong mutagenicity.

Fourth, while the Ames test served its purpose for decades, there are well-described problems including false positives, false negatives and critically, a lack of human metabolism that even supplementation with rodent S9 mix cannot always overcome.

Finally, a point also raised by the accompanying commentary to our publication is that perhaps the time has come to re-evaluate how mutagenicity assays are performed. Current assays cannot capture the genome-wide mutation patterns revealed by whole-genome sequencing in human cells, and as a community we should consider using unbiased, agnostic, modern genomic approaches capable of detecting all classes of mutational changes in human cells. This is not an attack on CX-5461; rather, it is a call to the community to consider re-evaluation of mutagenicity assays in drug development.

8. Unsubstantiated claims<br /> The claim of potential contaminants accounting for the mutagenic outcomes we and others have observed is speculative and unsupported. The fact that multiple studies1,6 observed the same mutagenic outcomes using CX-5461 from independent sources suggests that this is unlikely. The authors showed no analytic chemistry (LC-MS/MS) and/or spiking experiments to substantiate this claim.

9. Inadequate supporting material throughout <br /> There were many gaps in the methods/supporting information, including adequate clinical annotation, precise sampling times/total treatment cycle, and basic quality-control metrics. Experimental details (e.g., antibodies used for MACS sorting, essential for interpreting analysed subpopulations) were not provided. These omissions limit transparency, reproducibility, and the interpretability of the findings.

10. Beneficence, non-maleficence, autonomy, justice<br /> First, in academia and medicine, we are guided by the principle of doing no harm. In identifying mutagenesis in experimental systems (an incidental finding), we acted in the best interest of the community – reporting an observation that could have an impact on patients and acknowledging the limitations of our system. We have no role in the (dis)continuation of clinical trials; we simply presented our data transparently and highlighted potential risk. <br /> Second, while the authors chose to discontinue their trial, several others remained active (e.g., NCT04890613, NCT06606990, NCT07069699, NCT07147231, NCT07137416). Their decision was conservative, and in our view, scientifically prudent. We commend their caution. However, it does not justify criticism of those of us reporting safety concerns in good faith.<br /> Third, as a community, we serve society better by being aware of issues, addressing the problems with robust experiments rather than polarising into groups “for” or “against” a compound, so that truly beneficial compounds can get to patients as quickly as possible. <br /> Finally, safety concerns may extend beyond mutagenesis and include tumour promotion effects. CX-5461’s interaction with TOP2B, for example, has been linked to serious, late-emerging toxicities, including therapy-induced leukaemia and cardiotoxicity7-10.

Concluding remarks<br /> Given the experimental and analytical shortcomings outlined above, definitive conclusions regarding CX-5461’s in vivo mutagenicity cannot yet be drawn. The absence of evidence should not be taken as evidence of absence. Rigorous, longitudinal studies with appropriate controls and independent oversight are required to assess true medium- to long-term risks.

We share the authors’ view that thorough, transparent evaluation of anticancer agents is essential. Given the authors’ vested interest in finding a negative result, we suggest independent individuals be involved in performing the analysis/interpretation of their studies to negate potential conflicts of interest. We remain open to collaboration in this effort, in the shared interest of patient safety and scientific integrity.

1. Koh, G.C.C., Boushaki, S., Zhao, S.J., Pregnall, A.M., Sadiyah, F., Badja, C., Memari, Y., Georgakopoulos-Soares, I., and Nik-Zainal, S. (2024). The chemotherapeutic drug CX-5461 is a potent mutagen in cultured human cells. Nat Genet 56, 23-26. 10.1038/s41588-023-01602-9.
2. Abascal, F., Harvey, L.M.R., Mitchell, E., Lawson, A.R.J., Lensing, S.V., Ellis, P., Russell, A.J.C., Alcantara, R.E., Baez-Ortega, A., Wang, Y., et al. (2021). Somatic mutation landscapes at single-molecule resolution. Nature 593, 405-410. 10.1038/s41586-021-03477-4.
3. Machado, H.E., Mitchell, E., Obro, N.F., Kubler, K., Davies, M., Leongamornlert, D., Cull, A., Maura, F., Sanders, M.A., Cagan, A.T.J., et al. (2022). Diverse mutational landscapes in human lymphocytes. Nature 608, 724-732. 10.1038/s41586-022-05072-7.
4. Khot, A., Brajanovski, N., Cameron, D.P., Hein, N., Maclachlan, K.H., Sanij, E., Lim, J., Soong, J., Link, E., Blombery, P., et al. (2019). First-in-Human RNA Polymerase I Transcription Inhibitor CX-5461 in Patients with Advanced Hematologic Cancers: Results of a Phase I Dose-Escalation Study. Cancer Discov 9, 1036-1049. 10.1158/2159-8290.CD-18-1455.
5. Mitchell, E., Pham, M.H., Clay, A., Sanghvi, R., Williams, N., Pietsch, S., Hsu, J.I., Obro, N.F., Jung, H., Vedi, A., et al. (2025). The long-term effects of chemotherapy on normal blood cells. Nat Genet 57, 1684-1694. 10.1038/s41588-025-02234-x.
6. Ye, F.B., Hamza, A., Singh, T., Flibotte, S., Hieter, P., and O'Neil, N.J. (2020). A Multimodal Genotoxic Anticancer Drug Characterized by Pharmacogenetic Analysis in Caenorhabditis elegans. Genetics 215, 609-621. 10.1534/genetics.120.303169.
7. Pan, M., Wright, W.C., Chapple, R.H., Zubair, A., Sandhu, M., Batchelder, J.E., Huddle, B.C., Low, J., Blankenship, K.B., Wang, Y., et al. (2021). The chemotherapeutic CX-5461 primarily targets TOP2B and exhibits selective activity in high-risk neuroblastoma. Nat Commun 12, 6468. 10.1038/s41467-021-26640-x.
8. Zhang, W., Gou, P., Dupret, J.M., Chomienne, C., and Rodrigues-Lima, F. (2021). Etoposide, an anticancer drug involved in therapy-related secondary leukemia: Enzymes at play. Transl Oncol 14, 101169. 10.1016/j.tranon.2021.101169.
9. Cowell, I.G., Sondka, Z., Smith, K., Lee, K.C., Manville, C.M., Sidorczuk-Lesthuruge, M., Rance, H.A., Padget, K., Jackson, G.H., Adachi, N., and Austin, C.A. (2012). Model for MLL translocations in therapy-related leukemia involving topoisomerase IIbeta-mediated DNA strand breaks and gene proximity. Proc Natl Acad Sci U S A 109, 8989-8994. 10.1073/pnas.1204406109.
10. Zhang, S., Liu, X., Bawa-Khalfe, T., Lu, L.S., Lyu, Y.L., Liu, L.F., and Yeh, E.T. (2012). Identification of the molecular basis of doxorubicin-induced cardiotoxicity. Nat Med 18, 1639-1642. 10.1038/nm.2919.

On 2026-01-13 16:13:38, user Christine Stabell Benn wrote:

Comment on “Non-specific effects of vaccines on all-cause mortality: a meta-analysis of randomized controlled trials (RCTs) 2012–2025”<br /> Christine Stabell Benn, Frederik Schaltz-Buchholzer, Sebastian Nielsen, Peter Aaby<br /> We commend the authors for addressing the important and contentious question of non-specific effects (NSEs) of vaccines on all-cause mortality. However, we have several major concerns regarding the framing, completeness, methodology, and interpretation of the preprint. Collectively, these issues undermine the conclusions drawn.

1. Restricted research question and dismissal of large parts of the evidence baseThe authors explicitly restrict their review to randomized controlled trials (RCTs) published after the WHO review of non-specific effects(1). If the stated objective is to assess the evidence for NSEs on all-cause mortality in randomized trials, an updated meta-analysis incorporating all relevant RCTs, rather than an arbitrarily time-limited subset, would be more informative. The decision to exclude pre-2012 RCTs from the main analysis appears methodological rather than substantive and risks answering a narrow procedural question rather than addressing the broader scientific question.

More importantly, NSEs represent a research area in which randomized trials are inherently difficult or impossible to conduct at scale, because the vaccines in question are already part of routine immunization schedules. As in other areas of public health - such as smoking, breastfeeding, or nutrition - causal inference therefore relies on triangulation across multiple study designs, including observational studies and natural experiments, supported by biological and immunological evidence.<br /> If the intention is to provide a meaningful update on the state of the evidence for NSEs, a comprehensive synthesis that acknowledges the strengths and limitations of all relevant study designs - or at minimum a clear and balanced justification for excluding them - is required.

2. Incomplete identification of relevant randomized trialsDespite claiming a comprehensive search, the review misses several important randomized controlled trials that are directly relevant to NSEs, including recent RCTs published well within the stated search window (e.g. PubMed IDs: 39357573, 38350670, 33893799, 30256314). The omission of these trials raises concerns about the sensitivity of the search strategy and undermines confidence in the completeness of the evidence base.

3. Extreme clinical and methodological heterogeneity invalidates the pooled meta-analysis<br /> The meta-analysis combines trials of three different vaccines (BCG, measles vaccine, and OPV) administered at vastly different ages (birth to 59 months), with follow-up periods ranging from days to five years, and using different randomization schemes and outcomes structures. This is not merely “heterogeneity,” but fundamentally different interventions addressing different biological hypotheses.

Pooling these studies is not equivalent to combining “apples and bananas,” but rather apples and cars. The resulting pooled estimate does not correspond to a coherent causal treatment effect and is therefore not interpretable.

4. Non-adherence with the WHO meta-analysis methodologyBy pooling all vaccines together, and furthermore by not focusing on the time window where a given vaccine is the most recent, the authors of the new meta-analysis violates the principles set out in the WHO meta-analysis, which emphasized vaccine-specific analyses and the importance of the most recent vaccine exposure.

5. Overreliance on conservative confidence interval methods without adequate justificationThe authors emphasize the use of the Hartung-Knapp-Sidik-Jonkman (HKSJ) method as providing “more reliable and conservative control of type I error.” While HKSJ can be appropriate when few studies estimate the same underlying effect, its application here - given the very marked heterogeneity and conceptual incoherence of the pooled treatment effect - adds statistical conservatism without resolving the more fundamental problem of model misspecification. The resulting wide confidence intervals should not be interpreted as robust evidence against NSEs.

6. Misinterpretation of heterogeneity statistics (I²)The statement that an I² of ~44% indicates that “approximately half the differences in the results are due to actual variations between studies” is misleading in this context. I² is meaningful only when studies estimate the same underlying causal association. When fundamentally different interventions are pooled, I² no longer has the interpretation implied by the authors.

7. Speculation that early BCG effects are due to bias is unsubstantiatedThe manuscript repeatedly suggests that observed mortality reductions within the first 1–3 days after BCG vaccination may reflect bias due to lack of blinding. This speculation appears inconsistent with the design and reporting of the original trials. In Guinea-Bissau randomization occurred at discharge, and post-randomization care was not provided by study staff(2). In the Indian trial, the authors explicitly state that it is unlikely that the lack of blinding influenced the result. In previous open label randomized trials of BCG Russian strain in the same sites, no difference in neonatal mortality was found, which suggests that the lack of blinding did not bias the findings(3).

Given these safeguards, attributing early effects to bias is unsupported by trial evidence and suggests that the original studies were not carefully read or adequately considered.

8. Ignoring extensive mechanistic evidence for rapid BCG effectsThe authors further imply that effects within days are biologically implausible. This overlooks a substantial body of experimental and clinical evidence demonstrating that BCG induces trained innate immunity, including rapid functional reprogramming of myeloid cells and emergency granulopoiesis, which can occur within days and protect against severe infections such as sepsis(4, 5). These mechanisms provide a biologically coherent explanation for early effects and should have been discussed as plausible alternatives to bias.

9. Failure to engage with established explanations for heterogeneous measles vaccine effectsThe manuscript notes heterogeneity across measles vaccine trials but does not engage with recent work offering compelling explanations for these differences, including interactions with OPV campaigns and vaccination sequence effects(6). Ignoring this literature leads to an oversimplified interpretation in which heterogeneity is treated primarily as noise rather than as potentially informative signal.

10. Introduction of an a posteriori unifying hypothesisLate in the discussion, the authors invoke a new hypothesis that all live-attenuated vaccines should yield similar NSEs on all-cause mortality. This hypothesis appears post hoc and is not clearly justified biologically. It has never been a hypothesis within the NSE field and is biologically implausible, not least because baseline mortality differs substantially by age. Introducing this assumption only after the pooled analysis further weakens the inferential logic of the paper.

Overall assessmentThe manuscript raises an important question, but its conclusions are undermined by:<br /> • an artificially restricted scope,<br /> • incomplete inclusion of relevant RCTs,<br /> • inappropriate pooling across fundamentally different interventions,<br /> • speculative dismissal of biologically plausible findings,<br /> • and inconsistent use of hypotheses introduced after the analysis.<br /> As currently written, the preprint does not provide a reliable basis for concluding that NSEs of vaccines on all-cause mortality are absent or unimportant. A substantially revised analysis - grounded in a comprehensive evidence base, clearer causal questions, and vaccine-specific syntheses - would be required to support such claims.

References1. Higgins JP, Soares-Weiser K, Lopez-Lopez JA, Kakourou A, Chaplin K, Christensen H, et al. Association of BCG, DTP, and measles containing vaccines with childhood mortality: systematic review. BMJ. 2016;355:i5170.<br /> 2. Biering-Sorensen S, Aaby P, Lund N, Monteiro I, Jensen KJ, Eriksen HB, et al. Early BCG-Denmark and Neonatal Mortality Among Infants Weighing <2500 g: A Randomized Controlled Trial. Clin Infect Dis. 2017;65(7):1183-90.<br /> 3. Adhisivam B, Kamalarathnam C, Bhat BV, Jayaraman K, Namachivayam SP, Shann F, et al. Effect of BCG Danish and oral polio vaccine on neonatal mortality in newborn babies weighing less than 2000 g in India: multicentre open label randomised controlled trial (BLOW2). BMJ. 2025;390:e084745.<br /> 4. Kleinnijenhuis J, Quintin J, Preijers F, Joosten LA, Ifrim DC, Saeed S, et al. Bacille Calmette-Guerin induces NOD2-dependent nonspecific protection from reinfection via epigenetic reprogramming of monocytes. Proc Natl Acad Sci U S A. 2012;109(43):17537-42.<br /> 5. Brook B, Harbeson DJ, Shannon CP, Cai B, He D, Ben-Othman R, et al. BCG vaccination-induced emergency granulopoiesis provides rapid protection from neonatal sepsis. Sci Transl Med. 2020;12(542):eaax4517.<br /> 6. Nielsen S, Fisker AB, da Silva I, Byberg S, Biering-Sørensen S, Balé C, et al. Effect of early two-dose measles vaccination on childhood mortality and modification by maternal measles antibody in Guinea-Bissau, West Africa: A single-centre open-label randomised controlled trial. EClinicalMedicine. 2022;49:101467.

On 2025-12-19 17:14:16, user Cesar Ugarte wrote:

The preprint by Fajardo et al. addresses an important evidence gap regarding the utility of combined antigen–antibody tests for detecting acute HIV infection. Although the authors adopt a valuable global perspective, the interpretation and synthesis of the data would benefit from greater nuance to enhance clinical relevance. The authors' QUADAS-2 assessment shows High Risk of Bias regarding patient selection and Unclear Risk regarding the conduct of the index test. In diagnostic epidemiology, such findings are not just descriptive but also signal a huge spectrum effect and possible threshold bias. Therefore, the summary estimates presented in Figures 3 and 4 may reflect a statistical average of disparate clinical realities rather than a reliable indicator of test performance (for example in Figure 3 there are 10 studies with a sensitivity less than 10%, including some with 0%, so the evaluation in detail of these studies should be done to see if these studies can be combined with the other ones). Another issue is the inclusion of "obsolete" diagnostic platforms that have been withdrawn due to suboptimal performance. A sensitivity analysis or subgroup stratification should be restricted to tests currently on the market. This would enable the reader to distinguish between the historical evolution of the technology and the expected performance in contemporary clinical practice.

The interpretation of diagnostic performance also should be addressed in detail. Whereas sensitivity and specificity have usually been considered "intrinsic" to a test (so doesn´t depends on disease prevalence), evidence suggests significant variation across clinical settings. The underlying epidemiological status and operator expertise can affect the test’s accuracy. Finally, I agree with the authors that real-world evidence on cost-effectiveness and implementation barriers is lacking. However, we should be very careful to avoid having a biased meta-analytic estimate that leads to the premature abandonment of "imperfect" but viable diagnostic solutions. In the case of acute HIV infection, for which early detection is critical to ART initiation and reduction of secondary transmission, interpretation of this evidence needs to balance statistical rigor against the urgent public health need for early diagnosis.

[-1,2]; abs. max of y=4 at x=-1 abs.min of y=2 a x=1 - on this closed interval there is an abs. max + min..

I think this article is very helpful if you are learning about digital literacy. The examples used really helps to understand what we are expected to be able to do. This article solidified the fact that I think it is very important to be prepared in digital literacy.

这篇论文的核心内容可以概括为：

一、研究目的

提出一种基于GIS的地震损失评估方法，用于在地震发生前或发生后，快速评估城市中：

• 建筑损坏情况
• 人员伤亡
• 道路阻塞情况 并辅助灾害应急与城市规划决策。

二、主要方法

论文构建了一个综合评估流程，包括：

1. 地震强度计算 利用震级、断层距离和土壤条件，计算每个建筑位置的地震烈度（MMI）。

2. 建筑损伤评估 通过fragility曲线（脆弱性曲线），将地震烈度转换为建筑损坏比例。

3. 人员伤亡估计 根据建筑损伤程度、人口分布和建筑类型，计算死亡和受伤人数。

4. 道路阻塞分析 根据倒塌建筑产生的废墟体积，评估道路被阻塞的程度。

5. 避难可达性分析 判断哪些建筑在震后无法通过道路到达临时避难点。

三、案例研究

以Tehran某区域为例进行模拟：

• 预测结果显示：

• 建筑损坏最高可达约64%

• 死亡人数约占33%
• 严重受伤约27%
• 约22%的道路完全阻塞

四、验证与结论

• 使用2003 Bam earthquake进行验证，模型结果略偏保守（偏高估损失）

• 结论：

• GIS方法能够有效整合多源数据进行地震损失评估

• 该模型可用于：

  • 灾前风险评估
  • 城市规划（加固建筑、拓宽道路）
  • 灾后应急救援规划

一句话总结

这篇论文提出了一套基于GIS的地震损失评估模型，通过“地震强度 → 建筑损伤 → 人员伤亡 → 道路阻塞”的链式分析，实现城市级灾害风险预测与决策支持。

Crazy... like free to play white. You can also check soundscape studies from games like Fortnite in Playthrough Poetics, and it's crazy how little nature embeds the area. Compare that to Red Dead 2, Rain World, Flow, Death Stranding 2, Proteus, Caves of Qud, Everything, or Kenshi

1 filteren, 2 aandacht, 4 nieuwsgierigheid, 6 doelgerichtheid, 8/9 planmatig handelen, kiezen/beslissen, 12 metacognitie, 15 motivatie

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

Learn more at Review Commons

Reply to the reviewers

Reviewer #1

Figure 1D: It would be useful to indicate the number of embryos analyzed for these experiments (n = ?).

Number of embryos now included in figure legend

Figure 3B: The control condition for gcl⁻/⁻; ras-RNAi is labeled as "EV". This terminology (presumably "empty vector") is not defined in either the text or the figure legend. In addition, the magenta channel for the Ras-G37 condition appears to be flipped horizontally.

We replaced with “-“ in figure and figure legend

Page 7: The text states that "Ras-C40 activates the PI3K pathway," whereas the figure depicts Ras-C40 as activating the RalA pathway. This discrepancy could be confusing for the reader and should be corrected.

The diagram has been corrected

Figures 4 and 5: To facilitate interpretation, it may be helpful to include a schematic of the PI3K complex indicating the different subunits used in the study, along with information (potentially color-coded) about whether each construct primarily acts as an activator or inhibitor of PI3K function.

Figure 4E and Figure 5E were added

Figure 4A and 4B: For clarity and consistency with the text, the panels (and corresponding plots) for dp110-WT and dp110-CAAX could be placed before those for dp110-D954A and dp110-ΔRBD.

Order of constructs was rearranged

Figure 5C: The term "p60-TCEp3," which appears to correspond to the germ plasm-targeted p60-WT construct, is not defined in either the figure legend or the main text.

Clarification was added to the text (p.11, line 225)

Page 12: The reference "(Fig. S1A, Movie 1)" should be corrected to "(Fig. S2A, Movie 1)."

Corrected

Page 13: There is a missing word in the sentence "the biosensor appeared to be enrich to...", which should be corrected to "enriched."

Corrected

Figure 7A: Although the data presented are interesting and ultimately support the authors' conclusion that Torso regulates PIP3 levels, the results are somewhat counter-intuitive and may be confusing for readers. The authors might consider moving this panel to the Supplementary Figures. In addition, it could be informative to include PIP3 measurements for gcl⁻/⁻ (and possibly gcl⁺/⁻) pole buds in Figure 7B, as PIP3 appears particularly enriched in these conditions compared to wild type.

We agree that at first the findings in the early embryos were confusing, but we prefer including them in the main figure to demonstrate changes in PIP 3 distributions in torso mutants. We are now providing a possible explanation for these findings (p13 line 270-). The differences are quite clear in the older embryos and measurements shown in 7B-D. Pole bud measurements for gcl-/- and gcl+/- are shown in figure 6 E-G.

Reviewer #2

Fig. legends to 1C and 1D are swapped.

Corrected

Why is csw not necessary for PGC formation? It acts upstream of Ras. This is not discussed.

We now highlight this point in the text (and refer to studies on the sevenless kinase, which suggested a similar position of Csw parallel or downstream of Ras (page 6 line 107-).

Fig 3C. Consider changing the order of the ras-variants used: S35, G37, C40 instead of S35, C40, G37.

We changed the schematic in Figure 3C that should make the order of Ras variants more intuitive.

Fig 4A, B: Consider changing the order of the panels. Control, dp110-wt, dp110-CAAX, dp110-D954A, dp110-deltaRBD.

Order of constructs was rearranged

Fig S4 is mentioned in the text before S2 and S3. Consider changing the suppl. figure order.

Order of supplementary figures was rearranged

Page 12: Fig S1 A does not show PIP2 dynamics. Movie 1 is not available to this reviewer. The authors most likely refer to fig. S2.

Movie 1 was uploaded and figure calls were corrected

Page 13, 1st para: Why do the authors use glc heterozygous embryos to look at PIP3 and PIP2? Particularly so when they report later in the MS that glc+/- behave differently to wt controls in terms of PIP3 levels (Fig. 7C). By looking at gcl+/+, they might find that now PIP2 levels are different in gcl mutant embryos or that the differences between PIP3 levels in +/+ and -/- are larger than compared with +/-.

Since gcl+/- embryos form the same number of PGCs as WT but show a statistically significant increase in PI3K activity when comparing membrane to cytoplasm staining intensity, we favor using gcl+/- embryos, as these embryos may represent a more sensitive test for PIP2 and PIP3 levels.

Pages 15 and 16: revise figure calls in the text.

Figure calls were revised

M+M: How were gcl+/- and gcl-/- embryos identified?

Since all genetic manipulations in this alter the maternal contribution to the embryo, we us the term ‘mutant’ embryos referring to the maternal genotype (indicated on page 3 line 33 and more clearly stated in material and methods and reagent table). Embryos derived from mother of a specific maternal genotype are all identical, thus we can easily distinguish between embryos derived from homozygous mutant mothers (gcl-/-) or heterozygous mutant mothers (gcl-/+) In the reagents table we include the precise genotype description. “CyO” refers to the balancer chromosome commonly used to identify heterozygotes on the second chromosome. Flies with the CyO balancer have curly wings.

