DOI: 10.1101/gad.352485.124

Resource: (IMSR Cat# JAX_005557,RRID:IMSR_JAX:005557)

Curator: @scibot

SciCrunch record: RRID:IMSR_JAX:005557

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Bezpečnost na 1. místě Naše stany splňují požadavky normy EN 13782, která určuje požadavky na odolnost stanů vůči poryvům větru.

Proč zvolit stany od nás? Zaměřujeme se na 100% kvalitu, proto naše stany odolají rychlosti větru až do 100 km/h! Nepromokavá látka Vám pak zajistí komfort i při silné bouřce.

Typy stanů v nabídce:

expanding numeric systems is quite easy

Ha!

Con ADD también empezamos con la instancia del objeto que queremos manipular.

Desde que me lo presentaron, siempre me ha desagradado el Test Driven Design (TDD), pues me parecía absurdamente burocrático y contra flujo. Afortunadamente, gracias al podcast de Book Overflow, encontré un autor reconocido, John Ousterhout, creador de Tcl/Tk y "A Philosophy of software design", que comparte mi opinón respecto a escribir los test antes de escribir el código y dice que en el TDD no se hace diseño, sino que se depura el software hasta su existencia.

Mi enfoque, que podría llamarse Argumentative Driven Design o ADD es uno en el que el código se desarrolla para mostrar un argumento en favor de una hipótesis, y las pruebas de código se van creando en la medida en que uno necesita inspeccionar y manipular los objetos que dicho código produce.

En palabras práctica, esto quiere decir que los test y su configuración deberían hacerse cuando uno necesita hacer un "print" (para probar/inspeccionar/manipular un estado/elemento del sistema) y no antes, lo cual aumenta la utilidad, no interrumpe el flujo y responde preguntas similares a las de este apartado, respecto a de dónde provienen las pruebas y qué hacer con los resultados exitosos.

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Reply to the reviewers

Manuscript number: RC-2025-03195R

Point-by-Point Response to Reviewers

We thank the reviewers for their thoughtful and constructive evaluations, which have helped us substantially improve the clarity, rigor, and balance of our manuscript. We are grateful for their recognition that our integrated ATAC-seq and RNA-seq analyses provide a valuable and technically sound contribution to understanding soxB1-2 function and regenerative neurogenesis in planarians.

We have carefully addressed the reviewers' major points as follows:

1. Direct versus indirect regulation by SoxB1-2:____ In the revision, we explicitly acknowledge the limitations of inferring direct regulation from our current datasets and have revised statements throughout the Results and Discussion to emphasize that our findings are correlative.
2. Evidence for pioneer activity:____ Although the pioneer role of SoxB1 transcription factors in well established in other systems, we agree that additional binding or motif data would be required to formally demonstrate SoxB1-2 pioneer function. Accordingly, we performed motif analysis and revised the text throughout to frame SoxB1-2's proposed role as consistent with, rather than demonstrating transcriptional activator activity.
3. Motif enrichment and downstream regulatory interactions:____ In response to Reviewer #1's suggestion, we have included a new motif enrichment analysis in the supplement to contextualize possible co-regulators within the SoxB1-2 network.
4. Data reproducibility and peak-calling consistency:____ We have included sample correlations ____and peak overlaps for ATAC-seq samples in the revision, providing a clearer assessment of reproducibility.
5. Clarification of co-expression and downstream targets:____ We included co-expression plots for soxB1-2 with mecom and castor in the supplemental materials. These plots were generated from previously published scRNA-seq data and demonstrate that cells expressing soxB1-2 also express mecom and __ __We appreciate the reviewers' recognition that our methods are rigorous and our data accessible. We have incorporated all major revisions suggested and believe have strengthened the manuscript's precision, interpretations, and conclusions. Below, we respond to each comment in detail.

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

Summary

The authors of this interesting study take the approach of combining RNAi, RNA-seq and ATAC-seq to try to build a regulatory network surrounding the function of a planarian SoxB1 ortholog, broadly required for neural specification during planarian regeneration. They find a number of chromatin regions that differentially accessible (measured by ATAC-seq), associate these with potential genes by proximity to the TSS. They then compare this set of genes with those that are differentially regulated (using RNA-seq), after SoxB1 RNAi mediated knockdown. This allows them the authors some focus on potential directly regulated targets of the planarian SoxB1. Two of these downstream targets, the mecom and castor transcription factors are then studied in greater detail.

Major Comments

I have no suggestions for new experiments that fit sensibly with the scope of the current work. There are other analyses that could be appropriate with the ATAC-seq data, but may not make sense in the content of SoxB1 acting as pioneer factor.

I would like to see motif enrichment analysis under the set of peaks to see if SoxB1 is opening chromatin for a restricted set of other transcription factors to then bind. Much of this could be taken from Neiro et al, eLife 2022 (which also used ATAC-seq) and matched planarians TF families to likely binding motifs. This could add some breadth to the regulatory network. It could be revealing for example if downstream TF also help regulate other targets that SoxB1 makes available, this is pattern often seen for cell specification (as I am sure the authors are aware). Alternatively, it may reveal other candidate regulators.

Thank you for this suggestion. We agree with the reviewers that this analysis should be done. We ran the motif enrichment analysis using the same methods as outlined in Neiro et al. eLife, 2022. We have included a new motif enrichment analysis in the supplement to contextualize possible co-regulators within the SoxB1-2 network.

Overall peak calling consistency with ATAC-sample would be useful to report as well, to give readers an idea of noise in the data. What was the correlation between samples?

__Excellent point. In response to this comment, we ran a Pearson correlation test on replicates within gfp and soxB1-2 RNAi replicates to get an idea of overall correlation between replicates. Additionally, we calculated percent overlap of peaks for biological replicates and between treatment groups. __

While it is logical to focus on downregulated genes, it would also be interesting to look at upregulated genes in some detail. In simple terms would we expect to see the representation of an alternate set of fate decisions being made by neoblast progeny?

This is also an important point that we considered but initially did not pursue it due to the lack of tools to test upregulated gene function. However, the reviewer is correct that this is straightforward to perform computationally. Thus, we have performed Gene Ontology analysis on the upregulated genes in all RNA-seq datasets (soxB1-2 RNAi, mecom RNAi, and castor RNAi). Both mecom and castor datasets did not reveal enrichment within the upregulated portion of the dataset. Genes upregulated after soxB1-2 RNAi were enriched for metabolic, xenobiotic detoxification, potassium homeostasis, and endocytic programs. Rather than indicating a shift toward alternative lineages, including non-ectodermal fates, these signatures are consistent with stress-responsive and homeostatic programs activated following loss of soxB1-2. We did not detect enrichment patterns strongly associated with alternative cell fates. We conclude that this analysis does not formally exclude potential shifts in lineage-specific transcriptional programs, but does support our hypothesis that soxB1-2 functions as a transcriptional activator.

Can the authors be explicit about whether they have evidence for co-expression of SoxB1/castor and SoxB1/mecom? I could find this clearly and it would be important to be clear whether this basic piece of evidence is in place or not at this stage.

We included co-expression plots for soxB1-2 with mecom and castor in the supplemental material. These plots were generated from previously published scRNA-seq data and demonstrate that cells expressing soxB1-2 also express mecom and castor. We have not done experiments showing co-expression via in situ at this time.

Minor comments

Formally loss of castor and mecom expression does mean these cells are absent, strictly the cell absence needs an independent method. It might be useful to clarify this with the evidence of be clear that cells are "very probably" not produced.

We agree that loss of castor and mecom expression does not formally demonstrate the physical absence of these cells, and that independent methods would be required to definitively confirm their loss. In response, we have revised our wording to indicate that castor- and mecom-expressing cells are very likely not being produced, rather than stating that they are absent.

Reviewer #1 (Significance (Required)):

Significance

Strengths and limitations.

The precise exploitation of the planarian system to identify potential targets, and therefore regulatory mechanisms, mediated by SoxB1 is an interesting contribution to the fi eld. We know almost nothing about the regulatory mechanisms that allow regeneration and how these might have evolved, and this work is well-executed step in that direction.

Advance

The paper makes a clear advance in our understanding of an important process in animals (neural specification) and how this happens in the context in the context during an example of animal regeneration. The methods are state-of-the-art with respect to what is possible in the planarian system.

Audience

This will be of wide interest to developmental biologists, particularly those studying regeneration in planarians and other regenerative systems,and those who study comparative neurodevelopment.

Expertise

I have expertise in functional genomics in the context of stem cells and regeneration, particularly in the planarian model system

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

Review - Cathell, et al (RC-2025-03195)

Summary and Significance:

Understanding regenerative neurogenesis has been difficult due to the limited amount of neurogenesis that occurs after injury in most animal species. Planarians, with their adult neurogenesis and robust post-injury response, allow us to get a glimpse into regenerative neurogenesis. The Zayas laboratory previously revealed a key role for SoxB1-2 in maintenance and regeneration of a broad set of sensory and peripheral neurons in the planarian body. SoxB1-2 also has a role in many epidermal fates. Their previous work left open the tempting possibility that SoxB1-2 acts as a very upstream regulator of epidermal and neuronal fates, potentially acting as a pioneer transcription factor within these lineages. In the manuscript currently under review, Cathell and colleagues use ATAC-Seq and RNA-Seq to investigate chromatin changes after SoxB1-2(RNAi). With the experimental limitations in planarians, this is a strong first step toward testing their hypothesis that SoxB1-2acts as a pioneer within a set of planarian lineages. Beyond these cell types, this work is also important because planarian cell fates often rely on a suite of transcription factors, but the nature of transcription factor cooperation has been much less well understood. Indeed, the authors do show that loss of SoxB1-2 by RNAi causes changes in a number of accessible regions of the genome; many of these chromatin changes correspond to changes in gene expression of genes nearby these peaks. The authors also examine in more detail two genes that have genomic and transcriptomic changes after SoxB1-2(RNAi), mecom and castor. The authors completed RNA-Seq on mecom(RNAi) and castor(RNAi) animals, identifying genes downregulated after loss of either factor that are also seen in SoxB1-2(RNAi). The results in this paper are rigorous and very well presented. I will share two major limitations of the study and some suggestions for addressing them, but this work may also be acceptable without those changes at some journals.

Limitation 1:

The paper aims to test the hypothesis that SoxB1-2 is a pioneer transcription factor. Observation that SoxB1-2(RNAi) leads to loss of many accessible regions in the chromatin supports the hypothesis. However, an alternate possibility is that SoxB1-2 leads to transcription of another factor that is a pioneer factor or a chromatin remodeling enzyme; in either of these cases, the accessibility peak changes may not be due to SoxB1-2 directly but due to another protein that SoxB1-2 promotes. The authors describe how they can address this limitation in the future; in the meantime, is it known what the likely binding for SoxB1-2 would be (experimentally or based on homology)? If so, could the authors examine the relative abundance of SoxB1-2 binding sites in peaks that change after SoxB1-2(RNAi)? This could be compared to the abundance of the same binding sequence in non-changing peaks. Enrichment of SoxB1-2 binding sites in ATAC peaks that change after its RNAi would support the argument that chromatin changes are directly due to SoxB1-2.

We appreciate the feedback and agree that distinguishing between direct SoxB1-2 pioneer activity and indirect effects mediated through downstream regulators is an important consideration. While we did not perform a direct abundance analysis of potential chromatin-remodeling cofactors, we conducted a motif enrichment analysis following the approach of Neiro et al. (eLife, 2022), comparing control and soxB1-2(RNAi) peak sets. This analysis revealed that Sox-family motifs, particularly SoxB1-like motifs, were among the most enriched in regions that remain accessible in control animals relative to soxB1-2(RNAi) animals, consistent with a model in which SoxB1-2 directly contributes to establishing or maintaining accessibility at these loci. We have now included this analysis in the supplemental materials to further contextualize potential co-regulators and transcriptional partners within the SoxB1-2 regulatory network. We agree and acknowledge in the report that future studies assessing chromatin remodeling factor expression and abundance will be valuable to definitively separate direct and indirect pioneer activity.

Limitation 2:

The characterization of mecom and castor is somewhat preliminary relative to the deep work in the rest of the paper. I think this could be addressed with a few experiments. The authors could validate RNA-seq findings with ISH to show that cells are lost after reduction of either TF (this would support the model figure). The authors could also try to define whether loss of either TF causes behavioral phenotypes that might be similar to SoxB1-2(RNAi); this would be a second line of evidence that the TFs are downstream of key events in the SoxB1-2

pathway.

Thank you for this suggestion. We agree that additional validation of the mecom and castor RNA-seq results and further phenotypic characterization would strengthen this section. We are currently conducting in situ hybridization experiments to validate transcriptional changes in mecom and castor using the same experimental framework applied to soxB1-2 downstream candidates. We anticipate completing these studies within the next three months and will incorporate the results into future work.

Regarding behavioral phenotypes, we performed preliminary screening for robust behavioral responses, including mechanosensory responses, but did not observe overt defects. However, the lack of established, standardized behavioral assays in planarians presents a current limitation; such assays need to be developed de novo, and predicting specific behavioral phenotypes in advance remains challenging. We fully agree that functional behavioral assays represent an important next step and are actively exploring strategies to systematically develop and implement them going forward.

Other questions or comments for the authors:

Is it known how other Sox factors work as pioneer TFs? Are key binding partners known? I wondered if it would be possible to show that SoxB1-2 is co-expressed with the genes that encode these partners and/or if RNAi of these factors would phenocopy SoxB1-2. This is likely beyond the scope of this paper, but if the authors wanted to further support their argument about SoxB1-2 acting as a pioneer in planarians, this might be an additional way to do it.

In other systems, Sox pioneer factors often act together with POU family transcription factors (for example, Oct4 and Brn2) and PAX family members such as Pax6. In planarians, a POU homolog (pou-p1) is expressed in neoblasts and may represent an interesting candidate co-factor for future investigation in the context of SoxB1-2 pioneer activity. We have also previously examined the relationship between SoxB1-2 and the POU family transcription factors pou4-1 and pou4-2. Although RNAi of these factors does not fully phenocopy soxB1-2 knockdown, pou4-2(RNAi) results in loss of mechanosensation, suggesting that downstream POU factors may contribute to aspects of neural function regulated by SoxB1-2 (McCubbin et al. eLife 2025). We agree that co-expression and functional interaction studies with these candidates would be highly informative, and we view this as an exciting future direction beyond the scope of the current manuscript.

This paper is one of few to use ATAC-Seq in planarians. First, I think the authors should make a bigger deal of their generation of a dataset with this tool! Second, it would be great to know whether the ATAC-Seq data (controls and/or RNAi) will be browsable in any planarian databases or in a new website for other scientists. I believe that in addition to the data being used to test hypotheses about planarians, the data could also be a huge hypothesis generating resource in the planarian community, so I would encourage the authors to both self-promote their contribution and make plans to share it as widely and usably as possible.

Thank you very much for this encouraging feedback. We appreciate the suggestion and have strengthened the text to emphasize the significance of generating this ATAC-seq resource for the planarian field. We agree that these datasets represent a valuable community resource and are committed to making all control and soxB1-2(RNAi) ATAC-seq data publicly accessible.

Reviewer #2 (Significance (Required)):

This paper's strengths are that it addresses an important problem in regenerative biology in a rigorous manner. The writing and presentation of the data are excellent. The paper also provides excellent datasets that will be very useful to other researchers in the fi eld. Finally, the work is one of, if not the first to examine how the action of one transcription factor in planarians leads to changes in the cellular and chromatin environment that could then be acted upon by subsequent factors. This is an important contribution to the planarian fi eld, but also one that will be useful for other developmental neuroscientists and regenerative biologists.

I described a couple of limitations in the review above, but the strengths outweigh the weaknesses.

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

The authors investigated the role of soxB1-2 in planarian neural and epidermal lineage specification. Using ATAC-seq and RNA-seq from head fragments after soxB1-2 RNAi, they identified regions of decreased chromatin accessibility and reduced gene expression, demonstrating that soxB1-2 induces neural and sensory programs. Integration of the datasets yielded 31 overlapping candidate targets correlating ATAC-seq and RNA-seq. Downstream analyses of transcription factors that had either/or differentially accessible regulatory region or showed differential expression (castor and mecom) implicated these transcription factors in mechanosensory and ciliary modules. The authors combined additional techniques, such as in situ hybridization to support the observations based on the ATACseq/RNAseq data. The manuscript is clearly written as well as data presentation in the main and supplementary figures. The major claim of the manuscript is that SoxB1-2 is likely a pioneer transcription factor that alters the accessibility of the chromatin, which if true, would be one of the first demonstrations of direct transcriptional regulation in planarians. As described below, I am not certain that this interpretation of the data is more valid than alternative interpretations.

Major comments

1. Direct vs. indirect regulation. The current analysis does not distinguish between direct and indirect soxB1-2 targets, therefore, this analysis cannot indicate whether soxB1-2 functions as a pioneer transcription. ATAC-seq and RNA-seq, as performed here, do not determine whether reduced accessibility or downregulation of gene expression represents a change within existing cells or a reduction in the proportion of specific cell types in the libraries produced. This limitation should be explicitly recognized where causal statements are made. In fact, several pieces of information strongly suggest that indirect effects are abundant in the data: (1) the observed loss of accessibility and gene expression in late epidermal progenitors likely represent indirect effects, indicating that within the timeframe of the experiment, it is impossible (using these techniques) to distinguish between the scenarios. (2) The finding that castor knockdown reduces soxB1-2 expression likely reflects population loss rather than direct regulation, given overlapping expression domains. This further illustrates the difficulty in inferring directionality from such datasets. In order to provide evidence for a more direct association between soxB1-2 and the differentially accessible chromatin regions, a sequence(e.g., motif) analysis would be required. Other approaches to infer direct regulation would have been useful, but they are not available in planarians to the best of my knowledge.

We agree that distinguishing between direct SoxB1-2 pioneer activity and indirect chromatin changes mediated by downstream factors is an important consideration. As suggested, examining the enrichment of SoxB1-2 binding motifs in regions that lose accessibility following soxB1-2(RNAi) can provide supporting evidence for direct regulation.

While we did not conduct a direct abundance analysis of all potential chromatin-remodeling cofactors, we performed a motif enrichment analysis following the methodology of Neiro et al. (eLife, 2022), comparing control-specific and soxB1-2(RNAi)-specific accessible peak sets. Consistent with a direct role for SoxB1-2 in chromatin regulation, Sox-family motifs, particularly SoxB1-like motifs, were among the most significantly enriched in regions that maintain accessibility in control animals relative to soxB1-2(RNAi) animals.

Evidence for pioneer activity. The authors correctly acknowledge that they do not present direct evidence of soxB1-2 binding or chromatin opening. However, the section title in the Discussion could be interpreted as implying otherwise. The claim of pioneer activity should remain explicitly tentative until supported (at least) by motif or binding data.

We have performed suggested motif analysis and changed the language in this section to better fit the data.

Replication and dataset comparability. Both ATAC-seq and soxB1-2 RNA-seq were performed on head fragments, but the number of replicates differ between assays (ATAC-seq n=2 per group, RNA-seq n=4-6). This is of course acceptable, but when interpreting the results, it should be taken into consideration that the statistical power is different when using data collected using different techniques and having a varied number of replicates.

Thank you for raising this important point regarding replication and comparability across datasets. We agree that the differing number of biological replicates between the ATAC-seq and RNA-seq experiments results in different statistical power across assays. We have now clarified this consideration in the manuscript text.

Minor comments

"Thousands of accessible chromatin sites". Please state the number of peaks and the thresholds for calling them. Ensure consistency between text (264 DA peaks) and Figure 1 legend (269 DA peaks).

__We have clarified specific peak numbers and will include the calling parameters in the methods section. Additionally, we will fix the discrepancies between differential peaks. __

Specify the y-axis normalization units in all coverage plots.

We have specified this across plots.

Clarify replicate numbers consistently in the text and figure legends.

We have identified and corrected discrepancies in the figure legends vs text and correct them and ensured they are included consistently across datasets.

Referees cross commenting

The reviews are highly consistent. They recognize the value of the work, and raise similar points. The main shared view is that the current data do not distinguish direct from indirect effects, and claims about pioneer activity should be softened, and further analysis of the differentially accessible peaks could strengthen the link between SoxB1-2 and the chromatin changes.

-I don't think that it's necessary to further characterize experimentally mecom or castor (as suggested), but of course that it could have value.

We thank all three reviewers for their positive assessment of the value of our work aiming to elucidate mechanisms by which SoxB1-2 programs planarian stem cells. In the revision, we have improved the presentation and carefully edited conclusions about the function of SoxB1-2. Performing motif analysis and GO annotation of upregulated genes has strengthened our observation that SoxB1-2 acts as an activator and has revealed putative binding sites.

The preliminary revision does not yet include further characterization of mecom and castor downstream genes. In response to Reviewer #2, we appreciate that additional validation of the mecom and castor RNA-seq results and further phenotypic characterization would strengthen this section. Although we are currently conducting in situ hybridization experiments to validate transcriptional changes in mecom and castor using the same experimental framework applied to soxB1-2 downstream candidates, we also reconsidered, as we did in our first revision, whether this is necessary or better suited for future investigations.

In the revision, we noted that our Discussion points were not balanced and that we emphasized the mecom and castor results in a manner that distracted from the major focus of the work, likely contributing to the impression that additional experimental evidence was required. Therefore, we have revised the section accordingly and streamlined the Discussion to avoid repetitive statements and to focus on the insights gained into the mechanism of SoxB1-2 function in planarian neurogenesis. We remain open to including these additional experiments if the reviewers or handling editors consider them essential; however, we agree that their inclusion is not absolutely necessary.

Reviewer #3 (Significance (Required)):

General assessment. The study offers valuable observations by combining chromatin and transcriptional analysis of planarian neural differentiation. The integration with in situ validation convincingly demonstrates effects on neural tissues and provides a solid resource for future functional work. However, mechanistic interpretation remains limited, partly because of technical limitations of the system. The data support an important role for soxB1-2 in neural and epidermal lineage regulation, but not direct binding or chromatin-opening activity. The authors have previously published analysis of soxB1-2 in planarians, so the addition of ATAC-seq data contributes to solving another piece of the puzzle.

__Advance. __

This is one of the first studies to couple ATAC-seq and RNA-seq in planarian tissue to dissect regulatory logic during regeneration. It identifies new candidate regulators of sensory and epidermal differentiation and identifies soxB1-2 as a likely upstream factor in ectodermal lineage networks. The work extends previous studies on soxB1-2 activity and neural cell production by integrating chromatin and transcriptional layers. In that respect the results are very solid, although the study remains correlative at the mechanistic level.

Audience.

This work will potentially interest researchers interested in regeneration and transcriptional networks. The datasets and gene lists will be valuable references for follow-up studies on planarian ectodermal lineages, and therefore will appeal to this community.

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Referee #3

Evidence, reproducibility and clarity

The authors investigated the role of soxB1-2 in planarian neural and epidermal lineage specification. Using ATAC-seq and RNA-seq from head fragments after soxB1-2 RNAi, they identified regions of decreased chromatin accessibility and reduced gene expression, demonstrating that soxB1-2 induces neural and sensory programs. Integration of the datasets yielded 31 overlapping candidate targets correlating ATAC-seq and RNA-seq. Downstream analyses of transcription factors that had either/or differentially accessible regulatory region or showed differential expression (castor and mecom) implicated these transcription factors in mechanosensory and ciliary modules. The authors combined additional techniques, such as in situ hybridization to support the observations based on the ATACseq/RNAseq data. The manuscript is clearly written as well as data presentation in the main and supplementary figures. The major claim of the manuscript is that SoxB1-2 is likely a pioneer transcription factor that alters the accessibility of the chromatin, which if true, would be one of the first demonstrations of direct transcriptional regulation in planarians. As described below, I am not certain that this interpretation of the data is more valid than alternative interpretations.

Major comments

1. Direct vs. indirect regulation. The current analysis does not distinguish between direct and indirect soxB1-2 targets, therefore, this analysis cannot indicate whether soxB1-2 functions as a pioneer transcription. ATAC-seq and RNA-seq, as performed here, do not determine whether reduced accessibility or downregulation of gene expression represents a change within existing cells or a reduction in the proportion of specific cell types in the libraries produced. This limitation should be explicitly recognized where causal statements are made. In fact, several pieces of information strongly suggest that indirect effects are abundant in the data: (1) the observed loss of accessibility and gene expression in late epidermal progenitors likely represent indirect effects, indicating that within the timeframe of the experiment, it is impossible (using these techniques) to distinguish between the scenarios. (2) The finding that castor knockdown reduces soxB1-2 expression likely reflects population loss rather than direct regulation, given overlapping expression domains. This further illustrates the difficulty in inferring directionality from such datasets. In order to provide evidence for a more direct association between soxB1-2 and the differentially accessible chromatin regions, a sequence (e.g., motif) analysis would be required. Other approaches to infer direct regulation would have been useful, but they are not available in planarians to the best of my knowledge.
2. Evidence for pioneer activity. The authors correctly acknowledge that they do not present direct evidence of soxB1-2 binding or chromatin opening. However, the section title in the Discussion could be interpreted as implying otherwise. The claim of pioneer activity should remain explicitly tentative until supported (at least) by motif or binding data.
3. Replication and dataset comparability. Both ATAC-seq and soxB1-2 RNA-seq were performed on head fragments, but the number of replicates differ between assays (ATAC-seq n=2 per group, RNA-seq n=4-6). This is of course acceptable, but when interpreting the results, it should be taken into consideration that the statistical power is different when using data collected using different techniques and having a varied number of replicates.

Minor comments

"Thousands of accessible chromatin sites". Please state the number of peaks and the thresholds for calling them. Ensure consistency between text (264 DA peaks) and Figure 1 legend (269 DA peaks). Specify the y-axis normalization units in all coverage plots. Clarify replicate numbers consistently in the text and figure legends.

Referees cross commenting

The reviews are highly consistent. They recognize the value of the work, and raise similar points. The main shared view is that the current data do not distinguish direct from indirect effects, and claims about pioneer activity should be softened, and further analysis of the differentially accessible peaks could strengthen the link between SoxB1-2 and the chromatin changes.

• I don't think that it's necessary to further characterize experimentally mecom or castor (as suggested), but of course that it could have value.

Significance

General assessment. The study offers valuable observations by combining chromatin and transcriptional analysis of planarian neural differentiation. The integration with in situ validation convincingly demonstrates effects on neural tissues and provides a solid resource for future functional work. However, mechanistic interpretation remains limited, partly because of technical limitations of the system. The data support an important role for soxB1-2 in neural and epidermal lineage regulation, but not direct binding or chromatin-opening activity. The authors have previously published analysis of soxB1-2 in planarians, so the addition of ATAC-seq data contributes to solving another piece of the puzzle.

Advance. This is one of the first studies to couple ATAC-seq and RNA-seq in planarian tissue to dissect regulatory logic during regeneration. It identifies new candidate regulators of sensory and epidermal differentiation and identifies soxB1-2 as a likely upstream factor in ectodermal lineage networks. The work extends previous studies on soxB1-2 activity and neural cell production by integrating chromatin and transcriptional layers. In that respect the results are very solid, although the study remains correlative at the mechanistic level.

Audience. This work will potentially interest researchers interested in regeneration and transcriptional networks. The datasets and gene lists will be valuable references for follow-up studies on planarian ectodermal lineages, and therefore will appeal to this community.

One source that stood out to me was the StackOverflow study (t29) about how programming languages differ between wealthy and developing countries. The most interesting detail I learned from that article is that Python and R—two languages I always hear people hype up—are barely used in poorer countries. Meanwhile, older languages like PHP and Android development stay extremely common there. The study explains that it’s not because developers in those countries “prefer” outdated tech, but because the global tech industry is shaped around Silicon Valley’s needs. That really clicked for me. It shows how something as simple as a programming language choice is actually influenced by economics and access, not just technical preference. It made me rethink the whole idea that tech is some neutral, equal-opportunity field.

no hay definición de factores asociados

esto es confianza interpersonal, no confianza en instituciones

I agree that most teachers need influence and impact, NOT power and control from their leadership!

this is important with the work I often do with teachers who speak english as a second language. We have to clarify and not make assumptions of understanding.

La construction de ce néologisme est intéressante. Pourrait-on la décliner ?

Cette phrase fait un parallèle inquiétant : notre société évolue vers la même déshumanisation du lien que dans la fiction d’Asimov.

Il anticipe que la technologie va imiter les émotions humaines, ce qui pourrait encore modifier nos relations.

Janssen explique que le numérique complique la capacité à être “seul avec l’autre”, un élément clé du lien humain.

Le numérique devient une protection contre la relation réelle, vécue comme trop intrusive.

On attend de l’autre une disponibilité totale, ce qui fragilise la relation.

La “proximité virtuelle” donne une illusion de relation, mais sans engagement réel.

Janssen rappelle que virtuel et réel ne sont pas équivalents. C’est important pour comprendre la perte de profondeur du lien.

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Reply to the reviewers

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

In this manuscript, the authors employed fast MAS NMR spectroscopy to investigate the gel aggregation of longer repeat (48×) RNAs, revealing inherent folding structures and interactions (i.e., G-quadruplex and duplex). The dynamic structure of the RNA gel was not resolved at high resolution, and only the structural features-namely, the coexistence of G-quadruplexes and duplexes-were inferred. The 1D and 2D NMR spectra were not assigned to specific atomic positions within the RNA, which makes it difficult to perform molecular dynamics (MD) modeling to elucidate the dynamic nature of the RNA gel. The following comments are provided for the authors' consideration:

Reviewer #1, Comment 1:

Figure 2E and Figure 3A: The data suggest that Ca²⁺ promotes stronger G-quadruplex formation within the RNA gel compared with Mg²⁺. This observation is somewhat puzzling, as Mg²⁺ is generally known to stabilize G-quadruplex structures. The authors should clarify this discrepancy.

__Response: __Mg2+ is also a stabilizer of double-stranded RNA. In most cases, Mg²⁺ stabilizes RNA duplexes more significantly than it stabilizes G-quadruplexes. When Mg2+ is removed and replaced for Ca2+, RNA duplex is destabilized more than G4 structures. We have added a clarification regarding that to the Conclusions section.

Reviewer #1, Comment 2:

Figures 2 and 3: The authors use the chemical shift at δN 144.1 ppm to distinguish between G-quadruplex and duplex structures. How was the reliability of this assignment evaluated? Chemical shifts of RNA atoms can be influenced by various factors such as intermolecular interactions, conformational stress, and local chemical environment, not only by higher-order structures. This point should be substantiated by citing relevant references or by analyzing additional RNA structures exhibiting δN 144.1 ppm signals using NMR spectroscopy.