Reviewer #3

Figure 1B: The authors describe that embryos with OptoSos still form buds which protruded from the cortex, but PGCs largely fail to cellularize (described in pg. 5). I'm not sure what they meant by "fail to cellularize" as this is not obvious to me when looking at the figure. The authors should describe how they know it's cellularized in the controls and not in the OptoSos or change the wording to "suggesting a failure to cellularize".

We used the word ‘protruded’ to describe our live observations. PGCs were quantified in fixed embryos, immunostained with anti-Vasa antibody to count Vasa positive cells (Fig 1C and D. We observe a lack of Vasa-positive PGCs, only in the light-activated OptoSos condition.

Fig. 1B, lines 4-5: at what stage are these embryos? Cycle 9? Cycle 14? Both?

Nuclear cycles of embryos for each panel are noted on the left side of each panel

Fig. 4A: add dp110-CAAX results to Results section

dp110-CAAX results are included in the Results section (p.9. line 177)

Figure 5C: The hyper-clustered phenotype they describe is hard to visualize in this figure (described in pg. 11). The authors should describe what is meant by "hyper-clustered".

We agree and re-worded the description of this observation to be clearer, page 11, line 226-.

Figure 7: When comparing Fig. 7A and 7B torsoHH/WK images, we can see that in Fig. 7A that PIP3 pattern changes such that PIP3 is now at the most posterior end where PGC will eventually form (compared to control that has low PIP3 in this region), but then in Fig. 7B they are looking at the buds and they say PIP3 levels decrease, which does not correspond to Fig. 7A. Are these simply different stages and PIP3 levels change over time (looking at Fig. 7C, PIP3 does not seem to change a lot over time)?

The figure legend now states more clearly that embryos were of different ages. We also explain in the text the apparent discrepancy in the patterns before and during budding (page13 line 266). The time points in figure 7C span nuclear cycle 10, not earlier (page14 line 274). By measuring membrane to cytoplasmic distribution, a more accurate comparison is possible at this stage.

p. 5, line 5: "Optosos" is written "OptoSos" elsewhere (suggest using OptoSos throughout)

Corrected

Is it possible that inhibition of myosin II recruitment is due to conversion of PIP2 -> PIP3, thus loss of PIP2, or is it that myosin is specifically recruited to regions where PIP2 is high? This seems like a point that should be added to the discussion.

This point is now discussed on page 20, line 403

p. 5, line 6: suggest adding a comma after "Ras" for clarity

Corrected

p. 5, last line: the genotype is "w^1118" (with ^ indicating a superscript), not "w^-1118", and is italicized (this should be corrected throughout)

Corrected

p. 6, line 2: replace "cellularizing" with "cellularization"

Corrected

p. 6, lines 11-13: Where is it shown that knockdown of csw, dsor1 and rolled did not restore PGC formation? The data are not present in Fig. 2C (could include in supp fig?)

We added these data as Supplementary figure 1

p. 7, line 1: replace "interfere" with "interferes"

Corrected

p. 7, last three lines: what is stated here, "Ras-G37 [activates] both the RalA and the PI3K pathways, and Ras-C40 activates the PI3K pathway" is not consistent with what is diagrammed in Fig. 3C, where Ras-C40 is indicated as activating RalA (please correct either the text or the diagram)

We apologize and corrected the figure

p. 11, lines 1-2: the Pi3K21B gene and transcript should be italicized (note that Pi3K21B is the official gene name on FlyBase)

Gene name was italicized

p. 11, lines 6-10: it might be helpful to explain how the p60 construct was overexpressed (current lines 9-10) before describing the results (current lines 7-8)

Clarification on p60 construct was added to p.11, line 215-

p. 12, paragraph 2, line 2: the PIP2 biosensor should be written as "PLCgamma[PH]:mCherry" throughout, not "PLCy[PH]:mCherry"; this should be changed in the figures as well as the text (Symbol font can be used to turn "g" into lower-case "gamma", both in Word and in Illustrator)

Gamma symbol was added

It would also be helpful to show the overlap of the PIP2 and PIP3 signals in control vs. gcl mutants at different stages so the relative distribution and intensity of the signals can be better appreciated (consider adding this as a supplementary figure).

Our data show that PIP2 is not affected by lack of GCL (Fig 6 B-D). We thus do not think that simultaneous imaging of PIP2 and PIP3 in gcl-/- would add to our conclusions. Furthermore, these experiments would require a significant time investment to generate the respective genotypes. Thus, we agree with the reviewer that this is experiment is beyond the scope of the paper.

p. 12, paragraph 2, line 3: it does not appear that the two PIP markers were used "simultaneously" in Fig. 6A; however, this is evident from Fig. S2 and Movie 1 (consider placing callouts to these earlier in the paragraph or moving the description of simultaneous expression and observation of the two markers later in the paragraph to avoid confusion)

We did simultaneously image PIP2 and PIP3 sensors and have added this as Movie 1 and also in supplementary Figure S4, which are now clearly referred to in the text.

p. 12, paragraph 2, line 7: replace "Fig. S1A" with "Fig. S2" (this was confusing)

Figure call was updated

p. 16: change "Fig. 7G-I" to "Fig. 8G-I"

Figure call was updated

p. 20, Deming reference: there appears to be a stray asterisk in the title

Asterisk was removed from reference

Fig. 1D: need to explain that the colors in the graph indicate the numbers of PGCs formed (this could also be added as a label across the top of the graph); in addition, the number of embryos examined for each genotype should be included in the legend

We added a label at the top of the graph and ‘n’ were added to figure legend

Fig. 2B: spell out where csw, dsor1 and rolled data are shown; also, "n" is not defined; was this the number of embryos per genotype?

We added these data as Supplemental Figure 1

Fig. 3B: "EV" should be defined in the legend; is this "empty vector"?

We are using a “-“ to mark controls without transgene

Fig. 3C: see previous comment re: mistake in the diagram; I believe Ras-C40 was described as activating PI3K, not RalA

We apologize and corrected the figure

Fig. 4B, line 2: was the graph plotted from the data in panel (C) or panel (A)? panel (A) seems more likely, because the data in C is plotted in D; please correct the panel callout

Figure legend was updated to refer to the correct panel

Fig. 5C: describe "p60-TCEp3" in the legend

We added germplasm-targeting 3’UTR (TCEp3) to legend and the construct and reference are provided in Material and Methods section

Figure 6: In Fig. 6E-G, the "brightness" of PIP3 at the membrane corresponds to the images even with different views (posterior and orthogonal) and agrees with the graph.

However, when looking at Fig. 6B, it looks to me that PIP2 is brighter in gcl+/-, but the opposite is true when looking at Fig. 6D (i.e., PIP2 looks brighter in gcl-/-). The authors might want to comment on this.

We have updated the figure to better reflect our observations.

Fig. 6A: define "(fire)" here or in the first figure legend where this is used

We added an inset for the fire lookup table to clearly define the pseudcolor scheme used in the image

Figure 8 title: "Actin fluorescence is increased in gcl-/- pole buds",But their graph in Fig. 8B comparing actin in gcl+/- to -/- is not significant

Thanks for catching our mistake, myosin not actin is changed

Fig. 8I: replace "Scarlett" with "Scarlet"

Corrected

Fig. 8D-F: Although the plots in panel E agree with the images in panel D, it is unclear why those in panel F are not more concordant. In F, myosin appears enriched at the cortex relative to the cytoplasm in gcl-/- mutants, which is hard to reconcile with the data in D-E.

We have updated the figure to better reflect our observations.

Fig. S2A: define the three time points shown here, and clarify that these are shown left to right (if this is indeed the case)

We removed S2A and updated the movie to replace it

Fig. S4: change "P60" to "p60" in the figure title

Corrected

Movie: The movies showing PIP2 and PIP3 in whole embryos are nice, but it would also be helpful to also include merged images of the two channels, so the reader can examine the relative accumulation of the two PIPs over time.

Merged images panel was added to the movie.

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Referee #3

Evidence, reproducibility and clarity

Summary:

Although Torso is known to antagonize primordial germ cell (PGC) formation, the underlying mechanisms remain unclear. Canonical Torso signalling typically results in activation of Ras. However, the authors show that Ras-mediated suppression of PGC formation is independent of the Raf/MEK/ERK pathway. Instead, they uncover an unexpected role for Torso in activating phosphoinositide 3-kinase (PI3K) that promotes formation of PIP3 enriched posterior membrane domains. The resulting increase in PI3K activity disrupts PGC formation. Furthermore, they show that by promoting Torso degradation, the ubiquitin ligase adaptor Germ Cell-Less (GCL) primes the posterior membrane with reduced PIP3 to facilitate PGC formation. Lastly, the authors suggest a model where antagonistic relationship between GCL and Torso influences actomyosin contractility that may allow the bud to constrict for proper PGC formation.

Major comments:

Figure 1B: The authors describe that embryos with OptoSos still form buds which protruded from the cortex, but PGCs largely fail to cellularize (described in pg. 5). I'm not sure what they meant by "fail to cellularize" as this is not obvious to me when looking at the figure. The authors should describe how they know it's cellularized in the controls and not in the OptoSos or change the wording to "suggesting a failure to cellularize".

Figure 5C: The hyper-clustered phenotype they describe is hard to visualize in this figure (described in pg. 11). The authors should describe what is meant by "hyper-clustered".

Figure 6: In Fig. 6E-G, the "brightness" of PIP3 at the membrane corresponds to the images even with different views (posterior and orthogonal) and agrees with the graph. However, when looking at Fig. 6B, it looks to me that PIP2 is brighter in gcl+/-, but the opposite is true when looking at Fig. 6D (i.e., PIP2 looks brighter in gcl-/-). The authors might want to comment on this.

It would also be helpful to show the overlap of the PIP2 and PIP3 signals in control vs. gcl mutants at different stages so the relative distribution and intensity of the signals can be better appreciated (consider adding this as a supplementary figure).

Figure 7: When comparing Fig. 7A and 7B torsoHH/WK images, we can see that in Fig. 7A that PIP3 pattern changes such that PIP3 is now at the most posterior end where PGC will eventually form (compared to control that has low PIP3 in this region), but then in Fig. 7B they are looking at the buds and they say PIP3 levels decrease, which does not correspond to Fig. 7A. Are these simply different stages and PIP3 levels change over time (looking at Fig. 7C, PIP3 does not seem to change a lot over time)?

Page 15, last paragraph: "If myosin II recruitment is inhibited when PIP3 levels are high" Is it possible that inhibition of myosin II recruitment is due to conversion of PIP2 -> PIP3, thus loss of PIP2, or is it that myosin is specifically recruited to regions where PIP2 is high? This seems like a point that should be added to the discussion.

Overall, I think their claim that antagonistic activities of GCL and Torso is crucial for PGC formation is well justified. The combination of optogenetic tools with activation and lof mutants is nicely done. Some clarification regarding the PIP3 and PIP2 levels will be helpful to the reader (see my comments above). The myosin claim is less convincing (see my comment on Fig. 8D-F below).

Minor comments on the text:

p. 5, line 5: "Optosos" is written "OptoSos" elsewhere (suggest using OptoSos throughout) p. 5, line 6: suggest adding a comma after "Ras" for clarity p. 5, last line: the genotype is "w^1118" (with ^ indicating a superscript), not "w^-1118", and is italicized (this should be corrected throughout) p. 6, line 2: replace "cellularizing" with "cellularization" p. 6, lines 11-13: Where is it shown that knockdown of csw, dsor1 and rolled did not restore PGC formation? The data are not present in Fig. 2C (could include in supp fig?) p. 7, line 1: replace "interfere" with "interferes" p. 7, last three lines: what is stated here, "Ras-G37 [activates] both the RalA and the PI3K pathways, and Ras-C40 activates the PI3K pathway" is not consistent with what is diagrammed in Fig. 3C, where Ras-C40 is indicated as activating RalA (please correct either the text or the diagram) p. 11, lines 1-2: the Pi3K21B gene and transcript should be italicized (note that Pi3K21B is the official gene name on FlyBase) p. 11, lines 6-10: it might be helpful to explain how the p60 construct was overexpressed (current lines 9-10) before describing the results (current lines 7-8) p. 12, paragraph 2, line 2: the PIP2 biosensor should be written as "PLCgamma[PH]:mCherry" throughout, not "PLCy[PH]:mCherry"; this should be changed in the figures as well as the text (Symbol font can be used to turn "g" into lower-case "gamma", both in Word and in Illustrator) p. 12, paragraph 2, line 3: it does not appear that the two PIP markers were used "simultaneously" in Fig. 6A; however, this is evident from Fig. S2 and Movie 1 (consider placing callouts to these earlier in the paragraph or moving the description of simultaneous expression and observation of the two markers later in the paragraph to avoid confusion) p. 12, paragraph 2, line 7: replace "Fig. S1A" with "Fig. S2" (this was confusing) p. 16: change "Fig. 7G-I" to "Fig. 8G-I" p. 20, Deming reference: there appears to be a stray asterisk in the title

Minor comments on the figures and figure legends:

Fig. 1B, lines 4-5: at what stage are these embryos? Cycle 9? Cycle 14? Both? Fig. 1C: see previous comment about "w^1118" genotype nomenclature Fig. 1D: need to explain that the colors in the graph indicate the numbers of PGCs formed (this could also be added as a label across the top of the graph); in addition, the number of embryos examined for each genotype should be included in the legend Fig. 2B: spell out where csw, dsor1 and rolled data are shown; also, "n" is not defined; was this the number of embryos per genotype? Fig. 3B: "EV" should be defined in the legend; is this "empty vector"? Fig. 3C: see previous comment re: mistake in the diagram; I believe Ras-C40 was described as activating PI3K, not RalA Fig. 3E: fix "w^1118" as described above Fig. 4A: add dp110-CAAX results to Results section Fig. 4B, line 2: was the graph plotted from the data in panel (C) or panel (A)? panel (A) seems more likely, because the data in C is plotted in D; please correct the panel callout Fig. 5C: describe "p60-TCEp3" in the legend Fig. 6A: define "(fire)" here or in the first figure legend where this is used Figure 8 title: "Actin fluorescence is increased in gcl-/- pole buds",But their graph in Fig. 8B comparing actin in gcl+/- to -/- is not significant Fig. 8D-F: Although the plots in panel E agree with the images in panel D, it is unclear why those in panel F are not more concordant. In F, myosin appears enriched at the cortex relative to the cytoplasm in gcl-/- mutants, which is hard to reconcile with the data in D-E. Fig. 8I: replace "Scarlett" with "Scarlet" Fig. S2A: define the three time points shown here, and clarify that these are shown left to right (if this is indeed the case) Fig. S4: change "P60" to "p60" in the figure title

Movie: The movies showing PIP2 and PIP3 in whole embryos are nice, but it would also be helpful to also include merged images of the two channels, so the reader can examine the relative accumulation of the two PIPs over time.

Referees cross-commenting

I agree enthusiastically with the comments of the other reviewers, who often came to the same conclusion I did about the manuscript and the data, including some of the detailed points about the figures, etc.

Significance

General assessment:

The many strengths of this manuscript include elegant genetic and optogenetic approaches using well-designed transgenes.

The main weakness is the lack of experiments showing simultaneous live imaging of the PIP2 and PIP3 sensors in gcl-/- and other genetic backgrounds, which would help the reader better envision how regulators of this pathway affect phospholipid distribution at the level of whole embryos and prospective pole cells. Note that because of the time required, I do not insist that they do this.

Advance:

Study demonstrates for the first time an unexpected role of Torso in PI3K regulation

Audience:

germ cell afficionados, developmental biologists, cell biologists, PI3K researchers

My field of expertise:

Drosophila, germ cell development, genetics, cell biology, live imaging, phosphoinositides

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

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Referee #1

Evidence, reproducibility and clarity

This is an outstanding and elegant study that addresses an important question in developmental and germline biology: how the soma-germline boundary is established during embryogenesis. This represents one of the most fundamental cell-fate decisions during organismal development. The authors combine elegant genetics, optogenetics, quantitative live imaging, and lipid biosensors to provide a compelling mechanistic framework linking receptor degradation, lipid signaling, and cytoskeletal dynamics. They show that Torso signaling via PI3K and PIP3 antagonizes primordial germ cell (PGC) formation and promotes somatic cell fate. Furthermore, they demonstrate that GCL-mediated degradation of Torso at the posterior pole creates a PIP3-low membrane domain that permits myosin II recruitment and pole bud constriction, thereby enabling PGC formation. Together, these results clearly demonstrate how the soma-germline boundary is established.

We have only minor comments on the manuscript, primarily aimed at improving clarity for non-specialist readers:

1. Figure 1D: It would be useful to indicate the number of embryos analyzed for these experiments (n = ?).
2. Figure 3B: The control condition for gcl⁻/⁻; ras-RNAi is labeled as "EV". This terminology (presumably "empty vector") is not defined in either the text or the figure legend. In addition, the magenta channel for the Ras-G37 condition appears to be flipped horizontally.
3. Page 7: The text states that "Ras-C40 activates the PI3K pathway," whereas the figure depicts Ras-C40 as activating the RalA pathway. This discrepancy could be confusing for the reader and should be corrected.
4. Figures 4 and 5: To facilitate interpretation, it may be helpful to include a schematic of the PI3K complex indicating the different subunits used in the study, along with information (potentially color-coded) about whether each construct primarily acts as an activator or inhibitor of PI3K function.
5. Figure 4A and 4B: For clarity and consistency with the text, the panels (and corresponding plots) for dp110-WT and dp110-CAAX could be placed before those for dp110-D954A and dp110-ΔRBD.
6. Figure 5C: The term "p60-TCEp3," which appears to correspond to the germ plasm-targeted p60-WT construct, is not defined in either the figure legend or the main text.
7. Page 12: The reference "(Fig. S1A, Movie 1)" should be corrected to "(Fig. S2A, Movie 1)."
8. Page 13: There is a missing word in the sentence "the biosensor appeared to be enrich to...", which should be corrected to "enriched."
9. Figure 7A: Although the data presented are interesting and ultimately support the authors' conclusion that Torso regulates PIP3 levels, the results are somewhat counter-intuitive and may be confusing for readers. The authors might consider moving this panel to the Supplementary Figures. In addition, it could be informative to include PIP3 measurements for gcl⁻/⁻ (and possibly gcl⁺/⁻) pole buds in Figure 7B, as PIP3 appears particularly enriched in these conditions compared to wild type.

Significance

The biological question is highly interesting, the experimental design is very clear, and the data are convincing throughout. The imaging, quantification, and movies are of very high quality and strongly support the authors' conclusions. Overall, this manuscript represents a significant conceptual and technical advance and will be of broad interest to the fields of germline biology, membrane biology, and embryonic morphogenesis.

Multiple antibiotic classes are effective against both streptococci and staphylococci, with the optimal choice depending on whether methicillin-resistant Staphylococcus aureus (MRSA) is suspected and the severity of infection.

For methicillin-susceptible strains, beta-lactam antibiotics remain the mainstay of treatment. These include penicillinase-resistant penicillins (dicloxacillin, oxacillin, nafcillin), first-generation cephalosporins (cephalexin, cefazolin), and amoxicillin-clavulanate, all of which provide excellent coverage against both streptococci and methicillin-susceptible staphylococci (MSSA). [1-4]

Clindamycin is particularly valuable as it demonstrates activity against approximately 80% or more of community-associated MRSA strains while maintaining excellent activity against group A streptococcus, making it an attractive single-agent option for skin and soft tissue infections. [5] However, clindamycin resistance appears to be increasing. [5]

For MRSA coverage with dual activity, several options exist. Linezolid and tedizolid (oxazolidinones) have activity against both MRSA and streptococci. [2][6-7] Vancomycin provides coverage against both organisms and has been the traditional mainstay for MRSA infections. [2][7] When MRSA is suspected but streptococcal coverage is also needed, combination therapy with trimethoprim-sulfamethoxazole (TMP-SMX) plus a beta-lactam (such as cephalexin or penicillin) is recommended, as TMP-SMX alone has poorly defined activity against streptococci. [1][5]

Doxycycline and minocycycline are effective against most MRSA isolates but have less well-defined activity against group A streptococcus, so they may require combination with a beta-lactam when streptococcal infection cannot be excluded. [1][5]

Would you like me to provide specific dosing recommendations for any of these antibiotics or discuss treatment duration for particular infections?

1

endovascular stenting emerging as first-line therapy for rapid symptom relief, while definitive treatment targets the underlying cause

Failure of venous insufficiency ulcerations to heal is most often due to inconsistent use of first-line treatment methods. Ongoing control of edema is essential to prevent recurrent ulceration; the use of compression stockings following ulcer healing is critical, with recurrence rates 2–20 times higher if compression stockings are not used

Primary varicose veins result from a complex multifactorial process involving genetic predisposition and environmental factors. The pathophysiology involves initial structural weakness within the vein wall leading to vein dilation, or valve incompetence causing blood pooling and subsequent vein dilation. [1][3] Risk factors include family history (autosomal dominant inheritance with variable penetrance), female sex, multiparity, pregnancy, prolonged standing, obesity, and advanced age. [2-3] If both parents have varicose veins, offspring have a 90% chance of developing them. [2]

Secondary varicose veins develop through a distinct mechanism involving inflammation, thrombosis, and recanalization that results in venous wall damage, dilation, and valve insufficiency. [3] The clinical picture manifests as post-thrombotic syndrome, which can include pain, edema, skin changes, and venous leg ulcers. [3] The 2020 CEAP classification further subdivides secondary causes into intravenous (Esi) and extravenous (Ese) etiologies. [4] Intravenous causes include any condition causing venous wall or valve damage from within the lumen, such as DVT, traumatic arteriovenous fistulas, or primary intravenous sarcoma. Extravenous causes involve conditions affecting venous hemodynamics without direct wall or valve damage, such as central venous hypertension from obesity or heart failure, extrinsic compression from tumors or retroperitoneal fibrosis, or muscle pump dysfunction from paraplegia or arthritis

1. The Scaling Crisis (Why Reviews are "Dead")

• Throughput vs. Cognition: The fundamental problem is a mismatch in speed. AI agents (like Cursor, Claude Code, or GitHub Copilot) can generate code at a rate 10x–100x faster than a human. If a developer uses an agent to clear 20 tickets in a morning, the human reviewer becomes a permanent, overwhelmed bottleneck.
• The "Rubber Stamp" Failure: In high-volume AI environments, human reviewers stop actually reading the code and start "rubber-stamping" (approving without checking). This creates a false sense of security while letting "slop" (low-quality, redundant, or slightly buggy AI code) leak into the codebase.

2. The Shift to "Spec-Driven Development"

The author posits that the "checkpoint" for quality is moving "upstream." * Reviewing Intent, Not Implementation: Engineers will spend their time reviewing the Specification or the Prompt rather than the resulting lines of code. If the spec is correct and the constraints are tight, the implementation is treated as a disposable artifact. * Natural Language as the New Source Code: The high-level description of a feature becomes the primary source of truth. If a bug is found, the human doesn't "fix the code"; they "fix the spec" and have the agent regenerate the solution.

3. Verification over Review

The traditional manual "looking at code" is being replaced by automated verification loops: * Agentic Testing: Agents are now responsible for writing their own unit tests and integration tests. The human's job is to review the test plan and verify that the tests actually cover the business requirements. * Formal Methods & Type Safety: The article emphasizes using "bounded interfaces" (like Infrastructure-from-Code or strict APIs). When the architecture is strictly typed, the AI is physically unable to make certain classes of errors, reducing the need for human oversight of the "plumbing."

4. The Emergence of "Cognitive Debt"

A primary warning in the text is the rise of "Cognitive Debt"—a state where a codebase is so dominated by AI-generated logic that no single human understands how the entire system works. * Tech Debt vs. Cognitive Debt: While tech debt is "bad code that's hard to change," cognitive debt is "working code that is impossible to reason about." * The New Senior Role: Senior engineers must transition into "System Curators" who manage this debt by enforcing architectural simplicity and ensuring that AI-generated modules remain modular and replaceable.

5. Future Prediction

• 2025: The year human-written code became the minority.
• 2026: The year the "Pull Request" as a human-to-human review ritual officially becomes obsolete in high-performing teams, replaced by automated agent-to-agent validation and human-to-spec oversight.

I suggest swapping sections four and five to better reflect a logical progression of actions. Reading the document sequentially, it currently implies that decommissioning is the first step following a good-faith warning.

Decommissioning is quite a drastic action and has a massive knock-on effect for the whole fleet, not to mention the possible stress it may cause for the crew and the interrupting of an individual's progress towards command or any other goals they may have in the fleet.

I think here, stability should be the priority, and I think the EC's default should be that the current section five comes first, with decommissioning happening only in the most extreme circumstances.

I look at the Astraeus's decommissioning and from my perspective as a non-CC simmer at the time, I was left wondering why the first officer couldn't take over, or a new/experienced commanding officer couldn't be air dropped in like had been done with Denali.

Document de Synthèse : Réalités, Mécanismes et Enjeux de la Violence Conjugale

Résumé Exécutif

Ce document analyse les témoignages et les interventions documentés au sein de l'association Flora Tristan, mettant en lumière la complexité systémique des violences conjugales.

Les principaux points à retenir sont :

• La cyclicité de la violence : Le processus de l'emprise repose sur un cycle répétitif (tension, crise, transfert de responsabilité, lune de miel) qui rend le départ des victimes extrêmement difficile (5 à 7 tentatives en moyenne).

• L'impact sur les enfants : Les enfants ne sont pas de simples témoins mais des victimes directes, subissant des traumatismes durables qui affectent leur développement et leur sécurité.

• Les défaillances institutionnelles : Le manque de places d'hébergement d'urgence, la précarité des solutions hôtelières (115) et l'accueil parfois inadéquat des forces de l'ordre constituent des obstacles majeurs à la mise en sécurité.

• Le rôle vital des structures spécialisées : L'accompagnement pluridisciplinaire (juridique, psychologique, social) est indispensable pour permettre aux femmes de passer du statut de victime à celui de survivante et de reconstruire leur estime de soi.

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1. Les Mécanismes de l'Emprise et le Cycle de la Violence

La violence conjugale ne se limite pas aux coups physiques ; elle s'inscrit dans une stratégie de manipulation et d'isolement.

Le Cycle de la Violence

Le document identifie quatre phases distinctes qui s'enchaînent de manière circulaire :

• Phase de tension : Accumulation d'agressivité chez l'agresseur.

• Phase de crise (Explosion) : Passage à l'acte violent (physique, verbal ou sexuel).

• Phase de transfert de responsabilité : L'agresseur justifie ses actes en culpabilisant la victime ("C'est toi qui me rends violent").

• Phase de lune de miel : L'agresseur demande pardon, se montre aimant et promet de changer.

C'est durant cette phase que les victimes sont le plus enclines à retirer leur plainte, espérant une amélioration.

Typologie des Violences

Les interventions révèlent une multiplicité de formes de violence qui s'entrecroisent :

• Psychologique et verbale : Insultes, dénigrement permanent, menaces de mort prises à la "rigolade".

• Physique : Coups, strangulation, séquestration.

• Sexuelle : Rapports imposés par le chantage ou la force physique (viols conjugaux souvent non identifiés comme tels par les victimes elles-mêmes).

• Économique et Administrative : Contrôle des ressources, confiscation des documents, promesses fallacieuses (notamment dans le cadre de l'immigration).

• Isolement : Interdiction de voir la famille, les amis, d'utiliser les réseaux sociaux ou même d'ouvrir la porte au gardien.

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2. L'Impact sur les Victimes : Entre Culpabilité et Honte

Le traumatisme psychologique crée des barrières internes puissantes qui s'ajoutent aux menaces extérieures.

| Sentiment | Description et Conséquences | | --- | --- | | Culpabilité | Les victimes se demandent souvent si elles sont responsables de la situation ou si elles auraient pu "sortir par la porte ouverte". | | Déni de statut | Difficulté à se reconnaître comme "victime", surtout pour les femmes se percevant comme ayant un caractère fort. | | Honte | Un obstacle majeur à la parole, particulièrement vis-à-vis de la famille ou de la société, exacerbé par le sentiment d'avoir échoué à "sauver" le couple. | | Banalisation | L'accumulation de petits incidents ("C'est pas grave") qui finissent par former une "montagne" de violences graves. |

« On doit pas avoir honte, c'est à eux d'avoir honte. » – Citation d'une survivante lors d'une manifestation.

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3. Les Enfants : Victimes Directes et Enjeux de Protection

Le document souligne que l'exposition à la violence conjugale est un traumatisme majeur pour les enfants, avec des conséquences à long terme.

• Traumatismes visibles : Troubles du sommeil (insomnies, cauchemars sur la sécurité de la mère), agitation extrême, retards dans les apprentissages et anxiété profonde.

• Mécanismes de survie : Certains enfants adoptent le comportement de l'agresseur pour tenter de se protéger eux-mêmes ou pour s'identifier à la figure de pouvoir.

• Défaillances du système judiciaire :

  • Les juges privilégient souvent le maintien du lien parental (autorité parentale) au détriment de la sécurité réelle de l'enfant.

• Les procédures sont extrêmement longues (reports de délibérés), prolongeant l'insécurité.

• L'enfant est parfois utilisé comme une "arme" par l'agresseur pour continuer à détruire la mère après la séparation.

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4. Les Limites du Soutien Institutionnel et Social

Malgré l'existence de structures comme Flora Tristan, le système global présente des failles critiques.

Les Obstacles à la Plainte

L'accueil au commissariat est décrit comme parfois "odieux".

Les témoignages rapportent des policiers remettant en cause la parole des victimes, rappelant avec insistance la présomption d'innocence ou menaçant de sanctions en cas de "fausses déclarations", ce qui décourage le dépôt de plainte, notamment pour les violences sexuelles.

La Crise de l'Hébergement

• Saturations des structures : En une période donnée, 217 demandes d'hébergement d'urgence ont été refusées par une seule association faute de places.

• L'échec du "115" (Hébergement hôtelier) :

  • Conditions insalubres (rats, punaises de lit).

• Éloignement géographique (jusqu'à 2h30 des écoles ou du travail), mettant en péril l'emploi et la scolarité.

• Insécurité chronique poussant certaines femmes à retourner au domicile conjugal par désespoir.

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5. Le Chemin vers la Reconstruction

La sortie de la violence est un processus long qui nécessite une réappropriation de son corps et de son identité.

• Sécurité et Cohabitation : Les structures comme Flora Tristan offrent un répit immédiat, bien que la cohabitation avec d'autres familles soit un défi supplémentaire.

• Reconnexion au corps : Des activités comme la boxe professionnelle permettent de reprendre confiance en soi, d'extérioriser la colère et de transformer l'énergie négative en force positive.

• Autonomie retrouvée : Le succès passe par la reprise d'études, la recherche d'un emploi stable et l'obtention d'un logement social pérenne (processus pouvant durer 18 mois à 2 ans).

• Évolution de l'identité : Passer du statut exclusif de "mère protectrice" à celui de "femme libre" ayant droit à ses propres besoins et désirs.

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Conclusion : Un Appel à l'Action Politique

Le personnel encadrant et les survivantes s'accordent sur le fait que la lutte contre les violences conjugales nécessite une volonté politique forte se traduisant par :

• Un financement massif et pérenne de places d'hébergement spécialisées.

• Une réforme de la justice pour mieux prendre en compte la parole des enfants et protéger les victimes des procédures abusives.

• Une sensibilisation accrue pour supprimer la "violence étatique" qui s'ajoute à la violence conjugale lorsque les institutions échouent à protéger les plus vulnérables.

Briefing : Lutte contre les manipulations de l’information et les ingérences numériques étrangères

Résumé exécutif

Ce document synthétise l'audition de Nicolas Roche, Secrétaire général de la défense et de la sécurité nationale (SGDSN), portant sur la stratégie nationale de lutte contre les manipulations de l'information (LMI) pour la période 2026-2030. Face à une menace caractérisée par une « persistance stratégique » et une mutation technologique rapide, la France a consolidé son dispositif opérationnel via le service Viginum.

Les points clés de l'intervention soulignent :

• L'évolution de la menace : Une activité permanente, hors périodes électorales, menée par des acteurs étatiques (Russie, Chine, Iran) et non étatiques (sphère MAGA), utilisant l'intelligence artificielle pour massifier la diffusion.- Le rôle de Viginum : Un service technique axé sur la détection des infrastructures d'amplification inauthentique, et non sur le contrôle des contenus (« ministère de la vérité »).- La stratégie 2026-2030 : Fondée sur quatre piliers, elle donne la priorité à la résilience de la nation par l'éducation et la transparence, plutôt qu'à une simple réponse régalienne.- Le cadre européen : La nécessité d'une mise en œuvre plus agressive du Règlement sur les services numériques (DSA) pour imposer une transparence algorithmique aux grandes plateformes.

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I. Analyse de la menace : Une triple mutation

Le paysage des ingérences numériques étrangères (INE) a subi des transformations profondes selon trois axes majeurs :

1. Paramètre stratégique : La persistance

Les adversaires ne se limitent plus aux coups d'éclat lors des scrutins. Ils s'inscrivent dans une logique de continuité, cherchant à s'implanter durablement dans le débat numérique français en se faisant passer pour des acteurs authentiques. Les principaux acteurs identifiés incluent :

• États : Russie, Chine, Iran, Azerbaïdjan (ce dernier ciblant spécifiquement les Outre-mer).- Acteurs non étatiques : Notamment la sphère MAGA.

2. Paramètre technologique : L'impact de l'IA

L'intelligence artificielle transforme l'économie de l'information :

• Contenus synthétiques (Deepfakes) : Bien que spectaculaires, ils sont jugés moins préoccupants car plus faciles à détecter.- Massification et réplication : L'IA est surtout utilisée pour contourner la modération des plateformes par la technique du « copy-paste » modifié, permettant une diffusion massive et automatisée.- Pollution de modèles (Data poisoning) : Un sujet de recherche prospectif visant à biaiser les réponses des IA natives dès leur phase d'entraînement.

3. Paramètre économique : L'économie de l'attention

L'ingérence peut avoir une finalité strictement lucrative. La polarisation du débat génère des clics et donc des revenus publicitaires. L'émergence des influenceurs comme relais, volontaires ou non, complique davantage cet écosystème.

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II. Viginum : Un modèle technique et transparent

Créé en 2021, Viginum est le service de référence pour la détection des INE. Nicolas Roche définit sa mission autour de critères stricts :

| Critère | Description | | --- | --- | | Extranéité | Seuls les acteurs étrangers (étatiques ou non) sont ciblés. | | Inauthenticité | Focus sur l'amplification massive et délibérée via des infrastructures techniques. | | Gravité | L'attaque doit porter atteinte aux intérêts fondamentaux de la nation. | | Transparence | Service non-renseignement, soumis à la CNIL et à un comité éthique et scientifique. |

Évolution du cadre juridique (Décret de février 2026)

Le cadre juridique a été adapté pour répondre à l'évolution de la menace :

• Abaisser le seuil de collecte : Suppression du seuil de 5 millions d'utilisateurs/jour pour permettre la surveillance de plateformes plus petites où les opérations débutent souvent.- Conservation des données : Allongement des durées pour suivre la construction des infrastructures numériques sur le long terme.

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III. Retour d'expérience : Élections municipales 2026

Le dispositif de surveillance spécifique mis en place pour les municipales de 2026 a permis de tester la robustesse du modèle français.

• Constat : Quatre opérations majeures d'ingérence détectées.- Impact : Jugé « assez faible » en termes de visibilité. Le rapport coût-bénéfice pour les adversaires a été médiocre.- Méthodologie : Publication de 10 bulletins d'information hebdomadaires pour éviter tout emballement médiatique et informer les citoyens en temps réel.- Gouvernance : Création d'un réseau de coordination incluant le SGDSN, le ministère de l'Intérieur, mais aussi des autorités indépendantes (ARCOM, Commission de contrôle du financement de la vie politique).

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IV. La Stratégie Nationale 2026-2030

La stratégie repose sur quatre piliers fondamentaux, avec un changement de paradigme vers la société civile.

Pilier 1 : La Résilience (Priorité absolue)

L'État reconnaît qu'il ne peut pas lutter seul. La résilience passe par :

• L'Académie de lutte contre les manipulations de l'information : Un projet pour 2026 visant à transférer l'expertise technique de Viginum vers les journalistes, les enseignants et les élus.- L'Éducation aux médias : Partenariat avec le Clémi et le ministère de l'Éducation nationale pour outiller les lycéens et collégiens.

Pilier 2 : Régulation des plateformes et IA

• Application du DSA : Exigence de transparence algorithmique (Article 35) pour vérifier si les plateformes modifient leurs recommandations en période électorale.- Audit algorithmique : Nécessité d'objectiver scientifiquement les biais des algorithmes, souvent considérés comme des « boîtes noires ».

Pilier 3 : Capacités opérationnelles

Renforcement des moyens humains et techniques de Viginum pour maintenir l'avance technologique face aux méthodes d'anonymisation sophistiquées.

Pilier 4 : Coopération internationale

Il n'est pas envisagé de « Viginum européen ». La compétence opérationnelle reste nationale, mais l'Union européenne est essentielle pour sa puissance normative face aux plateformes américaines.

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V. Enjeux spécifiques et vulnérabilités

Les Outre-mer

Le SGDSN souligne une surexposition des territoires ultramarins. L'Azerbaïdjan a notamment été identifié comme ayant ciblé la Nouvelle-Calédonie et d'autres territoires via des modes opératoires informationnels documentés. Un guide spécifique a été transmis aux préfets et hauts-commissaires pour élever le niveau de vigilance local.

Les Jeunes

Nicolas Roche récuse l'idée d'une vulnérabilité intrinsèque des jeunes, notant que ces derniers sont souvent plus « natifs » et sensibilisés aux mécanismes numériques que les générations précédentes. La stratégie doit donc être segmentée de façon plus fine que par simple critère d'âge.

Le Droit comme arme

Des évolutions législatives sont envisagées pour :

• Étendre le « référé fake news » (L163-2 du code électoral) aux élections locales, actuellement limité aux scrutins nationaux.- Clarifier l'article L97 du code électoral pour mieux contrer les nouvelles formes d'ingérence.

« Notre travail ne consiste pas à dire ce qui est vrai ou faux. Notre travail consiste à préserver la souveraineté française. » — Nicolas Roche

Synthèse de l'Audition du Haut Conseil de la Santé Publique (HCSP) sur la Stratégie Nationale de Santé

Résumé Exécutif

L'audition du Haut Conseil de la santé publique (HCSP) devant la commission des affaires sociales de l'Assemblée nationale met en lumière un constat alarmant : le système de santé français fait face à une crise majeure et à un risque systémique.

Pour y répondre, le HCSP préconise une transformation profonde, passant d'un modèle quasi exclusivement centré sur le soin curatif à une approche systémique de santé publique sur le temps long (10 ans).

Les priorités identifiées incluent la refonte de l'articulation entre santé publique et soins, la lutte contre les maladies chroniques, la réduction des inégalités sociales et territoriales, et l'intégration de la "santé dans toutes les politiques".

Malgré l'urgence, les échanges révèlent un blocage opérationnel majeur : l'absence de décret publiant officiellement la Stratégie Nationale de Santé (SNS) 2023-2033, limitant ainsi la visibilité des acteurs et le pilotage territorial.

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1. Identité et Rôle du Haut Conseil de la Santé Publique (HCSP)

Le HCSP est une instance d'expertise pluridisciplinaire au service de l'État.

Ses caractéristiques clés sont les suivantes :

• Composition : 120 experts indépendants et bénévoles.

• Organisation : Cinq commissions spécialisées et des groupes de travail permanents transversaux coordonnés par un collège.

• Missions :

  • Contribuer à l'élaboration, au suivi et à l'évaluation de la SNS.

• Fournir des réflexions prospectives et des conseils sur les questions de santé publique.

• Répondre aux saisines ministérielles ou parlementaires et produire des auto-saisines sur des sujets d'intérêt public.

• Transparence : L'ensemble des avis et rapports est publié en accès libre.

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2. Diagnostic et Vision Stratégique 2023-2033

Le HCSP a publié en mars 2023 ses travaux pour la nouvelle SNS.

Le diagnostic est celui d'une crise majeure nécessitant un changement de paradigme.

Le choix de la décennie

Contrairement à la durée légale de 5 ans, le HCSP préconise une stratégie sur 10 ans pour permettre l'atteinte d'objectifs ambitieux à moyen et long terme, s'alignant ainsi sur les dynamiques européennes.

Les quatre grands objectifs cardinaux

Le HCSP structure sa vision autour de quatre piliers :

• Reconstruction du système : Articuler le système de santé publique et le système de soins (objectifs à 5 et 10 ans), renforcer l'attractivité des métiers et valoriser la territorialisation.

• Maladies chroniques : Diminuer le recours aux soins lié aux maladies chroniques (5 ans), ralentir l'augmentation de leur prévalence par la détection précoce (10 ans) et augmenter l'espérance de vie en bonne santé (15 ans).

• Réduction des inégalités : Renforcer la politique du "dernier kilomètre" via l'universalisme proportionné et les dispositifs d' "aller-vers" pour réduire la mortalité prématurée.

• Prévention systémique : Agir sur les déterminants de santé tout au long de la vie (petite enfance, milieu scolaire, travail, seniors) via une approche populationnelle.

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3. Les 12 Actions Stratégiques Préconisées

Le HCSP a détaillé 12 leviers pour transformer le système :

| Action | Description Key Point | | --- | --- | | 1\. Durée de 10 ans | Adopter un temps long pour la SNS. | | 2\. Évolution par étapes | Fixer des jalons à court, moyen et long terme. | | 3\. Santé dans toutes les politiques | Définir la santé comme paramètre intersectoriel et interministériel. | | 4\. Participation citoyenne | Impliquer les populations dans l'élaboration et la mise en œuvre territoriale. | | 5\. Politique de prévention intégrée | Approche socio-écologique par milieu de vie et développement de la littératie en santé. | | 6\. Environnement et Climat | Inscrire l'environnement comme déterminant majeur (approche One Health). | | 7\. Agilité territoriale | Valoriser les acquis numériques de la crise COVID et mobiliser les acteurs locaux. | | 8\. Plan Outre-mer spécifique | Rattraper le retard des indicateurs de santé dans les territoires ultramarins. | | 9\. Gouvernance et Financement | Réformer l'ONDAM et évaluer le retour sur investissement des dépenses de santé. | | 10\. Système d'information | Assurer l'interopérabilité des données et l'accès aux données en vie réelle. | | 11\. Recherche en santé publique | Loi de programmation avec financement pluriannuel sanctuarisé. | | 12\. Prospective | Développer les capacités de compréhension des forces futures pour guider l'action. |

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4. Thématiques Critiques et Enjeux de Société

Santé Mentale : Un secteur en "échec de soins"

Le constat des parlementaires, partagé par le HCSP, souligne une dégradation alarmante :

• Augmentation de 80 % des épisodes dépressifs chez les 18-24 ans en quatre ans.

• Chute de 34 % du nombre de pédopsychiatres entre 2010 et 2022.

• Le HCSP insiste sur le besoin de temps pour rattraper le retard structurel de ce secteur, malgré son statut de "grande cause nationale".

Territoires et Outre-mer

La stratégie doit s'adapter aux contextes locaux extrêmes :

• Mayotte : Crise de l'eau, pression migratoire, épidémies de choléra et manque de spécialistes.

• Guyane : Problématiques d'éloignement (forêt amazonienne) nécessitant des modes de prévention spécifiques.

• Principe : Le HCSP intègre systématiquement un volet ultramarin dans ses rapports pour éviter une approche standardisée inopérante.

Inégalités et Discriminations

L'audition a soulevé la question des stéréotypes racistes en médecine (ex: "syndrome méditerranéen").

Bien que non explicitement détaillé dans le rapport SNS du HCSP, l'instance rappelle que l'équité et l'égalité en droit sont des principes fondamentaux, et que ces sujets relèvent de la formation éthique et juridique des professionnels de santé.

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5. Analyse des Freins Politiques et Budgétaires

L'absence de décret SNS 2023-2033

Un point de tension majeur apparaît entre les élus et le HCSP : le gouvernement n'a toujours pas pris le décret actant la nouvelle stratégie.

• Conséquence : Un manque de visibilité pour le Parlement et les citoyens, ainsi qu'une difficulté pour les Agences Régionales de Santé (ARS) à actualiser leurs projets régionaux de santé (PRS).

• Position du HCSP : L'instance rappelle son rôle strictement consultatif, déclinant toute responsabilité quant à l'agenda réglementaire de l'exécutif.

Le défi du financement

Le passage d'un modèle curatif à un modèle préventif pose la question de l'allocation des ressources :

• Les dépenses de prévention représentent environ 8 % des 300 milliards d'euros de budget santé.

• Le HCSP suggère que le financement de la prévention ne doit pas reposer uniquement sur l'Assurance Maladie, mais doit être intersectoriel (éducation, sport, alimentation).

• Efficience : Des économies significatives (estimées à 300 000 € par an pour un service de 10 urologues à titre d'exemple cité par un député) pourraient être réalisées en améliorant la pertinence des actes et la coopération interprofessionnelle.

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Citations Clés

« Le diagnostic [...] est bien celui d'une crise majeure et d'un risque systémique de l'ensemble du système de soins et du système de santé. » — Véronique Ghileron, Présidente du HCSP

« La santé est bien un sujet politique et elle est largement produite par nos façons de vivre. Elle doit être abordée dans tous les secteurs. » — Véronique Ghileron

« Nous ne sommes plus face à un secteur en tension mais face à un secteur en échec de soins. » — Sandrine Rousseau, Députée (à propos de la santé mentale)

« La prévention n'est pas un sujet médical uniquement... c'est une politique de vie. » — HCSP

Harcèlement scolaire : Analyse des impacts durables et des processus de reconstruction

Synthèse

Le harcèlement scolaire ne constitue pas une simple phase transitoire de l'enfance, mais agit comme un traumatisme fondateur dont les répercussions s'étendent sur plusieurs décennies.

Le document met en lumière une « double peine » pour les victimes : au traumatisme initial s'ajoute un retard social et émotionnel persistant à l'âge adulte.

Les témoignages de Nathalie, Laurine et Samuel révèlent que les stigmates — qu'ils soient psychologiques (manque de confiance, anxiété, colère) ou sociaux (difficultés relationnelles et amoureuses) — forgent une identité marquée par la défiance et le sentiment de temps perdu.

Si des dispositifs modernes comme le programme « Phare » et le rôle des élèves ambassadeurs marquent un progrès institutionnel, la reconstruction demeure un processus long, laborieux et parfois inabouti.

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1. Profils des victimes et mécanismes de l'oppression

Le harcèlement s'appuie systématiquement sur la détection d'une différence ou d'une vulnérabilité, qu'elle soit physique, cognitive ou sociale.

Facteurs de vulnérabilité identifiés

• Particularités physiques et santé : L'acné sévère, la corpulence, ou des problèmes dentaires/maxillaires (cas de Laurine) servent de catalyseurs aux moqueries.