Response: The assignment was made by comparing the chemical shifts with published data and by comparing the obtained spectra with existing datasets in the lab. We have added an explanation to the Results section and cited the literature. The 144.1 ppm was an illustrative value selected for guiding the discussion and we noted that it could sound too specific. We modified Figure 2 to outline the regions of chemical shifts in accordance with our interpretation of spectra.

Reviewer #1, Comment 3:

The authors state that "Our findings demonstrate that fast MAS NMR spectroscopy enables atomic-resolution monitoring of structural changes in GGGGCC repeat RNA of physiological lengths." This claim appears overstated, as no molecular model was constructed to define atomic coordinates based on NMR restraints.

Response: We agree and we have rewritten the conclusions to be more precise in wording. The new text does not mention “atomic-resolution” anymore.

Reviewer #1, Comment 4: Figure 3B: The experiment using nuclear extracts supplemented with Mg²⁺ to study RNA aggregation via 2D NMR may not accurately reflect intracellular conditions. It would be informative to perform a parallel experiment using nuclear extracts without additional Mg²⁺ to better simulate the native environment for RNA folding.

__Response: __We agree that we have not yet approached physiological conditions and that it would be interesting to obtain data for conditions at physiological Mg2+ concentrations in the range between 0.5 mM – 1 mM. The buffer of purchased nuclear extracts does not contain MgCl2, so some MgCl2 would still need to be added. In our opinion, nuclear extracts are actually not the optimal way to move forward, since they still differ from real in cell environment with the caveat that their composition is not well controlled. Full reconstitution with recombinant proteins might be a better approach because stoichiometry can be better regulated.

__Reviewer #1 (Significance (Required)): __ In this manuscript, the authors employed fast MAS NMR spectroscopy to investigate the gel aggregation of longer repeat (48×) RNAs, revealing inherent folding structures and interactions (i.e., G-quadruplex and duplex). The dynamic structure of the RNA gel was not resolved at high resolution, and only the structural features-namely, the coexistence of G-quadruplexes and duplexes-were inferred. The 1D and 2D NMR spectra were not assigned to specific atomic positions within the RNA, which makes it difficult to perform molecular dynamics (MD) modeling to elucidate the dynamic nature of the RNA gel.

Response: We agree that constraints for molecular dynamics cannot be derived from these data. The focus of this work is methodological: to demonstrate how 1H-15N 2D correlation spectra can be used to characterize G-G pairing in RNA gels directly. Such spectra could be used to study effects of small molecules or interacting proteins for example.

__Reviewer #2 (Evidence, reproducibility and clarity (Required)): __ The manuscript by Kragelj et al. has the potential to become a valuable study demonstrating the role and power of modern solid-state NMR spectroscopy in investigating molecular assemblies that are otherwise inaccessible to other structural biology techniques. However, due to poor experimental execution and incomplete data interpretation, the manuscript requires substantial revision before it can be considered for publication in any journal.

__Reviewer #2, Major Concern __Inspection of the analytical gels of the transcribed RNA clearly shows that the desired RNA product constitutes only about 10% of the total crude transcript. The RNA must therefore be purified, for example by preparative PAGE, before performing any NMR or other biophysical studies. As it stands, all spectra shown in the figures represent a combined signal of all products in the crude mixture rather than the intended 48 repeat RNA. Consequently, all analyses and conclusions currently refer to a heterogeneous mixture of transcripts rather than the specific target RNA.

Response: The estimate of 10% 48xG4C2 on the gel is an overstatement. While multiple bands are visible, they correspond to dimers or multimers of the 48xG4C2 RNA. Transcripts that are longer than 48xG4C2 cannot occur in our transcription conditions. Bands at lower masses than expected are folded RNA. The high repeat length and the presence of Mg²⁺ during transcription promote multimerization, which is not fully reversed by denaturation in urea. If shorter transcripts had arisen from early termination they would be still substantially longer than 24 repeats based of what is visible on the gel and would thus remain within the pathological length range. Therefore, the observed NMR spectra primarily report on 48 repeat lengths.

__Reviewer #2, Specific Comments 1: __The statements: "We show that a technique called NMR spectroscopy under fast Magic Angle Spinning (fast MAS NMR) can be used to obtain structural information on GGGGCC repeat RNAs of physiological lengths. Fast MAS NMR can be used to obtain structural information on biomolecules regardless of their size." on page 1 are not entirely correct. Firstly, not only fast MAS NMR but MAS NMR in general can provide structural information on biomolecules regardless of their size. Fast MAS primarily allows for ¹H-detected experiments, improves spectral resolution, and reduces the required sample amount. Conventional ¹³C-detected solid-state MAS NMR can provide very similar structural information. A more thorough review of relevant literature could help address this issue.

Response: We have clarified the distinction between MAS NMR and Fast MAS NMR in the introduction.

__Reviewer #2, Specific Comments 2: __Secondly, MAS NMR has already been applied to systems of comparable complexity - for instance, the (CUG)₉₇ repeat studied by the Goerlach group as early as 2005. That work provided a comprehensive structural characterization of a similar molecular assembly. The authors are strongly encouraged to cite these studies (e.g., Riedel et al., J. Biomol. NMR, 2005; Riedel et al., Angew. Chem., 2006).

Response: We added a mention of that study in the introduction.

Reviewer #2, Experimental Description 1: The experimental details are poorly documented and need to be described in sufficient detail for reproducibility. Specifically: 1. What was the transcription scale? What was the yield (e.g., xx mg RNA per 1 mL transcription reaction)?

Response: Between 3.5 mg and 4.5 mg per 10 ml transcription reaction. We’ve added this information to the methods.

Reviewer #2, Experimental Description 2: 2. Why was the transcription product not purified? Dialysis only removes small molecules, while all macromolecular impurities above the cutoff remain. What was the dialysis cutoff used?

Response: RNA was purified using dialysis and phenol-chloroform precipitation. We have added the information about molecular weight cutoff for dialysis membranes to the methods.

Reviewer #2, Experimental Description 3: 3. How much RNA was used for each precipitation experiment? Were the amounts normalized? For example, if 10 mg of pellet were obtained, what fraction of that mass corresponded to RNA? Was this ratio consistent across all samples?

Response: In the test gel formations, we used 180.0 µg per condition. We used 108.0 µg of RNA for gelation test in the presence of nuclear extracts. We have not determined the water content in the gels. We added this information to methods and results section.

Reviewer #2, Experimental Description 4: 4. Why is there a smaller amount of precipitate when nuclear extract (NE) or CaCl₂ is added?

Response: The apparent difference in pellet size may reflect variations in water content rather than RNA quantity. While the Figure 1 might entice to directly compare pellet weights across different ion series tests, our primary goal was to determine the minimal divalent-ion concentrations required to reproducibly obtain gels. We have added a clarification in the Results section and in the Figure 1 caption regarding the comparability of conditions

Reviewer #2, Experimental Description 5: 5. The authors should describe NE addition in more detail: What is the composition of NE? What buffer was used (particularly Mg²⁺ and salt concentrations)? Was a control performed with NE buffer-type alone (without NE)?

Response: We have added the full description of NE buffer to the methods section. Its composition is: 40 mM Tris pH 8.0, 100 mM KCl, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT, 25 % glycerol. After mixing the nuclear extract with RNA, the target buffer was: 20 mM Tris pH 8.0, 90 mM KCl, 0.1 mM EDTA, 0.25 mM PMSF, 0.75 mM DTT, 12.5% glycerol, and 10 mM MgCl2.

We have not performed a control with NE buffer-type alone but we confirmed separately that glycerol does not affect gel formation.

Reviewer #2, Experimental Description 6: 6. How much pellet/RNA material was actually packed into each MAS rotor?

Response: Starting with a 5 mg pellet, we packed a rotor with a volume of 3 µl. We added this information to the methods section.

Reviewer #2, Additional Clarifications: P5. What is meant by "selective" in the phrase "We recorded a selective 1D-¹H MAS NMR spectrum of 48×G₄C₂ RNA gels"?

Response: That was a typo. We meant imino-selective. It is now corrected.

__Reviewer #2, Additional Clarifications: __ There are also several contradictions between statements in the text and the corresponding figures. For example: • Page 4: The authors write that "The addition of at least 5 mM Mg²⁺ was required for significant 48×G₄C₂ aggregation." However, Figure 1E shows significant aggregation already at 3 mM MgCl₂ (NE−), and in samples containing NE, aggregation appears even at 1 mM MgCl₂. Was aggregation already present in the sample containing NE but without any added MgCl₂?

Response: We changed text in the results section to more closely align with what’s depicted on the figure. There was some aggregation present in the nuclear extracts but it was of different quantity and quality. We clarified this in the results section.

__Reviewer #2 (Significance (Required)): __ The manuscript by Kragelj et al. has the potential to become a valuable study demonstrating the role and power of modern solid-state NMR spectroscopy in investigating molecular assemblies that are otherwise inaccessible to other structural biology techniques.

In its current form, tthe manuscript has significant experimental concerns - particularly the lack of RNA purification and inadequate description of materials and methods. The data therefore cannot support the conclusions presented. I recommend extensive revision and repetition of the experiments using purified RNA material before further consideration for publication.

__Response: __We’ve addressed the concerns about RNA purification within the response to the first comment (Major concern).

__Reviewer #3 (Evidence, reproducibility and clarity (Required)): __ This is an interesting manuscript reporting evidence for formation of both hairpins and G-quadruplexes within RNA aggregates formed by ALS expansion repeats (GGGGCC)n. This is in line with literature but never directly confirmed. Given the novelty of the method (NMR magic angle) and of the data (NMR on aggregate), I believe this manuscript should be considered for publication. I also trust the methods are appropriately reported and reproducible.

Below are my main points:

Major points:

__Reviewer #3, Comment 1: __ 1) RNA aggregation of the GGGGCCn repeat has been reported for expansion as short as 6-8 repeats (see Raguseo et al. Nat Commun 2023), so the authors might not see aggregation under the conditions they use for these shorter repeats but this can happen under physiological conditions . The ionic strengths and the conditions used can vary heavily the phase diagram and the authors therefore should tone down significantly their conclusions. They characterise one aggregate that is likely to contain both secondary structures under the conditions used (in terms of ion and pHs). However, it has been shown in Raguseo et al that aggregates can arise by both intermolecular G4s and hairpins (or a mixture of them) depending on the ionic conditions used. This means that what the authors report might not be necessarily relevant in cells, which should be caveated in the manuscript.

__Response: __We toned down our statements regarding aggregation of shorter repeats in the introduction. We added the citation to Raguseo et al. Nat Commun 2023, which indeed provides useful insights about aggregation of GGGGCC repeats. In Supplementary Figure 1, we had data on gel formation with 8x and 24x repeats which showed these repeat lengths form gels to some extent. We oversimplified our conclusion and said there were no aggregates which needs correction, especially considering other studies reported in the literature have observed in vitro aggregation of these repeat lengths. We modified the results section to reflect this nuance.

__Reviewer #3, Comment 2: __ 2) It would be important to perform perturbation experiments that might promote/disrupt formation of the G4 or hairpin and see if this affect RNA aggregation, which has been already reported by Raguseo et al, and wether this can be appreciated spectroscopically in their assay. This can be done by taking advantage of some of the experiments reported in the manuscript mentioned above, such as: PDS treatment (favouring monomolecular G4s and preventing aggregation), Li vs K treatment (favouring hairpin over G4s), NMM photo-oxidation (disassembling G4s) or addition of ALS relevant RNA binding proteins (i.e. TDP-43). Not all of these controls need to be performed but it would be good to reconcile how the fraction of G4 vs hairpin reflect aggregates' properties, since the authors offer such a nice technique to measure this.

Response: We appreciate the reviewer’s suggestions and we would be eager to do the perturbation experiments in the future. However, these experiments would require additional optimization and waiting for approval and availability of measurement time on a high-field NMR spectrometer. Given that the primary goal of this manuscript is reporting on the methodological approach, we think the current data adequately demonstrate the technique’s utility.

__Reviewer #3, Comment 3: __ 3) I disagree with the speculation of the monomolecular G4 being formed within the condensates, as the authors have no evidence to support this. It has been shown that n=8 repeat forms multimolecular G4s that are responsible of aggregation, so the authors need to provide direct evidence to support this hypothesis if they want to keep it in the manuscript, as it would clash with previous reports (Raguseo et al Nat Commun 2023)

Response: We agree that multimolecular G4s contribute to aggregation in our 48xG4C2 gels. We also realized, after reading this comment, that the original presentation of data and schematics may have unintentionally suggested the presence of monomolecular G4 in our RNA gels. To address this, we have added a clarification to the results section, we modified Figure 2 and 3, and we included a new Supplementary Figure 4. For clarification, both multimolecular and monomolecular G4s in model oligonucleotides produce imino 1H and 15N chemical shifts in the same region and cannot be distinguished by the experiments used in our study. Based on the observations reported in the literature, we believe that G4s in 48xG4C2 form primarily intermolecularly, although direct experimental proof is not available with the present data.

Minor points:

__Reviewer #3, Comment 4: __ 4) An obvious omission in the literature is Raguseo et al Nat Commun 2023, extensively mentioned above. Given the relevance of the findings reported in this manuscript for this study, this should be appropriately referenced for clarity.

Response: We’ve added the citation to Raguseo et al Nat Commun 2023 to the introduction where in vitro aggregation is discussed.

__Reviewer #3, Comment 5: __ 5) The schematic in Figure 3 is somehow confusing and the structures reported and how they relate to aggregate formation is not clear. Given that in structural studies presentation and appearance is everything, I would strongly recommend to the authors to improve the clarity of the schematic for the benefit of the readers.

Response: We thank you for your comment. We’ve modified the figure, and we hope it is now clearer.

Providing that the authors can address the criticisms raised, I would be supportive of publication of this fine study.

Reviewer #3 (Significance (Required)):

The main strength of this paper is to provide direct evidence of DNA secondary structure formation within aggregates, which is something that has not been done before. This is important as it reconcile with the relevance of hairpin formation for the disease (reported by Disney and co-workers) and the relevance of G4-formation in the process of aggregation through multimolecular G4-formation (reported by Di Antonio and co-workers). Given the significance of the findings in this context and the novelty of the method applied to the study of RNA aggregation, this reviewer is supportive for publication of this manuscript and of its relevance to the field. I would be, however, more careful in the conclusions reported and would add additional controls to strengthen the conclusions.

Response: We thank the reviewer for the comment. In the conclusion section, we have added a statement highlighting the potential roles of both double-stranded and G4 structures in gel formation, in line with what has been reported in previous studies.

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

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Referee #1

Evidence, reproducibility and clarity

In this manuscript, the authors employed fast MAS NMR spectroscopy to investigate the gel aggregation of longer repeat (48×) RNAs, revealing inherent folding structures and interactions (i.e., G-quadruplex and duplex).

The dynamic structure of the RNA gel was not resolved at high resolution, and only the structural features-namely, the coexistence of G-quadruplexes and duplexes-were inferred. The 1D and 2D NMR spectra were not assigned to specific atomic positions within the RNA, which makes it difficult to perform molecular dynamics (MD) modeling to elucidate the dynamic nature of the RNA gel. The following comments are provided for the authors' consideration:

1. Figure 2E and Figure 3A: The data suggest that Ca²⁺ promotes stronger G-quadruplex formation within the RNA gel compared with Mg²⁺. This observation is somewhat puzzling, as Mg²⁺ is generally known to stabilize G-quadruplex structures. The authors should clarify this discrepancy.
2. Figures 2 and 3: The authors use the chemical shift at δN 144.1 ppm to distinguish between G-quadruplex and duplex structures. How was the reliability of this assignment evaluated? Chemical shifts of RNA atoms can be influenced by various factors such as intermolecular interactions, conformational stress, and local chemical environment, not only by higher-order structures. This point should be substantiated by citing relevant references or by analyzing additional RNA structures exhibiting δN 144.1 ppm signals using NMR spectroscopy.
3. The authors state that "Our findings demonstrate that fast MAS NMR spectroscopy enables atomic-resolution monitoring of structural changes in GGGGCC repeat RNA of physiological lengths." This claim appears overstated, as no molecular model was constructed to define atomic coordinates based on NMR restraints.
4. Figure 3B: The experiment using nuclear extracts supplemented with Mg²⁺ to study RNA aggregation via 2D NMR may not accurately reflect intracellular conditions. It would be informative to perform a parallel experiment using nuclear extracts without additional Mg²⁺ to better simulate the native environment for RNA folding.

Significance

In this manuscript, the authors employed fast MAS NMR spectroscopy to investigate the gel aggregation of longer repeat (48×) RNAs, revealing inherent folding structures and interactions (i.e., G-quadruplex and duplex).

The dynamic structure of the RNA gel was not resolved at high resolution, and only the structural features-namely, the coexistence of G-quadruplexes and duplexes-were inferred. The 1D and 2D NMR spectra were not assigned to specific atomic positions within the RNA, which makes it difficult to perform molecular dynamics (MD) modeling to elucidate the dynamic nature of the RNA gel.

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Reviewer #3 (Public review):

Summary:

This important study combines in vitro and in vivo recording to determine how the firing of cortical and striatal neurons changes during a fever range temperature rise (37-40 oC). The authors found that certain neurons will start, stop, or maintain firing during these body temperature changes. The authors further suggested that the TRPV3 channel plays a role in maintaining cortical activity during fever.

Strengths:

The topic of how the firing pattern of neurons changes during fever is unique and interesting. The authors carefully used in vitro electrophysiology assays to study this interesting topic.

Weaknesses:

(1) In vivo recording is a strength of this study. However, data from in vivo recording is only shown in Fig 5A,B. This reviewer suggests the authors further expand on the analysis of the in vivo Neuropixels recording. For example, to show single spike waveforms and raster plots to provide more information on the recording. The authors can also separate the recording based on brain regions (cortex vs striatum) using the depth of the probe as a landmark to study the specific firing of cortical neurons and striatal neurons. It is also possible to use published parameters to separate the recording based on spike waveform to identify regular principal neurons vs fast-spiking interneurons. Since the authors studied E/I balance in brain slices, it would be very interesting to see whether the "E/I balance" based on the firing of excitatory neurons vs fast-spiking interneurons might be changed or not in the in vivo condition.

(2) The author should propose a potential mechanism for how TRPV3 helps to maintain cortical activity during fever. Would calcium influx-mediated change of membrane potential be the possible reason? Making a summary figure to put all the findings into perspective and propose a possible mechanism would also be appreciated.

(3) The author studied P7-8, P12-14, and P20-26 mice. How do these ages correspond to the human ages? it would be nice to provide a comparison to help the reader understand the context better.

Comments on revisions:

In this revised version, the authors nicely addressed my critiques. I have no more comments to make.

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public review):

The paper by Chen et al describes the role of neuronal themo-TRPV3 channels in the firing of cortical neurons at a fever temperature range. The authors began by demonstrating that exposure to infrared light increasing ambient temperature causes body temperature to rise to a fever level above 38{degree sign}C. Subsequently, they showed that at the fever temperature of 39{degree sign}C, the spike threshold (ST) increased in both populations (P12-14 and P7-8) of cortical excitatory pyramidal neurons (PNs). However, the spike number only decreased in P7-8 PNs, while it remained stable in P12-14 PNs at 39 degrees centigrade. In addition, the fever temperature also reduced the late peak postsynaptic potential (PSP) in P12-14 PNs. The authors further characterized the firing properties of cortical P12-14 PNs, identifying two types: STAY PNs that retained spiking at 30{degree sign}C, 36{degree sign}C, and 39{degree sign}C, and STOP PNs that stopped spiking upon temperature change. They further extended their analysis and characterization to striatal medium spiny neurons (MSNs) and found that STAY MSNs and PNs shared the same ST temperature sensitivity. Using small molecule tools, they further identified that themo-TRPV3 currents in cortical PNs increased in response to temperature elevation, but not TRPV4 currents. The authors concluded that during fever, neuronal firing stability is largely maintained by sensory STAY PNs and MSNs that express functional TRPV3 channels. Overall, this study is well designed and executed with substantial controls, some interesting findings, and quality of data. Here are some specific comments:

(1) Could the authors discuss, or is there any evidence of, changes in TRPV3 expression levels in the brain during the postnatal 1-4 week age range in mice?

This is an excellent question. To our knowledge, no published studies have documented changes in TRPV3 expression in the mouse brain during the first to fourth postnatal weeks. Research on TRPV3 expression has primarily relied on RT-PCR analysis of RNA from dissociated adult brain tissue (Jang et al., 2012; Kumar et al., 2018), largely due to the limited availability of effective antibodies for brain sections at the time. Furthermore, the Allen Brain Atlas does not provide data on TRPV3 expression in the developing or postnatal brain. To address this gap, we performed immunohistochemistry to examine TRPV3 expression at P7,

P14, and P21 (Figure 7). To confirm specificity, the TRPV3 antibody was co-incubated with a TRPV3 blocker (Figure 7A, top row, right panel). While immunohistochemistry is semiquantitative, we observed a trend toward increased TRPV3 expression in the cortex, striatum, hippocampus, and thalamus from P7 to P14.

(2) Are there any differential differences in TRPV3 expression patterns that could explain the different firing properties in response to fever temperature between the STAY- and STOP neurons?

This is another excellent question, and we plan to explore it in the future by developing reporter mice for TRPV3 expression and viral tools that leverage endogenous TRPV3 promoters to drive a fluorescent protein, enabling monitoring of cells with native TRPV3 expression. To our knowledge, such tools do not currently exist. Creating them will be challenging, as it requires identifying promoters that accurately reflect endogenous TRPV3 expression.

We have not yet quantified TRPV3 expression in STOP and STAY neurons. However, our analysis of evoked spiking at 30, 36, and 39 °C suggests that TRPV3 may mark a population of cortical pyramidal neurons that tend to remain active (“STAY”) as temperatures increase. While we have not directly compared TRPV3 expression between STAY and STOP neurons at feverrange temperatures, intracellular blockade of TRPV3 with forsythoside B (50 µM) significantly reduced the proportion of STAY neurons (Figure 9B). Consistently, spiking was also significantly reduced in Trpv3⁻/⁻ mice (Figure 10D).

In our immunohistochemical analysis, TRPV3 was detected in L4 barrels and in L2/3, where we observed a patchy distribution with some regions showing more intense staining (Figure 7B). It is possible that cells with higher TRPV3 levels correspond to STAY neurons, while those with lower levels correspond to STOP neurons. As we develop tools to monitor activity based on endogenous TRPV3 levels, we anticipate gaining deeper insight into this relationship.

(3) TRPV3 and TRPV4 can co-assemble to form heterotetrameric channels with distinct functional properties. Do STOP neurons exhibit any firing behaviors that could be attributed to the variable TRPV3/4 assembly ratio?

There is some evidence that TRPV3 and TRPV4 proteins can physically associate in HEK293 cells and native skin tissues (Hu et al., 2022).TRPV3 and TRPV4 are both expressed in the cortex (Kumar et al., 2018), but it remains unclear whether they are co-expressed and coassembled to form heteromeric channels in cortical excitatory pyramidal neurons. Examination of the I-V curve from HEK cells co-expressing TRPV3/4 heteromeric channels shows enhanced current at negative membrane potentials (Hu et al., 2022).

Currently, we cannot characterize cells as STOP or STAY and measure TRPV3 or TRPV4 currents simultaneously, as this would require different experimental setups and internal solutions. Additionally, the protocol involves a sequence of recordings at 30, 36, and 39°C, followed by cooling back to 30°C and re-heating to each temperature. Cells undergoing such a protocol will likely not survive till the end.

In our recordings of TRPV3 currents, which likely include both STOP and STAY cells, we do not observe a significant current at negative voltages, suggesting that TRPV3/4 heteromeric channels may either be absent or underrepresented, at least at a 1:1 ratio. However, the possibility that TRPV3/4 heteromeric channels could define the STOP cell population is intriguing and plausible.

(4) In Figure 7, have the authors observed an increase of TRPV3 currents in MSNs in response to temperature elevation?

We have not recorded TRPV3 currents in MSNs in response to elevated temperatures. Please note that the handling editor gave us the option to remove these data from the paper, and we elected to do so to develop them as a separate manuscript.

(5) Is there any evidence of a relationship between TRPV3 expression levels in D2+ MSNs and degeneration of dopamine-producing neurons?

This is an interesting question, though it falls outside our current research focus in the lab. A PubMed search yields no results connecting the terms TRPV3, MSNs, and degeneration. However, gain-of-function mutations in TRPV4 channel activity have been implicated in motor neuron degeneration (Sullivan et al., 2024) and axon degeneration (Woolums et al., 2020). Similarly, TRPV1 activation has been linked to developmental axon degeneration (Johnstone et al., 2019), while TRPV3 blockade has shown neuroprotective effects in models of cerebral ischemia/reperfusion injury in mice (Chen et al., 2022).

The link between TRPV activation and cell degeneration, however, may not be straightforward. For instance, TRPV1 loss has been shown to accelerate stress-induced degradation of axonal transport from retinal ganglion cells to the superior colliculus and to cause degeneration of axons in the optic nerve (Ward et al., 2014). Meanwhile, TRPV1 activation by capsaicin preserves the survival and function of nigrostriatal dopamine neurons in the MPTP mouse model of Parkinson's disease (Chung et al., 2017).

(6) Does fever range temperature alter the expressions of other neuronal Kv channels known to regulate the firing threshold?

This is an active line of investigation in our lab. The results of ongoing experiments will provide further insight into this question.

Reviewer #2 (Public review):

Summary:

The authors study the excitability of layer 2/3 pyramidal neurons in response to layer four stimulation at temperatures ranging from 30 to 39 Celsius in P7-8, P12-P14, and P22-P24 animals. They also measure brain temperature and spiking in vivo in response to externally applied heat. Some pyramidal neurons continue to fire action potentials in response to stimulation at 39 C and are called stay neurons. Stay neurons have unique properties aided by TRPV3 channel expression.

Strengths:

The authors use various techniques and assemble large amounts of data.

Weaknesses:

(1) No hyperthermia-induced seizures were recorded in the study.

The goal of this manuscript is to uncover age-related physiological changes that enable the brain to maintain function at fever-range temperatures, typically 38–40°C. Febrile seizures in humans are also typically induced within this temperature range. Given this context, we initially did not examine hyperthermia-induced seizures. However, as requested, we assessed the effects of reduced Trpv3 expression on hyperthermia-induced seizures in WT(Trpv3^+/+), heterozygous (Trpv3^+/-), and homozygous knockout (Trpv3^-/-) P12 pups. Please see figure 10.

While T_b at seizure onset and the rate of T_b increase leading to seizure were not significantly different among genotypes, the time to seizure from the point of loss of postural control (LPC), defined as collapse and failure to maintain upright posture, was significantly longer in Trpv3^+/- and Trpv3^-/- mice. Together, these results indicate that reduced TRPV3 function enhances resistance to seizure initiation and/or propagation under febrile conditions, likely by decreasing neuronal depolarization and excitability.

(2) Febrile seizures in humans are age-specific, extending from 6 months to 6 years. While translating to rodents is challenging, according to published literature (see Baram), rodents aged P11-16 experience seizures upon exposure to hyperthermia. The rationale for publishing data on P7-8 and P22-24 animals, which are outside this age window, must be clearly explained to address a potential weakness in the study.

As requested, we have added an explanation in the “Introduction” for our rationale in including age ranges that flank the period of susceptibility to hyperthermia-induced seizures (see lines 80–100). In summary, we emphasize that this design provides negative controls, allowing us to determine whether the changes observed in the P12–14 window are specific to this developmental period.

(3) Authors evoked responses from layer 4 and recorded postsynaptic potentials, which then caused action potentials in layer 2/3 neurons in the current clamp. The post-synaptic potentials are exquisitely temperature-sensitive, as the authors demonstrate in Figures 3 B and 7D. Note markedly altered decay of synaptic potentials with rising temperature in these traces. The altered decays will likely change the activation and inactivation of voltage-gated ion channels, adjusting the action potential threshold.

The activation and inactivation of voltage-gated ion channels can modulate action potential threshold. Indeed, we have identified channels that contribute to the temperature-induced increase in spike threshold, including BK channels and Scn2a. However, Figure 4B represents a cell with no inhibition at 39°C, and thus the observed loss of the late postsynaptic potential (PSP). This primarily contributes to the prolonged decay of the synaptic potentials. By contrast, cells in which inhibition is retained, when exposed to the same thermal protocol, do not exhibit such extended decay.

(4) The data weakly supports the claim that the E-I balance is unchanged at higher temperatures. Synaptic transmission is exquisitely temperature-sensitive due to the many proteins and enzymes involved. A comprehensive analysis of spontaneous synaptic current amplitude, decay, and frequency is crucial to fully understand the effects of temperature on synaptic transmission.

We did not intend to imply that E-I balance is generally unchanged at higher temperatures. Our statements specifically referred to observations in experiments conducted during the P20–26 age range in cortical pyramidal neurons. We are conducting a parallel line of investigation examining the differential susceptibility of E-I balance across age and temperature, and we have observed age- and temperature-dependent effects. Recognizing that our earlier wording may have been misleading, we have removed this statement from the manuscript.

(5) It is unclear how the temperature sensitivity of medium spiny neurons is relevant to febrile seizures. Furthermore, the most relevant neurons are hippocampal neurons since the best evidence from human and rodent studies is that febrile seizures involve the hippocampus.

Thank you for the opportunity to provide clarification. The goal of this manuscript is to uncover age-related physiological changes that enable the brain to maintain stable, non-excessive neuronal firing at fever-range temperatures (typically 38–40°C). We hypothesize that these changes are a normal part of brain development, potentially explaining why most children do not experience febrile seizures. By understanding these mechanisms, we may identify points in the process that are susceptible to dysfunction, due to genetic mutations, developmental delays, or environmental factors, which could provide insight into the rare cases when seizures occur between 2–5 years of age.

Our aim was not to establish a link between medium spiny neuron (MSN) function and febrile seizures. MSNs were included in this study as a mechanistic comparison because they represent a non-pyramidal, non-excitatory neuronal subtype, allowing us to assess whether the physiological changes observed in L2/3 excitatory pyramidal neurons are unique to these cells. Please note that the handling editor gave us the option to remove these data from the manuscript, and we chose to do so, developing these findings into a separate manuscript.