• Troubles neurodéveloppementaux : La dyspraxie (cas de Samuel) entraîne des difficultés motrices et une fatigue rapide, perçues par les harceleurs comme une forme de « saleté » ou de « maladie contagieuse ».

• Décalage cognitif et émotionnel : Une grande sensibilité, un tempérament « fantasque » ou un décalage entre les capacités intellectuelles précoces et une immaturité sociale (entrer en primaire à 4 ans) créent une cible isolée.

• Identité culturelle : L'usage d'une langue maternelle différente (l'alsacien pour Nathalie) et un accent prononcé ont été des vecteurs d'exclusion dès l'entrée au collège.

Typologie des actes de harcèlement

| Forme de harcèlement | Exemples concrets issus des témoignages | | --- | --- | | Physique | Tapes sur la tête, bousculades, jets de sucreries mâchées (carambars/malabars), vols de sacs, gifles dans les transports scolaires. | | Verbal et Psychologique | Surnoms dénigrants (« la calculatrice »), insultes sexistes ou homophobes, rumeurs de perversion ou d'inceste familial. | | Social et Environnemental | Violation de l'intimité (agressions dans les toilettes), exclusion des groupes de discussion (cyberharcèlement), mépris silencieux et regards condescendants. |

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2. La « Double Peine » : Conséquences à l'âge adulte

Les sources soulignent que le harcèlement « paramètre » les individus, altérant leur perception du monde et d'eux-mêmes bien après la fin de la scolarité.

Le retard social et relationnel

Les victimes font état d'un « coche raté » durant l'adolescence.

N'ayant pas appris les codes sociaux et amoureux au moment opportun par peur du rejet, elles développent :

• Une méfiance généralisée : Une difficulté à nouer des liens, percevant chaque soupir ou rire comme une moquerie potentielle.

• Un sentiment d'indignité : La conviction profonde d'être « dégoûtant » ou indigne d'être aimé, rendant les relations amoureuses catastrophiques ou inexistantes.

• L'atrophie de la vie sociale : Une tendance à l'isolement (« devenir une sauvage ») et une incapacité à se projeter dans l'avenir.

Impacts sur la santé mentale

• Traumatismes persistants : Présence d'une mémoire traumatique qui se réactive dans des contextes banals (prendre le tramway, passer devant son ancien collège).

• Pathologies lourdes : États dépressifs sévères, idées noires, recours aux antidépresseurs sur de longues durées et épisodes d'hospitalisation en psychiatrie.

• Érosion de l'estime de soi : Un manque de confiance en miettes, une haine du propre corps et une exigence envers soi-même parfois écrasante.

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3. Le rôle des institutions et de l'entourage

L'analyse montre une évolution historique entre la gestion passée, marquée par l'indifférence, et les tentatives actuelles de prise en charge.

La défaillance historique des adultes

Dans les années 80 et 90, le harcèlement était souvent minimisé par le personnel éducatif :

• Invisibilisation : Les adultes considéraient ces situations comme des « histoires entre gamins ».

• Impuissance apprise : Des cadres éducatifs (CPE, surveillants) refusaient d'intervenir sous prétexte que les faits se déroulaient « devant la grille » et non à l'intérieur.

• Injonction à l'affirmation : Les victimes se voyaient reprocher leur manque de caractère (« il fallait s'affirmer »), plaçant la responsabilité du harcèlement sur la proie.

Les dispositifs contemporains

• Programme Phare : Plan de lutte officiel de l'Éducation nationale visant à structurer la réponse au harcèlement.

• Élèves Ambassadeurs : Des élèves formés pour agir comme « veilleurs », repérer les situations de détresse et offrir un soutien entre pairs, inversant ainsi le rapport de force collectif.

• Limites persistantes : Le cyberharcèlement reste une frontière difficile à réguler, et la visibilité des situations de harcèlement demeure problématique pour les élèves qui ne savent pas toujours distinguer la théorie de la pratique.

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4. Processus de reconstruction et résilience

La reconstruction est décrite non pas comme un retour à l'état initial, mais comme une cicatrisation complexe.

• L'accompagnement thérapeutique : Le travail avec des psychologues ou psychiatres est jugé indispensable pour traiter la culpabilité et la colère.

Des exercices comme la rédaction de lettres aux harceleurs ou la reconstruction de la chronologie des faits aident à extérioriser le vécu.

• L'engagement professionnel : Certains anciens harcelés deviennent enseignants, voyant dans ce métier une évidence pour protéger les nouvelles générations, tout en restant conscients de leur propre fragilité émotionnelle face au sujet.

• Le sport et la réussite académique : Pour certains, le sport permet de reprendre possession de leur corps, tandis que l'excellence professionnelle (études de médecine) sert de moteur de reconstruction, bien que la personnalité reste « écorchée ».

• L'acceptation de la colère : Contrairement aux injonctions sociales, le pardon n'est pas toujours souhaité. La colère est parfois perçue comme un moteur de survie légitime face à l'injustice subie.

Conclusion

Le harcèlement scolaire est un préjudice global qui prive les individus de leur dignité et de leur capacité à choisir leur rythme de vie.

Si la résilience est possible par le dialogue et le soutien thérapeutique, les sources indiquent que l'on ne « guérit » jamais totalement : on apprend à vivre avec une identité forgée par l'adversité.

Le temps perdu, particulièrement les « amours de jeunesse » et l'insouciance, demeure une perte inéluctable.

L'enjeu majeur pour l'institution reste la détection précoce afin d'éviter que le « petit enfant harcelé » ne devienne systématiquement un « adulte abîmé ».

Synthèse de l'Audition du Président du Conseil d’Évaluation de l’École (CEE)

Résumé Exécutif

Ce document synthétise les points clés de l'audition du président du Conseil d’évaluation de l’école (CEE) devant la Commission de la culture et de l’éducation de l'Assemblée nationale.

Le CEE, succédant au Cnesco suite à la loi de 2019, a pour mission de structurer et d'analyser l'évaluation systémique des établissements scolaires français.

Les points saillants de l'audition incluent :

• Succès quantitatif : 100 % des établissements du second degré et 66 % du premier degré ont été évalués.

La France a comblé son retard par rapport aux autres pays de l'OCDE.

• Changement de paradigme : L'évaluation n'est pas un audit de conformité, mais un outil d'auto-évaluation visant à renforcer l'autonomie et le pouvoir d'agir des équipes locales.

• Phase de transformation : Le passage de la simple évaluation (Vague 1) à la transformation opérationnelle (Vague 2) est l'enjeu majeur.

Les projets d'établissement doivent devenir des « plans de réussite collectifs ».

• Pilotage par la donnée : Une culture de la donnée s'installe, avec des outils innovants comme le projet « Alumni » pour suivre le devenir des élèves 3 à 5 ans après leur sortie.

• Défis persistants : Les critiques parlementaires soulignent des risques de doublons administratifs, l'urgence face à la dégradation des conditions de travail des enseignants et l'inertie potentielle du système.

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I. Missions et Cadre Institutionnel du CEE

Le Conseil d’évaluation de l’école (CEE), créé par la loi de 2019 pour une école de la confiance, remplit quatre missions fondamentales définies par le législateur :

• Cohérence des évaluations : Veiller à l'unité des travaux portés par le ministère (notamment ceux de la DEPP) concernant les acquis des élèves et l'école inclusive.

• Expertise méthodologique : Donner un avis sur les outils et les résultats des évaluations du système éducatif.

• Mesure des inégalités : Proposer des méthodologies pour évaluer les disparités territoriales et formuler des recommandations pour les réduire (équité scolaire).

• Cadre de l'évaluation des établissements : Définir la méthodologie, les outils d'auto-évaluation et la fréquence des évaluations externes.

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II. Bilan des Évaluations d'Établissements

1. État d'avancement quantitatif

Le président souligne un « succès collectif » concernant le déploiement des évaluations depuis 2020 :

• Second degré : 100 % des établissements évalués à ce jour.

• Premier degré : 66 % de couverture atteinte cette année.

• Comparaison internationale : La France, précédemment dernière de l'OCDE (après la Bulgarie) en matière d'évaluation systémique, a rattrapé son retard.

2. La Philosophie de l'Évaluation

L'évaluation se distingue radicalement d'un audit de conformité.

Son but est d'interroger les décisions propres de l'établissement dans le cadre de ses marges d'autonomie.

• Auto-évaluation : Un diagnostic partagé par les équipes locales.

• Évaluation externe : Un regard extérieur pour challenger et objectiver le diagnostic initial.

• Objectif : Accompagner le « pouvoir d'agir » des acteurs de terrain plutôt que de vérifier l'application de directives verticales.

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III. Les Domaines Opérationnels de l'Évaluation

Pour simplifier la réflexion des équipes, le cadre de l'évaluation s'articule autour de 4 domaines principaux déclinés en 12 portes d'entrée :

| Domaine | Thématiques couvertes | | --- | --- | | Acquis des élèves | Parcours scolaires, orientation, maîtrise des fondamentaux (français/maths). | | Bien-être et Climat | Bien-être des élèves et personnels, lutte contre le harcèlement, climat scolaire. | | Organisation et Pédagogie | Choix pédagogiques, ressources (manuels), formation continue des personnels. | | Partenariats | Liaisons inter-degrés, relations avec les familles et partenaires extérieurs. |

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IV. Stratégie de Transformation et Perspectives

Le président du CEE présente une stratégie en trois phases pour assurer l'utilité des évaluations :

• Vague 1 (Évaluation) : Constat et massification des données.

• Vague 2 (Transformation) : L'évaluation doit servir de socle aux projets d'établissement.

Actuellement, moins de 20 % des projets d'établissement sont jugés opérationnels.

L'objectif est de les transformer en plans de réussite collectifs basés sur les résultats de l'évaluation.

• Vague 3 (Adaptation systémique) : Si la transformation est réelle, le système devra s'adapter pour répondre aux besoins spécifiques exprimés par les établissements (renversement du plan national de formation).

Innovations Clés :

• Projet Alumni : En partenariat avec l'ONICEP, mise à disposition d'indicateurs de suivi des anciens élèves pour mesurer la contribution réelle de l'établissement à l'égalité des chances et à l'insertion (ex: accès des filles aux filières scientifiques 5 ans après le collège).

• Indicateurs simplifiés : Dotation des chefs d'établissement de trois indicateurs simples de performance scolaire pour piloter sur le temps long.

• Leadership scolaire : Renforcement des compétences managériales des cadres (via l'IH2EF) pour passer d'une gestion administrative à un pilotage pédagogique et stratégique.

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V. Synthèse des Interventions Parlementaires

L'audition a donné lieu à des échanges critiques sur l'efficacité et la structure du CEE :

1. Critiques sur l'utilité et la structure (RN)

• Remise en question de la valeur ajoutée du CEE face à des organismes existants (DEPP, Inspection Générale, Cour des Comptes).

• Dénonciation d'un système qui est « administré » mais ni « dirigé », ni « piloté ».

2. Conditions de travail et implication des enseignants (Socialistes)

• Alerte sur l'épuisement professionnel : 70 % des enseignants ne recommanderaient pas leur métier.

• Nécessité absolue d'associer étroitement les enseignants aux conclusions de l'auto-évaluation pour qu'elles soient suivies d'effets.

3. Opérationnalité et délais (Droite Républicaine)

• Inquiétude sur le temps long : s'il a fallu 7 ans pour la phase d'évaluation, la transformation risque d'être trop lente.

• Besoin de garanties sur la traduction des rapports en décisions tangibles et en moyens.

4. Enjeux territoriaux et démographiques (Horizons, Députés individuels)

• Contradiction entre la fermeture de classes due à la baisse démographique et la dégradation du niveau scolaire.

• Questionnement sur la pertinence de l'évaluation nationale face aux spécificités locales et à la mixité sociale (ségrégation scolaire).

• Décrochage en mathématiques (classements PISA) et nécessité de réformer la formation continue.

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VI. Réponses du Président aux Interrogations

Le président a apporté les précisions suivantes :

• Sur la démographie : La réduction du nombre d'élèves par classe a un impact marginal sur la performance.

La solution réside dans l'organisation collective et le pilotage du parcours de l'élève (vision horizontale sur 15 ans).

• Sur les doublons : Le CEE est complémentaire. Contrairement à la DEPP ou l'Inspection, il est le seul à promouvoir l'auto-évaluation et à interroger le terrain sur ses marges de manœuvre.

• Sur la transparence : Depuis février 2024, le CEE a voté pour que les rapports d'évaluation soient accessibles à l'ensemble de la communauté éducative, incluant les parents et les élèves.

• Sur la confiance : La transformation nécessite de restaurer la confiance entre les enseignants et les directions (perte de 14 points de confiance en 10 ans). L'évaluation doit être un levier pour reconnaître la professionnalité des acteurs.

Briefing : Le traitement judiciaire des violences sexuelles incestueuses sur mineurs

Ce document synthétise les témoignages et analyses fournis par les représentants des syndicats de la magistrature (USM, Syndicat de la Magistrature, Unité Magistrat) lors de leur audition devant la commission d'enquête de l'Assemblée nationale.

Résumé exécutif

L'institution judiciaire française traverse une crise structurelle profonde qui entrave le traitement efficace des violences sexuelles incestueuses.

Malgré une libération de la parole et une augmentation des signalements, la réponse judiciaire reste marquée par un taux de classement sans suite alarmant (environ 70 % à 80 % dans les affaires de viols sur mineurs) et des délais de traitement inacceptables pouvant dépasser un an et demi entre le signalement et les premières mesures de garde à vue.

Les principaux points de blocage identifiés sont :

• Un manque criant de moyens humains et matériels : La France dispose de deux à quatre fois moins de magistrats et procureurs que la moyenne européenne, entraînant une gestion des flux au détriment de la qualité.

• Une faillite de la chaîne de protection : Les enquêtes sont ralenties par le sous-effectif des brigades spécialisées et la pénurie d'experts psychiatres qualifiés.

• Un besoin de réforme des outils civils : Les syndicats plaident pour un renforcement de l'ordonnance de protection, gérée par le juge aux affaires familiales (JAF), pour protéger l'enfant dès la phase de soupçon.

• Le poids du passé : Le traumatisme de l'affaire d'Outreau continue d'influencer la perception de la parole de l'enfant, oscillant entre déni et suspicion.

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1. Une institution sous tension : le constat des moyens

L'analyse des syndicats souligne un décalage majeur entre les attentes sociétales et la réalité budgétaire de la justice.

Déficit de personnel et comparaison européenne

Le système judiciaire français est sous-dimensionné par rapport à ses voisins européens (chiffres de la CEPEJ) :

| Catégorie | France (pour 100 000 hab.) | Moyenne Européenne | | --- | --- | --- | | Procureurs | 3,2 | 12,2 | | Juges | 11,3 | 21,9 |

Il manquerait environ 20 000 magistrats pour assurer une justice de qualité, alors que seulement 8 500 sont actuellement en poste.

Conséquences sur les enquêtes

Le manque d'enquêteurs spécialisés crée des stocks de dossiers "morts".

• À Nantes, 500 dossiers étaient en attente en mars 2024 avec seulement 9 enquêteurs.

• À Paris, le délai entre un signalement et une garde à vue est estimé entre 12 et 18 mois.

• La priorité est souvent donnée à la délinquance de voie publique (opérations "place nette") au détriment des enquêtes complexes sur l'inceste qui exigent du temps (auditions multiples, perquisitions informatiques).

Obsolescence des outils informatiques

Les magistrats travaillent encore en "silos".

Le logiciel des juges des enfants (non "webisé") ne permet pas de savoir si un parent fait l'objet d'une procédure dans une autre juridiction.

L'absence d'interopérabilité entre le civil et le pénal freine la circulation des informations cruciales pour la protection des mineurs.

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2. La parole de l'enfant et l'expertise judiciaire

Le traitement de la parole de l'enfant reste le point le plus critique de la procédure.

La formation des acteurs

Bien que la formation initiale à l'École Nationale de la Magistrature (ENM) intègre des modules sur les violences sexuelles (auditions filmées, protocoles NI-CHD), la formation continue est souvent délaissée par les magistrats en raison d'une charge de travail excessive.

• Statistique clé : 60 % des auditions de mineurs victimes ne sont toujours pas réalisées par des enquêteurs formés.

La crise de l'expertise

Le recours aux experts psychiatres et psychologues est décrit comme une "misère".

• Pénurie : Certaines cours d'appel n'ont aucun pédopsychiatre inscrit.

• Qualité variable : Des expertises cruciales tiennent parfois sur une seule page.

• Délais : Une demande de contre-expertise peut rallonger la procédure de 18 mois à 2 ans.- Coût : La faible rémunération des experts dissuade les professionnels les plus qualifiés de travailler pour la justice.

Le traumatisme d'Outreau

L'affaire d'Outreau est citée comme une "régression fatale".

Elle a instauré un climat de suspicion systématique envers la parole de l'enfant, poussant certains magistrats à exiger des preuves matérielles impossibles à obtenir dans des dossiers d'inceste (souvent "parole contre parole").

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3. Propositions de réformes et débats juridiques

Les syndicats proposent plusieurs leviers pour améliorer la protection et le jugement.

Élargissement du cadre légal

• Définition de l'inceste : Unanimité sur l'intégration des cousins et cousines dans la définition légale.

• Ordonnance de protection : Proposition de calquer l'ordonnance de protection des enfants sur celle des violences conjugales.

Elle permettrait au JAF de statuer en 6 jours sur la suspension de l'autorité parentale sur la base de la "vraisemblance" des faits, sans attendre la preuve pénale.

Opposition au "Plaider-coupable" criminel

Le projet de loi visant à instaurer un "plaider-coupable" pour les crimes (dont les viols incestueux) rencontre une vive opposition :

• Risque d'une justice "expéditive" et dégradée.

• Nécessité de l'audience publique pour poser l'interdit social.

• Doute sur la réalité de la reconnaissance des faits par les auteurs (souvent assortie d'une minimisation du type "elle était d'accord").

La question de l'imprescriptibilité

La majorité des intervenants est défavorable à l'imprescriptibilité.

L'argument principal est que cela "vend du rêve" aux victimes : plus le temps passe, plus les preuves se dégradent, rendant la condamnation quasi impossible et le procès déceptif.

Le Syndrome d'Aliénation Parentale (SAP)

Unité Magistrat et le Syndicat de la Magistrature alertent sur la persistance de ce concept "pseudo-scientifique".

Utilisé pour discréditer les mères protectrices en les accusant d'instrumentaliser l'enfant, le SAP contribuerait à masquer les situations réelles d'inceste.

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4. Dysfonctionnements systémiques et protection de l'enfant

Le classement "21" (infraction insuffisamment caractérisée)

Ce motif de classement est majoritaire.

Les syndicats précisent qu'un classement ne signifie pas que les faits n'ont pas eu lieu, mais que l'enquête n'a pas permis de réunir des preuves suffisantes pour un procès pénal.

Cela crée un sentiment de déni violent pour l'enfant.

La non-représentation d'enfant (NRE)

Le Syndicat de la Magistrature prône la dépénalisation de ce délit, souvent utilisé contre les mères qui refusent de confier leur enfant à un père suspecté de violences.

Actuellement, 83 % des personnes condamnées pour NRE sont des mères.

Manque de coordination "Transversalité"

Le besoin d'un "référent inceste" en juridiction est évoqué, mais la multiplication des casquettes de "référent" (VIF, mineurs, etc.) sans moyens dédiés est jugée inefficace.

La priorité doit être la circulation fluide des dossiers entre le procureur, le juge des enfants et le juge aux affaires familiales.

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Citations clés

« La justice n'est pas réparée [...]. Elle a besoin de cap clair quant aux priorités, car tout ne peut pas être prioritaire. » — Ludovic Fria (USM)

« Un classement ne signifie pas que des faits n'ont pas existé. C'est une vérité judiciaire, pas une vérité absolue. » — Ségolène Marquet (SM)

« Nous sommes obligés de prioriser les urgences entre elles. C'est compliqué quand on reçoit les victimes et qu'on leur dit : "Votre dossier n'a pas avancé car les enquêteurs ont 15 commissions rogatoires avant la vôtre". » — Stéphanie Caprin (USM)

« L'inceste est le tabou par excellence. Ce n'est pas un crime comme un autre. » — Béatrice Brugère (Unité Magistrat)

Briefing : Traitement judiciaire et médical des violences sexuelles incestueuses

Ce document de synthèse analyse les témoignages et expertises présentés devant la commission d'enquête parlementaire relative au traitement judiciaire des viols et agressions sexuelles incestueuses sur mineurs.

Il compile les observations de trois experts pédopsychiatres : les docteurs Maurice Berger, Françoise Fericelli et Myriam Pieron-Bertier.

Résumé exécutif

L'analyse des sources révèle une crise systémique dans la protection de l'enfance face à l'inceste en France.

Les points critiques identifiés sont les suivants :

• Terrorisation du corps médical : Les médecins hésitent à signaler les soupçons d'inceste par crainte de sanctions disciplinaires systématiques de la part du Conseil de l'Ordre des médecins (CNOM), souvent à la suite de plaintes déposées par les agresseurs présumés.

• Infiltration d'idéologies non scientifiques : Le "Syndrome d'Aliénation Parentale" (SAP), bien qu'exclu des classifications internationales (DSM-5, CIM-11), continue d'influencer massivement les experts et les magistrats, conduisant à discréditer la parole de l'enfant et du parent protecteur.

• Carence d'expertise spécialisée : Les expertises judiciaires sont trop souvent confiées à des psychiatres d'adultes sans formation en clinique de l'enfant, menant à des évaluations superficielles, voire traumatisantes.

• Déni spécifique chez le nourrisson : Il existe un déni massif concernant les agressions sexuelles sur les enfants de moins de trois ans, malgré une sémiologie clinique et psychosomatique précise.

• L'idéologie du "lien à tout prix" : La priorité donnée au maintien des liens familiaux et à la coparentalité, même en présence de violences avérées, met les enfants en danger immédiat de réexposition traumatique.

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1. Obstacles au signalement et rôle du Conseil de l'Ordre des médecins

Le système actuel entrave la détection précoce de l'inceste en raison de la vulnérabilité juridique des médecins signalants.

La "profession terrorisée"

• Les médecins libéraux (généralistes, pédiatres) sont les plus exposés aux attaques du Conseil de l'Ordre lorsqu'ils rédigent un certificat ou un signalement.

• Même l'utilisation des modèles officiels fournis par le CNOM ne garantit aucune immunité.

Un médecin a été condamné à 15 jours d'interdiction d'exercer malgré la validation préalable de son écrit par le président de son conseil départemental.

• Conséquence : Le taux de signalements provenant des médecins est alarmant (entre 1 % et 2 % selon les chiffres récents de l'UMJ de l'Hôtel-Dieu).

Nécessité d'une réforme structurelle

• Limitation du pouvoir disciplinaire : Les experts préconisent que le CNOM perde son pouvoir de sanction dans les affaires de signalements concernant les enfants, s'inspirant des modèles suisse ou anglais où ces questions relèvent d'instances mixtes incluant des magistrats et des représentants de la société civile.

• Sécurisation juridique : L'obligation de signalement ne peut être envisagée sans une protection préalable et absolue du médecin contre les poursuites ordinales (Préconisation 17 de la Civise).

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2. Défaillances et normes de l'expertise judiciaire

L'expertise est le pivot de la décision judiciaire, mais sa qualité est jugée erratique et souvent insuffisante.

Les lacunes de la pratique actuelle

• Absence de spécialisation : Des psychiatres d'adultes expertisent des enfants sans maîtriser le développement psycho-affectif spécifique à chaque âge.

• Conditions inadaptées : Entretiens trop courts (parfois 10 minutes), absence de matériel de jeu ou de dessin, et réception des enfants à des heures inappropriées (sieste, soirée).

• Manquements méthodologiques : Défaut d'observation des interactions parent-enfant (diades), omission de la lecture du carnet de santé et absence de contact avec les professionnels suivant l'enfant (enseignants, éducateurs).

Les critères d'une expertise de qualité

Une expertise rigoureuse devrait répondre aux standards suivants :

• Durée : Entre 12 et 25 heures de travail effectif pour une évaluation complète de la famille.

• Méthodologie : Analyse clinique, utilisation de protocoles validés (NICHD/Nich pour l'audition, SVA pour la crédibilité des déclarations).

• Formation : Exigence d'une pratique clinique de 5 à 10 ans en pédopsychiatrie avant l'inscription sur les listes d'experts.

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3. L'influence du "Syndrome d'Aliénation Parentale" (SAP)

Le SAP est décrit comme un "fourre-tout conceptuel" utilisé stratégiquement par les agresseurs pour obtenir l'impunité.

Un outil de disqualification

• Lorsqu'un enfant dénonce un inceste dans un contexte de séparation, l'agresseur accuse fréquemment la mère d'être "aliénante" ou "fusionnelle".

• Cette rhétorique conduit à une inversion accusatoire : la détresse normale d'un parent protecteur est pathologisée et utilisée pour justifier le transfert de la garde à l'agresseur présumé.

Une infiltration institutionnelle

• Bien que rejeté par l'OMS et le Parlement européen, le lobby du SAP a infiltré les manuels scolaires et a longtemps été enseigné à l'École Nationale de la Magistrature (ENM).

• Les experts soulignent que la "mère fusionnelle" est souvent une mère parfaitement adaptée aux besoins de protection d'un enfant traumatisé.

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4. Clinique de l'inceste chez le nourrisson (0-3 ans)

Les agressions sur les très jeunes enfants constituent un angle mort majeur du système judiciaire.

Sémiologie spécifique

L'inceste chez le bébé se manifeste par des signes cliniques précis que les experts non formés ignorent :

• Troubles psychosomatiques : Eczéma brutal, infections vulvovaginales ou angines à répétition, béance anale ou vaginale.

• Troubles du comportement : Masturbation compulsive, phobie des soins d'hygiène (change, douche), agitation extrême (souvent confondue avec un TDH) ou retrait relationnel massif.

• Données probantes : Le carnet de santé est un document biographique crucial qui révèle souvent des ruptures de courbes de croissance ou des hospitalisations suspectes.

La fiabilité de la parole précoce

• Les enfants de 2 ou 3 ans "ne mentent pas" car ils n'ont aucune connaissance préalable de la sexualité adulte.

• Leur vocabulaire est spécifique (ex: "manger le pipi" pour désigner des actes sexuels oraux).

Ignorer ces propos au motif de "l'âge de raison" (7 ans) est une erreur scientifique.

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5. Conséquences judiciaires et "Idéologie du lien"

La justice française est critiquée pour son attachement dogmatique au maintien du lien biologique, même au détriment de la sécurité de l'enfant.

La réexposition traumatique

• Visites médiatisées : Le maintien de contacts en présence de professionnels est souvent inefficace pour protéger l'enfant du retraumatisme.

Des cas de viols ont été signalés lors de ces rencontres.

• Syndrome de Stockholm : Le fait qu'un enfant se précipite vers son parent agresseur au tribunal ne prouve pas l'absence de violences, mais témoigne d'un mécanisme de survie et d'emprise.

Statistiques et faits saillants

| Catégorie | Donnée statistique | | --- | --- | | Profil des agresseurs | 94-96 % d'hommes (pères, oncles, grands-parents) ; 4-6 % de femmes. | | Risque corrélé | 6,5 fois plus de risques d'inceste dans les foyers marqués par des violences conjugales. | | Crédibilité | Les fausses allégations volontaires représentent seulement 0,5 % à 6 % des cas. | | Rétractations | 22 % des enfants se rétractent par peur, mais 93 % d'entre eux reviennent ensuite sur leur rétractation. | | Démographie médicale | Environ 600 pédopsychiatres en France, moyenne d'âge de 62 ans. |

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6. Préconisations concrètes

Pour remédier à ces dysfonctionnements, les experts proposent :

• Réforme de la déontologie : Sortir les signalements d'enfants de la compétence disciplinaire du Conseil de l'Ordre.

• Spécialisation des experts : Réserver les expertises de mineurs aux seuls professionnels justifiant d'une pratique clinique longue auprès des enfants (minimum 5 ans).

• Formation à l'ENM : Remplacer les enseignements basés sur le SAP par des modules sur la victimologie et le psychotraumatisme de l'enfant.

• Application des protocoles SVA/Nich : Généraliser ces outils de validation scientifique pour limiter l'arbitraire de l'expert.

• Remise en cause du lien absolu : Reconnaître juridiquement l'existence de "disparentalités définitives" et suspendre tout droit de visite dès lors que la santé psychique de l'enfant est gravement compromise.

Este tema de los ARN largos no codificantes (lncRNA) es de lo más avanzado en biología molecular actual, Gaby. Básicamente, son los "directores de orquesta" del genoma: no se convierten en proteínas, pero le dicen a todas las demás moléculas qué hacer.

Aquí te resumo los puntos clave de tu texto para que los entiendas de forma sencilla:

1. ¿Qué son? (Sus "credenciales") Tamaño: Tienen más de 200 nucleótidos (son grandes).

Aspecto: Engañan a la célula porque parecen un ARNm normal (tienen su Cap 5', su cola Poli-A y sufren splicing), pero no codifican proteínas.

Especificidad: Son muy "especiales"; no están en todas las células igual, sino que aparecen en momentos específicos (control temporal) y en células específicas.

1. ¿Cómo funcionan? (Sus 4 roles principales) El texto menciona que actúan de varias formas gracias a su estructura compleja:

Como Guías: Se pegan a una proteína y la llevan de la mano hasta el lugar exacto del ADN donde debe trabajar.

Como Escalafones (Andamios): Sirven como una plataforma donde varias proteínas se sientan juntas para poder interactuar entre ellas. Sin el lncRNA, estas proteínas estarían perdidas por la célula y no se encontrarían.

Como "Esponjas": Se pegan a los microARN (miARN) y los atrapan. Al "secuestrarlos", evitan que los miARN destruyan a otros mensajes. Es un guardaespaldas de los ARNm.

Control de la Cromatina: Se unen a complejos que metilan histonas (como la H3). Esto decide si el ADN se enrolla (se apaga) o se desenrolla (se enciende).

1. Importancia Médica (¿Por qué te lo preguntan en medicina?) Tu texto destaca procesos vitales donde los lncRNA son los protagonistas:

Inactivación del cromosoma X: En las mujeres, uno de los dos cromosomas X debe "apagarse" para no tener doble dosis de genes. Un lncRNA llamado Xist es el encargado de cubrir todo ese cromosoma y silenciarlo.

Diferenciación celular: Ayudan a que una célula madre sepa si debe ser neurona o hepatocito.

Patologías: Si estos lncRNA fallan o se expresan de más, pueden causar cáncer (ayudando a que los tumores crezcan), diabetes o fibrosis hepática.

osforilación: El "Interruptor" MaestroEs la adición de un grupo fosfato ($PO_4^{3-}$) a aminoácidos como la serina, treonina o tirosina.Enzimas: Las encargadas son las quinasas (lo pegan) y las fosfatasas (lo quitan).Función: Activa o desactiva proteínas al instante. Es la base de la comunicación celular (señalización).2. Acetilación y Metilación: El Control del ADNAunque ocurren en muchas proteínas, son famosísimas en las histonas (las proteínas que enrollan el ADN).Acetilación: Generalmente "abre" la cromatina para que los genes se puedan leer.Metilación: Puede activar o silenciar genes dependiendo de dónde se pegue.3. Hidroxilación: La Fuerza del ColágenoConsiste en añadir un grupo hidroxilo ($-OH$).Ejemplo médico: Es vital para el colágeno. Sin vitamina C (cofactor), la enzima no puede hidroxilar la prolina del colágeno, las fibras no se trenzan bien y da escorbuto (encías sangrantes, debilidad).4. Carboxilación: La Coagulación SangrientaSe añade un grupo carboxilo ($-COOH$).Ejemplo médico: Es esencial para los factores de coagulación (como la protrombina). Este proceso necesita vitamina K. Si no hay carboxilación, los factores no pueden unirse al calcio y no puedes dejar de sangrar.

Antienshittification

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Referee #3

Evidence, reproducibility and clarity

Summary

In this manuscript, the authors investigate the role of the tubulin polyglutamylase TTLL6 in maintaining colonic epithelial homeostasis and its potential role in colorectal cancer (CRC). Using transcriptomic analyses, mouse genetics, histology, and proteomics, the authors report that TTLL6 is highly expressed in colonic epithelial cells and decreases during CRC progression. Constitutive and epithelial-specific deletion of Ttll6 in mice leads to elongated colonic crypts, expansion of proliferative and stem cell compartments, and increased susceptibility to chemically induced colitis-associated carcinogenesis. Mechanistically, the authors identify the nucleic acid-binding protein PurA as a potential non-tubulin substrate of TTLL6. They propose that TTLL6-mediated polyglutamylation of PurA regulates its nuclear localization, thereby contributing to epithelial homeostasis in the colon. Together, the study suggests a TTLL6-PurA axis that may restrain early colorectal tumorigenesis.

Major comments

1. Evidence that PurA is a physiologically relevant TTLL6 substrate remains incomplete. A central conclusion of the manuscript is that PurA is a substrate of TTLL6 whose polyglutamylation regulates nuclear localization. While the authors present several lines of evidence (PolyE immunoprecipitation, co-transfection experiments, and mutagenesis of the PurA C-terminal glutamate residues), the physiological relevance of this modification remains somewhat indirect. For example, polyglutamylation of endogenous PurA in colonic epithelial cells is inferred but not directly demonstrated. The PolyE antibody detects glutamate chains but does not identify the specific modified protein in tissue. Direct evidence that PurA is polyglutamylated in vivo (e.g., MS identification of the modification site on PurA or PurA immunoprecipitation followed by PolyE detection) would strengthen the mechanistic claim. At present, the data convincingly show that TTLL6 can glutamylate PurA in an overexpression system, but the endogenous modification remains less clearly demonstrated.
2. Mechanistic link between PurA localization and the epithelial phenotype is not established. The authors propose that loss of TTLL6 disrupts PurA nuclear localization and thereby alters epithelial homeostasis. However, the manuscript does not establish a causal relationship between PurA localization and the observed crypt phenotypes. Specifically, it is not shown whether PurA loss phenocopies Ttll6 deficiency in the colon. No experiments test whether restoring nuclear PurA rescues the Ttll6 phenotype. Downstream transcriptional or signaling pathways regulated by PurA are not explored. Thus, while the TTLL6-PurA relationship is intriguing, the study remains largely correlative with respect to functional consequences.
3. Interpretation of the tumorigenesis data should be tempered. The authors conclude that Ttll6 deficiency promotes colon carcinogenesis. However, the tumor data appear somewhat limited. Increased tumor numbers are reported only at an early time point (day 40) and are described as a trend toward significance. By day 70, tumor numbers and sizes appear comparable between groups. The increased incidence of vimentin-positive crypts is interesting but does not clearly establish increased tumor burden. Given these results, the conclusion that TTLL6 restrains tumorigenesis may be stronger than supported by the data. The authors may wish to frame this as enhanced early tumor development or altered tumor progression rather than increased tumorigenesis per se.
4. Expansion of multiple epithelial cell populations requires clarification. The authors report that Ttll6-deficient colons exhibit expansion of stem/progenitor compartments as well as increased numbers of differentiated cells (e.g., goblet cells and enterocytes). While these findings are interesting, the biological interpretation is somewhat unclear. For example, expansion of stem/progenitor compartments typically accompanies reduced differentiation rather than increased differentiation. It is not clear whether the increased numbers of differentiated cells reflect overall crypt enlargement or altered lineage allocation. Quantification of cell-type proportions rather than absolute cell numbers would help clarify whether differentiation programs are altered.
5. Nuclear polyglutamylation requires further clarification The authors report nuclear PolyE staining in colonic epithelial cells and propose that this reflects polyglutamylation of non-tubulin substrates such as PurA. However, it is not clear whether other nuclear proteins could account for this signal. The specificity of the nuclear PolyE signal should be better validated. Additional controls (e.g., peptide competition or validation with alternative approaches) would strengthen the interpretation.

Minor comments

1. The manuscript would benefit from clearer distinction between tubulin vs non-tubulin glutamylation throughout the text.
2. Some conclusions in the Discussion appear slightly overstated relative to the data (e.g., the role of the TTLL6-PurA axis in tumor suppression).
3. The description of the Ttll6 mouse models (constitutive vs conditional deletion) could be clarified earlier in the Results section.
4. Quantification methods for histological analyses (crypt length, cell counts, marker-positive cells) should be described in greater detail in the Methods.
5. It would be useful to include representative images of PurA localization in control vs Ttll6-deficient colon tissue in the main figures.
6. Several minor typographical issues appear throughout the manuscript and should be corrected during revision.

Significance

General assessment

This study investigates the role of the polyglutamylase TTLL6 in intestinal epithelial biology and colorectal cancer. The identification of a potential non-tubulin substrate (PurA) is conceptually interesting and expands the emerging view that tubulin-modifying enzymes can regulate additional cellular proteins. The study combines mouse genetics, histological analysis, transcriptomic datasets, and proteomics, which together provide a substantial dataset supporting a role for TTLL6 in regulating crypt architecture and epithelial proliferation. However, the mechanistic link between TTLL6 activity, PurA modification, and epithelial homeostasis remains incompletely resolved. The tumorigenesis data also suggest only modest effects on carcinogenesis.

Advance relative to previous literature

Previous studies have linked members of the TTLL family primarily to microtubule regulation and ciliary biology. This work extends these findings by suggesting a tissue-specific function of TTLL6 in the colon, and the existence of non-tubulin substrates regulating epithelial biology. If further validated, the identification of PurA polyglutamylation could represent an interesting conceptual advance.

The manuscript will likely be of interest to researchers working in cytoskeletal post-translational modifications, intestinal epithelial biology, colorectal cancer biology

My expertise lies in cytoskeletal regulation, epithelial biology, and intestinal tissue organization, which are directly relevant to the central themes of this manuscript.

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Referee #2

Evidence, reproducibility and clarity

Summary: In this study, the authors investigate novel functions of the tubulin typrosine ligase-like protein 6 (TTLL6), which covalently adds glutamate residues to the C-terminus of a given protein. The authors have already published previously on this topic. In the current study, the role of TTLL6 in colon function and pathologies was investigated. The study consists of two major parts. In the first part, a mouse model is used to show that TTLL6 is expressed at elevated levels and activity in epithelial cells of the colon. A database search indicated that TTL6 expression positively correlates with prognosis of patients with colorectal cancer. The authors generated a TTLL6 KO mouse and showed that induced tumor growth at 40 days was more positive for vimentin in the crypts of these mice, which should correlate with tumor aggressiveness. This difference was not observed anymore after 70 days. Morphological analyses of the crypts showed that in TTLL6 KO mice the crypts increased in length, a difference in proliferation markers, and a change of cell types in the crypts was observed.<br /> In the second part, the authors used a modification-specific antibody to immunoprecipitate (IP) proteins modified by TTLLs. To identify TTLL6-dependently modified target proteins, they compared these results with IPs from TTLL6 KO mice. A total of 43 proteins were identified this way. Because of their similarity to the tubuline tail sequence, two of the enriched proteins, PurA and PurB, were further analyzed. The authors provide evidence that PurA but not PurB is modified by TTLL6, which as a result changes its subcellular localization. While the first part of this work provides convincing novel insights into TTLL6's function with potential pathological relevance, the second part raises some concerns. I would therefore tend to rate the quality of the first part significantly higher than the second part.

Major comments:

1) When considering the results of the induced colorectal cancer test, the only significant difference between WT and KO was the moderately higher expression of Vimentin (figure 5E-F). Since this is the main evidence for a pathological relevance of TTLL6 in cancer, it is important to understand how the quantification of Vimentin in the complex tissue shown in figure 5E was done. A detailed description of how these images were analysed and perhaps a table with raw data would be essential to convince the reader of the conclusions. In the currently presented form, I find the analyses not too convincing.

2) Figure 7A: It was somewhat surprising that two of the least significant (PurA is just below the cutoff) were used for further analyses. Although the authors explain that both proteins have strong sequence similarity to the know TTLL6 target, tubuline, the C-terminal, genomically encoded protein sequence of PurA and PurB already contain several glutamates. This raises the concern that the polyE antibody in the IP possibly detected the non-modified C-terminal tail of PurA and PurB and that both proteins may not be modified by TTLL6. Because of this and the lower significance than other candidates, the authors should consider focussing on other hits (OPTIONAL). Besides being much more significant, they lack an accumulation of glutamates in their C-terminus (at least the ones I looked at). Alternatively, the concern of having potentially IP-ed unmodified proteins should be addressed.

3) Figure 8A: this figure compares PurA with a modified PurA that lacks the C-terminal EEE stretch. The authors conclude that the subcellular localization is different between both and that the nuclear localization of WT PURA must be due to modification by the co-expressed TTLL6. There are two major concerns with this conclusion: Firstly, the expression of PurA without TTLL6 co-expression is a missing essential control. This would show if PurA itself is already predominantly located in the nucleus regardless of potential modifications (PurA seems to have different nucleocytoplasmic localization in different cell types). Secondly, both depicted cells look very different. In PurA the nuclei are much smaller and the cytoplasm seems also much smaller than in the PurA DDD-expressing cells. Furthermore, IF staining without proper quantification of several cells seem less than ideal for such conclusions. In case, the authors want to convincingly validate this conclusion such a quantification with several cells would be required. OPTIONAL: an alternative approach would be a nucleo-cytoplasmic fractionation experiment followed by a western blot.

Figure 8B: it seems that the contrast between the images of the upper and lower panel is very different. For this reason, I find it difficult to follow the conclusions. However, even when ignoring this aspect, I have great problems coming to the same conclusions as the authors.

Minor comments:

1) In figure 3A it would help if the legend describes what exactly "Control (+ or -)" means.

2) In figure 3E-F, a label inside of the figure (what is the red bar, what the blue) would help the reader to faster grasp the subfigures.

3) Figure 7C-D: these experiments are based on strong overexpression of TTLLs, which might result in unphysiological modifications of PurA. I would suggest to include a note of caution in the discussion that this is a possibility.

4) In the discussion (page 9, last paragraph), it is stated: "Our findings suggest that the polyglutamylation of PurA is essential for maintaining colonic homeostasis". I do not understand this statement, as this study does not provide any evidence that modification of PurA does play a functional role in the colon (expression itself is not an evidence for function importance or even being "essential"). I recommend to remove this statement.

5) Not all abbreviations are introduced properly (like CRC).

Significance

In general, this study addresses a very interesting aspect - i.e. the covalent addition of multiple glutamate residues to the C-terminus of a target gene by the enzyme TTLL6. The authors convincingly show that this protein regulates the morphology and composition of crypts in subregions of the colon. This is certainly a new and important finding that expands our knowledge about the functional breadth of this class of enzymes. If convincingly validated (see major concerns), also the pathological relevance of this enzyme for cancer progression would be of general interest. However, this statement has to be considered with a note of caution as this is not my area of expertise.

The validation of novel targets of TTLL6 after IP is - at this stage of the manuscript - not very convincing to me. In particular the claim that PurA does play a functional role in the TTLL6-dependent regulation (of crypts) is not justified by the data. However, given that the list of other candidates contains several important gene regulators, this work might have the potential to open up to open up the field for new research directions.

The reviewer's areas of expertise: cell biology, biochemistry, histology.

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Referee #1

Evidence, reproducibility and clarity

Summary:

This study shows that loss of TTLL6 affects colonic epithelial homeostasis (crypt architecture and proliferation/differentiation markers) and proposes that TTLL6 contributes to a nuclear glutamylation signal, with PurA presented as a candidate non-tubulin substrate. The authors also connect TTLL6 mRNA levels to human CRC progression and outcome. Overall, the observations are potentially interesting, but the manuscript currently does not establish a clear mechanistic link between TTLL6 activity, PurA, and the in vivo phenotype.

Major comments:

1. The "TTLL6-PurA link" framing is currently too strong. The pull-down data indicate multiple candidate substrates, and the study does not show that PurA is the key functional mediator of the epithelial phenotype. As written, the manuscript reads as though PurA is the central downstream effector, which is not yet supported. Requested change: Either add substrate-specific functional evidence (additional KO/rescue-type experiments) or soften the language throughout (title/abstract/discussion) to reflect that PurA is one candidate among several.
2. PurA glutamylation should be demonstrated directly by MS. PolyE/GT335 immunoblotting and enrichment in PolyE pull-downs are suggestive, but they do not conclusively establish glutamylation of PurA at the C-terminal end (antibody specificity and/or co-precipitating glutamylated proteins remain possible explanations). Essential experiment: MS/MS identification of glutamylated residue(s) on PurA, ideally with evidence that the modification is TTLL6-dependent (WT vs KO or epithelial-inducible KO).
3. Regional TTLL6 expression vs phenotype needs to be reconciled. TTLL6 expression is reported to be highest in distal colon, yet the strongest crypt-length phenotype appears in transverse colon (as presented). Proximal colon data are not shown in the main text. Requested revision: Provide complete regional analyses (proximal/transverse/distal) with consistent quantification and statistics, and discuss explicitly why TTLL6 expression levels and phenotype do (or do not) align.
4. Several internal inconsistencies and missing statistics.
   • Fig. 1A vs 1B: CEC enrichment appears ~80-fold in panel A and ~4-fold in the panel B; if these reflect the same enrichment workflow, this discrepancy needs a clear explanation (normalization, starting material, ....).
   • Fig. 2A: statistics are missing.
   • Fig. 5D: the effects appear borderline; the conclusions should match the statistical support/significance. Requested revision: Ensure complete statistical reporting in the manuscript (n, definition of replicates, test used, p-values/thresholds) and avoid interpretive language where differences are not significant.
5. PurA Localization claims would benefit from stronger imaging and quantification. For nuclear localization/redistribution conclusions (main Fig. 8 and related supplement), confocal imaging with Z-stacks (and orthogonal views) would be more convincing than representative single-plane images. In addition, conditions with PurA-only expression need a clear baseline description and quantification. Requested additions: confocal Z-stacks + blinded quantification of nuclear/cytosolic localization across replicates; ideally support with subcellular fractionation and quantitative immunoblotting.
6. Overexpression artifacts should be considered more carefully. If TTLL6 has been described as an elongase in prior work (Mahalingan, NSMB, 2020, DOI: 10.1038/s41594-020-0462-0) high-level overexpression may generate non-physiological modifications or localization patterns. Requested revision: Soften conclusions drawn from overexpression experiments and provide appropriate expression controls and/or supportive evidence in more physiological settings.
7. Mouse tumor data should be interpreted more cautiously relative to the human correlations. The human datasets suggest a correlation between TTLL6 mRNA levels and clinical features/outcome (including recurrence-free survival), which is potentially interesting. In contrast, the mouse CAC results appear modest/borderline and, in places, are interpreted as stronger evidence than the data support. Requested change: Avoid strong claims about TTLL6 promoting or suppressing tumor growth unless supported by robust, clearly significant differences and comprehensive burden metrics.

Minor comments:

• Every figure should clearly state n (biological vs technical), statistical test, and multiple-comparison correction where applicable.
• Where effects are segment-specific, the text should reflect that specificity and avoid global statements.
• The Discussion would benefit from a clearer separation of what is directly shown versus what is proposed (especially near the end).
• TTLL6 expression is largely presented at the transcript level; it would help to make this explicit throughout and avoid wording that implies protein-level validation where it is not shown.