(6) TRP3V3 data would be convincing if the knockout animals did not have febrile seizures.

We find that approximately equal numbers of excitatory neurons either start or stop firing at fever-range temperatures (typically 38–40 °C). Neurons that continue to fire (“STAY” cells), thus play a key role in maintaining stable, non-excessive network activity. While future studies will examine the mechanisms driving some neurons to initiate spiking, our findings suggest that a reduction in the number of STAY cells could influence more subtle aspects of seizure dynamics, such as time to onset, by decreasing overall network excitability. We assessed the effects of reduced Trpv3 expression on hyperthermia-induced seizures in WT(Trpv3^+/+), heterozygous (Trpv3^+/-), and homozygous knockout (Trpv3^-/-) P12 pups. As you stated, these mice have hyperthermic seizures, however, we noted that the time to seizure from the point of loss of postural control (LPC), defined as collapse and failure to maintain upright posture, was significantly longer in Trpv3^+/- and Trpv3^-/- mice. Normally, seizures happen shortly after this point, but notably, Trpv3^-/- mice took twice as long to reach seizure onset compared with wildtype mice. In an epileptic patient, this increased time may be sufficient for a caretaker to move the patient to a safer location, reducing the risk of injury during the seizure.

Consistent with findings that TRPV3 blockade using 50 µM forsythoside B reduces spiking in cortical L2/3 pyramidal neurons, we observed significantly reduced spiking in Trpv3^-/- mice as well (Figure 10D). Analysis of postsynaptic potentials in these neurons showed that, in WT mice, PSP amplitude increased with temperature elevation into the febrile range, whereas this temperature-dependent depolarization was absent in Trpv3^-/- mice (Figure 10E). Together, these results indicate that reduced TRPV3 function enhances resistance to seizure initiation and/or propagation under febrile conditions, likely by decreasing neuronal depolarization and excitability.

Reviewer #3 (Public review):

Summary:

This important study combines in vitro and in vivo recording to determine how the firing of cortical and striatal neurons changes during a fever range temperature rise (37-40 oC). The authors found that certain neurons will start, stop, or maintain firing during these body temperature changes. The authors further suggested that the TRPV3 channel plays a role in maintaining cortical activity during fever.

Strengths:

The topic of how the firing pattern of neurons changes during fever is unique and interesting. The authors carefully used in vitro electrophysiology assays to study this interesting topic.

Weaknesses:

(1) In vivo recording is a strength of this study. However, data from in vivo recording is only shown in Figures 5A,B. This reviewer suggests the authors further expand on the analysis of the in vivo Neuropixels recording. For example, to show single spike waveforms and raster plots to provide more information on the recording. The authors can also separate the recording based on brain regions (cortex vs striatum) using the depth of the probe as a landmark to study the specific firing of cortical neurons and striatal neurons. It is also possible to use published parameters to separate the recording based on spike waveform to identify regular principal neurons vs fast-spiking interneurons. Since the authors studied E/I balance in brain slices, it would be very interesting to see whether the "E/I balance" based on the firing of excitatory neurons vs fast-spiking interneurons might be changed or not in the in vivo condition.

As requested, we have included additional analyses and figures related to the in vivo recording experiments in Figure 5. Specifically, we added examples of multiunit and single-spike waveforms, as well as autocorrelation histograms (ACHs). ACHs were used because raster plots of individual single units would not be very informative given the long recording period. Additionally, Figure 5F was also aimed to replace raster plots as it helps to track changes in the firing rate of a single neurons over time.

Additionally, all recordings were conducted in the cortex at a depth of ~1 mm from the surface, and no recordings were performed in the striatum. Based on the reviewing editor’s suggestions, we decided to remove the striatal data from the manuscript and develop this aspect of the project for a separate publication.

Lastly, we used published parameters to classify recordings based on spike waveform into putative regular principal neurons and interneurons. To clarify this point, we have now included descriptions that were previously listed only in the “Methods” section into the “Results” section as well.

The paragraph below from the methods section describes this procedure.

“Following manual curation, based on their spike waveform duration, the selected single units (n= 633) were separated into putative inhibitory interneurons and excitatory principal cells (Barthóet al., 2004). The spike duration was calculated as the time difference between the trough and the subsequent waveform peak of the mean filtered (300 – 6000 Hz bandpassed) spike waveform. Durations of extracellularly recorded spikes showed a bimodal distribution (Hartigan’s dip test; p < 0.001) characteristic of the neocortex with shorter durations corresponding to putative interneurons (narrow spikes) and longer durations to putative principal cells (wide spikes). Next, k-means clustering was used to separate the single units into these two groups, which resulted in 140 interneurons (spike duration < 0.6 ms) and 493 principal cells (spike duration > 0.6 ms), corresponding to a typical 22% - 78% (interneuron – principal) cell ratio”.

As suggested, we calculated the E/I balance using the average firing rates of excitatory and inhibitory neurons in the in vivo condition. Our analysis revealed that the E/I balance remained unchanged (see Author response image 1). Nonetheless, following the option provided by the reviewing editor, we have chosen to remove the statement referencing E/I balance from the manuscript.

Author response image 1.

(2) The author should propose a potential mechanism for how TRPV3 helps to maintain cortical activity during fever. Would calcium influx-mediated change of membrane potential be the possible reason? Making a summary figure to put all the findings into perspective and propose a possible mechanism would also be appreciated.

Thank you for your helpful suggestion. In response, we have included a summary figure (Figure 11) illustrating the hypothesis described in the Discussion section. We agree with your assessment that Trpv3 most likely contributes to maintaining cortical activity during fever by promoting calcium influx and depolarizing the membrane potential.

(3) The author studied P7-8, P12-14, and P20-26 mice. How do these ages correspond to the human ages? it would be nice to provide a comparison to help the reader understand the context better.

Ideally, the mouse to human age comparison should depend on the specific process being studied. Per your suggestion, we have added additional references in the Introduction (Dobbing and Sands, 1973; Baram et al., 1997; Bender et al., 2004) to help readers better understand the correspondence between mouse and human ages.

Recommendations for the authors:

Reviewer #2 (Recommendations for the authors):

(3) Perform I-F curves to study the intrinsic properties of layer 2/3 neurons without the confound of evoked responses.

We performed F-I curve analyses (Figures 2H–I), as suggested by Reviewer 2, to study intrinsic properties of L2/3 neurons without evoked responses. Although rheobase increased at 39 °C compared to 30 °C, consistent with findings such as depolarized spike threshold and reduced input resistance, the mean number of spikes across current steps did not differ.

Reviewer #3 (Recommendations for the authors):

Some statistical descriptions are not clearly stated. For example, what statistical methods were used in Fig 2E? The effect size in Fig 2D seems to be quite small. The authors are advised to consider "nested analysis" to further increase the rigor of the analysis. Does each dot mean one neuron? Some of the data points might not be totally independent. The author should carefully check all figures to make sure the stats methods are provided for each panel.

We apologize for not including statistical details in Figure 2E. We have now added this information and verified that statistical descriptions are provided in all figure legends. In Figure 2D, each dot represents a cell, with measurements taken from the same cell at 30°C, 36°C, and 39°C. Given this design, the appropriate test is a one-way repeated-measures ANOVA.

Reviewer #3 (Public review):

Summary and Significance:

In this work, Cary and Hayashi address the important question of when, in evolution, certain mobile genetic elements (Ty3/gypsy-like non-LTR retrotransposons) associated with certain membrane fusion proteins (viral glycoprotein F or B-like proteins), which could allow these mobile genetic elements to be transferred between individual cells of a given host. It is debated in the literature whether the acquisition of membrane fusion proteins by non-LTR retrotransposons is a rather recent phenomenon that separately occurred in the ancestors of certain host species or whether the association with membrane fusion proteins is a much more ancient one, pre-dating the Cambrian explosion. Obviously, this question also touches upon the origin of the retroviruses, which can spread between individuals of a given host but seem restricted to vertebrates. Based on convincing data, Cary and Hayashi argue that an ancient association of non-LTR retrotransposons with membrane fusion proteins is most probable.

Strengths:

The authors take the smart approach to systematically retrieve apparently complete, intact, and recently functional Ty3/gypsy-like non-LTR retrotransposons that, next to their characteristic gag and pol genes, additionally carry sequences that are homologous to viral glycoprotein F (env-F) or viral glycoprotein B (env-B). They then construct and compare phylogenetic trees of the host species and individual encoded proteins and protein domains, where 3D-structure calculations and other features explain and corroborate the clustering within the phylogenetic trees. Congruence of phylogenetic trees and correlation of structural features is then taken as evidence for an infrequent recombination and a long-term co-evolution of the reverse transcriptase (encoded by the pol gene) and its respective putative membrane fusion gene (encoded by env-F or env-B). Importantly, the env-F and env-B containing retrotransposons do not form a monophyletic group among the Ty3/gypsy-like non-LTR retrotransposons, but are scattered throughout, supporting the idea of an originally ancient association followed by a random loss of env-F/env-B in individual branches of the tree (and rather rare re-associations via more recent recombinations).

Overall, this is valuable, stimulating, and important work of general and fundamental interest, but still also somewhat incompletely explored, imprecisely explained, and insufficiently put into context for a more general audience.

Weaknesses:

Some points that might be considered and clarified:

(1) Imprecise explanations, terms, and definitions:

It might help to add a 'definitions box' or similar to precisely explain how the authors decided to use certain terms in this manuscript, and then use these terms consistently and with precision.

a) In particular, these are terms such as 'vertebrate retrovirus' vs 'retrovirus' vs 'endogenized retrovirus' vs 'endogenous retrovirus' vs 'non-LTR retrotransposon' and 'Ty3/gypsi-like retrotransposon' vs 'Ty3/gypsy retrotransposon' vs 'errantivirus'.

b) The comment also applies to the term 'env' used for both 'env-F' and 'env-B', where often it remains unclear which of the two protein types the authors refer to. This is confusing, particularly in the methods, where the search for the respective homologs is described.

c) Other examples are the use of the entire pol gene vs. pol-RT for the definition of the Ty3/gypsy clade and for the generation of phylogenetic trees (Methods and Figure S1), and the names for various portions of pol that appear without prior definition or explanation (e.g., 'pro' in Figure 1A, 'bridge' in Figure S1C, 'the chromodomain' in the text and Figure 7).

d) It is unclear from the main text which portions of pol were chosen to define pol-RT and why. The methods name the 'palm-and-fingers', 'thumb', and 'connections' domains to define RT. In the main text, the 'connection' domain is called 'tether' and is instead defined as part of the 'bridge' region following RT, which is not part of RT.

(2) Insufficient broader context:

a) The introduction does not state what defines Ty3/gypsy non-LTR retrotransposons as compared to their closest relatives (Ty1/copia retrotransposons, BEL/pao retrotransposons, vertebrate retroviruses). This makes it difficult to judge the significance and generality of the findings.

b) The various known compositions of Ty3/gypsi-like retrotransposons are not mentioned and explained in the introduction (open reading frames, (poly-)proteins and protein domains, and their variable arrangement, enzymatic activities, and putative functions), and the distribution of Ty3/gypsi-like retrotransposons among eukaryotes remains unclear. The introduction does not mention that Ty3/gypsi-like retrotransposons apparently are absent from vertebrates, and Figure 7 is not very clear about whether or not it includes sequences from plants ('Chromoviridae').

c) The known association of Ty3/gypsi-like retrotransposons from different metazoan phyla with putative membrane fusion proteins (env-like) genes is mentioned in the introduction, but literature information, whether such associations also occur in the context of other retrotransposons (e.g., Ty1/ copia or BEL/pao), is not provided. The abstract is somewhat misleading in this respect. Finally, the different known types of env-like genes are not mentioned and explained as part of the introduction ('env-f', 'env-B', 'retroviral env', others?)

d) Some key references and reviews might be added:

- Pelisson, A. et al. (1994) https://www.embopress.org/doi/abs/10.1002/j.1460-2075.1994.tb06760.x<br /> (next to Song et al. (1994), for the identification of env in Ty3/gypsy)

- Boeke, J.D. et al. (1999)<br /> In Virus Taxonomy: ICTV VIIth report. (ed. F.A. Murphy),. Springer-Verlag, New York.<br /> (cited by Malik et al. (2000) - for the definition and first use of the term 'errantivirus')

- Eickbush, T.H. and Jamburuthugoda, V.K. (2008) https://doi.org/10.1016/j.virusres.2007.12.010<br /> (on the classification of retrotransposons and their env-like genes)

- Hayward, A. (2017) https://doi.org/10.1016/j.coviro.2017.06.006<br /> (on scenarios of env acquisition)

(3) Incomplete analysis:

a) Mobile genetic elements are sometimes difficult to assemble correctly from short-read sequencing data. Did the authors confirm some of their newly identified elements by e.g., PCR analysis or re-identification in long-read sequencing data?

b) The authors mention somewhat on the side that there are Ty3/gypsy elements with a different arrangement (gag-env-pol instead of gag-pol-env). Why was this important feature apparently not used and correlated in the analysis? How does it map on the RT phylogenetic tree? Which type of env is found with either arrangement? Is there evidence for a loss of env also in the case of gag-env-pol elements?

c) Sankey plots are insufficiently explained. How would inconsistencies between trees (recombinations) show up here? Why is there no Sankey plot for the analysis of env-B in Figure 5?

d) Why are there no trees generated for env-F and env-B like proteins, including closely related homologous sequences that do NOT come from Ty3/gypsy retrotransposons (e.g., from the eukaryotic hosts, from other types of retrotransposons (Ty1/copia or BEL/pao), from viruses such as Herpesvirus and Baculovirus)? It would be informative whether the sequences from Ty3/gypsy cluster together in this case.

e) Did the authors identify any other env-like ORFs (apart from env-F and env-B) among Ty3/gypsy retrotransposons? Did they identify other, non-env-like ORFs that might help in the analysis? It is not quite clear from the methods if the searches for env-F and env-B - containing Ty3/gypsy elements were done separately and consecutively or somehow combined (the authors generally use 'env', and it is not clear which type of protein this refers to).

f) Why was the gag protein apparently not used to support the analysis? Are there different, unrelated types of gag among non-LTR retrotransposons? Does gag follow or break the pattern of co-evolution between RT and env-F/env-B?

g) Data availability. The link given in the paper does not seem to work (https://github.com/RippeiHayashi/errantiviruses_2025/tree/main). It would be useful for the community to have the sequences of the newly identified Ty3/gypsy retrotransposons listed readily available (not just genome coordinates as in table S1), together with the respective annotations of ORFs and features.

AA children were considered to have less comprehension of the language

Il semble manquer un déterminant pour chaque terme :

"...sur l'islamophobie et le génocide".

Il est aussi possible de nuancer le propos ainsi :

"...sur la question / l'enjeu / le concept de l'islamophobie et du génocide"

"de jure" semble un peu éloigné de l'élément qu'il qualifie. Je proposerais de les rapprocher pour éclaircir le sens :

"...sont susceptibles d'être de la sorte justifiées de jure par un élément cognitif"

OU

"...sont de jure susceptibles d'être de la sorte justifiées par un élément cognitif"

Briefing : Le Rôle des Modèles dans le Développement de l'Enfant

Synthèse

Ce document de synthèse analyse le rôle complexe et multifacette des modèles dans le développement de l'enfant, en se basant sur les perspectives de psychologues, d'experts en développement et de témoignages personnels.

Il ressort que les parents constituent les modèles les plus fondamentaux, dont l'influence est primordiale durant les premières années.

Cependant, la recherche de la perfection parentale est contre-productive ; l'authenticité, la capacité à reconnaître ses erreurs et à s'excuser sont bien plus formatrices.

L'enfant n'imite pas aveuglément mais opère une sélection rigoureuse de ses modèles, privilégiant la compétence, la familiarité et la confiance.

Les modèles parentaux dysfonctionnels, marqués par l'addiction ou des troubles psychiques, ont des conséquences graves et durables sur la sécurité affective et l'estime de soi de l'enfant.

À l'adolescence, la recherche de modèles s'élargit au-delà du cercle familial pour construire une identité propre, un processus sain de différenciation qui peut inclure la rébellion et l'adhésion à des groupes de pairs.

Enfin, une perspective émergente et cruciale est mise en lumière : les enfants et adolescents ne sont pas de simples récepteurs passifs mais peuvent être de puissants modèles et des acteurs de changement, capables d'influencer positivement leur entourage, y compris leurs propres parents, et de façonner la société de demain.

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L'Imitation Sélective : Comment les Enfants Choisissent Leurs Modèles

Le processus par lequel un enfant choisit et imite un modèle est loin d'être passif.

Il repose sur des mécanismes neurologiques et psychologiques complexes qui démontrent une grande sélectivité dès le plus jeune âge.

• Bases Neurologiques : Selon Moritz Köster, professeur de psychologie du développement, lorsqu'un enfant observe quelqu'un agir, des séquences de mouvements similaires sont activées dans son propre cortex moteur au niveau cellulaire.

• Sélectivité Basée sur la Confiance : L'enfant n'imite pas tout ce qu'il voit. Son choix est nuancé par les émotions et une évaluation de la personne observée. Les principaux critères de sélection sont :

◦ La familiarité : Il préférera imiter une personne qu'il connaît.   

◦ La compétence : Il analyse si la personne a déjà fait des choses "intelligentes" ou des erreurs, et choisira d'imiter la personne jugée la plus compétente.   

◦ L'autorité : Pour tout ce qui est nouveau, l'enfant se tournera préférentiellement vers les adultes, qu'il perçoit comme des figures de confiance.

• Apprentissage des Normes : C'est principalement en observant le comportement des adultes et de leur entourage que les enfants apprennent et intègrent les valeurs et les normes sociales.

Lise, 8 ans : "Pour moi un modèle c'est quand on fait quelque chose de bien et que quelqu'un d'autre nous imite."

Les Parents : Les Premiers et Plus Influents Modèles

L'environnement familial, et plus particulièrement les parents, constitue la première et la plus puissante source de modèles pour un enfant, une influence que les parents ont souvent tendance à sous-estimer.

L'Influence Fondamentale de l'Environnement Familial

Durant les premières années de vie (1-2 ans), l'environnement de l'enfant est restreint aux parents et grands-parents.

Leur comportement façonne entièrement la compréhension initiale de l'enfant sur les interactions sociales.

• Apprentissage des Comportements Sociaux : La manière de gérer un conflit, d'éviter les disputes ou de présenter des excuses est directement apprise par l'observation des parents.

• Ancrage Émotionnel : Si les échanges familiaux sont marqués par la bienveillance et l'amour, l'enfant intègre ce modèle. Inversement, si les cris ou la violence sont la norme, il retiendra ce schéma comme référence.

• La Famille comme Microcosme : Au départ, l'enfant perçoit le monde entier comme fonctionnant selon les règles de sa propre famille. Ce n'est qu'à son entrée en maternelle qu'il découvre la diversité des modes de fonctionnement.

Le Piège du "Parent Parfait" et la Valeur de l'Authenticité

La psychologue Nora Imlau met en garde contre la volonté de certains parents de devenir "parfaits" après la naissance d'un enfant, la qualifiant de "très mauvaise idée".

• L'Inauthenticité : Les enfants ressentent très bien quand leurs parents ne sont pas authentiques, se mettent la pression et ignorent leurs propres besoins.

• Un Standard Inatteignable : Un enfant confronté à des modèles "parfaits" (qui ne se mettent jamais en colère, ne perdent jamais patience) n'a aucune chance de faire aussi bien.

Il sera sans cesse confronté à ses propres insuffisances.

• L'Importance de l'Erreur : Le fait que les parents commettent des erreurs est une opportunité d'apprentissage cruciale.

Cela permet à l'enfant d'apprendre comment on gère ses propres erreurs.

Présenter ses excuses à ses enfants pour des propos qui ont "dépassé notre pensée" est un acte modelant très puissant.

Nora Imlau, psychologue : "Ce que j'entends par parents parfaits, ce sont les parents qui ne se mettent jamais en colère, qui ne perdent jamais patience [...] ce qui est inhumain en soi."

Gérer les Émotions Parentales Difficiles

Le comportement d'un enfant est souvent le reflet de l'état d'âme inconscient de ses parents. Un enfant agité peut être le miroir d'un parent stressé ou préoccupé.

• La Gestion de la Tristesse : Quand un parent est triste et qu'un enfant vient le consoler, il est conseillé d'accepter cette aide dans un premier temps.

Cependant, il est crucial que le parent reprenne ensuite le contrôle et rassure l'enfant sur sa capacité à gérer la situation, afin de ne pas inverser les rôles et de préserver l'enfant de la charge de ses responsabilités d'adulte.

• La Vulnérabilité Assumée : Une mère souffrant de trouble bipolaire témoigne de sa capacité à être présente pour ses enfants même dans les phases de dépression, tout en ne cachant pas sa tristesse.

Cela illustre la possibilité de rester un parent fonctionnel malgré des difficultés psychiques.

Les Conséquences des Modèles Parentaux Dysfonctionnels

Lorsque les parents ne peuvent pas s'occuper correctement de leurs enfants, que ce soit à cause d'une dépendance ou d'un trouble psychique, les conséquences sur le développement de l'enfant sont multiples et profondes.

L'Impact sur le Développement de l'Enfant

Le témoignage de Mia, 16 ans, dont le père était alcoolique, illustre les dégâts d'un modèle parental défaillant.

• Rupture de la Confiance : Un parent souffrant de dépression ou d'addiction n'est plus en mesure d'interpréter correctement les signaux de son enfant et d'y réagir de manière adaptée.

L'enfant retient que ses besoins ne sont pas satisfaits.

• Attachement Insécurisant : La relation d'attachement parent-enfant ne devient pas sécurisante, ce qui entrave la construction de la confiance en soi.

Cette confiance initiale est pourtant la base essentielle du développement de l'autonomie.

• Hypervigilance de l'Enfant : L'enfant est constamment aux aguets, utilisant une énergie considérable pour anticiper les réactions de ses parents et adapter son propre comportement, ce qui peut entraîner des problèmes d'autonomie et de sentiment de sécurité à l'âge adulte.

Mia, 16 ans : "En fait il fallait toujours qu'on soit la famille parfaite, on parlait jamais des problèmes, on avait pas le droit d'en parler et ça c'est très mal."

La Recherche de Modèles Toxiques à l'Adolescence

Suite à la séparation de ses parents et à ses propres difficultés psychologiques, Mia a été confrontée à des "modèles toxiques" dans un cadre thérapeutique.

• Influence des Pairs : En observant des jeunes toxicodépendants, elle a perçu leur consommation comme un moyen de "déconnecter totalement" et de ne plus être accessible émotionnellement, un état qu'elle a alors désiré atteindre.

• Augmentation de la Consommation : Son exposition à ces modèles a directement influencé son propre comportement, entraînant une augmentation significative de sa consommation d'alcool.

L'Adolescence : Identité, Rébellion et Recherche de Nouveaux Modèles

L'adolescence est une période de questionnements identitaires intenses ("Qui suis-je ?") où la recherche de modèles s'intensifie et s'étend au-delà du cercle familial.

La Construction de Soi au-delà de la Famille

Selon la psychothérapeute Isabelle Filliozat, l'adolescent va "chercher des modèles un petit peu partout pour [s]'aider à se construire".

• Le Rôle du Groupe : Le désir d'appartenance à un groupe de pairs est très fort.

Le groupe offre un cadre identitaire ("dans mon groupe on fait les choses d'une certaine manière [...] je sais à peu près qui je suis").

• Gestion des Modèles Négatifs : Lorsqu'un enfant adhère à un modèle jugé "malsain" (agressif, délinquant), la réaction parentale la plus constructive n'est pas de chercher à changer le comportement extérieur, mais de s'intéresser aux besoins et aux émotions de l'enfant qui le poussent vers ce modèle.

En répondant à ces besoins profonds, l'enfant est plus susceptible d'abandonner de lui-même le modèle négatif.

Le Rôle Essentiel de la Rébellion

La révolte contre les parents à l'adolescence est un processus "sain et normal", une étape nécessaire du développement.

• Processus de Détachement : Les frictions parents-enfants font partie du processus de détachement et de la prise de conscience par l'adolescent qu'il est une personne à part entière, distincte de ses parents.

• Différenciation : Pour se construire, l'adolescent a besoin de s'opposer, de définir en quoi il est différent de ses parents (valeurs, mentalité) mais aussi en quoi il leur ressemble.

Ce processus est essentiel pour pouvoir, à terme, quitter le foyer et construire une nouvelle relation, d'adulte à adulte, avec ses parents.

Les Enfants comme Acteurs de Changement et Modèles d'Avenir

La vision traditionnelle du modèle descendant (adulte vers enfant) est de plus en plus complétée par une reconnaissance du rôle actif des jeunes comme modèles et agents d'influence.

L'Influence Ascendante : Des Enfants sur les Parents

Des recherches ont démontré que les enfants peuvent avoir une influence positive sur la manière de penser et sur le comportement de leurs parents.

• "L'Hypothèse des Anniversaires" : Dans des zones post-conflit, le fait que des enfants d'un groupe ethnique ou religieux invitent à leur anniversaire des enfants d'un groupe adverse force les parents des deux bords à entrer en contact.

Il a été observé que lorsque l'attitude des enfants envers "l'autre groupe" change, celle des parents change également.

• Acteurs de Paix : Les enfants peuvent ainsi devenir des acteurs clés de la promotion de la paix.

L'Engagement des Jeunes comme Nouveau Modèle

Des adolescents comme Noé Renard, 17 ans, s'imposent comme des modèles d'engagement pour leur génération.

• Rendre l'Engagement Accessible : En créant l'association "les engagés Marseille", son but est de montrer l'exemple et de permettre à d'autres jeunes de se mobiliser sur des enjeux locaux (inégalités, pollution, mobilité).

• Une Voix pour la Jeunesse : De nombreux jeunes partagent le sentiment de ne pas être suffisamment écoutés dans les institutions politiques.

Ils peuvent devenir des modèles pour leurs pairs mais aussi pour les chercheurs, comme l'illustre la mise en place d'un Conseil consultatif de la jeunesse à l'Université libre de Berlin.

Noé Renard, 17 ans : "Défendre des causes c'est pas le faire pour soi mais c'est plutôt le faire pour les autres et je pense que c'est ça qui est important c'est de pouvoir montrer aux autres que l'engagement c'est [...] surtout pour les autres et pour aider ceux qui en ont besoin."

La Nécessité d'une Participation Démocratique Précoce

Une critique est formulée quant au fait d'attendre la majorité pour accorder le droit de vote sans formation préalable aux règles de la démocratie.

• Apprentissage Précoce : Les experts plaident pour que les enfants apprennent beaucoup plus tôt comment fonctionne un consensus, comment on règle les conflits dans une démocratie, et qu'ils aient davantage d'influence sur leur vie quotidienne.

• Faire Confiance : Pour que les jeunes développent leur identité et leur capacité à prendre des responsabilités, les parents doivent apprendre à leur faire confiance et à les laisser expérimenter par eux-mêmes, même si c'est "à leur façon".

Argument pour : les résultats de cette étude, pourtant mondiale, ont indiqué que la majorité des étudiants n'ont pas réussi à déconnecter, ne serait-ce que 24h, et se sont même considérés comme dépendants. Ceci évoque un comportement proche d’un processus addictif (désir incontrôlable, auto-déclaration d’addiction).

Argument contre : dans l'addiction, on note une conduite compulsive, mais ici, l’usage excessif est expliqué par la compensation de besoins sociaux.

Argument contre : le texte montre que les effets négatifs d'une fréquentation intense d'internet (dépression, anxiété) sont liés à des comparaisons sociales et à des ruminations, des pensées négatives, mais pas à une dépendance.

Argument pour : La difficulté à résister à un usage numérique fait état d'impulsions généralement associées aux addictions comportementales.

Argument contre : l'addiction à internet est plutôt vue comme une pratique excessive que comme un véritable trouble mental. En tout cas, elle n'est pas reconnue par le DSM 5, on ne cite pas ici par exemple de critères cliniques.

klais kai klais kai klais kai epeidh den sou dinei kanenas shmasia stamatas na klais

Argument pour : à travers la notion de défi indiquée, internet peut-il donc influencer les comportements des individus ?

Argument contre : internet permet donc d'optimiser les ressources, en permettant à davantage de patient d'avoir accès à un professionnel.

Argument pour : les usages numériques ne pourraient-ils alors pas engendrer une fragilité possible, créant un terrain propice à des comportements inappropriés ?

Argument contre : cette position contredit l’idée que l'usage d'internet est nécessairement dangereux ou addictif : plusieurs arguments sont développés ici (accessibilité, disponibilité, possibilités financières …)

Argument contre : ici internet peut être envisagé comme un outil de soin, pas seulement un espace de risque.

Argument pour : même si on parle ici d'addiction "a fortiori", les critères de dépendance décrits y ressemblent fortement.

Argument contre : la notion d’addiction numérique reste controversée.

Argument pour : le vocabulaire de l’addiction est ici utilisé pour les activités numériques.

Écologie : Complexité, Paradoxes et Holisme — Synthèse de la Leçon Inaugurale de Franck Courchamp

Résumé Exécutif

Cette note de synthèse résume la leçon inaugurale de Franck Courchamp, titulaire de la chaire annuelle "Biodiversité et écosystèmes" au Collège de France.

La présentation articule l'étude de l'écologie autour de trois concepts fondamentaux : la complexité, les paradoxes et le holisme.

Franck Courchamp, directeur de recherche au CNRS et scientifique de renommée mondiale, démontre que la biodiversité est un système d'une richesse et d'une interconnexion extraordinaires, dont la compréhension ne peut être que partielle sans une approche globale.

Les points clés sont les suivants :

• La Biodiversité est une réalité multidimensionnelle et largement méconnue.

Définie à trois niveaux (spécifique, génétique, écosystémique), elle représente une richesse quantitative (potentiellement jusqu'à 10 milliards d'espèces de procaryotes) et qualitative (valeur utilitaire et intrinsèque) immense.

Cependant, la science n'a décrit qu'une infime fraction de cette diversité (2,3 millions d'espèces eucaryotes), alors même qu'un million d'espèces sont menacées d'extinction.

• La complexité est la caractéristique fondamentale des écosystèmes.

Le nombre vertigineux d'espèces (des dizaines de milliers dans une surface équivalente à une salle de conférence en forêt amazonienne) et la multitude d'interactions directes et indirectes entre elles et avec leur environnement créent des systèmes dynamiques et auto-organisés d'une complexité qui dépasse souvent l'intuition.