Significance

The manuscript has the potential to be of interest because it points to a possible role for TTLL6 in non-tubulin, nuclear glutamylation in the intestinal epithelium, and it links TTLL6 expression to human CRC datasets. At present, however, the broader impact is limited by (i) insufficient direct evidence that PurA is glutamylated in vivo and (ii) the lack of a causal connection between PurA and the epithelial phenotype. In addition, while the human data show correlations between TTLL6 expression and clinical parameters/outcome, the mouse CAC phenotype appears comparatively modest/borderline and should be interpreted with appropriate caution. With stronger biochemical validation (MS), improved localization quantification, and more restrained framing (or additional functional data), the work could appeal to readers in intestinal epithelial biology, post-translational modification biology, and CRC research.

Expertise: enzymology; post-translational modifications; microscopy; cancer mechanisms.

Rapport de Synthèse : Le Traitement Judiciaire de l’Inceste Parental sur Mineurs

Résumé Exécutif

Ce document synthétise les témoignages et analyses de Mme Hélène Romano (psychologue et docteure en droit) et de Mme Eugénie Izard (pédopsychiatre) lors de leur audition par la commission d'enquête sur l'inceste.

Le constat est celui d'un échec structurel du système français : seuls 8 % des enfants ayant révélé des faits d'inceste sont effectivement protégés, tandis que 95 % des procédures sont classées sans suite.

Les points critiques identifiés sont :

• L'institutionnalisation du doute : Un basculement idéologique post-affaire d'Outreau qui érige l'enfant en menteur de principe et le parent protecteur en manipulateur.

• L'absence de méthodologie scientifique : L'inexistence d'outils standardisés (comme le protocole SVA) pour évaluer la crédibilité de la parole de l'enfant.

• Le rôle délétère de l'Ordre des Médecins : Une institution accusée de réduire les médecins au silence et de sanctionner ceux qui signalent des maltraitances.

• La primauté de la famille sur l'enfant : Une sacralisation de la "coparentalité" et du lien biologique qui contraint les enfants à maintenir des liens avec leurs agresseurs présumés.

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1. Un Système Structurellement Défaillant

L'inceste n'est pas une accumulation de faits divers conjoncturels, mais un phénomène systémique et organisé.

Malgré une libération de la parole médiatique, les statistiques révèlent une impunité persistante.

Chiffres clés du traitement de l'inceste en France

| Indicateur | Donnée | | --- | --- | | Enfants victimes de violences sexuelles par an | 160 000 | | Part des violences étant d'ordre incestueux | 80 % | | Taux de protection des enfants après révélation | 8 % | | Taux de procédures classées sans suite | 95 % | | Condamnations annuelles | Environ 1 000 |

Le système actuel est décrit comme une "inversion perverse" où l'enfant qui parle n'est pas entendu et se voit souvent remis à la garde du parent désigné comme agresseur.

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2. L'Idéologie du Doute et l'Inversion de la Culpabilité

Depuis l'affaire d'Outreau, une "idéologie du mensonge" imprègne les institutions judiciaires et médico-sociales.

La disqualification du parent protecteur

Majoritairement des mères (95 % des auteurs d'inceste étant des hommes), les parents protecteurs subissent un processus de disqualification en plusieurs étapes :

• Disqualification psychologique : Mère jugée "trop fusionnelle", fatiguée ou déprimée.

• Biais de confirmation : L'épuisement de la mère face au système est utilisé pour confirmer son instabilité psychique.

• Accusations de pathologie : Recours au Syndrome d'Aliénation Parentale (SAP) ou au Syndrome de Münchhausen par procuration pour accuser la mère de manipuler l'enfant.

Note : Le SAP n'est pas reconnu par le DSM et son usage est proscrit par plusieurs instances internationales, mais il reste largement utilisé pour discréditer les signalements.

Les conséquences pour l'enfant

L'enfant est souvent réduit à l'état d'objet.

S'il ne parle pas (contexte de terreur, handicap, bas âge), son silence est utilisé contre lui.

S'il parle, il subit une "sur-violence" institutionnelle : répétition épuisante des récits (parfois plus de 20 fois), confrontations traumatisantes et absence de protection.

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3. Défaillances de l'Expertise et Absence de Méthodologie

La France souffre d'un manque criant de professionnels formés spécifiquement au psychotrauma de l'enfant et à l'évaluation de sa parole.

• Le "Chifoumi" judiciaire : En l'absence de méthodes validées, les décisions sont souvent le fruit de biais cognitifs et de stéréotypes (ex: "les enfants mentent", "l'inceste n'existe pas").

• L'outil SVA (State Validity Assessment) : Cette méthode, utilisée à l'étranger mais proscrite en France après Outreau, repose sur 19 critères permettant de distinguer un récit vécu d'un récit fabriqué (cohérence, détails atypiques, dialogues rapportés).

• Incompétence des experts : Des experts psychiatres d'adultes sont parfois mandatés pour des enfants sans avoir de pratique clinique pédiatrique, rendant des rapports basés sur des entretiens de quelques minutes.

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4. L'Ordre des Médecins : Un Obstacle à la Protection

Le témoignage du Dr Izard met en lumière une "silenciation" des médecins par leur propre institution.

• Pressions et sanctions : Des médecins sont interdits d'exercer pour avoir signalé des enfants en danger, l'Ordre invoquant souvent "l'immixtion dans les affaires de famille".

• Entrave à la loi : Bien que la loi de 2015 dégage les médecins de leur responsabilité en cas de signalement de bonne foi, l'Ordre est accusé d'ignorer la loi pénale pour sanctionner disciplinairement les praticiens.

• Résultat : Seul 1 % des signalements de violences sexuelles incestueuses proviennent de médecins, par crainte de représailles ordinales.

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5. La Sacralisation du Lien Familial et de la Coparentalité

Le système français privilégie le maintien du lien familial au détriment de la sécurité de l'enfant.

• Le paradigme de la réparation : L'enfant est souvent utilisé comme un "outil de réparation" pour le parent agresseur via des visites médiatisées forcées, au nom de la coparentalité.

• Le conflit vs la violence : Le terme "conflit parental" est abusivement utilisé pour invisibiliser les violences sexuelles.

Or, il ne peut y avoir de coparentalité dans une situation d'inceste.

• La violence judiciaire : Le cas de Mme Romano illustre l'extrême violence subie par les parents protecteurs : gardes à vue humiliantes, perquisitions, ruine financière (frais d'avocats s'élevant à plusieurs centaines de milliers d'euros) et condamnations pour "non-représentation d'enfant" même face à des preuves médicales de violences.

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6. Pistes de Réformes et Recommandations

Les intervenantes proposent plusieurs leviers pour transformer radicalement la prise en charge :

Réformes Procédurales et Judiciaires

• Création d'un crime d'inceste spécifique dans le code pénal, intégrant la notion de domination et de crime contre l'humanité de l'enfant.

• Reconnaissance de l'imprescriptibilité pour les crimes sexuels sur mineurs.

• Priorisation de l'ordonnance de protection immédiate dès la révélation.

• Saisine directe du Juge aux Affaires Familiales (JAF) par le procureur pour suspendre les droits du parent mis en cause pendant l'enquête.

• Suppression du délit de non-représentation d'enfant en cas de suspicion de violences.

Évaluation et Santé

• Usage de méthodologies standardisées (SVA, protocole NICHD) pour l'audition et l'expertise.

• Enregistrement audiovisuel systématique de toutes les expertises.

• Création de collèges d'experts spécialisés en psychotraumatologie infantile.

• Immunité disciplinaire effective pour les médecins signalant des maltraitances de bonne foi.

• Anonymat possible pour les professionnels signalant des faits graves.

Transformation Sociétale

• Sortir du déni de la domination masculine et patriarcale au sein de la famille.

• Former massivement tous les professionnels au contact des enfants (école, santé, social) au repérage des troubles spécifiques.

• Protéger l'enfant avant de protéger la famille, en cessant de considérer le lien biologique comme sacré au-delà de la sécurité physique et psychique.

DOI: 10.1038/s41419-026-08605-4

Resource: (Proteintech Cat# 10176-2-AP, RRID:AB_2224574)

Curator: @scibot

SciCrunch record: RRID:AB_2224574

What is this?

freundlish吸附线和其他等温吸附线判断吸附类型

Annotating this to keep the prompt handy. I am going to test this to see how the opinion is swayed.

By what measurement? I would expect them to be No 1 or 2 by BIP

Claude: Resources page shortening suggestions:

1. Remove duplicate tool cards — the Dashboard + Learn page cards are identical to what's on the About page. Replace with a compact one-liner: 'Interactive tools: Dashboard · How CM is Made · TEA Comparison · Technical Docs'

2. Add TEA Comparison link — the new compare.html page systematically compares Pasitka, Humbird, CE Delft, GFI, and Goodwin. This is directly relevant to the reading list context.

3. Keep the Shared Reading Collection — it serves a unique purpose (downloadable PDFs) not available elsewhere.

4. Keep the PQ Database section — it's the canonical link set.

Claude: Content trimming plan for this page (1,147 lines → ~700 target)

Proposed changes in priority order: 1. Fold FBS/serum-free section — historical context, no longer reflects current practice. One visible sentence + collapsed detail. 2. Move jurisdiction & 'Which market?' discussions to workshop — these are workshop topics, not dashboard content. Replace with one-line links. 3. Fold GF signaling diagram — educational but not cost-relevant. Keep the price/solutions tables visible. 4. Bold audit — reduce bold to headings + key numbers only (addresses multiple reviewer comments). 5. Trim Further Resources — link to workshop resources page for full list. 6. CSS fixes — larger diagrams, less whitespace around SVGs.

Optional: fold 2-3 SVG diagrams (cell banking, seed train) to reduce scroll length — the text already explains these steps.

Full plan at .private/content_trimming_plan.md. Feedback welcome here or in chat.

Author Response:

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

Public Reviews:

Reviewer #1 (Public review):

(1) The sample size for the ex vivo electrophysiology is small. Given the difficulty and complexity of the preparation, this is understandable. However, a larger sample size would have strengthened the authors' conclusions.

We appreciate that the sample size is small, but this was limited by the technical difficulty and relatively low yield with this preparation. From a total of 16 experiments, we were able to obtain successful recordings in 6 cases, and these provided the characterisation of the 11 cells reported in Figure 4. We believe that this is sufficient to “strongly suggest” that the cells with dense Trpm8 input correspond to cold-selective cells. We have toned down the statements in the abstract (line 23) and the Results section (line 246).

(2) The authors used tdTomato expression to identify brain targets innervated by these coldselective lamina I projection neurons. Since tdTomato is a soluble fluorescent protein that fills the entire cell, using synaptophysin reporters (e.g., synaptophysin-GFP) would have been more convincing in revealing the synaptic targets of these projection neurons.

As the Reviewer says, tdTomato labelling fills the entire cell. However, examination at high magnification reveals numerous varicosities along the labelled axons, presumably corresponding to synaptic boutons. We now illustrate this in Figure 6–figure supplement 2F.

In addition, we have provided further evidence that these varicosities correspond to (glutamatergic) synaptic boutons by immunostaining sections through the LPB for the postsynaptic density protein Homer1, and showing Homer1 puncta apposed to varicosities (Figure 6–figure supplement 2 G,H). This new information now appears in the Results section (lines 374-380).

(3) The summary cartoon shown in Figure 7 can be misleading because this study did not determine whether these cold - selective lamina I projection neurons have collateral branches to multiple brain targets or if there are anatomical subtypes that may project exclusively to specific targets. For example, a recent study (Ding et al., Neuron, 2025) demonstrated that there are PBN-projecting spinal neurons that do not project to other rostral brain areas. Furthermore, based on the authors' bulk labeling experiments, the three main brain targets are NTS, PBNrel, and cPAG. The VPL projection is very sparse and almost negligible.

We agree that branches to different brain nuclei may originate from specific subsets of ALS3 neurons and this is now stated in the figure legend. It is true that there are projections to other brain regions (including NTS). These are not included in the diagram, because their circuitry in relation to cold-sensing is less well understood. Although the projection to VPL from lumbar cord is sparse, this is likely to be explained by the very low proportion of lamina I projection neurons with axons that reach the thalamus. Our retrograde tracing data (e.g. Figure 6-figure supplement 4) had already revealed many cells in the C7 segment that were densely coated with Trpm8 afferents and retrogradely labelled from the lateral thalamus. We have carried out additional experiments in which AAV1.Cre^ON.td Tomato was injected into the cervical enlargement of Calb1^Cre mice.This resulted in much denser labelling in the VPL and PoT thalamic nuclei, supporting the suggestion that cold-selective lamina I neurons in the cervical enlargement project to these nuclei. This is now described in lines 381-387 and illustrated in Figure 6–figure supplement 3.

Reviewer #2 (Public review):

(1) In the characterization of recorded neurons in close contact or in the absence of this contact with TRPM8 afferents, the number of recorded neurons is relatively low. In addition, the strength of thermal stimuli is not very well controlled, preventing a more precise characterization of the connectivity.

We fully accept that the sample size is small (please see response to Reviewer 1 above). We also accept that the thermal stimulation was not that well controlled. Unfortunately, commercially available probes for controlling skin temperature are too large to apply to the skin in this preparation. For this reason, we have used application of hot and cold saline, as in our previous studies with this preparation.

(2) The authors could provide some sense of the effort needed to record from the 6 coldactivated neurons described. How many preparations were needed, etc?

We now state that 6 out of 16 experiments resulted in successful recordings for this part of the study (lines 858-861).

Reviewer #3 (Public review):

(1) While anatomical evidence for direct synaptic connectivity between Trpm8+ afferents and lamina I projection neurons is compelling, a physiological demonstration of strict monosynaptic transmission is not shown. The conclusion that these inputs are exclusively monosynaptic should be toned down. Similarly, the statement that "Lamina I ALS neurons that are surrounded by Trpm8 afferents are cold-selective" should also be toned down as only a few neurons have been tested and it cannot be excluded that other neurons with similar characteristics may be polymodal.

We have now carried out optogenetic experiments by expressing channelrhodopsin in Trpm8 afferents and retrogradely labelling ALS neurons with tdTomato. This has allowed us to directly demonstrate monosynaptic input. This is described in the Results section (lines 180-202) and the Methods section has been updated. As noted above, we have toned down the statement about lamina I neurons surrounded by Trpm8 afferents being coldselective (line 246).

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

(1) The patch innervation of Trpm8+ sensory neurons in lamina I of the spinal cord dorsal horn is interesting. Do they occupy specific areas within lamina I along the mediolateral axis, or are their placements random? Quantifying the distribution of these terminals in lamina I might be worthwhile.

Although we have not studied the mediolateral distribution systematically, it appears that the locations of the patches in the mediolateral axis is random, and they could be seen in medial, central and lateral parts of lamina I (as shown in Figure 2). We have added a comment to this effect in the Results section (lines 114-116). Quantifying Trpm8 terminals would be very labour-intensive, and we do not feel that this would be of great benefit.

(2) Quantification for the percentage of Trpm8+ boutons contacting Phox2a+ neurons that are vGlut3+

The main purpose of this part of the study was to provide a possible explanation for the finding by Li et al (2015) that some lamina I cells were associated with Vglut3-

immunoreactive boutons. We found that the percentages of Trpm8+ boutons that contained Vglut3 varied considerably from cell to cell, and this is now stated in the text (lines 133134). However, knowing exact proportions was not an important aspect of the study, we have therefore not carried out a detailed analysis.

(3) Quantification for the percentage of PBN projections neurons densely innervated by Trpm8+ axons that are calb1+.

As requested, we have carried out immunohistochemistry to determine the proportion of lamina I ALS neurons with dense Trpm8 input that are calbindin-immunoreactive. We examined 31 neurons from 3 different mice and found that all but 4 (i.e. 87%) were immunoreactive. This is now described (lines 287-293) and illustrated (Figure 5–figure supplement 1). We have now put the electrophysiological characterisation that was in this figure into a separate supplement (Figure 5–figure supplement 2).

(4) It might be helpful to confirm the brain projection targets of Cal1b+ lamina 1 projection neurons using AAV1-CreON-Synaptophysin-GFP (or other fluorescent proteins) injections

Please see our response to Public review Reviewer 1 comment 2 above. We have provided further evidence that the brain regions that received input from the Calb1+ cells contain axonal boutons (lines 374-380 and Figure 6–figure supplement 2F-H).

(5) Figure 6 - Figure Supplements 3 and 4 are duplicated

We apologise for this duplication, which was made in error in the version originally submitted to eLife. This has now been corrected.

Reviewer #2 (Recommendations for the authors):

(1) As mentioned, in the characterization of recorded neurons in close contact or in the absence of this contact with TRPM8 afferents, the number of recorded neurons is relatively low, some recorded in current clamp, a few in voltage clamp. This prevents any solid statistical evaluation of the findings

Please see response to response to the first point made by Reviewer 1 in the Public reviews. As stated above, we have toned down the statement about the relationship between cells with dense Trpm8 input and cold-selective cells (line 246).

(2) In addition, the strength of thermal stimuli is not very well controlled, preventing a more precise characterization of the synaptic connection between afferents and ALS projection neurons.

Please see our response to the Public review comment made by this Reviewer.

(3) Line 35. In the description of the anterolateral system and the effects of lesions, the species(s) should be specified since rodents and humans have a different anatomical distribution of spinal tracts.

We now state that while ALS axons ascend in the anterolateral quadrant in humans, they are located in the dorsolateral white matter in rodents (lines 40-42)

(4) To describe the semi-intact preparation used for recording and stimulation from the periphery, the authors cite a study by Julien Allard (reference 25). However, that study describes an in vivo preparation. I believe there is an error in the citation.

We thank the Reviewer for pointing this out – it has now been corrected.

(5) Line 726. Dorsal horn recordings were performed at 25 ºC. What is the temperature of the skin? How would this low temperature affect the excitability of cold afferents and their axons? Perhaps a comment about this issue would be appropriate.

The skin temperature in this preparation is the same as that of the spinal cord (25 °C). At this temperature, Trpm8 afferents would be active, but are likely to have adapted during the course of the experiment. Since this temperature is below 37 °C, it is likely that the conduction velocity of these afferents will be slower than in the in vivo situation. We have added a comment to this effect (lines 818-821).

(6) Line 401. The authors could not detect Trpv1-immunoreactivity in the central terminals of Trpm8Flp;RCE:FRT mice. Could they detect Trpv1 immunoreactivity in any central terminal? Do they have positive evidence that their immunostaining worked?

Trpv1 was readily detected in central terminals with the Trpv1 antibody. An example showing lack of detectable Trpv1-immunoreactivity in GFP-labelled (Trpm8-expressing) afferents is now shown in Figure 2–figure supplement 1K-M.

(7) Line 437. What is the expected anterograde transport time for YFP from the lumbar cord to the brainstem? Are 2-3 weeks not sufficient based on the literature? I noticed the authors are using longer survival times after intraspinal injections

In preliminary experiments for a previous study Substance P-expressing excitatory interneurons in the mouse superficial dorsal horn provide a propriospinal input to the lateral spinal nucleus | Brain Structure and Function we had found that a 2 week survival time after injection of AAV1.Cre^ON.GFP into the lumbar spinal cord of Tac1^Cre mice was not sufficient to label axons in the brain, although at 4 weeks we saw brain labelling. We have also found that extending survival times from 4 to 6 weeks gives greatly improved labelling, especially in the thalamus.

(8) Figure 5A. Many of the labelled cells appear to have the somas in the white matter, which makes little sense. It seems the reference section to plot the cells is not optimal

The placement of cells is accurate. Many spinal projection neurons are present outside the main region of grey matter (i.e. laminae I-X). These cells are found in 2 main regions – the lateral spinal nucleus (LSN) and the lateral reticulated part of lamina V. These two regions are intermediate between grey and white matter – i.e. they contain scattered cell bodies amongst a dense collection of axons. For this reason they appear outside the grey/white border as it is conventionally shown on diagrams of this type. This has been reported in numerous studies, e.g. see Figure 2 in The cells of origin of the spinothalamic tract of the rat: a quantitative reexamination - PubMed.

(9) Recent transcriptomic studies suggest the presence of more than one subpopulation of Trpm8-expressing DRG or trigeminal neurons. It is unclear to what extent the Trpm8-Flp line is capturing this diversity.

We are aware that there are at least 3 transcriptomic subsets of Trpm8-expressing primary sensory neurons. However, we are not aware of any suitable molecular markers that would allow us to discriminate between them, and therefore address this point.

(10) Could the patchy distribution of Trpm8 afferents in lamina I reflect incomplete recombination; the empty spaces could be occupied by unmarked afferents?

In theory it could, but this seems unlikely. The Trpm8^Flp line (crossed with RCE:FRT) captures ~83% of Trpm8-positive cell bodies, and it seems very unlikely that the remaining 17% of Trpm8-expressing afferents would fill the spaces between GFP bundles that we see in lamina I. This is now stated in the Results section (lines 116-120).

Reviewer #3 (Recommendations for the authors):

(1) It would be a nice addition to the validation of the Trpm8-Flp line to specify what ages (if multiple) have been analysed and whether there are any differences. In addition, is labelling different at different levels of the spinal cord, and is there any labeling in supraspinal regions?

The tissue used for this part of the study was obtained from mice aged 5-9 weeks and this is now stated (lines 78-79). We did not observe any differences with age, but we did not look at this in detail. Labelling was similar at different levels of the spinal cord, and this is stated (lines 108-109). We have added a brief account of the distribution of GFP labelling in the brain (lines 140-144).

(2) Line 169. It is not clear how ALS neurons are labeled. It is explained in the material and methods (I believe it is AAV9.mCherry into the LPB or CVLM). Although I could not find a mention of a tdTomato AAV, maybe I missed it. In any case, it would be great to have the experimental strategy briefly explained in the text. For the same reason, I would recommend moving Figure 4 Supplement 1A and 1B schematics to the main figure, very helpful for understanding the experiment.

We thank the Reviewer for this suggestion. We now explain in the Results section how the ALS neurons were labelled (lines 209-212), and as the Reviewer recommends we have put the schematic diagrams from Figure 4–figure supplement 1 into the main Figure. As noted in the text, the tdTomato labelling resulted from injection of an AAV coding for Cre into mice that contained the Ai9 allele. We have also updated the descriptions of brain injections in the Methods section to cover the new experiments (optogenetics, and calbindin immunohistochemistry).

(3) Line 184. "Figure 4" would be good to specify the panels; I believe it should be 4A-C. Same for line 194, 4D-F?

We apologise that this was omitted from the original version – we have now specified the panels.

(4) Line 179. It would be great to specifiy in the text and figures the temperature used for hot and warm water. In addition, would the responses be different using different temperatures? Can you test ramps? These would go a great way to compare with responses shown in vivo by Ran and colleagues.

We now specify the hot and cold saline temperatures used to stimulate the skin in the semiintact preparation in the legend for Figure 4 and in the Results section (lines 222-223). As noted above, it is difficult to use more accurate thermal stimuli in this preparation. Please see response to Reviewer 2 public comment 1.

(5) Figure 4-Figure supplement 1F. It looks like these are very slow responses (1 sec?) for monosynaptic connectivity.

In this figure (now part 1D) the action potential frequency was determined from counts of APs in 1 sec bins, and this is now stated in the legend. This might have given the impression of slow responses.

(6) Line 203. I would tone down the statement, as only 6 cells "that were clearly associated with numerous GFP-labelled afferents" have been tested. Thus, it cannot be excluded that other cells with similar anatomical characteristics may also respond to other stimuli

As requested, we have toned down this statement (line 246).

(7) Line 230. Here AAV11.CreON.td Tomato is used, in previous retrograde experiments, AAV9 has been used (Figure 4), why the switch to 11? Is the tropism the same? Is it possible that because you are using a different serotype, you are targeting different neurons?

We have found that although AAV9 coding for fluorescent proteins is very good for retrograde labelling, AAV9 coding for Cre-dependent constructs (e.g. AAV.Cre^ON.tdTomato) gives very poor recombination in spinal projection neurons, for reasons that we do not understand. We recently became aware of the AAV11 serotype, which was recommended as being suitable for retrograde transport AAV11 enables efficient retrograde targeting of projection neurons and enhances astrocyte-directed transduction | Nature Communications. We have found that this works very well for labelling ALS cells throughout the spinal cord when using Cre-dependent constructs. We have added a reference to this paper at this point in the text. We are not able to say whether tropism is the same or different, but in each case many ALS neurons (including many of those in lamina I) are captured.

(8) Line 234. Is there any positional organization for the "tdTomato-labelled cells densely innervated byTrpm8 afferents", do they preferentially cluster in some position of lamina I?

These cells are found throughout the mediolateral extent of the dorsal horn, and this is now stated (lines 279-280).

(9) Line 237. The actual number of cells/mm would be informative.

This would be difficult to estimate, as the sections were cut in the horizontal plane, which means that lamina I can appear on a variable number of sections.

(10) Line 249. From the figures, the action potentials of the Calb+ neurons seem to have a delayed onset (at the end of cold saline treatment, Figure 5, Supplement 1l) compared to lamina I ALS neurons recorded in Figure 4, Supplement 1f. If real, it is an interesting difference in the time-course of response that could indicate different coding properties e.g., response to cooling (general neurons) vs. response to absolute temperature (calb + neurons).

As for Fig 4-figure supplement 4 (see response to point #5 above), action potential frequency was determined from APs counted in 1 sec bins, and this is now stated in the legend.

(11) Figure 7. In the model, the disynaptic pathway should also be shown

We have added a comment to the legend stating that there may also be indirect (“polysynaptic”) input from Trpm8 afferents to ALS3 neurons.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

CCK is the most abundant neuropeptide in the brain, and many studies have investigated the role of CCK and inhibitory CCK interneurons in modulating neural circuits, especially in the hippocampus. The manuscript presents interesting questions regarding the role of excitatory CCK+ neurons in the hippocampus, which has been much less studied compared to the well-known roles of inhibitory CCK neurons in regulating network function. The authors adopt several methods, including transgenic mice and viruses, optogenetics, chemogenetics, RNAi, and behavioral tasks to explore these less-studied roles of excitatory CCK neurons in CA3. They find that the excitatory CCK neurons are involved in hippocampal-dependent tasks such as spatial learning and memory formation, and that CCK-knockdown impairs these tasks.

However, these questions are very dependent on ensuring that the study is properly targeting excitatory CCK neurons (and thus their specific contributions to behavior). There needs to be much more characterization of the CCK transgenic mice and viruses to confirm the targeting. Without this, it is unclear whether the study is looking at excitatory CCK neurons or a more general heterogeneous CCK neuron population.

Strengths:

This field has focused mainly on inhibitory CCK+ interneurons and their role in network function and activity, and thus, this manuscript raises interesting questions regarding the role of excitatory CCK+ neurons, which have been much less studied.

Weaknesses:

(1a) This manuscript is dependent on ensuring that the study is indeed investigating the role of excitatory CCK-expressing neurons themselves and their specific contribution to behavior. There needs to be much more characterization of the CCK-expressing mice (crossed with Ai14 or transduced with various viruses) to confirm the excitatory-cell targeting. Without this, it is unclear whether the study is looking at excitatory CCK neurons or a more general heterogeneous CCK neuron population.

Thank you for this constructive comment. Indeed, the current study lacks comprehensive strategies to unequivocally distinguish excitatory CCK neurons from heterogeneous CCK neuronal populations. Nevertheless, we provide multiple lines of evidence supporting the distribution of CaMKIIα/Vglut1-expressing CCK⁺ neurons in the hippocampus (Figure 1F), using complementary approaches including transgenic mouse models as well as viral and antibody-based labeling (Figure 1A, Figure 1H-I). In addition, we demonstrate that 635 nm light reliably evokes field excitatory postsynaptic potentials (fEPSPs) at CA3-Schaffer collateral synapses expressing DIO-CaMKIIα-ChrimsonR in vitro (Figure 2A-F). Importantly, these light-evoked excitatory synaptic responses are abolished by AMPA and NMDA receptor antagonists (CNQX and APV), confirming the excitatory nature of the DIO-CaMKIIα-ChrimsonR-expressing synapses. To demonstrate the future works that can further support our findings and conclusions, we have added the strategies that can be conducted in the Discussion section in the revision:

“Due to technical limitations at the current stage, we were unable to perform whole-cell recordings or pharmacological manipulations using CCK receptor antagonists. In future studies, the application of these approaches to directly record and selectively block EPSPs from excitatory CCK neurons in the hippocampus will further strengthen and validate our conclusions.” (Line 265 - line 269 in the revision).

(1b) For the experiments that use a virus with the CCK-IRES-Cre mouse, there is no information or characterization on how well the virus targets excitatory CCK-expressing neurons. (Additionally, it has been reported that with CaMKIIa-driven protein expression, using viruses, can be seen in both pyramidal and inhibitory cells.

We thank the reviewer for this insightful comment regarding the specificity of viral targeting in CCK-IRES-Cre mice.

To address this concern, we performed additional characterization of viral expression in CA3. We found that DIO-CaMKIIα-mCherry expression showed a high degree of colocalization with CaMKIIα immunoreactivity, indicating preferential targeting of excitatory neurons (sFigure 1A-B; sFigure 2A-B; sFigure 3A-B). We showed an example to confirmed the high specificity of the AAV for infecting the excitatory CCK neurons in CA3 area.

Besides, we acknowledge prior reports showing that CaMKIIα-driven viral expression can, in some cases, be detected in a small subset of inhibitory neurons. However, because CA3-Schaffer collateral projections to CA1 arise exclusively from excitatory CA3 pyramidal neurons, any potential expression in inhibitory CCK⁺ interneurons are unlikely to directly contribute to the recorded CA1 synaptic responses in our electrophysiological experiments. That said, we cannot fully exclude the possibility that a minor population of inhibitory CCK⁺ neurons could indirectly modulate CA3 pyramidal neuron activity via local circuit mechanisms, particularly in experiments involving optogenetic manipulation or shRNA expression. We now explicitly acknowledge this limitation in the revised manuscript:

“Importantly, to further improve cell-type specificity, we propose an intersectional genetic strategy using CCK-IRES-Cre × VGlut1-Flp mice combined with a Cre-On/Flp-On (Con/Fon) AAV, which would restrict expression exclusively to excitatory CCK-expressing neurons and eliminate potential contributions from inhibitory CCK⁺ cells. This approach will be implemented in future studies to refine circuit specificity.” (Line 269 - line 273 in the revision).

(2) The methods and figure legends are extremely sparse, leading to many questions regarding methodology and accuracy. More details would be useful in evaluating the tools and data. More details would be useful in evaluating the tools and data. Additionally, further quantification would be useful-e.g. in some places, only % values are noted, or only images are presented.

Thank you for these constructive comments. We have expanded the methodological descriptions in both the Methods section and the figure legends to provide sufficient detail for evaluating the experimental tools and data accuracy. In addition, we have added quantitative analyses where previously only representative images or percentage values were shown. Specifically, quantification has now been included for each AAV condition in the corresponding figures in the revised manuscript.

(3) It is unclear whether the reduced CCK expression is correlated, or directly causing the impairments in hippocampal function. Does the CCK-shRNA have any additional detrimental effects besides affecting CCK-expression (e.g., is the CCK-shRNA also affecting some other essential (but not CCK-related) aspect of the neuron itself?)? Is there any histology comparison between the shRNA and the scrambled shRNA?

Recent studies from our lab demonstrated that knockout the CCK gene expression significantly attenuates the hippocampal-dependent spatial learning and CA3-CA1 LTP, indicating CCK plays a critical role in modulating the hippocampal functions[1,2]. Additionally, CCK-shRNA or CCK-scramble did not significantly affect the excitatory synaptic transmission in the CA3-CA1 projections, hinting that CCK-shRNA may exhibits no obvious adverse effect on other neural components.

Finally, we have provided the histology comparison between the shRNA and the scrambled shRNA regrading the expression level of the CCK protein (Pro-CCK) in the revision. Our result shows that CCK-shRNA (left panel) significantly reduced CCK expression in CA3^CCK-positive neurons compared with the CCK-Scramble group (right panel).

Citation:

(1) Wang, J. L., Sha, X. Y., Shao, Y., Zhang, Z. H., Huang, S. M., Lin, H., ... & Sun, J. P. (2025). Elucidating pathway-selective biased CCKBR agonism for Alzheimer’s disease treatment. Cell.

(2) Zhang, N., Sui, Y., Jendrichovsky, P., Feng, H., Shi, H., Zhang, X., ... & He, J. (2024). Cholecystokinin B receptor agonists alleviates anterograde amnesia in cholecystokinin-deficient and aged Alzheimer's disease mice. Alzheimer's research & therapy, 16(1), 109.

https://doi.org/10.7554/eLife.109001.1.sa2

Reviewer #2 (Public review):

Summary:

In this study, the authors have demonstrated, through a comprehensive approach combining electrophysiology, chemogenetics, fiber photometry, RNA interference, and multiple behavioral tasks, the necessity of projections from CCK+ CAMKIIergic neurons in the hippocampal CA3 region to the CA1 region for regulating spatial memory in mice. Specifically, authors have shown that CA3-CCK CAMKIIergic neurons are selectively activated by novel locations during a spatial memory task. Furthermore, authors have identified the CA3-CA1 pathway as crucial for this spatial working memory function, thereby suggesting a pivotal role for CA3 excitatory CCK neurons in influencing CA1 LTP. The data presented appear to be well-organized and comprehensive.

Strengths:

(1) This work combined various methods to validate the excitatory CCK neurons in the CA3 area; these data are convincing and solid.

(2) This study demonstrated that the CA3-CCK CAMKIIergic neurons are involved in the spatial memory tasks; these are interesting findings, which suggest that these neurons are important targets for manipulating the memory-related diseases.

(3) This manuscript also measured the endogenous CCK from the CA3-CCK CAMKIIergic neurons; this means that CCK can be released under certain conditions.

Weaknesses:

(1) The authors do not mention which receptors of the CCK modulate these processes.

We appreciate the reviewer for raising this important question. Based on our recent work, CCK-B receptors are the primary neural components mediating CCK functions in the hippocampus at both the synaptic plasticity and behavioral levels (Su et al., 2023; Zhang et al., 2024; Wang et al., 2025). To clarify this mechanism, we have added the following content to the revised manuscript:

“Based on our recent work, CCK signaling in the hippocampus is predominantly mediated by CCK-B receptors, which play a critical role in regulating synaptic plasticity and spatial memory-related behaviors.” (Line 105 - line 106 in the revision).

(2) This author does not test the CCK gene knockout mice or the CCK receptor knockout mice in these neural processes.

Thank you for this insightful comment. We previously tested these experiments in an earlier study. Our results showed that high-frequency electrical stimulation failed to induce significant LTP in the CA3-CA1 pathway in both CCK gene knockout (CCK-KO) mice and CCK-B receptor knockout (CCK-BR-KO) mice in vitro (Su et al., 2023; Zhang et al., 2024; Wang et al., 2025). These findings indicate that CCK mediates its synaptic effects predominantly through CCK-B receptors in the CA3-CA1 pathway. Accordingly, we have added this description to the revised manuscript.

“Additionally, high-frequency electrical stimulation fails to induce LTP in the CA3-CA1 pathway in both CCK-KO and CCK-BR-KO mice, indicating that CCK-dependent synaptic plasticity in this circuit is primarily mediated by CCK-B receptors.” (Line 170 - line 173 in the revision).

(3) The author does not test the source of CCK release during the behavioral tasks.

We thank the reviewer for raising this important point. In our previous work, we directly monitored CCK release in the hippocampus during an object-exploration task using a GPCR-based CCK-BR sensor combined with fiber photometry (Su et al., 2023). During object exploration, we observed a rapid and robust increase in CCK-BR sensor fluorescence, indicating activity-dependent CCK release in the hippocampus. Based on these findings, we deduced that hippocampal CCK release plays a critical role in hippocampus-dependent behavioral tasks.

We acknowledge that hippocampal neurons receive CCK-positive projections from multiple brain regions, making it technically challenging to isolate and monitor the precise source of CCK release in the CA1 area during behavioral tasks in vivo. One potential strategy to address this limitation is selective overexpression of CCK in CA3 neurons (e.g., AAV-CCK delivery), followed by assessment of CCK-BR sensor responses during hippocampal-dependent behaviors. We have added this discussion to the revised manuscript to clarify the source and functional relevance of CCK release during behavioral tasks.

“Besides, using a GPCR-based CCK-BR sensor combined with fiber photometry, our previous work demonstrated rapid, activity-dependent CCK release in the hippocampus during object-exploratory behavior, supporting a functional role for hippocampal CCK signaling in cognitive tasks (Su et al., 2023). Given that hippocampal neurons receive CCK-positive projections from multiple brain regions, it remains technically challenging to precisely identify the cellular source of CCK release in CA1 during behavior. Future studies employing selective CCK overexpression in CA3 neurons, together with CCK-BR sensor recordings, may help further delineate the contribution of CA3-derived CCK to hippocampal-dependent behaviors.” (Line 313 - line 321 in the revision).

Citation:

(1) Wang, J. L., Sha, X. Y., Shao, Y., Zhang, Z. H., Huang, S. M., Lin, H., ... & Sun, J. P. (2025). Elucidating pathway-selective biased CCKBR agonism for Alzheimer’s disease treatment. Cell.

(2) Zhang, N., Sui, Y., Jendrichovsky, P., Feng, H., Shi, H., Zhang, X., ... & He, J. (2024). Cholecystokinin B receptor agonists alleviates anterograde amnesia in cholecystokinin-deficient and aged Alzheimer's disease mice. Alzheimer's research & therapy, 16(1), 109.

(3) Su, J., Huang, F., Tian, Y., Tian, R., Qianqian, G., Bello, S. T., ... & He, J. (2023). Entorhinohippocampal cholecystokinin modulates spatial learning by facilitating neuroplasticity of hippocampal CA3-CA1 synapses. Cell Reports, 42(12).

https://doi.org/10.7554/eLife.109001.1.sa1

Reviewer #3 (Public review):

Summary:

Fengwen Huang et al. used multiple neuroscience techniques (transgenetic mouse, immunochemistry, bulk calcium recording, neural sensor, hippocampal-dependent task, optogenetics, chemogenetics, and interfer RNA technique) to elucidate the role of the excitatory cholecystokinin-positive pyramidal neurons in the hippocampus in regulating the hippocampal functions, including navigation and neuroplasticity.

Strengths:

(1) The authors provided the distribution profiles of excitatory cholecystokinin in the dorsal hippocampus via the transgenetic mice (Ai14::CCK Cre mice), immunochemistry, and retrograde AAV.

(2) The authors used the neural sensor and light stimulation to monitor the CCK release from the CA3 area, indicating that CCK can be secreted by activation of the excitatory CCK neurons.

(3) The authors showed that the activity of the excitatory CCK neurons in CA3 is necessary for navigation learning.

(4) The authors demonstrated that inhibition of the excitatory CCK neurons and knockdown of the CCK gene expression in CA3 impaired the navigation learning and the neuroplasticity of CA3-CA1 projections.

Weaknesses:

(1) The causal relationship between navigation learning and CCK secretion?

Thank you for pointing out this important issue. Previous studies have shown that CCK can be rapidly secreted during exploratory behaviors, as detected by the CCK-BR sensor. In parallel, CCK-positive neurons have been demonstrated to play a critical role in the precise execution of hippocampus-dependent spatial learning. Together, these findings suggest that exploratory behavior induces CCK secretion, which in turn contributes to the accuracy of hippocampal-dependent learning and memory processes. Based on this evidence, we propose that CCK secretion serves as a functional link between behavioral exploration and spatial learning. We have added these explanations in the revised manuscript to better clarify the causal relationship between behavioral exploration and CCK secretion:

“Besides, using a GPCR-based CCK-BR sensor combined with fiber photometry, our previous work demonstrated rapid, activity-dependent CCK release in the hippocampus during object-exploratory behavior, supporting a functional role for hippocampal CCK signaling in cognitive tasks (Su et al., 2023). Given that hippocampal neurons receive CCK-positive projections from multiple brain regions, it remains technically challenging to precisely identify the cellular source of CCK release in CA1 during behavior. Future studies employing selective CCK overexpression in CA3 neurons, together with CCK-BR sensor recordings, may help further delineate the contribution of CA3-derived CCK to hippocampal-dependent behaviors.” (Line 313 - line 321 in the revision)

(2) The effect of overexpression of the CCK gene on hippocampal functions?

We thank the reviewer for this comment. In fact, an earlier study from our laboratory demonstrated that intraperitoneal injection of exogenous CCK-4 significantly improved performance in hippocampus-dependent spatial learning tasks in both CCK gene knockout (CCK-KO) mice and Alzheimer’s disease (AD) mouse models. These findings suggest that enhancing CCK signaling can ameliorate hippocampal dysfunction at both the behavioral and synaptic plasticity levels (Zhang et al., 2024; Wang et al., 2025). Accordingly, although direct genetic overexpression of CCK in the hippocampus has not yet been extensively characterized, the observed benefits of exogenous CCK delivery support the notion that increased CCK availability positively modulates hippocampal function and spatial learning. We have cited this study in the revised manuscript to support this interpretation.

“Interestingly, an earlier study demonstrated that intraperitoneal injection of exogenous CCK-4 significantly improved performance in hippocampus-dependent spatial learning tasks in both CCK gene knockout (CCK-KO) mice and Alzheimer’s disease (AD) mouse models (Zhang et al., 2024). These findings suggest that enhancing CCK signaling can ameliorate hippocampal dysfunction at both the behavioral and synaptic plasticity levels.” (Line 291 - line 297 in the revision)

(3) What are the functional differences between the excitatory and inhibitory CCK neurons in the hippocampus?

In the hippocampus, CCK-expressing neurons consist of two major populations with distinct functions: excitatory (glutamatergic) and inhibitory (GABAergic) neurons. Excitatory CCK neurons are relatively sparse and intermingled with pyramidal cells. By releasing glutamate, they directly contribute to excitatory transmission and are thought to participate in synaptic plasticity and information processing related to learning and memory. In contrast, inhibitory CCK neurons are more abundant and include well-characterized interneuron subtypes such as CCK-positive basket cells. These neurons release GABA and primarily target the perisomatic region of pyramidal neurons, providing strong control over neuronal firing. Notably, inhibitory CCK interneurons are highly sensitive to neuromodulatory signals, particularly endocannabinoids via CB1 receptors, enabling dynamic regulation of inhibitory tone and network activity. Together, excitatory CCK neurons mainly support hippocampal excitation and plasticity, whereas inhibitory CCK neurons regulate network dynamics and spike timing. As the focus of the present study is on excitatory CCK neurons, a detailed comparison between these two populations was not included in the original manuscript.

(4) Do CCK sources come from the local CA3 or entorhinal cortex (EC) during the high-frequency electrical stimulation?

Thank you for this insightful comment. Our data indicate that the CCK detected during high-frequency stimulation originates from CA3 neurons rather than the entorhinal cortex (EC). As shown in Figure 2, we used an optogenetic approach combined with a GPCR-based CCK sensor to selectively examine CCK release from the CA3-CA1 pathway. ChrimsonR was specifically expressed in CA3 neurons projecting to CA1, restricting light stimulation to CA3 axon terminals. In parallel, the CCK sensor was locally expressed in CA1, allowing real-time detection of CCK release at CA3 presynaptic sites. High-frequency light stimulation robustly evoked CCK signals in CA1, demonstrating activity-dependent CCK release from CA3 terminals. Importantly, EC inputs were neither genetically targeted nor optically stimulated in this experiment, excluding the EC as a source of the detected CCK. Together, these results support the conclusion that CCK released during high-frequency stimulation is derived from local CA3 projections to CA1. Similarly, as the focus of the present study is on excitatory CCK neurons in the CA3 area, a detailed comparison between these two CCK sources was not included in the original manuscript.

Citation:

(4) Wang, J. L., Sha, X. Y., Shao, Y., Zhang, Z. H., Huang, S. M., Lin, H., ... & Sun, J. P. (2025). Elucidating pathway-selective biased CCKBR agonism for Alzheimer’s disease treatment. Cell.

(5) Zhang, N., Sui, Y., Jendrichovsky, P., Feng, H., Shi, H., Zhang, X., ... & He, J. (2024). Cholecystokinin B receptor agonists alleviates anterograde amnesia in cholecystokinin-deficient and aged Alzheimer's disease mice. Alzheimer's research & therapy, 16(1), 109.

(6) Su, J., Huang, F., Tian, Y., Tian, R., Qianqian, G., Bello, S. T., ... & He, J. (2023). Entorhinohippocampal cholecystokinin modulates spatial learning by facilitating neuroplasticity of hippocampal CA3-CA1 synapses. Cell Reports, 42(12).

Reviewer #1 (Public review):

Summary:

The authors test whether the frog buccal ventilatory rhythm generator behaves as a discrete, anatomically localized oscillator or as a distributed, state-dependent network. They combine reduced preparations (segment/subsegment work), systematic extracellular unit surveys over a defined grid, and local AMPA/GABA microinjections in a hemisected brainstem preparation. Based on these approaches, the authors conclude that mild global excitation (bath AMPA) broadens the distribution of rhythmically active units and renders a previously defined "buccal area" functionally non-identifiable as a unique necessary/sufficient locus.

The central idea is plausible, and the overall experimental strategy is appropriate for the question being asked. However, in its current form, the manuscript overstates the strength of inference supporting the "expansion" and "loss of necessity/sufficiency" conclusions. This is primarily due to (a) statistical treatment of unit-mapping data that does not respect clustering by preparation/animal, (b) inconsistent statistical reporting across sections, and (c) limited interpretability of focal inhibitory perturbations under a globally excited state.

Strengths:

(1) The manuscript addresses a clear mechanistic question with broader relevance: whether rhythm generation is best conceptualized as a localized kernel or as an emergent distributed property that changes with excitatory state.

(2) The authors use convergent approaches (reduced preparations, mapping, and necessity/sufficiency-style pharmacological perturbations), which is appropriate for circuit-level inference.

(3) A strong element is the within-unit analysis supporting state-dependent changes in phase coupling for a subset of units ("lung" units adopting a buccal-like pattern). The authors' offline PCA-based spike sorting (with cluster-quality selection via silhouette score) provides some reassurance that the reported pre/post injection changes are not simply driven by unit misidentification.

Weaknesses:

(1) Pseudoreplication in unit-survey statistics undermines the main mapping inference. The Methods state that "Units were pooled from multiple preparations" and that chi-squared tests were used to compare proportions across conditions (baseline vs 60 nM AMPA). The Results similarly report proportion changes (e.g., 110 units pooled from three preparations vs 137 units pooled from three additional animals) analyzed with chi-squared tests. Because many units come from the same preparation/animal, independence is unlikely to hold; therefore, inference about state-dependent reorganization at the systems level should be made at the preparation/animal level or via hierarchical models that explicitly account for clustering.

(2) Statistical methods are inconsistently described and need harmonization. In the segment dose-response "Analysis," values are described as compared to zero using a "One-sample t-test." Yet Table 1 is titled as using a "Wilcoxon One-sample Test." These discrepancies must be resolved throughout (Methods, Results, figure legends, and tables), including clear reporting of the unit of n and exact test statistics.