• De cette complexité naissent des paradoxes écologiques. De nombreux phénomènes observés en écologie sont contre-intuitifs.

Par exemple, l'ajout d'engrais peut appauvrir la diversité végétale, la prévention des incendies peut engendrer des méga-feux, et la réintroduction de prédateurs comme le loup peut paradoxalement rendre les routes plus sûres en modifiant le comportement de leurs proies.

• L'approche holistique est indispensable pour comprendre et agir.

Seule une vision globale de l'écosystème, intégrant toutes ses composantes et interactions, permet de déchiffrer ces paradoxes et d'éviter des interventions de conservation aux conséquences inverses de celles escomptées.

L'exemple de la réintroduction des loups à Yellowstone, qui a modifié jusqu'au cours des rivières, illustre parfaitement la puissance des effets en cascade qu'une approche holistique peut révéler.

La conférence conclut que ces trois concepts — complexité, paradoxes, holisme — sont des outils intellectuels essentiels pour naviguer dans le champ de l'écologie.

Ils formeront le fil conducteur des cours à venir, qui se concentreront sur la biologie des invasions, en adoptant une perspective résolument interdisciplinaire.

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Introduction : Contexte et Présentation de Franck Courchamp

La leçon inaugurale a été prononcée dans le cadre de la cinquième édition de la chaire annuelle "Biodiversité et écosystèmes" du Collège de France, une initiative soutenue par la Fondation Jean-François de Clermont-Tonnerre.

Cette chaire vise à promouvoir la recherche et à éclairer le débat public sur les enjeux du vivant.

Le titulaire de la chaire, Franck Courchamp, est une figure de premier plan dans le domaine de l'écologie. Ses qualifications incluent :

• Positions académiques : Directeur de recherche première classe au CNRS, il dirige une équipe à l'Université Paris-Saclay et est titulaire de la chaire AXA "Biologie des invasions".

• Reconnaissance internationale : Auteur de plus de 200 publications, il est l'un des scientifiques les plus cités au monde dans son domaine et contribue aux travaux de panels intergouvernementaux majeurs comme le GIEC et l'IPBES.

• Distinctions : Il a reçu de nombreuses récompenses, dont la médaille d'argent du CNRS (2011), a été nommé à l'Académie européenne des sciences (2014) et fait chevalier de l'Ordre national du Mérite (2021).

• Vulgarisation : Reconnu pour son talent de communicant, il a participé à des documentaires (notamment la série Une espèce à part sur Arte), et a publié des ouvrages grand public tels que L'Écologie pour les nuls et la bande dessinée La Guerre des fourmis.

Thème I : Définition et Importance de la Biodiversité

Les Trois Niveaux de la Biodiversité

La biodiversité, contraction de "diversité biologique", est classiquement analysée selon trois échelles interdépendantes :

1. La biodiversité spécifique : Le nombre d'espèces présentes dans un espace donné (ex. : 160 000 à 180 000 espèces de papillons dans le monde). C'est le niveau le plus couramment étudié.

2. La biodiversité génétique : La diversité au sein d'une même espèce (ex. : les 340 races de chiens). Une faible diversité génétique, comme chez le guépard, rend une espèce très vulnérable.

3. La biodiversité écosystémique : La variété des écosystèmes dans un paysage (ex. : un paysage avec forêt, lac et prairie a une plus grande diversité écosystémique qu'un récif corallien, même si ce dernier a une très grande diversité spécifique).

L'Étendue de la Biodiversité : Connue et Inconnue

L'ampleur de la biodiversité sur Terre reste largement sous-estimée.

• Espèces décrites : La science a formellement décrit 2,3 millions d'espèces eucaryotes (animaux, plantes, champignons, protistes).

• Espèces inconnues : Les estimations suggèrent que la grande majorité des espèces reste à découvrir. Le tableau suivant, évoqué dans la conférence, illustre ce déficit de connaissance :

Groupe Taxonomique

Pourcentage d'Espèces Inconnues (estimation)

Mammifères

Près de 10 %

Poissons

Près de 90 %

Insectes

Près de 90 %

Algues

Près de 90 %

Champignons

Plus de 90 %

Franck Courchamp souligne : "Nous vivons, sans le savoir, dans un monde de champignons et d'insectes."

De plus, les eucaryotes ne sont qu'une infime partie du vivant ; les procaryotes (bactéries et archées) pourraient représenter jusqu'à 10 milliards d'espèces.

La Double Valeur de la Biodiversité

La biodiversité est importante pour l'humanité de deux manières distinctes :

• La valeur utilitaire : Elle fournit des "biens" et des "services" essentiels.

◦ Biens : Alimentation (seulement 12 espèces végétales fournissent 75% de la nourriture mondiale), matériaux (bois, coton, laine), et médicaments (deux tiers des molécules pharmaceutiques proviennent directement des plantes).  

◦ Services : Pollinisation (près de 80% de nos cultures), purification de l'eau et de l'air, fertilisation des sols et biodégradation.

• La valeur intrinsèque : Chaque espèce, écosystème ou individu possède une valeur propre, indépendamment de son utilité pour l'être humain.

Une Richesse Menacée

Cette richesse est en péril. Le rapport de l'IPBES de 2019 a établi qu'un million d'espèces animales et végétales sont menacées d'extinction au cours des prochaines décennies, avec une accélération notable du rythme des extinctions récentes.

Thème II : L'Écologie, Science des Interactions du Vivant

L'écologie est la discipline scientifique qui étudie les interactions entre les organismes et leur environnement. Elle est intrinsèquement liée à la science de l'évolution. Comme le formule Franck Courchamp : "L'écologie observe la danse des espèces dans leur environnement [...]. L'évolution raconte l'histoire de cette danse."

Des Systèmes Simples aux Réseaux Complexes

L'écologie analyse des systèmes à différentes échelles, des individus à la biosphère. L'étude de la dynamique des populations offre une porte d'entrée.

L'exemple classique des cycles prédateur-proie entre le lynx et le lièvre arctique, documenté grâce aux registres de la Compagnie de la Baie d'Hudson, montre comment des modèles mathématiques simples (comme le modèle de Lotka-Volterra) peuvent décrire des dynamiques complexes.

Cependant, la réalité est celle de réseaux trophiques où chaque espèce interagit avec de nombreuses autres, créant des systèmes d'une complexité immense, auxquels s'ajoutent les interactions non-vivantes (cycles biogéochimiques du carbone, de l'azote, etc.).

Thème III : Les Concepts Clés pour Appréhender l'Écologie

Franck Courchamp propose une grille de lecture de l'écologie fondée sur trois concepts interdépendants.

La Complexité : Le Fondement de l'Écologie

La biodiversité est un système caractérisé par une richesse, une dynamicité et un nombre d'interactions extraordinairement élevés.

Un exercice de pensée illustre ce point : sur une surface équivalente à celle de la salle de conférence, une forêt amazonienne peut abriter entre 10 000 et 20 000 espèces différentes, dont 5 000 à 10 000 espèces d'insectes.

L'ensemble des interactions directes et indirectes entre ces milliers d'acteurs forme un système dynamique, auto-organisé (autopoïétique) et multiscalaire.

Les Paradoxes : Les Conséquences Contre-Intuitives de la Complexité

De cette complexité émergent des résultats qui défient l'intuition. Ces paradoxes sont omniprésents en écologie.

• Paradoxes généraux :

◦ Écologie des communautés : L'ajout d'engrais peut "tuer" les plantes en favorisant quelques espèces dominantes au détriment de la diversité globale, rendant l'écosystème moins stable.  

◦ Écologie forestière : La suppression systématique des feux de faible intensité mène à l'accumulation de combustible et à des "méga-feux" dévastateurs.  

◦ Biologie de la conservation : Le retour des loups dans certaines régions des États-Unis a réduit de près d'un quart les accidents de voiture impliquant des cerfs, non pas en diminuant leur population, mais en modifiant leur comportement (création d'un "paysage de la peur").

• Paradoxes issus des recherches de Franck Courchamp :

◦ Épidémiologie : Les chats infectés par le VIF (sida du chat) vivent plus longtemps, car le virus se transmet lors de combats entre les individus les plus dominants et les plus robustes.  

◦ Effet Allee : Pour certaines espèces sociales (suricates, lycaons), c'est l'incapacité à coopérer en dessous d'un certain seuil d'effectif qui cause l'extinction, et non la compétition.  

◦ Paradoxe de la rareté : La rareté d'une espèce augmente sa valeur sur le marché (chasse, collection), ce qui intensifie son exploitation et accélère sa disparition dans une boucle de rétroaction positive.  

◦ Espèces charismatiques : Elles sont à la fois les plus aimées, les plus menacées, et leur omniprésence culturelle nous fait croire à tort qu'elles sont communes, ce qui freine les efforts de conservation.

L'Holisme : La Nécessité d'une Approche Globale

La clé pour comprendre ces paradoxes et agir efficacement est l'adoption d'une approche holistique, qui considère l'écosystème dans son ensemble.

• Pour Comprendre : L'Exemple des Loups à Yellowstone La réintroduction du loup, un prédateur apical, a déclenché une cascade d'effets dans tout l'écosystème :

1. Contrôle des élans : Diminution de la pression de broutage sur la végétation.  

2. Régénération de la végétation : Les saules et les peupliers ont pu repousser.  

3. Retour des castors : Avec plus de bois, les populations de castors ont explosé, créant des barrages.  

4. Modification des rivières : Les barrages ont modifié l'hydrologie et la morphologie des cours d'eau, créant des habitats pour d'autres espèces (poissons, amphibiens, oiseaux). Cet exemple montre qu'une seule action peut avoir des répercussions profondes et inattendues sur l'ensemble du système.

• Pour Agir : Biologie de la Conservation Une vision non-holistique peut mener à l'échec. La surprotection des éléphants dans certaines réserves, sans tenir compte du reste de l'écosystème, a conduit à la dégradation de la végétation et a nui à d'autres herbivores.

De même, l'interdiction totale du commerce de l'ivoire, bien qu'intentionnée, a créé un marché noir qui a pu intensifier le braconnage dans certaines zones.

Conclusion et Perspectives

La complexité, les paradoxes et le holisme ne sont pas de simples concepts académiques, mais des outils essentiels pour déchiffrer le fonctionnement du vivant et orienter l'action humaine.

Ces principes formeront la structure des cours à venir de Franck Courchamp, qui se concentreront sur la biologie des invasions.

Chaque cours sera enrichi par un séminaire mené par un spécialiste d'une autre discipline (économie, philosophie, épidémiologie, etc.), soulignant la nécessité d'une approche interdisciplinaire pour relever les défis environnementaux actuels.

La leçon se termine sur une citation de Carl Sagan, rappelant que la nature recèle encore d'innombrables merveilles à découvrir : "Quelque part, quelque chose d'incroyable attend d'être connu."

La parola kānaka maoli per indicare l'acqua è wai, mentre quella hawaiana per indicare la ricchezza è waiwai, a indicare che l'acqua è fonte di benessere e ricchezza

[https://imagesofoldhawaii.com/ka-wai-ola-a-kane/]

La coltivazione dell'igname in Giamaica utilizza il metodo dello “stick” (bastone), con materiale di piantagione chiamato “yam heads” (teste di igname) sepolto in singoli cumuli di terra chiamati “yam hills” (collinette di igname).

[https://it.wikipedia.org/wiki/Igname]

pas besoin d'être un pro, WALL_ter est à la portée de tous

Mettre plus en forme les section « en savoir plus » parce que la c’est un bloc de texte assez indigeste. Scinder en paragraphe et donner plus de forme au texte

Peut être pas la meilleure formule possible

New to me!

pressure and altitude aren't exactly exponential because temperature has na impact Pressure decreases when the atmosphere is warmer as the density is lower, and increases when the temperature is lower as the density is higher These variations aren't huge because the temp range in kelvin is small, about 213-288K over the troposphere

Reduction is donating electrons Oxidation is accepting electrons Reducing gases are hydrogen or contain hydrogen (methane, ammonia) Oxidising gases include oxygen We can see past atmospheres through records in rocks and seidements this has led to division of geological time into four eons: Haden 4.6-4 billion, Archea 4-2.5, Proterozic 2.5-540 and Phanerozoic

Dati palesemente falsi e non validati da nessuno studio scientifico N.B.: Il risparmio riportato per il vaccino meningococcico B e quello per la varicella zoster sono identici: € 38 759 608, il che è veramente ridicolo Credibilità = 0**

successioni cronologiche prive di fondamenta scientifiche e la cui validità è non dimostrata. La ripresa dell'incidenza di malattie gravi o potenzialmente mortali (che sarebbero facilmente evitabili tramite semplici vaccinazioni) è arbitraria e gratuita...

Je sais pas si j'ai déjà vu des notes de bas de page dans les proposition pour des colloques. La parenthèse probablement est mieux. Je rajouterais la collaboration avec le PURH.

Contendido de la forma relativa del valor, nos sugiere que debemos encontrar ese ALGO que nos permita transformar formas de trabajo diferentes, mercancías de diferente uso, cualitativamente, donde podamos equipararlas, cuantitativamente.

Aquí se nos esta introduciendo al hecho de como es posible equiparar dos mercancías que son cualitativamente diferentes. Es por medio de la medición de la duración del TRABAJO puesto a su creación. Mas abajo en breve se nos insiste que las mercancías en el solo representan la cantidad de trabajo en ella contenida (duración del trabajo util en ellas contenida).

DOI: 10.1016/j.xcrm.2025.102466

Resource: None

Curator: @scibot

SciCrunch record: RRID:AB_469228

What is this?

DOI: 10.1016/j.xcrm.2025.102466

Resource: (BD Biosciences Cat# 566967 (also 566968), RRID:AB_2869977)

Curator: @scibot

SciCrunch record: RRID:AB_2869977

What is this?

L'Égalité des Genres : Analyse des Origines du Patriarcat et des Modèles Alternatifs

Résumé

Ce document de synthèse analyse la thèse selon laquelle le patriarcat n'est pas une loi naturelle et immuable, mais une construction historique.

S'appuyant sur des exemples historiques, archéologiques et anthropologiques, il démontre que les relations entre les genres ont pris des formes très diverses au cours de l'histoire humaine.

L'égalité a non seulement existé, mais elle persiste dans certaines sociétés matrilinéaires contemporaines.

L'analyse révèle que l'émergence des premiers États a été un facteur décisif dans l'institutionnalisation et la propagation mondiale du patriarcat comme outil de contrôle démographique et social.

Le cas de l'Islande illustre que l'égalité moderne est une conquête récente et fragile, fruit d'une lutte collective déterminée, et non un retour à un état originel.

En conclusion, la reconnaissance de la mutabilité des structures sociales ouvre la voie à la possibilité de construire un avenir égalitaire, en comprenant que l'ordre social actuel n'est pas une fatalité.

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1. La Remise en Question du Patriarcat comme Ordre Naturel

La perception commune présente la lutte pour les droits des femmes comme un combat sans fin contre un patriarcat qui serait une constante de l'histoire humaine. Cette vision postule une rébellion perpétuelle contre l'exclusion du pouvoir, le travail domestique non rémunéré et la violence.

Le documentaire remet fondamentalement en cause cette narration en posant la question centrale : « les femmes et les hommes n'ont-ils jamais été égaux ? ».

Il suggère que loin d'être une "loi naturelle", l'organisation patriarcale n'est qu'une des nombreuses façons dont les sociétés humaines ont structuré les relations de genre au fil du temps.

2. La Lutte Moderne pour l'Égalité : Le Cas de l'Islande

L'Islande est souvent citée comme un modèle de l'égalité des genres au 21e siècle, avec une égalité salariale inscrite dans la loi, un congé parental largement adopté par les pères, et des femmes aux plus hautes fonctions politiques. Cependant, cette situation est le résultat d'une lutte récente et intense.

• Le Contexte d'Inégalité : Dans les années 1970-80, la situation était radicalement différente.

L'anthropologue Sigridur Duna Christmunir, cofondatrice du premier parti féministe islandais en 1983, rapporte qu'à l'époque, les femmes gagnaient à peine 60 % du salaire de leurs collègues masculins.

Elle compare la frustration grandissante des femmes à une « éruption volcanique ».

• La Grève Historique du 24 octobre 1975 : Face à cette inégalité, 90 % des femmes islandaises ont refusé de travailler lors du « jour de vacances des femmes » (gena Frida Urine).

Cette grève concernait à la fois le travail rémunéré et les tâches domestiques (cuisine, garde d'enfants, ménage).

◦ Impact : La société a été « totalement paralysée », créant un « état d'urgence total ».

Sigridur Duna Christmunir se souvient :

« Je sentais l'odeur de la viande brûlée dans les rues. Les hommes faisaient la cuisine [...]. L'odeur de la viande brûlée me rappelle toujours cette journée. »

• Conséquences Politiques et Législatives : L'événement a provoqué une accélération spectaculaire des réformes :

◦ 1976 : Entrée en vigueur de la loi sur l'égalité salariale.  

◦ 1980 : Élection de Vigdís Finnbogadóttir, première femme au monde élue présidente démocratiquement.  

◦ Par la suite, l'entrée au parlement de la « Liste des femmes », dont faisait partie Sigridur Duna, a « révolutionné la politique islandaise ».

3. Relecture de l'Histoire : Des Vikings à la Préhistoire

L'analyse historique et archéologique révèle des indices d'organisations sociales non patriarcales, contredisant l'idée d'une domination masculine universelle.

A. Le Statut des Femmes Viking : Entre Mythe et Réalité

Les sagas et les découvertes archéologiques nuancent l'image d'une société viking strictement patriarcale.

• Droits et Autonomie : Les sagas du 13e siècle, comme la Saga de Laxdæla, dépeignent des femmes de la classe supérieure comme intelligentes et volontaires.

Le premier recueil de lois islandais, les Grágás, confirme que les femmes vikings pouvaient divorcer et, en tant que veuves, hériter et gérer leur propre fortune.

• Limites du Pouvoir : Ce statut ne s'appliquait pas à toutes.

Il concernait principalement l'élite et excluait les esclaves.

Surtout, les femmes n'avaient aucun pouvoir politique direct et n'avaient pas voix au chapitre au Þing, l'assemblée populaire. Leur influence était indirecte, via leurs liens avec des hommes puissants.

• La Guerrière de Birka : La découverte en 2017 que la tombe d'un guerrier viking de haut rang, découverte en 1878 en Suède, contenait en réalité le squelette d'une femme (prouvé par l'ADN) a forcé une réévaluation des préjugés sur les rôles de genre, illustrant comment les idées actuelles sont projetées sur le passé.

B. Indices d'Égalité dans les Sociétés Préhistoriques

L'archéologie préhistorique suggère fortement l'existence de sociétés égalitaires.

• Pratiques Funéraires : Dans les tombes somptueuses de l'Âge du Fer, des femmes étaient enterrées avec les mêmes trésors (chars, armes, bijoux) que les hommes, indiquant un statut social potentiellement égal dans la mort comme dans la vie.

• Le Cas de Çatalhöyük : Ce site anatolien, l'une des plus anciennes cités connues (9000 ans), offre des preuves frappantes.

L'analyse des résidus pulmonaires et des squelettes a montré que les hommes et les femmes passaient autant de temps à l'intérieur qu'à l'extérieur et que leur différence de taille était minime.

La journaliste scientifique Angela Saini, qui a étudié le site, rapporte la conclusion des archéologues : « dans les plus anciennes colonies humaines, les hommes et les femmes menaient à peu de choses près la même vie [...] sur un pied d'égalité ».

4. Le Débat sur le Matriarcat et la Matrilinéarité

Le concept de matriarcat est souvent mal interprété. L'anthropologie lui préfère le terme de société matrilinéaire pour décrire des modèles sociaux non patriarcaux.

• Critique du Concept de Matriarcat : L'archéologue Brigitte Röder considère les termes « matriarcat » et « patriarcat » comme des « catégories scientifiques non appropriées » car elles reposent sur un modèle binaire des genres, produit de la société bourgeoise du 18e siècle.

• La Théorie de Marija Gimbutas : Dans les années 70, l'archéologue Marija Gimbutas a postulé l'existence de cultures matriarcales pacifiques en Europe primitive, centrées sur le culte d'une déesse mère, qui auraient été détruites par des tribus de cavaliers patriarcaux.

Cette théorie a été critiquée pour son interprétation très libre des données archéologiques, de nombreux artefacts étant ambigus (la "déesse" pouvant être un phallus).

• Les Sociétés Matrilinéaires : Il existe des preuves de l'existence de plus de 160 cultures matrilinéaires, où la filiation, l'héritage et le statut social se transmettent par la mère.

◦ L'Exemple des Mosuo (Chine) : Ce groupe ethnique vivant autour du lac Lugu offre un exemple contemporain.      

▪ Organisation Sociale : La grand-mère est la chef de famille. Tous les membres de la lignée maternelle vivent ensemble. Les femmes gèrent les finances et les affaires importantes.       

▪ Relations et Filiation : Les hommes restent vivre dans la maison de leur mère.

Les relations amoureuses prennent la forme du « mariage par visite », où l'homme rend visite à la femme la nuit mais ne vit pas avec elle.

Le frère de la mère assume le rôle de père social pour les enfants.     

▪ Stabilité : Selon Jiong Zhidui, directeur du musée des Mosuo, ce modèle familial est « le plus stable qui soit », car l'homogénéité familiale limite les conflits.

5. L'Émergence et l'Imposition du Patriarcat

Le patriarcat ne s'est pas imposé comme une défaite unique et soudaine du genre féminin, mais comme un processus graduel et insidieux, étroitement lié à la naissance des États.

• Le Rôle Clé de l'État : L'émergence des premiers États en Mésopotamie (environ 5000 ans avant notre ère) a été un tournant.

La gestion de larges populations a nécessité un contrôle démographique et une organisation stricte de la société.

• La Codification des Rôles de Genre : Les élites étatiques ont établi une répartition claire des rôles (qui combat, qui s'occupe des enfants, qui travaille) et les ont inscrits dans des listes classées par genre.

Une fois ces différences « gravées dans le marbre », elles ont commencé à être perçues comme naturelles.

• Un Instrument de Contrôle : Le patriarcat est devenu un instrument efficace pour contrôler la population.

Comme le souligne Angela Saini : « Les systèmes de domination ne tirent pas seulement leur pouvoir de la force brute, ils déploient également leur puissance en imposant des idées ».

• L'Expansion Mondiale : Ce modèle s'est répandu à travers le monde par l'expansion des États, qui ont supplanté d'autres formes d'organisation sociale.

Les lois sur le mariage, le divorce et l'adultère sont devenues de plus en plus strictes pour les femmes, légitimant et solidifiant un ordre social qui avantageait une élite masculine au sommet du pouvoir.

6. Conclusion : L'Égalité, un Horizon Possible

L'analyse des différentes formes d'organisation sociale à travers l'histoire humaine mène à une conclusion fondamentale : il n'existe pas de forme "naturelle" de cohabitation entre hommes et femmes.

• La Mutabilité des Sociétés : La diversité des modèles observés prouve que les structures sociales sont des constructions culturelles et peuvent changer. Le patriarcat lui-même est une construction.

• Le Mécanisme du Patriarcat : Son ressort le plus efficace est de « monter les uns contre les autres et nous faire oublier que les sociétés peuvent changer ».

L'idée d'une opposition fondamentale entre hommes et femmes est un produit de ce système.

• Une Lutte Continue : Même dans un pays avancé comme l'Islande, des problèmes comme la violence domestique et la misogynie persistent.

Sigridur Duna Christmunir conclut : « Je me demande s'il y aura un jour une égalité parfaite quelque part. Peut-être n'est-ce qu'un mythe. Quoi qu'il en soit, il reste encore beaucoup à faire. »

• Regarder vers l'Avenir : Il n'est pas nécessaire de prouver l'existence d'un passé parfaitement égalitaire pour imaginer un futur égalitaire. Il suffit de comprendre que ce qui est considéré comme "normal" n'est pas immuable.

La lutte pour les droits des femmes appartient au présent.

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Le Programme pHARe : Stratégie et Mise en Œuvre de la Lutte Contre le Harcèlement Scolaire

Synthèse

Ce document présente une analyse exhaustive de la politique française de lutte contre le harcèlement scolaire, axée sur le programme pHARe.

Initié à titre expérimental en 2019 et renforcé par le plan interministériel de septembre 2023, le programme pHARe constitue une réponse systémique et globale déployée de l'école primaire au lycée.

Il s'articule autour de trois ambitions majeures : la prévention, la détection et l'apport de solutions concrètes.

La stratégie repose sur une "responsabilité collective" mobilisant l'ensemble de la communauté éducative : personnels, élèves et parents.

Les données issues d'une enquête annuelle à grande échelle révèlent que si le harcèlement au sens strict concerne 3 à 5 % des élèves, les situations de vulnérabilité et de violences répétées touchent une part bien plus large, atteignant jusqu'à 20 % et 30 % des élèves respectivement.

Les piliers du programme pHARe incluent la formation de l'ensemble des personnels, la mise en place d'équipes ressources spécialisées, le déploiement de plus de 120 000 élèves ambassadeurs et l'organisation d'un questionnaire annuel pour tous les élèves du CE2 à la terminale.

Une nouveauté majeure permet désormais aux élèves de renseigner leur identité sur ce questionnaire pour faciliter une prise en charge directe.

L'implication des parents est un axe stratégique, évoluant d'une simple information à une participation active via des ateliers de sensibilisation et le nouveau dispositif de parents ambassadeurs, visant à renforcer la prévention et le dialogue.

De multiples ressources, telles que la plateforme en ligne "Des clés pour les familles", les protocoles de traitement des situations et le numéro national 30 18, sont mises à disposition pour outiller chaque acteur.

L'objectif final est de construire une "alliance éducative" solide pour garantir un climat scolaire sécurisant, condition essentielle à l'épanouissement et aux apprentissages de chaque élève.

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1. Contexte et Ampleur du Phénomène de Harcèlement

La politique de lutte contre le harcèlement scolaire s'inscrit dans une démarche de longue haleine, mais a connu une accélération significative face à un phénomène perçu comme "s'approfondissant".

• Historique et Cadre Politique : Le programme pHARe a été lancé à titre expérimental dès 2019.

La politique a été renforcée et dotée de moyens nouveaux par le plan interministériel de septembre 2023, structuré autour de trois axes : prévention, détection et solutions.

Cette politique s'intègre dans une vision plus large de la protection de la santé physique et psychique des élèves, considérée par le ministère comme l'un des deux piliers de l'école, avec l'instruction.

• Mesure du Phénomène : Pour mieux connaître et combattre le harcèlement, le ministère s'appuie sur une enquête annuelle d'envergure menée par la DEPP (Direction de l'évaluation, de la prospective et de la performance) auprès de plus de 30 000 élèves, du CE2 à la terminale.

Données Clés sur le Harcèlement Scolaire

Catégorie de Harcèlement

Population Concernée

Taux

Harcèlement au sens strict

Écoliers

3 %

Collégiens

5 %

Lycéens

3 %

Situations de vulnérabilité ou de fragilité

Écoliers

Près de 20 % (17 % spécifiquement mentionné)

Violences répétées (insultes, etc.)

Tous niveaux

Jusqu'à 30 % des élèves (victimes d'au moins deux types de violence plusieurs fois dans l'année)

Le ministère adopte une "vision extensive du phénomène", considérant non seulement le harcèlement strict mais aussi toutes les formes de violence et de mal-être pour calibrer son action.

2. Le Programme pHARe : Une Approche Structurée et Globale

L'objectif central du programme pHARe est de doter chaque école, collège et lycée d'un plan de prévention du harcèlement structuré et efficient.

Il repose sur la mobilisation de tous les acteurs et se décline à travers un système de labellisation progressif.

2.1. Les Piliers du Programme

1. Formation des Adultes : Formation de l'ensemble des personnels pour repérer les signaux faibles, comprendre les mécanismes du harcèlement et savoir prendre en charge les situations.

2. Sensibilisation des Élèves : Organisation de séances de sensibilisation pour tous les élèves, afin qu'ils comprennent ce qu'est le harcèlement et comment réagir.

3. Élèves Ambassadeurs : Au collège et au lycée, des élèves volontaires sont formés et encadrés pour être des relais attentifs auprès de leurs pairs et mener des actions de prévention.

4. Implication des Parents : Les parents sont considérés comme des partenaires essentiels, avec une implication croissante à chaque niveau du programme.

2.2. Le Système de Labellisation

L'engagement des établissements est structuré par un label à trois niveaux, qui vient récompenser leur degré d'implication.

Niveau de Label

Exigences Clés

Statut

Niveau 1

- Constitution d'une équipe ressource formée (au niveau de la circonscription pour le primaire, de l'établissement pour le secondaire).<br>\

• Participation à la journée nationale (9 novembre) avec passation du questionnaire annuel par tous les élèves (CE2-Terminale).<br>\

• Information des parents sur le programme.<br>- Mise en place d'élèves ambassadeurs (secondaire).

Obligatoire pour 100% des écoles et établissements. Environ 80% sont officiellement dans ce niveau via la plateforme de suivi.

Niveau 2

Inclut les critères du niveau 1 et ajoute l'organisation d'un atelier de sensibilisation à destination des parents sur une thématique liée au harcèlement.

Volontaire

Niveau 3

Inclut les critères des niveaux 1 et 2 et ajoute la mise en place du dispositif de parents ambassadeurs.

Volontaire

3. Les Acteurs Clés et Leurs Rôles

La réussite du programme repose sur une répartition claire des rôles et une collaboration active entre les différents acteurs.

3.1. Les Équipes Ressources et les Coordinateurs

Dans chaque collège et lycée, un coordinateur pHARe est nommé par le chef d'établissement.

Il est chargé de piloter l'équipe ressource, composée de 5 personnes formées, et de déployer l'ensemble des actions du programme.

Pour le premier degré, cette équipe est mutualisée au niveau de la circonscription.

Ces équipes sont les expertes du traitement des situations et suivent un protocole précis.

3.2. Les Élèves Ambassadeurs

• Nombre : Plus de 120 000 élèves ambassadeurs sont actifs dans les collèges et lycées.

• Sélection : Ils sont choisis sur la base du volontariat.

• Rôle : Formés et encadrés par des adultes, leur mission est d'être attentifs à leurs pairs, de relayer les situations préoccupantes aux adultes et de mener des actions de sensibilisation.

• Visibilité : Leur identité est connue de tous les élèves via des trombinoscopes, des badges ou des présentations en classe pour qu'ils soient facilement identifiables.

3.3. Les Parents Ambassadeurs

Ce dispositif, correspondant au niveau 3 de la labellisation, est un axe de développement prioritaire.

• Initiative : La démarche est initiée par l'établissement, en concertation avec les parents.

• Rôle : Leur mission n'est pas de résoudre les situations de harcèlement, ce qui reste la responsabilité de l'établissement. Leur rôle est centré sur la prévention :

◦ Sensibiliser les autres familles.   

◦ Aider à identifier les signes de harcèlement.  

◦ Orienter les parents vers les bons interlocuteurs.    ◦ Promouvoir une communication constructive avec l'établissement.

• Cadre : Une "charte d'engagement mutuel" formalise la relation de confiance entre les parents ambassadeurs et l'établissement. Il n'est pas nécessaire d'être un parent élu pour devenir parent ambassadeur.

4. Outils et Ressources Pratiques

Un ensemble d'outils concrets est déployé pour soutenir la politique de lutte contre le harcèlement.

• Le Questionnaire Annuel : Passé par tous les élèves du CE2 à la terminale entre le 6 et le 21 novembre.

Depuis cette année, il offre la possibilité aux élèves d'inscrire leur nom et prénom pour permettre une aide plus directe et rapide.

• Les Protocoles de Traitement : Des documents méthodologiques "pas à pas" sont fournis aux personnels pour les guider depuis le signalement d'une situation jusqu'à sa résolution.

Ces protocoles sont publics et téléchargeables sur le site du ministère, garantissant la transparence de la démarche. La politique est qu'« aucune situation ne doit rester sans réponse ».

• Plateforme "non au harcèlement - des clés pour les familles" : Créée avec le CNED, cette plateforme propose un parcours d'auto-formation gratuit d'une heure en quatre modules.

Elle explique le phénomène du harcèlement et les actions mises en œuvre dans les établissements.

• Site Ministériel (education.gouv.fr) : Centralise les informations institutionnelles, les campagnes de communication (comme le clip annuel "tous différents, jamais indifférent"), et les coordonnées des lignes d'assistance académiques.

• Le Numéro 30 18 : Plateforme nationale gratuite et confidentielle, ouverte 7j/7 de 9h à 23h.

Gérée par l'association e-Enfance, elle offre une écoute, des conseils et, si nécessaire, transmet les signalements de harcèlement scolaire aux responsables académiques qui saisissent l'établissement concerné.

5. Recommandations Pratiques pour les Parents

Comment Signaler une Situation

La chaîne de signalement recommandée est la suivante :

1. Contact Direct avec l'Établissement : C'est le premier et principal interlocuteur.

Les parents doivent s'adresser à l'équipe de direction, au coordinateur pHARe, ou à tout adulte de confiance au sein de l'école ou de l'établissement.

2. Lignes d'Assistance Académiques : Si le contact direct est difficile ou n'aboutit pas, chaque académie dispose d'une ligne téléphonique dédiée dont les numéros sont disponibles sur les sites du ministère et des académies.

3. Le 30 18 : En dernier recours ou pour un conseil extérieur, ce numéro national prend en charge le signalement et assure le relais vers l'Éducation nationale.

Suivi du Protocole

Une fois un signalement effectué, le protocole est déclenché rapidement.

L'établissement assure la mise en protection de l'élève victime et engage un dialogue avec toutes les parties concernées.

Les parents sont tenus informés de la mise en œuvre du protocole par l'équipe qui prend en charge la situation, typiquement le coordinateur pHARe.

Devenir Parent Ambassadeur

Pour devenir parent ambassadeur, il faut se rapprocher de la direction de l'établissement de son enfant pour savoir si la démarche est engagée ou pour proposer de l'initier.

Le processus repose sur le volontariat et une discussion avec l'équipe de direction pour s'accorder sur les objectifs et les modalités, formalisés par la charte d'engagement.

Notre capacité de concentration : Déclin ou Adaptation ?

Résumé

Ce document de synthèse analyse l'état actuel de la capacité de concentration humaine à l'ère numérique, en se basant sur des perspectives historiques, psychologiques et neuroscientifiques.

Loin de l'idée répandue d'un déclin généralisé, les données suggèrent une adaptation profonde de notre cerveau aux nouvelles exigences environnementales.

La capacité attentionnelle fondamentale, soit la faculté de traiter simultanément un nombre limité d'informations (entre un et quatre éléments), demeure stable depuis les années 1960.

Les tests objectifs montrent même une amélioration de la performance en attention sélective au cours des dernières décennies.

La découverte centrale est que l'attention n'est pas un état constant, mais un processus rythmique et oscillatoire.

Notre cerveau alterne à une fréquence très élevée (toutes les 250 millisecondes) entre un état de concentration sensorielle intense et un état moteur, plus propice à l'action et à la distraction.

Ce mécanisme, hérité d'une évolution de plus de 22 millions d'années, confère une flexibilité cognitive essentielle.

L'environnement numérique, avec son flux constant de notifications et de contenus, n'a pas détruit notre capacité de concentration mais a favorisé le développement de nouvelles compétences, comme le passage rapide d'une tâche à l'autre et un filtrage plus efficace de l'information.

La véritable question n'est donc pas celle d'une perte de capacité, mais celle de l'autodétermination : qui, ou quoi, contrôle notre attention ?

La capacité à maintenir une concentration prolongée n'est pas perdue ; elle peut être réapprise et renforcée par un entraînement ciblé, démontrant la plasticité continue de notre cerveau.

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1. Le Mythe du Déclin de l'Attention

L'idée que notre capacité de concentration se dégrade est une préoccupation récurrente, mais elle manque de fondement scientifique solide.

• Une anxiété historique : Le débat sur la concentration n'est pas nouveau.

Il a émergé au 19ème siècle avec l'industrialisation, qui exigeait une attention soutenue pour maximiser la productivité et la sécurité.

La psychologie naissante s'est alors emparée de l'étude de l'attention pour optimiser le recrutement de la main-d'œuvre.

• La fable du poisson rouge : En 2015, une affirmation largement relayée prétendait que la durée d'attention humaine (8 secondes) était devenue inférieure à celle d'un poisson rouge (9 secondes).

Cette donnée provient d'une étude de Microsoft mesurant le temps passé sur une page web.

Plutôt qu'une dégradation, ce chiffre peut indiquer une amélioration de notre efficacité à filtrer l'information en ligne.

Comme le souligne le document, "être attentif c'est sélectionner l'information".

• Les paniques morales : Chaque nouvelle technologie a suscité des craintes similaires.

Au 18ème siècle, le roman était jugé dangereux ; au 20ème, le cinéma.

Aujourd'hui, les réseaux sociaux et le streaming sont les boucs émissaires.

2. La Nature Fondamentale de la Concentration

Les mécanismes de base de notre attention sont bien étudiés et révèlent une capacité stable et multifactorielle.

• Une capacité de base stable : Des tests de laboratoire, reproduits régulièrement depuis les années 1960, démontrent que notre capacité attentionnelle fondamentale est limitée et stable.

Nous pouvons nous concentrer sur un à quatre éléments simultanément, selon leur complexité.

• Les deux fonctions essentielles : L'attention remplit un double rôle crucial :

1. Traitement sélectif : Focaliser nos ressources cognitives sur l'information pertinente.   

2. Filtrage : Occulter les stimuli parasites, qu'ils soient externes (bruits, lumières) ou internes (pensées, émotions).

• Les conditions de l'état de "Flow" : Le psychologue Mihaly Csikszentmihalyi a décrit le "flow" comme un état de concentration totale et sans effort, où l'on est absorbé par une tâche qui procure satisfaction.

Cet état optimal est atteint lorsque la difficulté d'une tâche est parfaitement équilibrée :

◦ Ni trop facile : pour éviter l'ennui et la divagation des pensées.  

◦ Ni trop difficile : pour éviter le sentiment d'être dépassé et l'abandon.   

◦ La motivation intrinsèque est également une composante essentielle.

3. Le Rythme Caché de notre Cerveau

Des recherches récentes révèlent que l'attention est un processus dynamique et non un état statique.

• Une oscillation permanente : L'attention n'est pas uniforme. Elle suit un rythme ondulatoire rapide. Des expériences montrent qu'elle "croit et décroit" en permanence.

• L'alternance Sensoriel/Moteur : Notre cerveau alterne constamment entre deux états à une fréquence d'environ 250 millisecondes :

◦ État sensoriel : Un pic de concentration, où nous sommes plus focalisés et absorbons plus d'informations.  

◦ État moteur : Un creux où notre système moteur est plus actif, nous rendant plus facilement distraits mais aussi plus prompts à l'action.

• Une flexibilité cognitive évolutive : Ce rythme est un mécanisme évolutif fondamental, retrouvé chez les macaques, ce qui suggère une origine remontant à au moins 22 millions d'années.

Cette "alternance attention-action" nous permet à la fois de nous concentrer intensément et de réagir rapidement à de nouvelles informations pertinentes.

La distraction est donc une composante intrinsèque de la concentration ; elles sont "les deux faces d'une même pièce".

• L'illusion de la maîtrise totale : L'idée que l'attention est un acte purement volontaire est une illusion.

L'effet "cocktail party" illustre que des informations subjectivement pertinentes (comme notre prénom) peuvent percer notre filtre attentionnel de manière quasi-automatique, redirigeant notre "projecteur" attentionnel.

4. L'Adaptation à l'Ère Numérique

Contrairement aux idées reçues, les données objectives ne soutiennent pas une thèse de dégradation, mais plutôt celle d'une adaptation.

• Une performance en hausse : Une méta-analyse menée entre 1990 et 2021 sur le test d'attention D2 (un test standardisé d'attention sélective) a révélé que la performance moyenne des participants a augmenté au fil des ans.

Cela indique qu'il n'y a "aucune raison de basculer dans le catastrophisme".

• De nouvelles compétences : L'environnement numérique agit comme un entraînement intensif pour certaines facultés :

◦ Les utilisateurs de médias numériques et les joueurs de jeux vidéo développent une grande habileté à passer rapidement d'une tâche à l'autre.   

◦ Ils affinent leur capacité à détecter les signaux pertinents (visuels, textuels).   

◦ Il s'agit d'un "gain, une adaptation nécessaire de notre cerveau à ce qu'il doit faire à un moment donné".

• Les défis de l'environnement moderne : Si notre capacité de base n'a pas diminué, le contexte a changé.

◦ L'effet "Brain Drain" : La simple présence d'un smartphone peut réduire la capacité de concentration et de mémorisation disponible.   

◦ Des alternatives attractives : Les médias numériques offrent des distractions puissantes, particulièrement alléchantes lorsque nous sommes confrontés à des tâches routinières ou ennuyeuses.

5. Le Spectre de l'Attention et la Question du Pouvoir

La discussion sur la concentration dépasse la simple mesure de performance pour toucher à des questions de neurodéveloppement et de contrôle personnel.

• Les extrêmes du spectre : Les troubles de l'attention (TDAH) peuvent être compris comme une défaillance du cycle rythmique de l'attention.

◦ L'hyperactivité : Les individus sont bloqués dans le "creux" du rythme, l'état moteur, passant constamment d'une activité à l'autre.   

◦ L'hyperfixation : Les individus sont bloqués dans le "pic" du rythme, l'état sensoriel, incapables de se détacher de leur objet de concentration.  

◦ L'attention est qualifiée de "mère de toutes les fonctions cognitives", et ses défaillances ont des impacts dramatiques.

• La question de l'autodétermination : Le véritable enjeu contemporain n'est pas la capacité, mais le contrôle.

• La possibilité de réapprentissage : La capacité de concentration prolongée n'est pas perdue, mais simplement moins sollicitée.

Elle peut être réentraînée. Des activités comme lire un livre ou apprendre un instrument de musique permettent de réapprendre à maintenir son attention.

Cela "demandera beaucoup de travail et d'entraînement, mais ce n'est pas perdu pour toujours".

Conclusion

Notre capacité de concentration n'a pas diminué ; elle a évolué pour s'adapter à un monde hyper-connecté.

Le discours alarmiste ignore la remarquable plasticité de notre cerveau et les nouvelles compétences que nous développons.

Le monde moderne n'est "ni mieux ni pire", il est simplement "différent".

Le défi pour chacun est de devenir plus conscient et volontaire dans la gestion de cette ressource précieuse, en trouvant un équilibre personnel entre les sollicitations externes et les objectifs internes.

La question fondamentale qui demeure est : à quoi choisissons-nous d'accorder notre attention ?

Synthèse des Expériences sur les Préjugés et le Racisme Inconscient

Résumé

Ce document de synthèse analyse une émission d'investigation sociale qui, à travers une série d'expériences en caméra cachée, démontre comment les préjugés et les stéréotypes raciaux influencent de manière inconsciente les comportements, les jugements et même la perception de la réalité.

Cinquante participants, croyant participer à une émission sur "les mystères de notre cerveau", sont confrontés à des situations de la vie quotidienne conçues pour révéler des biais automatiques.

Les résultats sont unanimes : des mécanismes cognitifs comme la catégorisation sociale poussent les individus à privilégier la similarité, à juger plus sévèrement les minorités visibles, et à percevoir une menace accrue en leur présence.

Les expériences révèlent également que ces biais sont acquis dès l'enfance et peuvent mener à une internalisation des stéréotypes par les groupes minoritaires eux-mêmes.

Le contexte s'avère crucial, capable d'atténuer ou de renforcer les stéréotypes.

Finalement, l'émission conclut que si ces mécanismes sont universels, la prise de conscience, l'éducation et la rencontre avec l'autre sont des leviers puissants pour les déconstruire, rappelant que ce qui rassemble les êtres humains est fondamentalement plus fort que ce qui les divise.

1. Dispositif Expérimental et Concepts Fondamentaux

L'émission met en scène 50 volontaires qui ignorent le véritable sujet de l'étude : le racisme.

Le faux titre, "Les mystères de notre cerveau", vise à garantir la spontanéité de leurs réactions.

Leurs comportements sont observés et analysés par la présentatrice Marie Drucker, le comédien et réalisateur Lucien Jean-Baptiste, et le psychosociologue Sylvain Delouvée.

L'analyse repose sur plusieurs concepts clés de la psychologie sociale :

• La Catégorisation Sociale : Mécanisme mental naturel et "paresseux" par lequel le cerveau classe les individus en groupes (hommes/femmes, jeunes/vieux, noirs/blancs) pour simplifier la complexité du monde.

Ce processus entraîne une perception accrue des ressemblances au sein de son propre groupe ("nous") et des différences avec les autres groupes ("eux"), pouvant générer méfiance et rejet.

• Le Stéréotype : Défini comme "un ensemble d'idées préconçues que l'on va appliquer à un individu du simple fait de son appartenance à un groupe."

Les stéréotypes ont un caractère automatique et sont intégrés culturellement (médias, éducation, etc.).

• Le Préjugé : C'est l'attitude, positive ou négative, que l'on développe envers un groupe sur la base de stéréotypes.

• La Discrimination : Le comportement qui découle des préjugés, comme le fait d'écarter une personne d'un emploi ou d'un logement.

Sylvain Delouvée souligne que "toutes les expériences que nous allons voir s'appuient sur des études scientifiques parfaitement documentées" et que les mécanismes étudiés (misogynie, sexisme, homophobie, etc.) reposent sur les mêmes fondements.

2. Le Biais de Similarité et le Jugement Spontané

Les premières expériences démontrent une tendance instinctive à favoriser les individus qui nous ressemblent et à porter des jugements hâtifs basés sur l'apparence physique.

Expérience 1 : La Salle d'Attente

• Dispositif : Les participants entrent un par un dans une salle d'attente où sont assis deux complices, un homme noir (Jean-Philippe) et un homme blanc (Florian), habillés identiquement. Une chaise vide est disponible de chaque côté.

• Résultats : La quasi-totalité des participants choisit de s'asseoir à côté de l'homme blanc.

Même lorsque les complices échangent leurs places pour éliminer un biais lié à la configuration de la pièce, le résultat reste le même.

• Analyse : Selon Sylvain Delouvée, ce comportement n'est pas "raciste en tant que tel" mais relève d'une recherche de similarité.

"On va chercher les gens qui nous ressemblent."

C'est un mécanisme presque "reptilien", hérité des tribus primitives qui se méfiaient de la différence.

Lucien Jean-Baptiste souligne les conséquences dramatiques de ce biais dans des contextes comme "l'accès au logement" ou la recherche d'emploi.

Expérience 2 : Le Procès Fictif

• Dispositif : Les participants agissent en tant que jurés et doivent attribuer une peine de prison (de 3 à 15 ans) à un accusé pour "coups et blessures volontaires ayant entraîné la mort sans l'intention de la donner".

Le crime et le contexte sont identiques pour tous, mais la moitié des participants juge un accusé blanc, l'autre moitié un accusé d'origine maghrébine.

• Résultats : L'accusé d'origine maghrébine écope en moyenne d'une peine de prison plus lourde.

Fait marquant, les participants ont été 5 fois plus nombreux à lui infliger la peine maximale de 15 ans.

• Analyse : Les commentaires des participants révèlent leurs biais : "Il a une bonne tête, il n'a pas l'air d'être violent" pour l'accusé blanc ; "Il n'y a pas de perpétuité ?" pour l'accusé maghrébin.

Delouvée explique que ce jugement est influencé par un "fameux biais intégré" via la culture et les médias, qui associent certaines catégories de personnes à la délinquance.

3. La Perception de la Menace et de la Culpabilité

Les expériences suivantes illustrent comment les stéréotypes raciaux activent automatiquement une perception de danger ou de culpabilité, menant à des réactions discriminatoires.

Expérience 3 : Le Vol de Vélo

• Dispositif : En caméra cachée dans la rue, trois comédiens (un homme blanc, Johan ; un homme d'origine maghrébine, Bachir ; une jeune femme blonde, Urielle) scient tour à tour l'antivol d'un vélo.

• Résultats :

◦ Johan (blanc) : Les passants sont indifférents ou bienveillants. Une femme lui dit même qu'il a "une tête de type honnête".  

◦ Bachir (maghrébin) : Les réactions sont immédiates et hostiles ("C'est pas bien, de faire ça").

Les passants l'interpellent et appellent la police, qui intervient réellement, forçant l'équipe de tournage à s'interposer.  

◦ **Urielle (blonde) :

** Plusieurs hommes s'arrêtent spontanément pour lui proposer leur aide, sans jamais remettre en question la propriété du vélo.

• Analyse : Cette expérience démontre un comportement discriminatoire flagrant.

Le stéréotype s'active automatiquement ("fait-il partie de mon groupe ?"), entraîne un préjugé ("j'ai confiance ou non") et déclenche une action (l'appel à la police).

Lucien Jean-Baptiste témoigne : "Il m'est arrivé combien de fois de rentrer dans des halls d'immeuble et qu'on me demande : 'Qu'est-ce que vous faites là ?'".

Expérience 4 : Le Laser Game (Le Biais du Tireur)

• Dispositif : Les participants, armés d'un pistolet de laser game, doivent neutraliser le plus rapidement possible des figurants armés qui surgissent, tout en évitant de tirer sur ceux qui tiennent un téléphone.

Les figurants sont de différentes origines (blancs, noirs, maghrébins).

• Résultats :

1. Les participants ont tiré près de 4 fois plus sur les figurants désarmés noirs ou d'origine maghrébine que sur les figurants désarmés blancs.    

1. Face à un dilemme où un homme blanc et un homme maghrébin surgissent simultanément armés, ils ont été 4 fois plus nombreux à tirer en priorité sur le figurant d'origine maghrébine.

• Analyse : Cette expérience, inspirée de recherches sur les forces de police américaines, illustre le "biais du tireur".

Elle ne signifie pas que les participants sont racistes, mais met en évidence "l'ancrage fort et automatique d'un stéréotype".

Face à une situation menaçante, le cerveau s'accroche aux stéréotypes pour agir, percevant la scène comme "encore plus menaçante qu'elle ne l'est".

4. La Genèse des Préjugés chez l'Enfant

Ces expériences démontrent que les stéréotypes raciaux sont absorbés et intégrés très tôt, non pas de manière innée, mais par observation et modélisation du monde adulte.

Expérience 5 : Les Marionnettes

• Dispositif : Des enfants de 5 à 6 ans assistent à un spectacle de marionnettes où le goûter de Vanessa a été volé. Deux suspects leur sont présentés : Kevin (blanc) et Moussa (noir).

On demande aux enfants de désigner le coupable.

• Résultats : Une majorité d'enfants désigne spontanément Moussa comme le voleur le plus probable.

• Analyse : "Ça commence très tôt", réagit Lucien Jean-Baptiste.

Delouvée précise que cela "ne prouve pas que les enfants sont enclins naturellement à la discrimination" mais qu'ils sont très sensibles aux normes sociales et "incorporent les stéréotypes, les préjugés de leur entourage".

Expérience 6 : Le Test de la Poupée

• Dispositif : L'émission présente les résultats d'une réplication du célèbre test des psychologues Kenneth et Mamie Clark (années 1940), issue du documentaire "Noirs en France".

On présente à de jeunes enfants, y compris des enfants noirs, une poupée blanche et une poupée noire et on leur pose des questions ("Quelle est la plus jolie ?", "La moins jolie ?").

• Résultats : Les enfants, y compris les enfants noirs, désignent majoritairement la poupée blanche comme la plus jolie et la poupée noire comme la moins jolie. Une petite fille noire déclare :

"Parce qu'elle est noire... quand je serai grande, je mettrai de la crème pour devenir blanche."

• Analyse : Ce test illustre tragiquement l'internalisation du stéréotype, où les membres d'un groupe minoritaire finissent par incorporer les préjugés négatifs qui leur sont attribués.

Le résultat, constant à travers les décennies, montre la puissance des modèles culturels et de l'entourage.

5. Stéréotypes, Contexte et Raccourcis Cognitifs

Cette section regroupe des expériences montrant comment les stéréotypes fonctionnent comme des raccourcis mentaux, comment le contexte peut les moduler et comment même les préjugés "positifs" sont problématiques.

Expérience 7 : La Reconnaissance des Visages ("Ils se ressemblent tous")

• Dispositif : Six comédiens (quatre blancs, deux asiatiques) jouent une courte scène.

Les participants doivent ensuite réattribuer chaque réplique au bon comédien via une application.

• Résultats : Les participants ont fait quasiment deux fois plus d'erreurs en attribuant les répliques aux comédiens d'origine asiatique qu'aux comédiens blancs.

• Analyse : Ce phénomène illustre que le cerveau perçoit moins les différences "intracatégorielles" pour les groupes qui ne sont pas le nôtre.

Comme l'explique Delouvée, "à partir du moment où nous catégorisons les individus en groupe, ce biais apparaît, cette tendance à voir les membres d'un groupe qui n'est pas le nôtre comme se ressemblant."

Expérience 8 : Les Accents des Conférenciers

• Dispositif : Trois groupes de participants assistent à la même conférence sur l'IA, mais donnée par trois "experts" différents.

1. Groupe 1 : Un comédien blanc prenant un fort accent allemand.    

1. Groupe 2 : Le même comédien prenant un accent marseillais.    

2. Groupe 3 : Un véritable professeur d'université noir, M. Diallo.

• Résultats :

◦ Accent allemand : Jugé "très compétent", "sérieux", mais "moyennement chaleureux".   

◦ Accent marseillais : Jugé "moins compétent", "pas convaincant", mais "sympathique" et "très chaleureux".    ◦ Professeur noir :

Les participants sont perplexes, peinent à le qualifier et expriment des doutes sur sa légitimité ("Pour moi, il s'agit d'un comédien").

• Analyse : L'accent active un stéréotype qui devient le critère principal de jugement.

L'Allemand est perçu comme rigoureux, le Marseillais comme sympathique mais peu sérieux.

Le professeur noir, lui, ne correspond à aucun stéréotype clair dans l'esprit des participants, ce qui crée une dissonance cognitive.

Le fait qu'il soit le seul véritable expert est la conclusion ironique de l'expérience.

Expérience 9 : Les Sprinteurs (Le Préjugé Positif)

• Dispositif : On demande aux participants qui, d'un sprinteur noir ou blanc, a le plus de chances de gagner une course.

• Résultats : Une majorité répond le sprinteur noir, se basant sur le cliché "les Noirs courent plus vite".

• Analyse : L'émission déconstruit ce stéréotype, expliquant qu'il n'a aucune base scientifique fiable.

Sa persistance est liée à des facteurs historiques (le corps noir associé au labeur physique durant l'esclavage) et socio-culturels (le sport comme l'un des rares modèles de réussite pour les jeunes noirs).

Delouvée qualifie ce type de croyance de "préjugé positif très problématique", car il "retire le mérite aux coureurs noirs de gagner", réduisant leur succès à une essence biologique plutôt qu'à leur travail.

Expérience 10 : L'Association de Mots (Le Rôle du Contexte)

• Dispositif : Trois groupes voient une photo d'une même femme asiatique dans trois contextes différents et doivent donner le premier mot qui leur vient à l'esprit.

1. Photo 1 : Mangeant avec des baguettes.  

2. Photo 2 : Se maquillant.  

3. Photo 3 : Portant une blouse blanche avec un stéthoscope.

• Résultats :

◦ Photo 1 : Les réponses évoquent l'origine ("Asie", "sushi", "femme asiatique").   

◦ Photo 2 : Les réponses évoquent la féminité ("maquillage", "rouge à lèvres", "belle femme").  

◦ Photo 3 : Les réponses évoquent la profession ("médecin", "infirmière", "hôpital").

• Analyse : L'expérience démontre que le contexte est capable d'effacer ou de renforcer un stéréotype.

Lorsque le contexte fournit une information plus saillante (le métier, la féminité), l'origine ethnique passe au second plan.

6. L'Impact Neurologique et Mémoriel des Préjugés

Ces expériences finales explorent les fondements biologiques et cognitifs des préjugés, montrant comment ils peuvent altérer l'empathie et même réécrire les souvenirs.

Expérience 11 : L'Empathie et la Douleur

• Dispositif : L'émission rapporte une étude neurologique où l'on mesure la réaction cérébrale de sujets (blancs et noirs) regardant une main se faire piquer par une aiguille.

• Résultats :

◦ Le cerveau d'un sujet blanc réagit (empathie, "freezing") en voyant une main blanche se faire piquer, mais pas en voyant une main noire.   

◦ Inversement, le cerveau d'un sujet noir réagit à la douleur d'une main noire, mais pas d'une main blanche.   

◦ Étonnamment, quand la main est de couleur violette (un groupe pour lequel aucun préjugé n'existe), les cerveaux des sujets blancs et noirs réagissent tous les deux avec empathie.

• Analyse : C'est la seule expérience basée sur la neurologie. Elle révèle que "nos préjugés effacent notre empathie à l'égard de personnes différentes de nous".

Le cerveau est plastique, et c'est "par la rencontre, l'éducation" que l'on peut développer une empathie plus universelle.

Expérience 12 : La Photo Contre-Stéréotypique et le Bouche-à-Oreille

• Dispositif : Les participants observent une photo de rue où un homme d'origine maghrébine donne une pièce à un homme blanc faisant la manche.

Puis, on teste leur mémoire.

Dans un second temps, une chaîne de bouche-à-oreille est créée pour voir comment l'information se transmet.

• Résultats :

1. Test de mémoire : Près de la moitié des participants décrivent la scène en inversant les rôles, affirmant avoir vu un homme blanc donner de l'argent à un SDF maghrébin.

Un participant, décrivant la scène correctement, la qualifie de "très perturbante".   

2. Bouche-à-oreille : Même lorsque la première personne décrit la scène correctement, l'information se déforme rapidement au fil de la transmission.

Les rôles s'inversent, et la scène d'aumône se transforme même en "une altercation".

• Analyse : La photo est "contre-stéréotypique" : elle contredit les attentes du cerveau.

Pour simplifier, le cerveau "corrige" la réalité pour la faire correspondre au stéréotype (le Maghrébin en situation de précarité).

L'expérience du bouche-à-oreille, basée sur une étude classique sur les rumeurs (Allport & Postman, 1940), montre comment "nos croyances et stéréotypes nous permettent de lire cette scène" et de la transformer.

7. Révélation Finale et Humanité Partagée

À la fin de la journée, le véritable titre de l'émission, "Sommes-nous tous racistes ?", est révélé aux participants, provoquant choc et prise de conscience.

L'objectif, leur explique-t-on, n'était pas de juger mais de montrer que "nous avons toutes et tous les mêmes mécanismes qui se déclenchent dans nos têtes".

L'ultime expérience vise à déconstruire les divisions.

Répartis en groupes de couleurs distinctes, les participants sont invités à avancer au centre s'ils se sentent concernés par une série de questions, allant du léger ("Qui a déjà revendu des cadeaux de Noël ?") au profondément intime.

• "Qui, parmi vous, se sent très seul ?" Plusieurs personnes, de groupes différents, se rejoignent au centre, partageant une vulnérabilité commune.

• "Qui, parmi vous, a été harcelé pendant sa scolarité ?"

Un grand nombre de participants avancent, partageant des témoignages émouvants sur le harcèlement lié à la couleur de peau ou à d'autres différences.

Cette dernière séquence démontre visuellement que malgré les appartenances à des groupes différents, les expériences humaines fondamentales (joie, amour, solitude, souffrance) sont partagées.

La conclusion de l'émission est un appel à la reconnaissance de cette humanité commune :

"Ce qui nous rassemble est toujours plus fort que ce qui nous divise."

Étant donnée que l'opposition est souvent écrite de la façon suivante :

"symétrie / asymétrie"

Est-ce qu'on ne devrait pas l'uniformiser dans le titre aussi ?

Est-ce que la section de la bibliographie est en général considérée comme un titre de niveau 2, ou bien est-ce qu'il y a une façon spécifique de la baliser ?

Titre de niveau 3 ?

Titre de niveau 3 ?

Titre de niveau 3 ?

Titre de niveau 3 ?

Titre de niveau 3 ?

Généralement, on ajoute la virgule avant la conjonction :

"...formelle, mais"

Ici, ça dépend du sens voulu. Est-ce qu'on parle des débats de nomination en général ? Auquel cas, il faudrait écrire ainsi :

"...un grand nombre de débats de nomination"

Ou bien, est-ce qu'on parle d'un ensemble plus spécifique des débats de nomination ? Auquel cas, on garde la formulation actuelle.

Il semble y avoir une rupture dans la syntaxe de la phrase. Est-ce que le propos original se rapprochait de ceci :

"...les adversaires de l'interdiction"

Ce titre ne devrait-il pas être balisé comme un titre (de niveau 2) ? À date, le texte a seulement la caractéristique d'être en gras.

Les AESH : Pilier Méconnu et Précaire de l'École Inclusive

Résumé Exécutif

Ce document de synthèse analyse les conditions de travail, le rôle et le manque de reconnaissance des Accompagnants d'Élèves en Situation de Handicap (AESH), un métier jugé indispensable au projet de l'école inclusive en France.

Il ressort une tension fondamentale : alors que les AESH sont essentiels à la scolarisation de près de 500 000 élèves et expriment une grande fierté pour leur mission, ils subissent une maltraitance institutionnelle systémique.

Cette situation se caractérise par une précarité salariale extrême, une absence de formation qualifiante, une hiérarchie floue et un manque de reconnaissance symbolique et matérielle.

Le "bricolage" permanent et le flou entourant leurs missions, bien que pratiques pour l'institution, abîment non seulement les professionnels mais compromettent également l'idéal de l'école inclusive, en faisant peser sur les AESH la responsabilité de compenser les défaillances du système.

L'analyse met en lumière que la négligence envers cette profession est intrinsèquement liée à la négligence envers les élèves qu'ils accompagnent.

1. Définition et Complexité du Métier d'AESH

Le métier d'AESH, bien que central pour l'application des lois de 2005 et 2019 sur l'école inclusive, demeure mal connu et peu défini. Il s'inscrit dans la tradition des métiers du "care" (soin à la personne) mais peine à trouver sa place en tant que profession éducative à part entière.

• Trois Axes Fondamentaux : Le travail s'articule autour de trois missions principales :

1. Aide à l'accès aux apprentissages.    2. Aide à la socialisation et à l'intégration dans le groupe-classe.    3. Aide dans les gestes de la vie quotidienne.

• Dimension Relationnelle Centrale : Au-delà de ces missions, le métier est profondément relationnel.

L'AESH est en interaction constante non seulement avec l'élève (souvent en relation duelle), mais aussi avec les enseignants et les autres adultes de l'établissement pour adapter l'environnement aux besoins de l'élève.

• Un Rôle d'Interface : Les AESH agissent comme une "passerelle" ou un "tampon" entre l'élève, le groupe-classe et les enseignants. Ils sont souvent amenés à "absorber les dysfonctionnements du système" pour permettre la scolarisation.

• Des Tâches Dépassant le Cadre Défini : Dans la pratique, les missions peuvent s'étendre bien au-delà du cadre officiel, incluant la surveillance de classes entières ou la réalisation de gestes de soin complexes (comme changer la canule de trachéotomie d'un élève) sans formation adéquate, les transformant de fait en "soignantes".

2. Une Profession en Proie à la Maltraitance Institutionnelle

Un thème majeur est le paradoxe vécu par les AESH : une grande fierté tirée du travail accompli et de son utilité sociale, juxtaposée à un sentiment de maltraitance et de mépris de la part de l'institution.

• Le Manque de Reconnaissance Symbolique : Cette maltraitance se manifeste par des "micro-mises à l'écart" quotidiennes :

◦ Invisibilisation : Oubli systématique dans les communications officielles de la hiérarchie (par exemple, les vœux de vacances).  

◦ Exclusion des Espaces Communs : Des "salles des profs" qui ne sont pas renommées en "salles des adultes" ou "des personnels", excluant symboliquement les AESH.   

◦ Absence aux Réunions Clés : Les AESH sont souvent "évincées" des Équipes de Suivi de la Scolarisation (ESS), alors que leur parole est cruciale pour l'évaluation des besoins de l'élève.

• Une Hiérarchie Floue et Oppressante : La structure hiérarchique est mal définie, créant une situation inconfortable. Une AESH résume ce sentiment par la phrase :

"Dans mon école, tout le monde est mon chef."

• Le Poids des Injonctions Paradoxales : Les AESH doivent constamment arbitrer entre des valeurs contradictoires.

Par exemple, leur mission est de lutter contre la stigmatisation de l'élève, tout en faisant elles-mêmes partie d'un dispositif (ULIS, accompagnement individualisé) qui est de fait stigmatisant.

3. Précarité Salariale et Pénibilité du Travail

Les conditions matérielles des AESH sont marquées par une précarité extrême qui reflète la faible valeur accordée à leur travail par l'institution.

Aspect

Description

Rémunération

Payées au SMIC horaire, avec des contrats à temps incomplet qui placent beaucoup d'entre elles sous le seuil de pauvreté.

Pluri-activité

La majorité des AESH sont contraintes de cumuler plusieurs emplois (cantine, aide aux devoirs, aide à domicile) pour subvenir à leurs besoins.

Primes

L'accès aux primes REP/REP+ (éducation prioritaire) est très récent (2023) et d'un montant faible (environ 80 €).

Pénibilité Physique

Le métier engendre des troubles musculosquelettiques, notamment lors de la prise en charge d'élèves (toilette, déplacements) dans des bâtiments non adaptés.

Charge Émotionnelle

La charge mentale et émotionnelle est immense, liée à la gestion de crises, à la crainte permanente de l'incident ("l'accident"), à l'attachement aux élèves et à l'incertitude sur leur avenir.

4. Le Déficit Criant de Formation Professionnelle

L'absence de formation adéquate est un point de critique central, perçu comme un signe de mépris et une source de difficultés professionnelles.

• Une "Adaptation à l'Emploi" Insuffisante : La formation officielle se résume à 60 heures d'adaptation à l'emploi, un héritage des anciens contrats aidés.

Elle est décrite comme une simple transmission d'informations via des diaporamas, et non une véritable formation professionnelle.

De nombreux AESH n'ont même jamais reçu cette formation.

• L'Autoformation comme Norme : Face à la diversité des handicaps (autisme, dyslexie, comorbidités, etc.), les AESH sont contraintes de s'autoformer sur leur temps personnel, en lisant des ouvrages ou en cherchant des informations pour s'adapter aux besoins spécifiques de chaque élève.

• Revendication d'un Statut Professionnel : Les syndicats, comme le SNES-FSU, revendiquent la création d'une véritable formation diplômante de niveau Bac+2, sur le modèle du CAPPEI pour les enseignants spécialisés, afin de reconnaître et de structurer le métier.

5. L'École Inclusive : Entre Idéal et "Bricolage"

Vingt ans après la loi fondatrice de 2005, le projet de l'école inclusive repose en grande partie sur le "bricolage" et le dévouement des AESH, ce qui fragilise l'ensemble du système.

• Des Chiffres Alarmants : Près de 50 000 élèves ayant une notification pour un accompagnement ne sont pas suivis, faute de moyens.

• Un Système Organisé pour Dysfonctionner : Selon Frédéric Grimaux, "si on voulait que l'école inclusive disfonctionne, on s'y prendrait pas autrement".

Le flou des missions, le manque de temps de concertation et la non-reconnaissance du travail collaboratif comme un travail en soi organisent l'échec.

• Exemples d'Indignité : Des situations dégradantes sont rapportées, comme celle d'un élève changé sur des sacs poubelles à l'arrière d'une classe, derrière un paravent improvisé avec des rideaux, illustrant "l'indignité totale de l'enfant, des travailleurs et de l'institution scolaire".

• La Mutualisation (PIAL) : Les Pôles Inclusifs d'Accompagnement Localisés (PIAL) ont accentué la mutualisation des moyens, menant à des situations où des AESH doivent accompagner plusieurs élèves simultanément ou effectuer des missions sur des sites géographiquement éloignés, au détriment de la qualité de l'accompagnement.

6. Le Poids du Langage et de la Stigmatisation

Le vocabulaire utilisé à l'école révèle les tensions et les préjugés entourant le handicap.

• La Prolifération des Sigles : Le jargon institutionnel (AESH, AVS, ULIS, ESS, GEVASCO, MDPH) est souvent incompréhensible pour les non-initiés, y compris les familles et les élèves.

• L'Infantilisation : Le fait d'appeler "les enfants" des adolescents au collège contribue à une infantilisation des élèves en situation de handicap.

• La Stigmatisation par le Langage : Le terme "Ulis" devient une insulte dans la cour de récréation ("T'es un Ulis").

Des mots comme "mongol" ou "autiste" sont encore couramment utilisés de manière péjorative, montrant que les mentalités évoluent lentement.

• La Persistance de la "Normalité" : Le concept de "normalité" reste prégnant, y compris chez certains professionnels de l'éducation, ce qui va à l'encontre de la philosophie d'une école inclusive qui devrait valoriser les différences.

7. Évolutions Récentes et Inquiétudes Futures

La situation des AESH pourrait se dégrader davantage avec les réformes à venir, notamment le Pôle d'Appui à la Scolarité (PAS).

Ce dispositif prévoit d'étendre les missions des AESH à l'ensemble des élèves à besoins éducatifs particuliers (enfants du voyage, allophones, élèves "dys", etc.), et pas seulement ceux en situation de handicap.

Cette évolution fait craindre une augmentation considérable de la charge de travail et de la charge mentale, sans formation ni revalorisation correspondantes, en s'appuyant une fois de plus sur le "dévouement" de ces professionnels.

Author response:

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

Reviewer #1 (Public review):

Summary:

From a forward genetic mosaic mutant screen using EMS, the authors identify mutations in glucosylceramide synthase (GlcT), a rate-limiting enzyme for glycosphingolipid (GSL) production, that result in EE tumors. Multiple genetic experiments strongly support the model that the mutant phenotype caused by GlcT loss is due to by failure of conversion of ceramide into glucosylceramide. Further genetic evidence suggests that Notch signaling is comprised in the ISC lineage and may affect the endocytosis of Delta. Loss of GlcT does not affect wing development or oogenesis, suggesting tissue-specific roles for GlcT. Finally, an increase in goblet cells in UGCG knockout mice, not previously reported, suggests a conserved role for GlcT in Notch signaling in intestinal cell lineage specification.

Strengths:

Overall, this is a well-written paper with multiple well-designed and executed genetic experiments that support a role for GlcT in Notch signaling in the fly and mammalian intestine. I do, however, have a few comments below.

Weaknesses:

(1) The authors bring up the intriguing idea that GlcT could be a way to link diet to cell fate choice. Unfortunately, there are no experiments to test this hypothesis.

We indeed attempted to establish an assay to investigate the impact of various diets (such as high-fat, high-sugar, or high-protein diets) on the fate choice of ISCs. Subsequently, we intended to examine the potential involvement of GlcT in this process. However, we observed that the number or percentage of EEs varies significantly among individuals, even among flies with identical phenotypes subjected to the same nutritional regimen. We suspect that the proliferative status of ISCs and the turnover rate of EEs may significantly influence the number of EEs present in the intestinal epithelium, complicating the interpretation of our results. Consequently, we are unable to conduct this experiment at this time. The hypothesis suggesting that GlcT may link diet to cell fate choice remains an avenue for future experimental exploration.

(2) Why do the authors think that UCCG knockout results in goblet cell excess and not in the other secretory cell types?

This is indeed an interesting point. In the mouse intestine, it is well-documented that the knockout of Notch receptors or Delta-like ligands results in a classic phenotype characterized by goblet cell hyperplasia, with little impact on the other secretory cell types. This finding aligns very well with our experimental results, as we noted that the numbers of Paneth cells and enteroendocrine cells appear to be largely normal in UGCG knockout mice. By contrast, increases in other secretory cell types are typically observed under conditions of pharmacological inhibition of the Notch pathway.

(3) The authors should cite other EMS mutagenesis screens done in the fly intestine.

To our knowledge, the EMS screen on 2L chromosome conducted in Allison Bardin’s lab is the only one prior to this work, which leads to two publications (Perdigoto et al., 2011; Gervais, et al., 2019). We have now included citations for both papers in the revised manuscript.

(4) The absence of a phenotype using NRE-Gal4 is not convincing. This is because the delay in its expression could be after the requirement for the affected gene in the process being studied. In other words, sufficient knockdown of GlcT by RNA would not be achieved until after the relevant signaling between the EB and the ISC occurred. Dl-Gal4 is problematic as an ISC driver because Dl is expressed in the EEP.

This is an excellent point, and we agree that the lack of an observable phenotype using NRE-Gal4 could be due to delayed expression, which may result in missing the critical window required for effective GlcT knockdown. Consequently, we cannot rule out the possibility that GlcT also plays a role in early EBs or EEPs. We have revised the manuscript to soften this conclusion and to include this alternative explanation for the experiment.

(5) The difference in Rab5 between control and GlcT-IR was not that significant. Furthermore, any changes could be secondary to increases in proliferation.

We agree that it is possible that the observed increase in proliferation could influence the number of Rab5+ endosomes, and we will temper our conclusions on this aspect accordingly. However, it is important to note that, although the difference in Rab5+ endosomes between the control and GlcT-IR conditions appeared mild, it was statistically significant and reproducible. In our revised experiments, we have not only added statistical data and immunofluorescence images for Rab11 but also unified the approaches used for detecting Rab-associated proteins (in the previous figures, Rab5 was shown using U-Rab5-GFP, whereas Rab7 was detected by direct antibody staining). Based on this unified strategy, we optimized the quantification of Dl-GFP colocalization with early, late, and recycling endosomes, and the results are consistent with our previous observations (see the updated Fig. 5).

Reviewer #2 (Public review):

Summary:

This study genetically identifies two key enzymes involved in the biosynthesis of glycosphingolipids, GlcT and Egh, which act as tumor suppressors in the adult fly gut. Detailed genetic analysis indicates that a deficiency in Mactosyl-ceramide (Mac-Cer) is causing tumor formation. Analysis of a Notch transcriptional reporter further indicates that the lack of Mac-Ser is associated with reduced Notch activity in the gut, but not in other tissues.

Addressing how a change in the lipid composition of the membranes might lead to defective Notch receptor activation, the authors studied the endocytic trafficking of Delta and claimed that internalized Delta appeared to accumulate faster into endosomes in the absence of Mac-Cer. Further analysis of Delta steady-state accumulation in fixed samples suggested a delay in the endosomal trafficking of Delta from Rab5+ to Rab7+ endosomes, which was interpreted to suggest that the inefficient, or delayed, recycling of Delta might cause a loss in Notch receptor activation.

Finally, the histological analysis of mouse guts following the conditional knock-out of the GlcT gene suggested that Mac-Cer might also be important for proper Notch signaling activity in that context.

Strengths:

The genetic analysis is of high quality. The finding that a Mac-Cer deficiency results in reduced Notch activity in the fly gut is important and fully convincing.

The mouse data, although preliminary, raised the possibility that the role of this specific lipid may be conserved across species.

Weaknesses:

This study is not, however, without caveats and several specific conclusions are not fully convincing.

First, the conclusion that GlcT is specifically required in Intestinal Stem Cells (ISCs) is not fully convincing for technical reasons: NRE-Gal4 may be less active in GlcT mutant cells, and the knock-down of GlcT using Dl-Gal4ts may not be restricted to ISCs given the perdurance of Gal4 and of its downstream RNAi.

As previously mentioned, we acknowledge that a role for GlcT in early EBs or EEPs cannot be completely ruled out. We have revised our manuscript to present a more cautious conclusion and explicitly described this possibility in the updated version.

Second, the results from the antibody uptake assays are not clear.: i) the levels of internalized Delta were not quantified in these experiments; ii) additionally, live guts were incubated with anti-Delta for 3hr. This long period of incubation indicated that the observed results may not necessarily reflect the dynamics of endocytosis of antibody-bound Delta, but might also inform about the distribution of intracellular Delta following the internalization of unbound anti-Delta. It would thus be interesting to examine the level of internalized Delta in experiments with shorter incubation time.

We thank the reviewer for these excellent questions. In our antibody uptake experiments, we noted that Dl reached its peak accumulation after a 3-hour incubation period. We recognize that quantifying internalized Dl would enhance our analysis, and we will include the corresponding statistical graphs in the revised version of the manuscript. In addition, we agree that during the 3-hour incubation, the potential internalization of unbound anti-Dl cannot be ruled out, as it may influence the observed distribution of intracellular Dl. We therefore attempted to supplement our findings with live imaging experiments to investigate the dynamics of Dl/Notch endocytosis in both normal and GlcT mutant ISCs. However, we found that the GFP expression level of Dl-GFP (either in the knock-in or transgenic line) was too low to be reliably tracked. During the three-hour observation period, the weak GFP signal remained largely unchanged regardless of the GlcT mutation status, and the signal resolution under the microscope was insufficient to clearly distinguish membrane-associated from intracellular Dl. Therefore, we were unable to obtain a dynamic view of Dl trafficking through live imaging. Nevertheless, our Dl antibody uptake and endosomal retention analyses collectively support the notion that MacCer influences Notch signaling by regulating Dl endocytosis.

Overall, the proposed working model needs to be solidified as important questions remain open, including: is the endo-lysosomal system, i.e. steady-state distribution of endo-lysosomal markers, affected by the Mac-Cer deficiency? Is the trafficking of Notch also affected by the Mac-Cer deficiency? is the rate of Delta endocytosis also affected by the Mac-Cer deficiency? are the levels of cell-surface Delta reduced upon the loss of Mac-Cer?

Regarding the impact on the endo-lysosomal system, this is indeed an important aspect to explore. While we did not conduct experiments specifically designed to evaluate the steady-state distribution of endo-lysosomal markers, our analyses utilizing Rab5-GFP overexpression and Rab7 staining did not indicate any significant differences in endosome distribution in MacCer deficient conditions. Moreover, we still observed high expression of the NRE-LacZ reporter specifically at the boundaries of clones in GlcT mutant cells (Fig. 4A), indicating that GlcT mutant EBs remain responsive to Dl produced by normal ISCs located right at the clone boundary. Therefore, we propose that MacCer deficiency may specifically affect Dl trafficking without impacting Notch trafficking.

In our 3-hour antibody uptake experiments, we observed a notable decrease in cell-surface Dl, which was accompanied by an increase in intracellular accumulation. These findings collectively suggest that Dl may be unstable on the cell surface, leading to its accumulation in early endosomes.

Third, while the mouse results are potentially interesting, they seem to be relatively preliminary, and future studies are needed to test whether the level of Notch receptor activation is reduced in this model.

In the mouse small intestine, Olfm4 is a well-established target gene of the Notch signaling pathway, and its staining provides a reliable indication of Notch pathway activation. While we attempted to evaluate Notch activation using additional markers, such as Hes1 and NICD, we encountered difficulties, as the corresponding antibody reagents did not perform well in our hands. Despite these challenges, we believe that our findings with Olfm4 provide an important start point for further investigation in the future.

Reviewer #3 (Public review):

Summary:

In this paper, Tang et al report the discovery of a Glycoslyceramide synthase gene, GlcT, which they found in a genetic screen for mutations that generate tumorous growth of stem cells in the gut of Drosophila. The screen was expertly done using a classic mutagenesis/mosaic method. Their initial characterization of the GlcT alleles, which generate endocrine tumors much like mutations in the Notch signaling pathway, is also very nice. Tang et al checked other enzymes in the glycosylceramide pathway and found that the loss of one gene just downstream of GlcT (Egh) gives similar phenotypes to GlcT, whereas three genes further downstream do not replicate the phenotype. Remarkably, dietary supplementation with a predicted GlcT/Egh product, Lactosyl-ceramide, was able to substantially rescue the GlcT mutant phenotype. Based on the phenotypic similarity of the GlcT and Notch phenotypes, the authors show that activated Notch is epistatic to GlcT mutations, suppressing the endocrine tumor phenotype and that GlcT mutant clones have reduced Notch signaling activity. Up to this point, the results are all clear, interesting, and significant. Tang et al then go on to investigate how GlcT mutations might affect Notch signaling, and present results suggesting that GlcT mutation might impair the normal endocytic trafficking of Delta, the Notch ligand. These results (Fig X-XX), unfortunately, are less than convincing; either more conclusive data should be brought to support the Delta trafficking model, or the authors should limit their conclusions regarding how GlcT loss impairs Notch signaling. Given the results shown, it's clear that GlcT affects EE cell differentiation, but whether this is via directly altering Dl/N signaling is not so clear, and other mechanisms could be involved. Overall the paper is an interesting, novel study, but it lacks somewhat in providing mechanistic insight. With conscientious revisions, this could be addressed. We list below specific points that Tang et al should consider as they revise their paper.

Strengths:

The genetic screen is excellent.

The basic characterization of GlcT phenotypes is excellent, as is the downstream pathway analysis.

Weaknesses:

(1) Lines 147-149, Figure 2E: here, the study would benefit from quantitations of the effects of loss of brn, B4GalNAcTA, and a4GT1, even though they appear negative.

We have incorporated the quantifications for the effects of the loss of brn, B4GalNAcTA, and a4GT1 in the updated Figure 2.

(2) In Figure 3, it would be useful to quantify the effects of LacCer on proliferation. The suppression result is very nice, but only effects on Pros+ cell numbers are shown.

We have now added quantifications of the number of EEs per clone to the updated Figure 3.

(3) In Figure 4A/B we see less NRE-LacZ in GlcT mutant clones. Are the data points in Figure 4B per cell or per clone? Please note. Also, there are clearly a few NRE-LacZ+ cells in the mutant clone. How does this happen if GlcT is required for Dl/N signaling?

In Figure 4B, the data points represent the fluorescence intensity per single cell within each clone. It is true that a few NRE-LacZ+ cells can still be observed within the mutant clone; however, this does not contradict our conclusion. As noted, high expression of the NRE-LacZ reporter was specifically observed around the clone boundaries in MacCer deficient cells (Fig. 4A), indicating that the mutant EBs can normally receive Dl signal from the normal ISCs located at the clone boundary and activate the Notch signaling pathway. Therefore, we believe that, although affecting Dl trafficking, MacCer deficiency does not significantly affect Notch trafficking.

(4) Lines 222-225, Figure 5AB: The authors use the NRE-Gal4ts driver to show that GlcT depletion in EBs has no effect. However, this driver is not activated until well into the process of EB commitment, and RNAi's take several days to work, and so the author's conclusion is "specifically required in ISCs" and not at all in EBs may be erroneous.

As previously mentioned, we acknowledge that a role for GlcT in early EBs or EEPs cannot be completely ruled out. We have revised our manuscript to present a more cautious conclusion and described this possibility in the updated version.

(5) Figure 5C-F: These results relating to Delta endocytosis are not convincing. The data in Fig 5C are not clear and not quantitated, and the data in Figure 5F are so widely scattered that it seems these co-localizations are difficult to measure. The authors should either remove these data, improve them, or soften the conclusions taken from them. Moreover, it is unclear how the experiments tracing Delta internalization (Fig 5C) could actually work. This is because for this method to work, the anti-Dl antibody would have to pass through the visceral muscle before binding Dl on the ISC cell surface. To my knowledge, antibody transcytosis is not a common phenomenon.

We thank the reviewer for these insightful comments and suggestions. In our in vivo experiments, we observed increased co-localization of Rab5 and Dl in GlcT mutant ISCs, indicating that Dl trafficking is delayed at the transition to Rab7⁺ late endosomes, a finding that is further supported by our antibody uptake experiments. We acknowledge that the data presented in Fig. 5C are not fully quantified and that the co-localization data in Fig. 5F may appear somewhat scattered; therefore, we have included additional quantification and enhanced the data presentation in the revised manuscript.

Regarding the concern about antibody internalization, we appreciate this point. We currently do not know if the antibody reaches the cell surface of ISCs by passing through the visceral muscle or via other routes. Given that the experiment was conducted with fragmented gut, it is possible that the antibody may penetrate into the tissue through mechanisms independent of transcytosis.

As mentioned earlier, we attempted to supplement our findings with live imaging experiments to investigate the dynamics of Dl/Notch endocytosis in both normal and GlcT mutant ISCs. However, we found that the GFP expression level of Dl-GFP (either in the knock-in or transgenic line) was too low to be reliably tracked. During the three-hour observation period, the weak GFP signal remained largely unchanged regardless of the GlcT mutation status, and the signal resolution under the microscope was insufficient to clearly distinguish membrane-associated from intracellular Dl. Therefore, we were unable to obtain a dynamic view of Dl trafficking through live imaging. Nevertheless, our Dl antibody uptake and endosomal retention analyses collectively support the notion that MacCer influences Notch signaling by regulating Dl endocytosis.

(6) It is unclear whether MacCer regulates Dl-Notch signaling by modifying Dl directly or by influencing the general endocytic recycling pathway. The authors say they observe increased Dl accumulation in Rab5+ early endosomes but not in Rab7+ late endosomes upon GlcT depletion, suggesting that the recycling endosome pathway, which retrieves Dl back to the cell surface, may be impaired by GlcT loss. To test this, the authors could examine whether recycling endosomes (marked by Rab4 and Rab11) are disrupted in GlcT mutants. Rab11 has been shown to be essential for recycling endosome function in fly ISCs.

We agree that assessing the state of recycling endosomes, especially by using markers such as Rab11, would be valuable in determining whether MacCer regulates Dl-Notch signaling by directly modifying Dl or by influencing the broader endocytic recycling pathway. In the newly added experiments, we found that in GlcT-IR flies, Dl still exhibits partial colocalization with Rab11, and the overall expression pattern of Rab11 is not affected by GlcT knockdown (Fig. 5E-F). These observations suggest that MacCer specifically regulates Dl trafficking rather than broadly affecting the recycling pathway.

(7) It remains unclear whether Dl undergoes post-translational modification by MacCer in the fly gut. At a minimum, the authors should provide biochemical evidence (e.g., Western blot) to determine whether GlcT depletion alters the protein size of Dl.

While we propose that MacCer may function as a component of lipid rafts, facilitating Dl membrane anchorage and endocytosis, we also acknowledge the possibility that MacCer could serve as a substrate for protein modifications of Dl necessary for its proper function. Conducting biochemical analyses to investigate potential post-translational modifications of Dl by MacCer would indeed provide valuable insights. We have performed Western blot analysis to test whether GlcT depletion affects the protein size of Dl. As shown below, we did not detect any apparent changes in the molecular weight of the Dl protein. Therefore, it is unlikely that MacCer regulates post-translational modifications of Dl.

Author response image 1.

To investigate whether MacCer modifies Dl by Western blot,(A) Four lanes were loaded: the first two contained 20 μL of membrane extract (lane 1: GlcT-IR, lane 2: control), while the last two contained 10 μL of membrane extract (B) Full blot images are shown under both long and shortexposure conditions.

(8) It is unfortunate that GlcT doesn't affect Notch signaling in other organs on the fly. This brings into question the Delta trafficking model and the authors should note this. Also, the clonal marker in Figure 6C is not clear.

In the revised working model, we have explicitly described that the events occur in intestinal stem cells. Regarding Figure 6C, we have delineated the clone with a white dashed line to enhance its clarity and visual comprehension.

(9) The authors state that loss of UGCG in the mouse small intestine results in a reduced ISC count. However, in Supplementary Figure C3, Ki67, a marker of ISC proliferation, is significantly increased in UGCG-CKO mice. This contradiction should be clarified. The authors might repeat this experiment using an alternative ISC marker, such as Lgr5.

Previous studies have indicated that dysregulation of the Notch signaling pathway can result in a reduction in the number of ISCs. While we did not perform a direct quantification of ISC numbers in our experiments, our Olfm4 staining—which serves as a reliable marker for ISCs—demonstrates a clear reduction in the number of positive cells in UGCG-CKO mice.

The increased Ki67 signal we observed reflects enhanced proliferation in the transit-amplifying region, and it does not directly indicate an increase in ISC number. Therefore, in UGCG-CKO mice, we observe a decrease in the number of ISCs, while there is an increase in transit-amplifying (TA) cells (progenitor cells). This increase in TA cells is probably a secondary consequence of the loss of barrier function associated with the UGCG knockout.

On the erosion of middle class America. Poverty line is around 140k if actual costs taken into account. The 1960s benchmark assumed cost of food to be 1/3 of overall costs. Now it's 7% or so, meaning 1/15 of overall costs. This pushes up the poverty line to 5 times the level used, or some 150k USD pa

Example of a proxy being used as 'measurement' and the assumptions in a proxy never re-evaluated.

Author response:

The following is the authors’ response to the previous reviews

Reviewer #3 (Recommendations for the authors):

The authors have done an excellent job of addressing most comments, but my concerns about Figure 5 remain. I appreciate the authors' efforts to address the problem involving Rs being part of the computation on both the x and y axes of Figure 5, but addressing this via simulation addresses statistical significance but overlooks effect size. I think the authors may have misunderstood my original suggestion, so I will attempt to explain it better here. Since "Rs" is an average across all trials, the trials could be subdivided in two halves to compute two separate averages - for example, an average of the even numbered trials and an average of the odd numbered trials. Then you would use the "Rs" from the even numbered trials for one axis and the "Rs" from the odd numbered trials for the other. You would then plot R-Rs_even vs Rf-Rs_odd. This would remove the confound from this figure, and allow the text/interpretation to be largely unchanged (assuming the results continue to look as they do).

We have added a description and the result of the new analysis (line #321 to #332), and a supplementary figure (Suppl. Fig. 1) (line #1464 to #1477). 

“We calculated 𝑅_𝑠 in the ordinate and abscissa of Figure 5A-E using responses averaged across different subsets of trials, such that 𝑅_𝑠 was no longer a common term in the ordinate and abscissa. For each neuron, we determined 𝑅_𝑠1 by averaging the firing rates of 𝑅_𝑠 across half of the recorded trials, selected randomly. We also determined 𝑅_𝑠2 by averaging the firing rates of 𝑅_𝑠 across the rest of the trials.  We regressed (𝑅 − 𝑅_𝑠1 )  on (𝑅_𝑓 − 𝑅_𝑠2) , as well as (𝑅_𝑠 - 𝑅_𝑠2)  on (𝑅_𝑓 − 𝑅_𝑠1), and repeated the procedure 50 times. The averaged slopes obtained with 𝑅_𝑠 from the split trials showed the same pattern as those using 𝑅_𝑠 from all trials (Table 1 and Supplementary Fig. 1), although the coefficient of determination was slightly reduced (Table 1). For ×4 speed separation, the slopes were nearly identical to those shown in Figure 5F1. For ×2 speed separation, the slopes were slightly smaller than those in Figure 5F2, but followed the same pattern (Supplementary Fig. 1). Together, these analysis results confirmed the faster-speed bias at the slow stimulus speeds, and the change of the response weights as stimulus speeds increased.”

An additional remaining item concerns the terminology weighted sum, in the context of the constraint that wf and ws must sum to one. My opinion is that it is non-standard to use weighted sum when the computation is a weighted average, but as long as the authors make their meaning clear, the reader will be able to follow. I suggest adding some phrasing to explain to the reader the shift in interpretation from the more general weighted sum to the more constrained weighted average. Specifically, "weighted sum" first appears on line 268, and then the additional constraint of ws + wf =1 is introduced on line 278. Somewhere around line 278, it would be useful to include a sentence stating that this constraint means the weighted sum is constrained to be a weighted average.

Thanks for the suggestion. We have modified the text as follows. Since we made other modifications in the text, the line numbers are slightly different from the last version. 

Line #274 to 275: 

“Since it is not possible to solve for both variables, 𝑤_𝑠 and 𝑤_𝑓, from a single equation (Eq. 5) with three data points, we introduced an additional constraint: 𝑤_𝑠 + 𝑤_𝑓 =1. With this constraint, the weighted sum becomes a weighted average.”

Also on line #309:

“First, at each speed pair and for each of the 100 neurons in the data sample shown in Figure 5, we simulated the response to the bi-speed stimuli (𝑅_𝑒) as a randomly weighted average of 𝑅_𝑓 and 𝑅_𝑠 of the same neuron. 

in which 𝑎 was a randomly generated weight (between 0 and 1) for 𝑅_𝑓, and the weights for 𝑅_𝑓 and 𝑅_𝑠 summed to one.”

Gălûñ′lătĭ, secondo la mitologia Cherokee, è un mondo creato prima dell'esistenza della Terra.

[https://www.cherokeepins.org/post/cherokee-stories-and-the-birth-of-a-new-world-the-first-fire]

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Reply to the reviewers

Reviewer #1 (Evidence, reproducibility and clarity (Required)): The authors map the ZFP36L1 protein interactome in human T cells using UltraID proximity labeling combined with quantitative mass spectrometry. They optimize labeling conditions in primary T cells, profile resting and activated cells, and include a time course at 2, 5, and 16 hours. They complement the interactome with co-immunoprecipitation in the presence or absence of RNase to assess RNA dependence. They then test selected candidates using CRISPR knockouts in primary T cells, focusing on UPF1 and GIGYF1/2, and report effects on global translation, stress, activation markers, and ZFP36L1 protein levels. The work argues that ZFP36L1 sits at the center of multiple post-transcriptional pathways in T cells (which in itself is not a novel finding) and that UPF1 supports ZFP36L1 expression at the mRNA and protein level. The main model system is primary human T cells, with some data in Jurkat cells.

The core datasets show thousands of identified proteins in total lysates and enriched biotinylated fractions. Known partners from CCR4-NOT, decapping, stress granules, and P-bodies appear, with additional candidates like GIGYF1/2, PATL1, DDX6, and UPF1. Time-resolved labeling suggests shifts in proximity during early activation. Co-IP with and without RNase suggests both RNA-dependent and RNA-independent contacts. CRISPR loss of UPF1 or GIGYF1/2 increases translation at rest and elevates activation markers, and UPF1 loss reduces ZFP36L1 protein and mRNA while MG132 does not rescue protein levels; UPF1 RIP enriches ZFP36L1 mRNA.

Among patterns worth noting are that the activation state drives the principal variance in both proteome and proximity datasets. Deadenylation, decapping, and granule proteins are consistently near ZFP36L1 across conditions, while some contacts dip at 2 hours and recover by 5 to 16 hours. Mitochondrial ribosomal proteins become more proximal later. UPF1 and GIGYF1 show time-linked behavior and RNase sensitivity that fits roles in mRNA surveillance and translational control. These observations support a dynamic hub model, though they remain proximity-based rather than direct binding maps.

We thank the reviewer for their careful reading and thoughtful summary. Please find our point-to point response below.

Major comments

1) The key conclusions are directionally convincing for a broad and dynamic ZFP36L1 neighborhood in human T cells. The data robustly recover established complexes and add plausible candidates. The time-course and RNase experiments strengthen the claim that interactions shift with activation state and RNA context. The functional tests around UPF1 and GIGYF1/2 point to biological relevance. That said, some statements could be qualified. The statement that ZFP36L1 "coordinates" multiple pathways implies mechanism and directionality that proximity data alone cannot prove. I suggest reframing as "positions ZFP36L1 within" or "supports a model where ZFP36L1 sits within" these networks.

We thank this reviewer for considering our data ‘directionally convincing, and robust, adding new plausible candidates as interactors with ZFP36L1’. We agree that the proposed wording is more appropriate and will change it accordingly.

2) UPF1, as an upstream regulator of ZFP36L1 expression, is a promising lead. The reduction of ZFP36L1 protein and mRNA in UPF1 knockout, the non-rescue by MG132, and the UPF1 RIP on ZFP36L1 mRNA together argue that UPF1 influences ZFP36L1 transcript output or processing. This claim would read stronger with one short rescue or perturbation that pins the mechanism. A compact test would be UPF1 re-expression in UPF1-deficient T cells with wild-type and helicase-dead alleles. This is realistic in primary T cells using mRNA electroporation or virus-based systems. Approximate time 2 to 3 weeks, including guide design check and expansion. Reagents and sequencing about 2 to 4k USD depending on donor numbers. This would help separate viability or stress effects from a direct role in ZFP36L1 mRNA handling.

We agree that a rescue experiment with wild-type and helicase-dead UPF1 in UPF1-deficient primary T cells would be interesting. Unfortunately, however, UPF1 knockout T cells are less viable and divide less (Supp Figure 6B), making further manipulations such as re-expression by viral transduction technically impossible. We will clarify this limitation in the Discussion and will more explicitly indicate that UPF1 promotes ZFP36L1 mRNA and protein expression, while acknowledging that the precise mechanistic contribution of UPF1 (e.g. to transcript processing, export, or surveillance) remain to be fully resolved.

3) The inference that ZFP36L1 proximity to decapping and deadenylation complexes reflects pathway engagement is reasonable and, frankly, expected. Still, where the manuscript moves from proximity to function, the narrative works best when supported by orthogonal validation. Two compact additions would raise confidence without opening new lines of work. First, a small set of reciprocal co-IPs for PATL1 or DDX6 at endogenous levels in activated T cells, run with and without RNase, would tie the RNase-class assignments to biochemistry. Second, a short-pulse proximity experiment using a reduced biotin dose and shorter labeling window in activated cells would address whether long incubations drive non-specific labeling. Both are feasible in 2 to 3 weeks with minimal extra cost for antibodies and MS runs if the facility is in-house.

We fully agree with the reviewer that orthogonal biochemical validation is valuable. Therefore, we already combined time-resolved proximity labeling (between 0-2h, 2-5h, and 5-16 hours) with time-resolved ZFP36L1 co-IPs ± RNase, to address the dynamic behavior and potential temporal broadening of the interactome.

As to running reciprocal co-IPs for PATL1 or DDX6: we had in fact already considered to follow up on PATL1. However, we failed to identified specific antibodies, revealing many unspecific bands (see below). As to DDX6, antibodies suitable for IP have been reported, and we can therefore offer such reciprocal IP as requested.

To further address the raised points, we will (i) clarify how we define and interpret RNase-sensitive versus RNase-resistant classes (ii) emphasize that some key factors (including PATL1) are already detected in shorter labeling conditions (2 h) in activated T cells (Fig 4C); and (iii) better highlight that the our data provide strong candidates and pathway hypotheses that warrant further mechanistic experimentation in follow-up studies, when moving from proximity to function.

As to the suggested lowering dose of biotin: As described in Figure S1, this appeared unsuccessful. We owe it to the reported dependence and use of biotin in primary T cells (Ref’s 31-33 of this manuscript). This also included that we could not culture T cells in biotin-free medium prior to labeling, as most protocols would do in cell lines.

The reviewer also suggested shorter labeling times. Please be advised that the labeling times chosen were based on the reported protein induction and activity on target mRNAs: 1) ZFP36L1 expression peaks at 2h of T cell activation (Zandhuis et al. 2025; 0.1002/eji.202451641, Petkau et al. 2024; 10.1002/eji.202350700), 3) shows the strongest effects on T cell function between 4-5h, and displays a late phase of activity at 5-16h (Popovic et al. Cell Reports 2023; 10.1016/j.celrep.2023.112419). We realize that additional explanation is warranted for this rationale, which we will provide.

4) Reproducibility is helped by donor pooling, repeated T-cell screens, Jurkat confirmation, and detailed methods including MaxQuant, LIMMA, and supervised patterning. Deposition of MS data is listed. The authors should consider adding a brief, stand-alone analysis notebook in SI or on GitHub with exact filtering thresholds and "shape" definitions, since the supervised profiles are central to claims. This would let others reproduce figures from raw tables with the same code and workflows.

We thank the reviewer for his or her suggestion and we have done as suggested. We will include the following link in the manuscript: https://github.com/ajhoogendijk/ZFP36L1_UltraID

5) Replication and statistics are mostly adequate for discovery proteomics. The thresholds are clear, and PCA and correlation frameworks are appropriate. For functional readouts in edited T cells, please make the number of donors and independent experiments explicit in figure legends, and indicate whether statistics are paired by donor. Where viability differs (UPF1), note any gating strategies used to avoid bias in puromycin or activation marker measurements. These clarifications are quick to add.

Please be advised that the current figure legends already contain the requested information at the bottom (which test used, donor number etc). To highlight this better, we will indicate this point more explicitly in the methods section.

Minor comments 6) The UltraID optimization in primary T cells is useful, but the long 16-hour labeling and high biotin should be framed as a compromise rather than a standard. A short statement about potential off-target labeling during extended incubations would set expectations and justify the RNase and time-course controls.

Please be advised that 1) high biotin was required because primary T cells depend on biotin and 2) increase biotin absorption a 2-7-fold upon activation (Ref 31-33 from the paper). For better time resolution, we included a labeling of 2h (from 0-2h of activation), 3h (from 2-5h) and 9h (from 5-16h) of T cell activation. Nevertheless, we agree that we cannot exclude the risk of off-target labeling, which in fact is inherent to any labeling and pulldown method. We will include such statement in the discussion.

7) The overlap across T-cell screens and with HEK293T APEX datasets is discussed, but a compact quantitative reconciliation would help. A table that lists shared versus cell-type-specific interactors with brief notes on known expression patterns would make this point concrete.

We thank the reviewer for this suggestion. We agree and we will include such table.

8) Figures are generally clear. Where proximity and total proteome PCA are shown, consider adding sample-wise annotations for donor pools and activation time to help readers link variance to biology. Ensure all volcano plots and heatmaps display the exact cutoffs used in text.

We agree that sample-wise annotations would be a nice addition. However, when testing this for e.g. FIgure 1D&E, such differentiation into individual donors becomes illegible due to the many different variables already present. We therefore decided against it.

9) Prior work on ZFP36 family roles in decay, deadenylation via CCR4-NOT, granules, and translational control is cited within the manuscript. In a few places, recent proximity and interactome papers could be more explicitly integrated when comparing overlap, especially where conclusions differ by cell type. A concise paragraph in Discussion that lays out what is truly new in primary T cells would help clarify the contribution of this work to the field.

We appreciate this suggestion and will revise the Discussion accordingly. As to what is new in primary T cells, we would also like to mention that adding H2O2 (required for APEX labeling) to T cells results in immediate cell death can therefore not be employed on T cells. This technical limitation further underscores the valuable contribution of the UltraID-based approach we present here.

Reviewer #1 (Significance (Required)):

Nature and type of advance. The study is a technical and contextual advance in mapping ZFP36L1 proximity partners directly in human primary T cells during activation. The combination of time-resolved labeling and RNase-class assignments is informative. The CRIS PR perturbations provide an initial functional bridge from proximity to phenotype, especially for UPF1.

Context in the literature. ZFP36 family proteins have long been linked to ARE-mediated decay, CCR4-NOT recruitment, and granule localization. The present work confirms those cores and extends them to include decapping and GIGYF1/2-4EHP scaffolds in primary T cells with temporal resolution. The UPF1 link to ZFP36L1 expression adds a plausible surveillance angle that merits follow-up. The cell-type specificity analysis versus HEK293T underscores that proximity networks vary with context.

Audience. Readers in RNA biology, T-cell biology, and proteomics will find the dataset valuable. Groups studying post-transcriptional regulation in immunity can use the resource to prioritize candidate nodes for mechanistic work.

Expertise and scope. I work on post-transcriptional regulation, RNA-protein complexes, and T-cell effector biology. I am comfortable evaluating the conceptual claims, experimental design, and statistical treatment. I am not a mass spectrometry specialist, so I rely on the presented parameters and deposited data for MS acquisition specifics.

To conclude, the manuscript delivers a substantive proximity map of ZFP36L1 in human T cells, with useful temporal and RNA-class information. The UPF1 observations are promising and would benefit from a compact rescue to secure causality. A few minor additions for biochemical validation and transparency in replication would further strengthen the paper.

We thank the reviewer for this comprehensive and constructive assessment. We agree that our study primarily provides a substantive and well-annotated proximity map of ZFP36L1 in human T cells, including temporal and RNA-class information, and that the UPF1 observations constitute a promising lead that merits more detailed mechanistic analysis in follow-up studies.

Reviewer #2 (Evidence, reproducibility and clarity (Required)): The manuscript by Wolkers and colleagues describes the protein interactome of the RNA-binding protein ZFP36L1 in primary human T-cells. There is inherent value in the use of primary cells of human origin, but there is also value in that the study is quite complete, as it is performed in a variety of conditions: T-cells that have been activated or not, at different time points after activation, and by two methods (co-IP and proximity labeling). One might imagine that this basically covers all what can be detected for this protein in T-cells. The authors report a large amount of new interactors involved at all steps in post-transcriptional regulation. In addition, the authors show that UPF1, a known interactor of ZFP36L1, actually binds to ZFP36L1 mRNA and enhances its levels. In sum, the work provides a valuable resource of ZFP36L1 interactors. Yet, how the data add to the mechanistic understanding of ZFP36L1 functions and/or regulation of ZFP36L1 remains unclear.

We thank the reviewer for this enthusiasm on our experimental setups, considering the use of primary T cells of inherent value and our study with the variety of conditions complete.

Major comments: 1) Fig 2: It is confusing that the Pearson correlation to define ZFP36L1 interactors is changed depending on figure panel. In panels A-C, a correlation {greater than or equal to} 0.6 is used, while panel D uses a correlation > 0.5, which changes the nº of interactors. Then, this is changed again in Fig 3A for some cell types but not for others. Why has this been done? It would be better to stick to the same thresholds throughout the manuscript.

Please be advised that different correlation thresholds arise from the composition of the individual datasets: they in depth, number of controls, and the overall dynamic range. The initial proximity labeling experiment (Figure 2A–C) had a higher depth and a larger number of suitable control samples, which allowed us to apply a stricter cutoff (r ≥ 0.6). The time-course experiment and some of the cross-cell-type comparisons have fewer controls and somewhat lower depth, which then required a more permissive threshold (e.g. r > 0.5) to retain known core interactors.

We fully agree that this rationale needs to be explicit. In the revised manuscript we (i) clearly state for each dataset which correlation cutoff is used (ii) emphasize that these thresholds are somewhat arbitrary and should not be directly compared across experiments, and (iii) highlight that our key biological conclusions do not depend on the exact boundary chosen but rather on the consistent enrichment of core complexes and pathways across .

2) Fig 3A: It would be nice to have the information of this Figure panel as a Table (protein name, molecular process(es), known or novel, previously detected in which cells) in addition to the figure.

We agree that this would increase the value of our work as a resource to the community, and we will include such table and merge it with the table Reviewer 1 asked about.

3) Fig 6: To what extent are the effects of UPF1 and GIGFYF1 knock-out on translation and T-cell hyper-activation mediated by ZFP36L1? If deletion of ZFP36L1 itself has no effect on these processes, it seems unlikely that it is involved. In this respect, I am not sure that Fig 6 contributes to the understanding of ZFP36L.

We appreciate this conceptual question. In our dataset, ZFP36L1 knockout affects T-cell activation markers, but does not recapitulate the increased global translation observed upon UPF1 or GIGYF1/2 deletion. We will discuss this finding more explicitly in the Results and Discussion. We discuss the possibility that other ZFP36 family members (e.g. ZFP36/TTP, ZFP36L2) may partially compensate for the absence of ZFP36L1 in some readouts1. Moreover, we will emphasize that at this point it is not clear whether ZFP36L1’s contribution to UPF1 and GIGYF1 protein levels is direct or indirect.

We nonetheless consider Fig. 6 an important component of the story, as it demonstrates that proximity partners emerging from the interactome (UPF1, GIGYF1/2) have measurable functional consequences on T cell activation and translational control, thereby illustrating how the resource can guide mechanistic hypotheses. We will now more carefully phrase this as “first indications of mechanism” and avoid implying that these phenotypes are mediated exclusively via ZFP36L1.

4) Fig 7E: Differences in ZFP36L1 mRNA expression are claimed as a consequence of UPF1 deletion, and indeed there is a clear tendency to reduction of ZFP36L1 mRNA levels upon UPF1 KO. Yet the difference is statistically non-significant. Please, repeat this experiment to increase statistical significance. In addition, a clear discussion on how UPF1 -generally associated to mRNA degradation- contributes to increase ZFP36L1 mRNA levels would be appreciated.

We would like to refrain from including repeats for increasing statistical power. We find similar trends with n=3 at 0h as with n=7 at 3h of activation (Fig. 7E). We rather would like to stress that despite the width overall expression levels which most probably stems from using primary human material, the overall levels of ZFP36L1 mRNA are lower in UPF1 KO T cells. We will include a point on how UPF1 possibly may contribute to the decreased ZFP36L1 mRNA levels, as suggested.

5) Fig 6A: The decrease in global translation by GIGFYF1 knock-out upon activation claimed by the authors is not clear in Fig 6A and is non-significant upon quantification. Please, modify narrative accordingly.

Indeed, this was not phrased well. We will correct our description to match the statistical analysis.

6) Page 6: The authors state 'This included the PAN2/3 complex proteins which trim poly(A) tails prior to mRNA degradation through the CCR4/NOT complex'. To the best of my knowledge, the CCR4/NOT complex does not degrade the body of the mRNA. Both PAN2/3 and CCR4/NOT are deadenylases that function independently.

We thank the reviewer for highlighting this inaccuracy. PAN2/3 and CCR4–NOT are indeed both deadenylase complexes that function independently rather than one acting strictly upstream of the other in degrading the mRNA body. We will correct this statement to that PAN2/3 and CCR4–NOT cooperate in poly(A) tail shortening and do not themselves degrade the mRNA body, which is instead handled by the downstream decay machinery.

7) Please, label all Table sheets. Right now one has to guess what is being shown in most of them. Furthermore, it would be convenient to join all Tables related to the same Figure in one unique Excel with several sheets, rather than having many Tables with only one sheet each.

We appreciate this suggestion. In the revised supplementary files all table sheets will be clearly labeled to indicate the corresponding figure and dataset, and combined into a single excel file when multiple tables relate to the same figure. We have already done so.

Minor comments: 8) Fig 1E: Shouldn't there be a better separation by biotinylation in the UltraID IP principal component analysis? In theory, only biotinylated proteins should be immunoprecipitated.

In theory this should indeed be the case. However, in practice, pull down experiments always suffer from background stickiness of proteins to tubes, beads etc. Combined, these known background issues highlight the critical addition of control samples, allowing for unequivocal call of proteins that are above background.

In addition, as we indicated in the manuscript, primary T cells depend on Biotin. This prohibited us to use biotin-free medium, even for a short culture period (it resulted in cell death). Such biotin-free culture steps are included in proximity labeling assays performed in cell lines. Owing to the continuous addition of biotin, some of the ‘background’ biotinylation signal may even be ‘real’. Nevertheless, the higher levels of biotin we added during the labeling results in increased signals, and statistical analysis with these controls identifies which of the proteins are above background, irrespective from the source. We will include a short note on this in the manuscript

9) Fig 3B-E: Is the labeling not swapped, top (always +) is Biotin and bottom (- or +) is aCD3/aCD28?

We thank the reviewer for catching this mistake- we have corrected it

10) Fig 7A data is from another paper, so I suggest to move this panel to Supplementary materials.

We respectfully disagree. Please be advised that we reanalysed data from published datasets, that resulted in this figure. Re-analysis is a widely accepted method and certainly used for main figure panels. Our re-analysis from Bestenhorn et al 2025; (10.1016/j.molcel.2025.01.001) confirms that ZFP36L1 interacts with UPF1 and GIGYF1/2 in the RAW 264.7 macrophage cell line, which we consider an important consolidation of our findings. To highlight that this table is a re-analysis of published data, we will include this information (including the reference) below the data. As ‘extracted from Bestenhorn et al'

11) Fig S1A: Why is there so much labeling in the UltraID only lane without biotin?

This is a phenomenon also reported by others (Kubitz et al. 2022; 10.1038/s42003-022-03604-5: Figure 5A). UltraID alone is a small protein of (19.7KD), comparable to TurboID or others (Kubitz et al. 2022; 10.1038/s42003-022-03604-5). If not tethered to a specific compartment, these proximity labeling moieties can diffuse through the cytoplasm, biotinylating any protein they ‘bump’ into. Please be advised that we included this control to show this effect, to substantiate why we use GFP-UltraID- as control, to limit such background effects. To highlight this point better, we will better articulate this reasoning in the results section.

12) Fig S1E: Please, explain better. What is WT?

We thank the reviewer for catching this inconsistency. We will explicitly define “WT” as wild-type primary T cells (non-edited, non-transduced) and clarify how this relates to the other conditions.

13) Fig S4B: Please, explain the labels on top of the shapes.

We will update the figure, explaining how the labels above each shape are chosen (e.g. indicating specific clusters, functional categories, or experimental conditions, as appropriate). This should make the reading more intuitive.

14) Page 3: A time-course of incubation with biotin is lacking in Fig S1B, and thereby it is confusing that the authors direct readers to this figure when an increased to 16h incubation is claimed to be better.

Please be advised that short labeling times yielded disappointing results in primary human T cells. Therefore all first analyses were performed with 16h biotinylation, as depicted in Figure S1B). Only after achieving good results (presented in Figure 1B), we performed time course experiments (presented in __Figure 4, __lowering incubation times to 2h, 3h and 9h). We realize that this is confusing and we will rephrase this point in page 3.

Reviewer #2 (Significance (Required)): Strengths: A thorough repository of ZFP36L1 interactors in primary human T-cells. A valuable resource for the community. Weaknesses: There is little mechanistic insight on ZFP36L1 function or regulation.

We would like to highlight that the purpose of our study was to provide a comprehensive interactome of ZFP36L1, and to study the dynamics of these interactions. In addition to known interactors, we identified novel putative interactors of ZFP36L1. We have indeed not followed up on all interactions, which we consider beyond the scope of this manuscript. Rather, we consider our study as a toolbox for the community, that helps in their studies.

Nevertheless, in Fig 6-7, we show first indications of mechanistic insights on ZFP36L1 interactors, exemplifying how the findings of this resource paper can be used by the community.

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

The authors have analyzed the interactome of ZFP36L1 in primary human T cells using a biotin-based proximity labeling method. In addition to proteins that are known to interact with ZFP36L1, the authors defined a multitude of novel interactions involved in mRNA decapping, mRNA degradation pathways, translation repressors, stress granule/p-body formation, and other regulatory pathways. Time-lapse proximity labeling revealed that the ZFP36L1 interactome undergoes remodeling during T cell activation. Co-IP for ZFP36L1 executed in the presence/absence of RNA further revealed the interactome and possible regulators of ZFP36L1, including the helicase UPF1. In addition to interacting with ZFP36L1, UPF1 promotes the ZFP36L1 protein expression, seemingly by binding to the ZFP36L1 mRNA transcript, and in some way stabilizing it. This comprehensive interactome map highlights the widespread interactions of ZFP36L1 with proteins of many types, and its potential roles in diverse T cell processes. Although somewhat descriptive, rather than hypothesis-testing, this work represents an important contribution to understanding the potential roles of the ZFP36 family proteins, and sets up many future experiments which could test molecular details.

We thank the reviewer for these thoughtful points, and for recognizing our paper as an important contribution for the field as resource, that should support future experiments.

Major points: 1) Can the authors discuss the specificity of the antibody for ZFP36L1 used in the Co-IP experiments? The antibody listed in Appendix A is abcam catalog number ab42473, although the catalog number for this antibody (unlike the others major ones used) is not listed in the Methods section - please add this to the Methods to make it easier for readers to find this detail. Could this antibody also be immunoprecipitating ZFP36 or ZFP36L2? Other antibodies have had cross-reactivity for the different family members. It is also notable that this antibody has been discontinued by the manufacturer (https://www.abcam.com/en-us/products/unavailable/zfp36l1-antibody-ab42473). Have the authors tried the current abcam anti-ZFP36L1 antibody being sold, catalog number ab230507?

We appreciate the opportunity to clarify this important technical point. We have now added the catalog number (ab42473, Abcam) of the anti-ZFP36L1 antibody used for co-IP to the Methods section, in addition to Appendix A, to facilitate reproducibility. The antibody ab42473 has indeed been discontinued by the manufacturer. We have contacted the manufacturer on multiple occasions with no luck.

We have evaluated multiple alternative anti-ZFP36L1 antibodies, including the currently available Abcam antibody ab230507. In our hands, these alternatives showed weaker or less specific detection of ZFP36L1 compared to the original ZFP36L1 antibody. Only antibody 1A3 recognized ZFP36L1. We therefore used this antibody for the Co-IP. Importantly, even though the signal is lower than the original antibody we used, the migration patterns observed with ab42473 in our co-IP experiments match the expected molecular weight of ZFP36L1 and do not suggest substantial cross-reactivity with ZFP36 or ZFP36L2, which display distinct sizes (we will add the sizes to the WB in figures). We discuss this point briefly in the revised Methods/Results.

2) On this point, the authors report interactions between ZFP36L1 and its related proteins ZFP36 and ZFP36L2 in the Co-IP experiment (Supp 5C). Did these proteins interact in the proximity labeling? Ideally this could be discussed in the Discussion section.

ZFP36 and ZFP36L2 were indeed detected as co-precipitating with ZFP36L1 in the co-IP experiments but were not found as high-confidence interactors in the UltraID proximity labeling datasets. Also in the APEX proximity labeling of Bestehorn et al. In RAW macrophage cells, they did not find ZFP36 or ZFP36L1 to interact with ZFP36L1. * *We now explicitly mention this in the Results and discuss it in the Discussion.

3) Can the authors discuss more fully the limited overlap in identified interactors across the two proximity labeling screens performed in primary T cells (Fig 2C)? Likewise, can the authors comment on the very limited overlap between the screens in T cells and the published ZFP36L1-APEX proximity labelling experiment performed in the HEK293T cell line by Bestehorn et al. (ref 42)? Only 6.8% of proteins found in either T cell screen were found as interactors in this cell line. The authors comment that this may be because "...either expression of certain proteins is cell-type specific, or [because] ZFP36L1 has cell-type specific protein interactions, in addition to its core interactome". While I agree that cell-type specific interactions may be at play, I would think most of the interactors found in the T cell screens are widely expressed proteins necessary for central cell functions.

First, the apparent overlap percentage depends on depth and filtering. As noted above and now detailed in a new Supplementary table, a core set of decapping, deadenylation, and granule-associated factors is consistently recovered across our T-cell screens and the HEK293T APEX dataset. However, beyond this core protein, overlap is reduced, reflecting several factors: (i) differences in expression levels of many interactors between HEK293T cells and primary T cells; (ii) the activation-dependent nature of ZFP36L1 function in T cells, which cannot be fully mimicked in HEK293T; (iii) different proximity labeling enzymes and fusion constructs (APEX vs UltraID, different tags, expression levels); and (iv) distinct experimental designs and control strategies, which influence statistical filtering and the effective “depth” of each interactome.

In the revised Discussion and in the new comparative table, we now emphasize that while many of the ZFP36L1 proximity partners identified in T cells are indeed widely expressed, their effective labeling and enrichment are strongly context dependent. We therefore interpret the relatively limited overlap as highlighting both a robust core interactome and substantial context-specific remodeling, rather than as evidence of artifacts in one or the other dataset.

Minor comments: 4) In Figure 3D, the legend states that black circles indicate significantly enriched proteins in biotin samples, while grey circles indicate non-significant enrichment. However, some genes, including DCP1A, DDX6, YBX1, have black circles in the -biotin group and grey in the +biotin group, which creates confusion in interpretation.

We thank the reviewer for this comment. We have accidentally switched the labeling of biotin and activation as pointed out by reviewer 2. Once this is fixed, this comment will also be fixed.

5) Did the authors find any interactors whose expression is known to be specific to CD4 or CD8 T cells?

In our current dataset we did not identify interactors whose presence was clearly restricted to CD4 or CD8 T-cells. We agree that differential ZFP36L1 interactomes in defined T-cell subsets represent an interesting avenue for future targeted studies and will outline this is the discussion.

Reviewer #3 (Significance (Required)):

The authors present the first comprehensive analysis of the ZFP36L1 interactome in primary T cells. The use of biotin-based proximity labeling enables detection of physiologically relevant interactions in live cells. This approach revealed many novel interactors.

Strengths include the overall richness of the dataset, and the hypothesis-provoking experiments that could follow in the future. Limitations include somewhat limited overlap with a published proximity labeling dataset from performed in a different cell line, suggesting that there may be artifacts in one or both datasets.

The audience for this article would include those interested broadly in RNA binding proteins and those interested in post-transcriptional and translational regulation.

I have immunology expertise on T cell activation and differentiation and expertise on transcriptional and post-transcriptional regulation of gene expression in T cells.

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

Learn more at Review Commons

Referee #2

Evidence, reproducibility and clarity

The manuscript by Wolkers and colleagues describes the protein interactome of the RNA-binding protein ZFP36L1 in primary human T-cells. There is inherent value in the use of primary cells of human origin, but there is also value in that the study is quite complete, as it is performed in a variety of conditions: T-cells that have been activated or not, at different time points after activation, and by two methods (co-IP and proximity labeling). One might imagine that this basically covers all what can be detected for this protein in T-cells. The authors report a large amount of new interactors involved at all steps in post-transcriptional regulation. In addition, the authors show that UPF1, a known interactor of ZFP36L1, actually binds to ZFP36L1 mRNA and enhances its levels. In sum, the work provides a valuable resource of ZFP36L1 interactors. Yet, how the data add to the mechanistic understanding of ZFP36L1 functions and/or regulation of ZFP36L1 remains unclear.

Major comments:

1) Fig 2: It is confusing that the Pearson correlation to define ZFP36L1 interactors is changed depending on figure panel. In panels A-C, a correlation {greater than or equal to} 0.6 is used, while panel D uses a correlation > 0.5, which changes the nº of interactors. Then, this is changed again in Fig 3A for some cell types but not for others. Why has this been done? It would be better to stick to the same thresholds throughout the manuscript.

2) Fig 3A: It would be nice to have the information of this Figure panel as a Table (protein name, molecular process(es), known or novel, previously detected in which cells) in addition to the figure.

3) Fig 6: To what extent are the effects of UPF1 and GIGFYF1 knock-out on translation and T-cell hyper-activation mediated by ZFP36L1? If deletion of ZFP36L1 itself has no effect on these processes, it seems unlikely that it is involved. In this respect, I am not sure that Fig 6 contributes to the understanding of ZFP36L.

4) Fig 7E: Differences in ZFP36L1 mRNA expression are claimed as a consequence of UPF1 deletion, and indeed there is a clear tendency to reduction of ZFP36L1 mRNA levels upon UPF1 KO. Yet the difference is statistically non-significant. Please, repeat this experiment to increase statistical significance. In addition, a clear discussion on how UPF1 -generally associated to mRNA degradation- contributes to increase ZFP36L1 mRNA levels would be appreciated.

5) Fig 6A: The decrease in global translation by GIGFYF1 knock-out upon activation claimed by the authors is not clear in Fig 6A and is non-significant upon quantification. Please, modify narrative accordingly.

6) Page 6: The authors state 'This included the PAN2/3 complex proteins which trim poly(A) tails prior to mRNA degradation through the CCR4/NOT complex'. To the best of my knowledge, the CCR4/NOT complex does not degrade the body of the mRNA. Both PAN2/3 and CCR4/NOT are deadenylases that function independently.

7) Please, label all Table sheets. Right now one has to guess what is being shown in most of them. Furthermore, it would be convenient to join all Tables related to the same Figure in one unique Excel with several sheets, rather than having many Tables with only one sheet each.

Minor comments:

8) Fig 1E: Shouldn't there be a better separation by biotinylation in the UltraID IP principal component analysis? In theory, only biotinylated proteins should be immunoprecipitated.

9) Fig 3B-E: Is the labeling not swapped, top (always +) is Biotin and bottom (- or +) is aCD3/aCD28?

10) Fig 7A data is from another paper, so I suggest to move this panel to Supplementary materials.

11) Fig S1A: Why is there so much labeling in the UltraID only lane without biotin?

12) Fig S1E: Please, explain better. What is WT?

13) Fig S4B: Please, explain the labels on top of the shapes.

14) Page 3: A time-course of incubation with biotin is lacking in Fig S1B, and thereby it is confusing that the authors direct readers to this figure when an increased to 16h incubation is claimed to be better.

Significance

Strengths: A thorough repository of ZFP36L1 interactors in primary human T-cells. A valuable resource for the community.

Weaknesses: There is little mechanistic insight on ZFP36L1 function or regulation.

Document d'Information : Le Traitement Médiatique des Violences Faites aux Femmes

Résumé Exécutif

Ce document d'information synthétise les discussions d'une table ronde sur le traitement médiatique des violences faites aux femmes, réunissant une journaliste d'investigation, une vulgarisatrice et une militante féministe.

Il ressort que si la médiatisation de ce sujet sociétal est croissante, elle est entachée de biais significatifs et de pratiques problématiques. Les points essentiels sont les suivants :

• Le Rôle Ambivalent des Médias : Les médias jouent un rôle crucial en rendant publiques des violences souvent cantonnées à la sphère privée, ce qui permet de faire évoluer les mentalités et de reconnaître le caractère systémique du problème.

Chaque avancée sociétale sur le sujet est liée à la médiatisation d'une affaire emblématique (Mazneff, Depardieu, etc.).

• Critiques Principales du Traitement Médiatique : La couverture médiatique est critiquée pour sa tendance à racialiser les agresseurs, servant un agenda politique raciste en surreprésentant les agresseurs étrangers ou racisés contre des victimes blanches.

On observe également une différence de traitement majeure entre la presse nationale, qui aborde parfois le sujet sous un angle systémique, et la presse locale (PQR), qui le confine souvent au sensationnalisme du "fait divers".

• Éthique Journalistique et Protection des Victimes : Le traitement rigoureux d'une affaire de violence sexiste et sexuelle (VSS) repose sur des principes déontologiques stricts.

La priorité est de croire et de protéger la victime, notamment par l'anonymat, et de respecter son choix de parler ou non.

L'enquête doit être irréprochable pour éviter les risques de diffamation et garantir la crédibilité du récit, ce qui inclut la vérification des faits et la procédure du "contradictoire" (contacter l'agresseur présumé).

• Les Angles Morts de la Médiatisation : De nombreuses formes de violences demeurent largement invisibles.

C'est le cas des violences psychologiques (contrôle, harcèlement numérique via traceurs) et surtout des violences visant les populations les plus marginalisées : les enfants, les travailleuses du sexe et les femmes trans, dont les agressions sont souvent ignorées, voire justifiées par un traitement médiatique transphobe et déshumanisant.

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1. Introduction et Définitions Clés

La discussion établit un cadre conceptuel pour analyser le traitement médiatique des violences faites aux femmes, un sujet de plus en plus présent dans le débat public, souvent à travers le prisme d'affaires très médiatisées impliquant des personnalités publiques (PPDA, Gérard Depardieu, Léo Grasset).

Définition du Patriarcat et de la Notion de "Femme"

Pour analyser les violences, les intervenantes adoptent une approche matérialiste et sociologique.

• Femme : Dans ce contexte, une "femme" n'est pas définie par sa biologie ou son identité de genre, mais comme une personne subissant des conditions sociales spécifiques, notamment le sexisme, les violences et l'exploitation par le système patriarcal.

• Patriarcat : Il est défini comme un système social qui hiérarchise les groupes sociaux "hommes" et "femmes".

Ce système organise l'exploitation (notamment économique via le travail domestique) et l'oppression des femmes, et sanctionne toute personne déviant des normes qu'il impose (ex: hétéronormativité, sanctionnée par l'homophobie).

2. Les Formes de Violence et le Rôle des Médias

Typologie des Violences Sexistes et Sexuelles (VSS)

Les VSS englobent une large gamme de violences, souvent sous-représentées dans leur diversité.

• Violences les plus médiatisées : Le viol et les agressions sexuelles sont les plus visibles médiatiquement, car perçus comme les plus graves.

Les violences conjugales physiques sont également mentionnées, mais les violences psychologiques restent largement ignorées.

• Statistiques et Binarité : Les statistiques disponibles sur les VSS sont majoritairement binaires (hommes/femmes), ce qui invisibilise les victimes non-binaires.

Pauline Bouty souligne que si la plupart des victimes sont des femmes et la plupart des auteurs des hommes, il est crucial de rappeler que des personnes de tous genres peuvent être victimes.

Il est rappelé que près de 90 % des victimes connaissent leur agresseur, qui est souvent un membre de la famille ou le conjoint, contredisant le mythe de l'agresseur inconnu dans une ruelle sombre.

L'Importance Cruciale du Rôle des Médias

Le traitement médiatique des VSS est considéré comme un enjeu public majeur et non une affaire privée.

• Le "5ème Pouvoir" : Jade Bourgerie, journaliste, qualifie les médias de "5ème pouvoir" dont le rôle est de refléter les maux de la société.

Traiter une affaire de VSS relève de l'intérêt public, car ces violences sont le symptôme d'une "société malade".

• Visibilité et Existence : Selon Pauline Bouty, "ce qu'on ne voit pas n'existe pas".

La médiatisation permet au public de prendre conscience de l'existence et de l'ampleur de ces violences.

Chaque progression dans la compréhension de ce phénomène est directement liée à la couverture médiatique d'une affaire symbolique.

• Déconstruire les Stéréotypes : La médiatisation aide à humaniser les victimes et les agresseurs, brisant l'image du "monstre".

Elle montre que l'agresseur peut être "votre voisin, votre frère, votre oncle", une personne perçue comme sympathique en société.

3. Pratiques et Éthique Journalistiques dans le Traitement des VSS

La journaliste Jade Bourgerie détaille les règles déontologiques qu'elle s'impose pour traiter ces sujets sensibles, en l'absence de règles formelles universelles dans la profession.

Les Règles Déontologiques et la Rigueur de l'Enquête

1. Respecter et Croire la Victime : Le point de départ est de croire la parole de la victime et de respecter ses volontés.

2. Rigueur de l'Enquête : L'article doit être "parfait" et "solide".

Cela implique de vérifier méticuleusement chaque élément fourni par la victime pour construire un dossier inattaquable et se prémunir contre les accusations de diffamation.

Exemple donné : retrouver une gynécologue consultée par une victime dans les années 90 pour corroborer une partie de son récit.

3. Le Contradictoire : Une étape essentielle consiste à contacter la personne mise en cause (l'agresseur présumé) pour lui exposer les faits recueillis et lui donner la possibilité de se défendre.

Le Rôle de l'Anonymat pour la Protection des Victimes

L'anonymat est un outil de protection essentiel pour les victimes, en particulier dans les milieux professionnels restreints (ex: musique classique) où tout le monde se connaît. Il permet à la victime d'éviter :

• D'être durablement étiquetée comme "victime de viol".

• De subir des représailles professionnelles ou sociales dans une société encore peu avancée sur ces questions.

4. Critiques Majeures du Traitement Médiatique Actuel

Plusieurs problèmes récurrents dans la couverture des VSS sont identifiés par les intervenantes.

La Racialisation des Récits

Lou Girard dénonce un biais racial majeur : les médias, en particulier ceux détenus par des groupes de droite et d'extrême-droite (citant les "empires Bolloré et Drahi"), tendent à surreprésenter les affaires où des femmes blanches sont agressées par des hommes racisés ou migrants.

Ce traitement sert un "narratif raciste" qui présente "la femme blanche, pure, la Française" comme étant attaquée par "le migrant, l'étranger".

Cela occulte la réalité statistique : la grande majorité des violences sont intra-communautaires et intrafamiliales.

Disparités entre Presse Nationale et Presse Quotidienne Régionale (PQR)

Un clivage important existe entre les types de médias.

Critère

Presse Nationale (ex: Le Monde, Libération)

Presse Quotidienne Régionale (PQR) (ex: La Dépêche)

Traitement

Tendance à traiter les affaires sous un angle plus systémique, souvent liées à des personnalités connues ou à des faits de grande ampleur.

Traitement majoritairement sous le prisme du fait divers et du sensationnalisme.

Biais Racial

Le narratif racialisant est "assez absent" des grands médias nationaux.

Le schéma "femme blanche victime d'un agresseur racisé" est beaucoup plus fréquent.

Causes

Journalistes plus jeunes, formés aux enjeux actuels des VSS dans les écoles de journalisme.

Journalistes souvent en poste depuis des décennies, moins formés à ces problématiques spécifiques.

L'Évolution du Vocabulaire : Du "Crime Passionnel" au "Féminicide"

Le langage utilisé a évolué, mais des termes problématiques persistent.

• Progrès : Le terme "féminicide" a émergé et s'est démocratisé après le mouvement #MeToo. Son usage est politique : il souligne que la victime a été tuée parce qu'elle est une femme, et non dans le cadre d'un simple homicide.

• Persistance : Des termes euphémisants ou inappropriés comme "crime passionnel" ou la description de viols comme des "relations sexuelles imposées" sont encore utilisés, minimisant la notion de violence et de domination.

5. Les Violences Invisibilisées et les Critères de Médiatisation

Violences Psychologiques et Violences contre les Populations Marginalisées

Certaines violences sont systématiquement absentes de la couverture médiatique.

• Violences Psychologiques : Le contrôle insidieux, qui ne "laisse pas de bleu", est très peu représenté. Pauline Bouty cite le documentaire Traquée de Marine Périn sur les hommes installant des traceurs sur les téléphones de leurs compagnes.

Ce contrôle peut aussi être financier ou social.

• Violences contre les enfants : Les enfants sont particulièrement vulnérables car dépendants des adultes qui sont souvent leurs agresseurs.

• Violences contre les femmes trans : Lou Girard souligne leur vulnérabilité extrême. "En tant que femme on a peur d'être violé, en tant que femme trans on a peur d'être violé puis tué."

Le traitement médiatique, quand il existe, est souvent abominable, utilisant des termes transphobes ("homme travesti") et présentant l'agression comme un fait divers "presque marrant".

Les victimes sont mégenrées, même après leur mort.

• Violences contre les travailleuses du sexe : Leurs agressions sont souvent invisibilisées ou justifiées par leur profession, niant la notion de consentement.

Les Critères de Médiatisation d'une Affaire

Pour qu'une affaire soit traitée médiatiquement de manière solide, plusieurs critères sont souvent nécessaires du point de vue journalistique :

• Avoir plusieurs victimes : Cela permet d'éviter la situation de "parole contre parole".

• Au moins une victime acceptant de parler à visage découvert : Cela renforce la crédibilité du récit.

• Des faits documentables avec des preuves : Une affaire reposant uniquement sur un témoignage sans plainte ni preuve est quasiment impossible à traiter pour un journaliste.

• Le consentement de la victime : Le respect de la parole de la victime est primordial. De nombreuses affaires ne sortent pas car les victimes ne souhaitent pas parler, un choix qui doit être absolument respecté.

6. L'Impact sur les Victimes et la Question du Langage

Le Manque de Couverture sur les Conséquences pour les Victimes

Les médias se concentrent sur les faits et les agresseurs, mais très rarement sur l'impact à long terme des violences sur la vie des victimes (psychologique, social, professionnel).

• Analyse Politique : Lou Girard analyse ce manque comme un choix politique.

S'intéresser à la "carrière brisée" de l'agresseur est commun, mais parler des "conséquences terribles du viol" sur la vie des femmes serait un acte "hautement féministe" que beaucoup de médias évitent.

• Le Rôle des Livres : Pauline Bouty nuance en affirmant que ce n'est peut-être pas le rôle des journalistes de parler à la place des victimes de leur ressenti.

Elle défend l'importance des espaces où les victimes peuvent s'exprimer avec leur propre voix, comme les livres (citant Florence Porcel) ou les films (Les Chatouilles).

L'Importance de la Précision Terminologique

L'usage de termes précis est un enjeu politique.

• Pédocriminalité vs. Pédophilie : Il est crucial de différencier la pédophilie (une paraphilie, un attrait) de la pédocriminalité (le passage à l'acte).

La plupart des personnes ayant des attirances pédophiles ne passent pas à l'acte et se font suivre. Un pédocriminel cherche avant tout à exercer une emprise et n'est pas nécessairement "pédophile".

• La Voix Active : Il est recommandé d'utiliser la voix active pour nommer l'agresseur et sa responsabilité : "un homme a violé une femme" plutôt que "une femme s'est fait violer".

Présenter les faits est un choix politique : soit on le fait avec des euphémismes, soit on nomme la violence telle qu'elle est.

Author response:

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

Reviewer #1 (Public review):

The authors focus on the molecular mechanisms by which EMT cells confer resistance to cancer cells. The authors use a wide range of methods to reveal that overexpression of Snail in EMT cells induces cholesterol/sphingomyelin imbalance via transcriptional repression of biosynthetic enzymes involved in sphingomyelin synthesis. The study also revealed that ABCA1 is important for cholesterol efflux and thus for counterbalancing the excess of intracellular free cholesterol in these snail-EMT cells. Inhibition of ACAT, an enzyme catalyzing cholesterol esterification, also seems essential to inhibit the growth of snail-expressing cancer cells.

However, It seems important to analyze the localization of ABCA1, as it is possible that in the event of cholesterol/sphingomyelin imbalance, for example, the intracellular trafficking of the pump may be altered.

The authors should also analyze ACAT levels and/or activity in snail-EMT cells that should be increased. Overall, the provided data are important to better understand cancer biology.

We thank the reviewer for recognizing the significance of our study. Consistent with the hypothesis that ABCA1 contributes to chemoresistance in hybrid E/M cells, we agree that demonstrating the localization of ABCA1 at the plasma membrane is important, and we have included additional experiments to address this point.

We also examined the expression of the major ACAT isoform in the kidney, SOAT1, across RCC cell lines. However, its expression did not correlate with that of Snail (Figure 4B), suggesting that SOAT1 is constitutively expressed at a certain level regardless of Snail expression. The details of these additional experiments are provided in the point-by-point responses below.

Reviewer #2 (Public review):

Summary:

In this study, the authors discovered that the chemoresistance in RCC cell lines correlates with the expression levels of the drug transporter ABCA1 and the EMT-related transcription factor Snail. They demonstrate that Snail induces ABCA1 expression and chemoresistance, and that ABCA1 inhibitors can counteract this resistance. The study also suggests that Snail disrupts the cholesterol-sphingomyelin (Chol/SM) balance by repressing the expression of enzymes involved in very long-chain fatty acid-sphingomyelin synthesis, leading to excess free cholesterol. This imbalance activates the cholesterol-LXR pathway, inducing ABCA1 expression. Moreover, inhibiting cholesterol esterification suppresses Snail-positive cancer cell growth, providing potential lipid-targeting strategies for invasive cancer therapy.

Strengths:

This research presents a novel mechanism by which the EMT-related transcription factor Snail confers drug resistance by altering the Chol/SM balance, introducing a previously unrecognized role of lipid metabolism in the chemoresistance of cancer cells. The focus on lipid balance, rather than individual lipid levels, is a particularly insightful approach. The potential for targeting cholesterol detoxification pathways in Snail-positive cancer cells is also a significant therapeutic implication.

Weaknesses:

The study's claim that Snail-induced ABCA1 is crucial for chemoresistance relies only on pharmacological inhibition of ABCA1, lacking additional validation. The causal relationship between the disrupted Chol/SM balance and ABCA1 expression or chemoresistance is not directly supported by data. Some data lack quantitative analysis.

We thank the reviewer for his/her insightful and constructive comments. In response, we have performed additional experiments using complementary approaches to further substantiate the contribution of Snail-induced ABCA1 expression to chemoresistance. Furthermore, to clarify the causal relationship between reduced sphingomyelin biosynthesis and ABCA1 expression, we conducted new experiments showing that supplementation with sphingolipids attenuates ABCA1 upregulation (Figure 3H). The details of these additional experiments are described in the point-by-point responses below.

Reviewer #1 (Recommendations for the authors):

In this paper, the authors reveal that snail expression in EMT-cells leads to an imbalance between cholesterol and sphingomyelin via a transcriptional repression of enzymes involved in the biosynthesis of sphingomyelin.

This paper is interesting and highlights how the imbalance of lipids would impact chemotherapy resistance. However, I have a few comments.

In Figure 2 in Eph4 cells, while filipin staining appears exclusively at the plasma membrane in the case of EpH4-snail cells filipin staining is also intracellular. It seems plausible that all filipin-positive intracellular staining is not exclusively in LDs, authors should therefore try to colocalize filipin with other intracellular markers. To this aim, authors might want to use topfluocholesterol-probe for instance.

We examined the distribution of TopFluor-cholesterol in hybrid E/M cells (Figure 2H) and found that TopFluor-cholesterol colocalizes with lipid droplets. In addition, we analyzed the colocalization between intracellular filipin signals and organelle-specific proteins, ADRP (lipid droplets) and LAMP1 (lysosomes) (Figure 2I). Since filipin binds exclusively to unesterified cholesterol, filipin signals did not colocalize with ADRP. Instead, we observed colocalization of filipin with LAMP1, suggesting that cholesterol accumulates in hybrid E/M cells in both esterified and unesterified forms.

In Figure 3, the authors reveal that the exogenous expression of the snail alters the ratio of cholesterol to sphingomyelin. The authors should reveal where is found the intracellular cholesterol and intracellular sphingomyelin within these cells Eph4-snail.

To investigate the lipid composition of the plasma membrane, we utilized lipid-binding protein probes, D4 (for cholesterol) and lysenin (for sphingomyelin) (Figures 2L and 2M). We found that the plasma membrane cholesterol content was not affected by EMT, whereas sphingomyelin levels were markedly decreased. In addition, intracellular cholesterol was visualized (Comment 1-1; Figures 2E–2K). On the other hand, because visualization of intracellular sphingomyelin is technically challenging, we were unable to include this analysis in the present study. We consider this an important direction for future investigation.

Regarding the model described in panel K of Figure 3. I would expect that the changes in lipid-membrane organization depicted in panel K should affect the pattern of GM1 toxin for instance or the motility of raft-associated proteins for instance. The authors could perform these experiments in order to sustain the change of lipid plasma membrane organization.

We attempted staining with FITC–cholera toxin to visualize GM1, but both EpH4 and EpH4–Snail cells exhibited very low levels of GM1, resulting in minimal or no detectable staining (data not shown). Instead, to assess the impact of decreased sphingomyelin on the overall biophysical properties of the plasma membrane, we used a plasma membrane–specific lipid-order probe, FπCM–SO₃ (Figures 2N–2P and Figure 2—figure supplement 3). We found that the plasma membrane of EpH4–Snail cells was more disordered (fluidized), suggesting that the overall properties of the plasma membrane are altered by ectopic expression of Snail.

Another issue is the intracellular localization of ABCA1 in Eph4-Snail cells. Knowing that a change in the cholesterol/sphingomyelin ratio can also modify intracellular protein trafficking, it seems important to analyze the intracellular localization of ABCA1 in EPh4-Snail cells.

We performed immunofluorescence microscopy for ABCA1 and found that ABCA1 was mainly localized at the plasma membrane in EpH4–Snail cells (Figure 1M).

As for the data on ACAT inhibition, we expect an increase in ACAT activity and protein levels in EMT cells overexpressing Snail. The authors should also investigate this point.

As noted in our response to the public review, we examined the expression of the major ACAT isoform in the kidney, SOAT1, across RCC cell lines. However, its expression did not correlate with Snail (Figure 4B), suggesting that SOAT1 is expressed at sufficient levels even in cells with low Snail expression. We agree that measuring ACAT activity would be important, as ACATs are regulated at multiple levels. However, we consider this to be beyond the scope of the present study and plan to address it in future work.

Minor comments

I do not understand why in the text, Figure S1 appears after Figure S2. The authors might want to change the numbering of these two figures.

We thank the reviewer for pointing this out. We have corrected the numbering of the supplementary figures so that Figure S1 now appears before Figure S2 in both the text and the revised figure legends.

Page 5, lane 20 Figure 1I instead of 1H.

Page 6, lane 2, Figure 1J instead of 1I, and lane 9 Figure 1H instead of 1I.

We thank the reviewer for carefully checking the figure references. We have corrected the figure numbering errors in the text as suggested.

Reviewer #2 (Recommendations for the authors):

For Figures 1B, 1H, 1J, 2B, 2C, 3G, S3A, and S3B, to enhance data reliability, it is necessary to conduct a quantitative analysis of the Western blot data. The average values from at least three biological replicates should be calculated, with statistical significance assessed.

We have conducted quantitative analyses of the Western blot data for Figures 1B, 1H, 1J, 2B, 2C, 3G, S3A, and S3B. Band intensities from at least three independent biological replicates were quantified, and the mean values with statistical significance are now presented in the revised figures.

For Figures 1D, 2A, 2D, and S2, the images of cells or tissues should not rely solely on selected fields. Quantitative analysis is required, and the mean values from at least three biological replicates should be provided with statistical significance testing.

We have performed quantitative analyses for Figures 1D, 2A, 2D, and S2. The quantification was based on data from at least three independent biological replicates, and the mean values with statistical significance are now included in the revised figures.

For Figures 1A, 1G, 4, and S5, evaluating ABCA1's involvement in drug resistance based solely on CsA treatment is insufficient. Demonstrating the loss of drug resistance through ABCA1 knockdown or knockout is necessary.

We generated ABCA1 knockout EpH4–Snail cells and examined their resistance to nitidine chloride. However, knockout of ABCA1 alone did not affect resistance to the compound (Figure 2 - figure supplement 2). This may be due to secondary metabolic alterations induced by ABCA1 loss or compensatory upregulation of other LXR-induced cholesterol efflux transporters. Instead, we demonstrated that treatment with the LXR inhibitor GSK2033 reduced the nitidine chloride resistance of EpH4–Snail cells (Figure 2C), supporting the idea that enhanced efflux of antitumor agents through the LXR–ABCA1–mediated cholesterol efflux pathway contributes to nitidine chloride resistance.

For Figure 3, to establish a causal relationship between changes in the Chol/SM balance and ABCA1 expression, it is important to test whether modifying cholesterol and SM levels to disrupt this balance affects ABCA1 expression.

Regarding causality, as shown in Figure 2, we have already demonstrated that reducing cholesterol levels in EpH4–Snail cells decreases ABCA1 expression. To further explore this relationship, we examined whether increasing sphingomyelin levels by adding ceramide to the culture medium—thereby restoring the sphingomyelin-to-cholesterol ratio—would reduce ABCA1 expression (Figure 3H). Indeed, supplementation with C22:0 ceramide decreased ABCA1 expression, suggesting that downregulation of the VLCFA-sphingomyelin biosynthetic pathway triggers ABCA1 upregulation. Collectively, these findings support a causal relationship between the Chol/SM balance and ABCA1 expression.

In Figure 3, if there is any information on differences in cholesterol affinity between LCFA-SM and VLCFA-SM, it would be beneficial to include it in the manuscript.

Differences in cholesterol affinity between LCFA-SM and VLCFA-SM in cellular membranes remain controversial and have yet to be fully elucidated. The decrease in cell surface sphingomyelin content, evaluated by lysenin staining (Figure 2L), was more pronounced than that of total sphingomyelin (Figure 3A). Given that VLCFA-SMs have been suggested to undergo distinct trafficking during recycling from endosomes to the plasma membrane (Koivusalo et al. Mol Biol Cell 2007), their reduction may lead to decreased plasma membrane sphingomyelin content by altering its intracellular distribution. We have added this discussion to the revised manuscript.

In Figure 3F, it is recommended to assess housekeeping gene expression as a control. Quantitative real-time PCR should be performed, and the average values from at least three biological replicates should be presented.

We have performed quantitative RT-PCR analysis. The average values from at least three independent biological replicates are presented in Figure 3G.

For Figure 3F, to show whether the reduction of CERS3 or ELOVL7 affects the Chol/SM balance and ABCA1 expression, it is necessary to investigate the phenotypes following the knockdown or knockout of these enzymes.

We fully agree that phenotypic analyses of epithelial cells lacking CerS3 or ELOVL7 would provide valuable insights. However, we consider such investigations to be beyond the scope of the present study and plan to pursue them in future work.

Clarifying whether similar phenotypes are induced by other EMT-related transcription factors, or if they are specific to Snail, would be beneficial.

We agree that examining whether similar phenotypes are induced by other EMT-related transcription factors would be highly valuable for understanding the broader EMT network. However, as the focus of the present study is on lipid metabolic alterations associated with EMT—particularly the imbalance between sphingomyelin and cholesterol—we consider this investigation to be beyond the scope of the current work and plan to address it in future studies.

There are errors in figure citations within the text that need correction:

p.9 l.18 Fig. 3D → Fig. 3G

p.9 l.22 Fig. 3I → Fig. 3H

p.9 l.23 Fig. S2 → Fig. S4

p.10 l.6 Fig. 3J → Fig. 1J

p.10 l.8 Fig. 3J → Fig. 1J

p.10 l.9 Fig. 3K → Fig. 3I

p.10 l.12 Fig. 3H → Fig. 3J

p.10 l.14 Fig. 2D and Fig. S4 → Fig. 2G and Fig. S4D

We thank the reviewer for carefully pointing out these citation errors. We have corrected all figure references in the text as suggested.

simple past tense di "swim", in senso letterale "gli occhi gli nuotavano nella testa", in senso letterato "così stordito, così confuso da avere la vista appannata"

[https://dictionary.cambridge.org/it/dizionario/inglese-italiano/swim]

Author response:

The following is the authors’ response to the previous reviews

Reviewer #1 (Public review):

This paper presents a computational model of the evolution of two different kinds of helping ("work," presumably denoting provisioning, and defense tasks) in a model inspired by cooperatively breeding vertebrates. The helpers in this model are a mix of previous offspring of the breeder and floaters that might have joined the group, and can either transition between the tasks as they age or not. The two types of help have differential costs: "work" reduces "dominance value," (DV), a measure of competitiveness for breeding spots, which otherwise goes up linearly with age, but defense reduces survival probability. Both eventually might preclude the helper from becoming a breeder and reproducing. How much the helpers help, and which tasks (and whether they transition or not), as well as their propensity to disperse, are all evolving quantities. The authors consider three main scenarios: one where relatedness emerges from the model, but there is no benefit to living in groups, one where there is no relatedness, but living in larger groups gives a survival benefit (group augmentation, GA), and one where both effects operate. The main claim is that evolving defensive help or division of labor requires the group augmentation; it doesn't evolve through kin selection alone in the authors' simulations.

This is an interesting model, and there is much to like about the complexity that is built in. Individual-based simulations like this can be a valuable tool to explore the complex interaction of life history and social traits. Yet, models like this also have to take care of both being very clear on their construction and exploring how some of the ancillary but potentially consequential assumptions affect the results, including robust exploration of the parameter space. I think the current manuscript falls short in these areas, and therefore, I am not yet convinced of the results. In this round, the authors provided some clarity, but some questions still remain, and I remain unconvinced by a main assumption that was not addressed.

Based on the authors' response, if I understand the life history correctly, dispersers either immediately join another group (with 1-the probability of dispersing), or remain floaters until they successfully compete for a breeder spot or die? Is that correct? I honestly cannot decide because this seems implicit in the first response but the response to my second point raises the possibility of not working while floating but can work if they later join a group as a subordinate. If it is the case that floaters can have multiple opportunities to join groups as subordinates (not as breeders; I assume that this is the case for breeding competition), this should be stated, and more details about how. So there is still some clarification to be done, and more to the point, the clarification that happened only happened in the response. The authors should add these details to the main text. Currently, the main text only says vaguely that joining a group after dispersing " is also controlled by the same genetic dispersal predisposition" without saying how.

In each breeding cycle, individuals have the opportunity to become a breeder, a helper, or a floater. Social role is really just a state, and that state can change in each breeding cycle (see Figure 1). Therefore, floaters may join a group as subordinates at any point in time depending on their dispersal propensity, and subordinates may also disperse from their natal group any given time. In the “Dominance-dependent dispersal