(3) Unit classification and operational definitions raise interpretational concerns. The unit classification scheme defines "buccal units" as those firing during buccal bursts as well as lung bursts, and explicitly notes that "no units were found which fired only during buccal bursts." This is a consequential result, and it currently reads more like a limitation of detection/classification (or state-space sampled) than a robust biological conclusion. Without additional evidence, it weakens claims about a distinct buccal rhythmogenic module and complicates the interpretation of "buccal identity" changes under excitation.

(4) Microinjection mapping: high exclusion rate and alternative explanations for 'loss of necessity' under excitation. The manuscript reports that 15 experiments were conducted, but 9 were excluded because the buccal area was not found or the preparation was "overdriven." This exclusion rate is too high to leave implicit; it raises concerns about selection bias and demands transparent accounting. Moreover, under baseline conditions, GABA (or AMPA-GABA) microinjections reliably reduce/abolish buccal bursts, but under bath 60 nM AMPA, the same injections produce no significant change in instantaneous frequency. This pattern can be interpreted as network redistribution, but it can also reflect state-dependent changes in gain, dynamic range, or local pharmacological impact (e.g., inhibition being comparatively underpowered in the globally excited state). Additional controls/analyses are required to distinguish these explanations.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors investigate the response of the amphibian respiratory rhythm generator under varying excitability conditions. They use pharmacological agents to increase and/ or decrease synaptic excitability and demonstrate the resilience of buccal rhythms under different conditions. They employ these results to formulate their primary thesis, that there is no obligatory locus of the buccal respiratory rhythm in the frog, and that their respiratory rhythmogenic mechanisms should be considered diffuse and anatomically distributed across a larger brainstem region.

Strengths:

This manuscript is well written, with a sufficiently large number of experiments, for which the authors should be congratulated.

Weaknesses:

The presented results don't support the authors' main conclusions, and the interpretation of the data is heavily biased toward their hypothesis. This impregnates an unsubstantiated narrative in the Abstract, Introduction, and Discussion of this manuscript, which must be reexamined with the following points in consideration:

(1) The authors seem to confuse degeneracy with redundancy. For instance, at line 54, they state, "These findings support the broader hypothesis that respiratory rhythm-generating circuits can switch to being diffuse and redundant, with discrete oscillators quickly drowning in a sea of excitations."

Redundancy means having the same component repeated multiple times to buffer the failure of any single component, whereas degeneracy means different functional components that compensate for one another under perturbations (Goaillard and Marder, ARN 2021)

Since the premotor-lung units get converted to buccal units under high excitability, this suggests a degenerate mechanism for respiratory rhythm generation- rather than a redundant mechanism, where there should be multiple buccal units that get recruited under different excitability conditions.

(2) Line 83, "but the essential requirement for a discrete, rudimentary buccal oscillator is also lost".

This statement is not supported by the data presented in this study. How does the expansion of the buccal unit imply that the essential requirement for discreteness is lost? Under increased excitability, does the burst/rhythm initiation zone also expand? Or does it still remain centered around the location of buccal units under physiological conditions? Increased excitability can lead to recruitment of a larger area, without a change in the location of the rhythmogenic kernel.

(3) Line 86, "... oscillators should be viewed as promiscuous flexible functional entities that expand or contract...".

Oscillators can be regarded as promiscuous only if, under physiological conditions, they switch positions. Under high excitability, only the flexibility argument holds, which has been established in mammals before (e.g., CA Del Negro, K Kam, JA Hayes, JL Feldman, The Journal of physiology 587 (6), 1217-1231; CA Del Negro, C Morgado-Valle, JL Feldman,Neuron 34 (5), 821-830; NA Baertsch, LJ Severs, TM Anderson, JM Ramirez, Proceedings of the National Academy of Sciences 116 (15), 7493-7502; NA Baertsch, HC Baertsch, JM Ramirez Nature communications 9 (1), 843).

Results:

(4) Interpretation of data in Figure 6.

How does the Buccal activity and L2 Power stroke change with 60nm AMPA (in CN5)? Does the increase in the Buccal neurons and decrease in power stroke neurons also reflect in the CN5 activity? Also see comments on Figure 9 data below.

(5) Interpretation of data in Figure 7.

Here, classifying buccal neurons solely by spiking may obscure the fact that the 'silent' neurons under baseline conditions were part of the rhythmic network but could not spike due to subthreshold inputs. 60 nM AMPA increased their firing in response to previously subthreshold synchronous inputs during the buccal burst. Intracellular recordings are required to negate this possibility and establish that the neuronal classification is robust.

(6) Interpretation of data in Figure 8.

"Lung units can transform into buccal units under excitation".<br /> CN5 buccal and lung bursts need to be compared before and after AMPA injection. From Figure 8 A-D, it is apparent that the example Unit2's activity increases during the buccal bursts, after AMPA injection. However, they are also present in buccal burst pre-AMPA, albeit with less frequency.

It is striking that the pre-AMPA epoch (panel A) is less than half of the post-AMPA epoch. This would, in itself, lead to a biased estimate of lung units that are active under the baseline condition during the buccal bursts.

Figure 8G, meta-analysis of lung units spiking during the baseline buccal bursts is warranted to interpret the main claim of this figure. Similarly, analysis of spiking per lung burst for the post-AMPA condition is essential for comparing the lung unit's contribution under high excitability.

(7) Interpretation of data in Figure 9

"Buccal area loses importance under increased excitation."

This interpretation is not fully supported by the data presented in this manuscript. Under 60 nm AMPA, does the ratio of lung burst to buccal burst change in CN5? This analysis is crucial for determining whether the lung units are indeed converted into buccal bursts at the expense of lung activity or whether their appearance during buccal bursts is incidental due to increased excitability. In the baseline, there are 4-5 buccal bursts per lung burst, whereas under high excitability, there are 2-3 buccal bursts per lung burst (Figure 9 A-B). This seems inconsistent with the conclusion that increased excitability converts lung units into buccal units (Figures 6 &7).

Could the authors comment on the connectivity between the lung and the buccal units? Results in Figure 9A-B indicate that lung units may receive an efference copy of buccal units, and under high excitability, their spikes may generate negative feedback onto the buccal units, terminating their bursts. This could explain the decrease in the buccal-to-lung burst in high-AMPA conditions. This type of circuit interaction resembles the mammalian breathing CPG, in which the parafacial/RTN (which controls the abdominal muscles) and preBötC (which controls the diaphragm) interact and cross-inhibit each other.

(8) Line 382.

"Buccal-like bursting produced from two independent slices".

The two "independent" slices have portions of the same anatomical kernel, the buccal rhythm generator. This experiment is like the sandwich slice preparation of preBötC (Del Negro Lab), in which two thinner slices exhibit rhythmic activity. Thus, the two slices are not independent; they are anatomically adjacent and functionally overlapping.

Author response:

Reviewer #1 (Public review):

Hierarchical Inference (Unit Survey)

We agree that pooling units across preparations can overstate the strength of inference if preparation-level clustering is ignored. We will therefore reanalyze the unit-survey dataset using a hierarchical approach in which the preparation/animal is treated as the unit of inference. Our pooled dataset was derived from three chunk preparations exposed to AMPA and three baseline preparations, allowing us to report per-preparation proportions and variability as requested.

A preliminary reanalysis of the buccal segment preparations is summarized below. In this analysis, the unit of inference is shifted from individual recorded units to the preparation level (n = 3 baseline; n = 3 at 60 nM AMPA), thereby accounting for potential within-preparation dependence.

Author response table 1.

The distribution of units for each of the three preparations per condition is as follows:

Using the proportion of buccal units per preparation as the dependent variable:

Baseline (n = 3): mean proportion of buccal units = 6.5% (SD 5.7%).

60 nM AMPA (n = 3): mean proportion of buccal units = 53.2% (SD 6.0%).

Absolute difference in proportions = 46.7% (95% CI 33.4% to 59.8%).

Independent-samples t-test on per-preparation proportions: t(4) = 9.77, p = 0.0006.

Thus, this preliminary hierarchical reanalysis indicates that the observed recruitment is consistent across preparations and is not driven by outlier data from a single animal. These results support substantial expansion of the buccal oscillator with excitation.

Statistical Standardization: In the revision, we will better justify our use of parametric and non-parametric versions of the one-sample tests and review usage in the Methods, Table 1, and figure legends for consistency.

Exclusion criteria for microinjection experiments: We will extend the description of these experiments by including a flow diagram summarizing the 15 attempted microinjection experiments and documenting the technical reasons for the 9 exclusions. These exclusions reflected the technical requirements of the preparation: (a) the buccal area had to be localized before AMPA excitation so that the effects of buccal-area manipulation during excitation could be interpreted reliably, which was not always possible; and (b) preparations had to exhibit sufficiently sustained periods of consecutive buccal bursting to permit quantification of buccal burst frequency, whereas some preparations expressed motor patterns dominated by lung bursts.

Pharmacological Potency and Necessity: We will revise the wording of this section to make the causal interpretation more precise. Our data already show that local GABA microinjections can reverse the excitatory effects of local AMPA microinjections, providing an internal control for local pharmacological efficacy of GABA when the local network is excited. Notably, the local AMPA concentration used in these experiments (5 µM) is nearly two orders of magnitude greater than the 60 nM concentration used in bath application. We therefore interpret the failure of focal GABA inhibition to abolish rhythm during global excitation as being consistent with expansion of rhythmogenic capacity beyond the spatial reach of the local injection, rather than with failure of the GABA manipulation itself.

Finding an inhibitory site that remains sensitive in bath applied AMPA is an interesting experiment but this would require identifying the anatomical substrate of a brainstem circuit for a non-ventilatory circuit in Rana that is guaranteed not to undergo reconfiguration with AMPA. This is beyond the scope of the current manuscript; based on our work to identify the neuronal substrate for ventilation in Rana, this would take at least five years to complete. In addition, having identified such a circuit there would be no guarantee that AMPA would not cause reconfiguration in this case too. With regards to transection boundaries and location of injections, we agree these would be useful refinements. We used the location of nerves as reliable landmarks to guide transections and located the buccal area using stereotactic coordinates to guide micropipette insertion and functional criteria (AMPA and GABA sufficiency and necessity tests) to locate the exact position based on our previous work.

Unit Classification: We will review the nomenclature we use to define units to ensure it does not cause confusion and provide more explicit criteria for unit classes. This will include clarification of the absence of “buccal-only” units as currently defined. Specifically, when both buccal and lung rhythms are present, units active during buccal bursts are also active during lung bursts in our preparation. This does not conflict with the multiple interacting oscillator model we have proposed previously. Rather, recruitment of buccal-area neurons during lung bursts is consistent with a model in which the lung oscillator excites the buccal oscillator. It is also consistent with prior evidence that lung bursts persist after buccal-area ablation. In addition, burst frequency during lung episodes exceeds buccal burst frequency during intervening buccal periods. We will revise the text to make this logic clearer.

Reviewer #2 (Public review):

(1) Degeneracy vs. Redundancy

We agree that degeneracy is the more precise term for the phenomenon our data demonstrate, in which structurally and functionally distinct neurons (lung units) acquire the capacity to participate in buccal rhythm generation under excitation. The Discussion already uses this language (e.g., "necessity and sufficiency may not work in a large degenerate network where rhythm generation is distributed across many elements"), but we used the word "redundant" in the Key Points Summary and Abstract in the broader sense of distributed robustness that a wider readership could grasp. Nonetheless, we recognize the distinction drawn by Goaillard and Marder (2021) and, considering the reviewers concerns, we will revise the Abstract and Key Points to adopt the degeneracy framework consistently.

(2) Loss of Essential Requirement for a Discrete Oscillator

The reviewer asks whether expansion of the rhythmically active region necessarily implies loss of the rhythmogenic kernel. We believe our necessity and sufficiency experiments (Figure 9) directly address this. Under baseline conditions, GABA microinjection into the buccal area reliably abolishes buccal bursting; under 60 nM bath AMPA, the same injection at the same location and volume has no significant effect on buccal frequency. If the kernel remained essential and the surrounding recruitment were merely supplementary, local inhibition of the kernel should still slow or abolish the rhythm. It does not. We interpret this as evidence that the essential requirement for the discrete buccal area is lost under excitation, not merely that a larger area has been recruited around a still-critical core. We acknowledge, however, that the word "lost" could be read as implying permanent elimination rather than state-dependent suspension, and we will temper this language in the revision.

(3) Novelty Relative to Mammalian Studies

We appreciate the reviewer drawing attention to the cited mammalian literature (Del Negro et al., 2002, 2009; Baertsch et al., 2018, 2019), which we discuss in detail in the manuscript. However, we respectfully note that our findings extend this literature in several ways that the public review does not acknowledge. First, Baertsch et al. demonstrated recruitment of tonic or silent neurons to become phasically active during inspiration; we show that neurons already assigned to one oscillator phase (lung) can be dynamically reassigned to another (buccal), which represents a qualitatively different form of reconfiguration. Second, we developed a novel approach to functionally ablate motor neuron pools using high-frequency nerve stimulation, enabling the unit survey to be interpreted at the premotor level which was not achieved in the mammalian studies cited. Third, our data provide the first demonstration of state-dependent oscillator expansion in a non-mammalian tetrapod, offering evolutionary context that strengthens the generality of the principle. We will revise the term "promiscuous" if it overstates the claim, but we maintain that our data support the conclusion that oscillator boundaries are flexible, which goes beyond what has been shown in mammals.

(4) Figure 6, CN5 Output Under AMPA

The reviewer asks whether the shift in premotor unit composition is reflected in CN5 motor output. This is a reasonable question. As noted in the manuscript, 60 nM AMPA produces only minor changes in the overt motor pattern as recorded from CN5, which is precisely why we interpret the premotor changes as a reorganization of the network's internal architecture that is not readily apparent from motor output alone. This is in sharp contrast to observations of substantive network reconfiguration in mammals in which eupnea is replaced by the pathological condition of gasping. We will add quantification of CN5 burst parameters (amplitude, duration, frequency) under baseline and 60 nM AMPA to make this point explicit.

(5) Subthreshold Recruitment vs. Network Expansion

The reviewer suggests that neurons classified as newly rhythmic under AMPA may have been part of the rhythmic network all along, receiving subthreshold inputs at baseline. We are grateful to the reviewer for highlighting this and hope they would agree that the literature clearly demonstrates that all respiratory neurons receive subthreshold phasic inputs of one kind or another, perhaps providing a clue that reconfiguration is a common feature of respiratory networks generally. Regardless of the implications for other animals, we agree this is likely the mechanism at work in the frog, and indeed our manuscript states that "this increase in the number and proportion of premotor buccal units is due in part to recruitment of sub-threshold buccal neurons that, under low excitability, only fire during lung bursts," citing intracellular evidence from Kogo and Remmers (1994) that lung neurons in this region receive subthreshold buccal-timed input. We note that this observation does not diminish our conclusion and likely explains the mechanism by which network expansion occurs. Whether one calls these neurons "newly recruited" or "pushed above threshold," the functional consequence is the same: a larger population of neurons is now rhythmically active during buccal bursts, and the necessity of the original buccal area is lost. We will clarify this reasoning in the revision and acknowledge the limitation that additional intracellular recordings from our preparation would be needed to fully characterize the subthreshold dynamics.

(6) Figure 8, Epoch Length and Meta-analysis

The reviewer notes that the pre-AMPA epoch appears shorter than the post-AMPA epoch in Figure 8A, which could bias unit classification. We will address this in the revision by reporting epoch durations explicitly and addressing its implication on spike counts where appropriate. Regarding the request for meta-analysis of lung unit spiking during baseline buccal bursts: this analysis is part of the rationale for the phase-recruitment panels, and we will expand Figure 8 to include the requested cross-condition comparisons (lung unit activity during baseline buccal bursts, and during post-AMPA lung bursts) as also suggested by Reviewer 3.

(7) Figure 9, Buccal-to-Lung Burst Ratio

The reviewer observes that the ratio of buccal to lung bursts decreases from approximately 4-5:1 under baseline to 2-3:1 under 60 nM AMPA, and suggests this is inconsistent with conversion of lung units into buccal units. We do not believe this is inconsistent. The buccal-to-lung burst ratio reflects the overt motor pattern, which is determined by the interaction of multiple oscillators and is influenced by AMPA at both buccal and lung levels. A change in this ratio does not speak to whether individual premotor units have acquired buccal-timed activity; the unit survey and the single-unit transformation data (Figure 8) address that question directly. Regarding the alternative model involving efference copy and cross-inhibition: this is an interesting hypothesis, but it is speculative and not tested by the current dataset. We are happy to discuss lung-buccal interactions more fully in the revision, including the parallels to parafacial/preBötC interactions in mammals, but we note that our data on unit transformation are better explained by network reconfiguration than by a feedback model that remains to be tested.

(8) "Independent" Slices

The reviewer compares our Level 2 transection to the preBötC sandwich slice preparation and argues the two resulting slices are not independent. We take the reviewer's point that "independent" may be read as implying no shared developmental or functional origin, which is not our intent. By "independent" we mean that the two physically separated slices can each generate rhythmic output without being synaptically connected to each other. This is, in fact, our central point: rhythmogenic capacity is distributed across a region broad enough to endow two separated slices with independent rhythm-generating capability when excited. We note that the analogy to the sandwich slice is imperfect because in our Level 1 cuts, only the rostral slice containing the buccal area generates rhythm -- the caudal slice does not -- whereas Level 2 cuts that bisect the buccal area produce rhythmicity in both halves, consistent with distributed capacity specifically within the buccal region. We will revise the wording to clarify what we mean by "independent" in this context.

Reviewer #3 (Public review):

Physiological Parallels: We will expand the Discussion to place these findings in a broader comparative context, including the eupnea-to-gasping transition in mammals as an example of state-dependent reconfiguration of respiratory networks. This will also allow us to clarify two advances that may otherwise be missed when comparing our work to that in mammals: (a) we developed a novel approach to functionally eliminate motor neurons, allowing mapped units to be interpreted as premotor; and (b) the state-dependent reconfiguration of the buccal oscillator occurred without qualitative changes in the overt lung-buccal motor pattern.

Unit Transformation Analysis: We will revise Figure 8 to improve clarity around the observed lung-to-buccal transformation by expanding the phase-recruitment panels as suggested and will revisit the operational definitions of lung and buccal unit identity to reduce ambiguity. The central observation is that some units active only during lung bursts under one condition become active during buccal bursts when network excitation is increased.

Saturation vs. Network Expansion: We will directly address the possibility that 60 nM bath-applied AMPA simply pushes the network toward a frequency ceiling. Two observations strongly argue against this interpretation: (a) 60 nM global AMPA produced only mild changes in buccal frequency, whereas local AMPA injection at much higher concentrations produced larger effects; and (b) local GABA was sufficient to reverse the effects of high-concentration local AMPA microinjections but insufficient to abolish rhythm during low-concentration global AMPA application. Together, these findings are more consistent with global AMPA endowing the network with distributed rhythm-generating capacity than with simple saturation of a discrete local oscillator. Notwithstanding these arguments, we will attempt to extend AMPA/GABA dose response experiment as suggested or add the lack of such experiments as a caveat to our interpretation.

Figure 9C Correction: We will correct the statistical markings in Figure 9C to align with the text in the Results regarding the significance of frequency changes under 60 nM AMPA.

In total, we believe these revisions will improve the rigor and clarity of the manuscript while preserving the central conclusion supported by the data: that the organization of the frog respiratory rhythmogenic network is state dependent and becomes more distributed under excitation.

Reviewer #1 (Public review):

This manuscript investigates how dentate gyrus (DG) granule cell subregions, specifically suprapyramidal (SB) and infrapyramidal (IB) blades, are differentially recruited during a high cognitive demand pattern separation task. The authors combine TRAP2 activity labeling, touchscreen-based TUNL behavior, and chemogenetic inhibition of adult-born dentate granule cells (abDGCs) or mature granule cells (mGCs) to dissect circuit contributions.

This manuscript presents an interesting and well-designed investigation into DG activity patterns under varying cognitive demands and the role of abDGCs in shaping mGC activity. The integration of TRAP2-based activity labeling, chemogenetic manipulation, and behavioral assays provides valuable insight into DG subregional organization and functional recruitment. However, several methodological and quantitative issues limit the interpretability of the findings. Addressing the concerns below will greatly strengthen the rigor and clarity of the study.

Major points:

(1) Quantification methods for TRAP+ cells are not applied consistently across panels in Figure 1, making interpretation difficult. Specifically, Figure 1F reports TRAP+ mGCs as density, whereas Figure 1G reports TRAP+ abDGCs as a percentage, hindering direct comparison. Additionally, Figure 1H presents reactivation analysis only for mGCs; a parallel analysis for abDGCs is needed for comparison across cell types.

(2) The anatomical distribution of TRAP+ cells is different between low- and high-cognitive demand conditions (Figure 2). Are these sections from dorsal or ventral DG? Is this specific to dorsal DG, as itis preferentially involved in cognitive function? What happens in ventral DG?

(3) The activity manipulation using chemogenetic inhibition of abDGCs in AsclCreER; hM4 mice was performed; however, because tamoxifen chow was administered for 4 or 7 weeks, the labeled abDGC population was not properly birth-dated. Instead, it consisted of a heterogeneous cohort of cells ranging from 0 to 5-7 weeks old. Thus, caution should be taken when interpreting these results, and the limitations of this approach should be acknowledged.

(4) There is a major issue related to the quantification of the DREADD experiments in Figure 4, Figure 5, Figure 6, and Figure 7. The hM4 mouse line used in this study should be quantified using HA, rather than mCitrine, to reliably identify cells derived from the Ascl lineage. mCitrine expression in this mouse line is not specific to adult-born neurons (off-targets), and its expression does not accurately reflect hM4 expression.

(5) Key markers needed to assess the maturation state of abDGCs are missing from the quantification. Incorporating DCX and NeuN into the analysis would provide essential information about the developmental stage of these cells.

Minor points:

(1) The labeling (Distance from the hilus) in Figure 2B is misleading. Is that the same location as the subgranular zone (SGZ)? If so, it's better to use the term SGZ to avoid confusion.

(2) Cell number information is missing from Figures 2B and 2C; please include this data.

(3) Sample DG images should clearly delineate the borders between the dentate gyrus and the hilus. In several images, this boundary is difficult to discern.

(4) In Figure 6, it is not clear how tamoxifen was administered to selectively inhibit the more mature 6-7-week-old abDGC population, nor how this paradigm differs from the chow-based approach. Please clarify the tamoxifen administration protocol and the rationale for its specificity.

Comments on revisions:

I appreciate the authors' careful and thorough revisions. They have addressed all of my previous concerns satisfactorily, and the manuscript is now significantly strengthened. I have no further concerns.

Reviewer #3 (Public review):

This study examines the role of dentate gyrus neuronal populations, reflecting neurogenesis and anatomical location (suprapyramidal vs infrapyramidal blade), in a mnemonic discrimination task that taxes the pattern separation functions of the dentate. The authors measure dentate gyrus activity resulting from cognitive training and test whether adult neurogenesis is required for both the anatomical patterns of activity and performance in the cognitive task. The authors find that more cognitively challenging variants of the task evoked more dentate activity, but also distinct patterns of activity (more activity in the suprapyramidal blade, less in the infdrapyramidal blade). Using chemogenetic approaches they silence mature vs immature dentate gyrus neurons and find that only mature neurons (either the general population or specifically mature adult-born neurons), and not immature adult-born neurons, are required for the difficult version of the task. Inhibition of mature adult-born neurons furthermore increased overall activity in the dentate and reduced the biased pattern of activity across the blades, consistent with evidence that adult-born neurons broadly regulate dentate gyrus activity.

Comments on revisions: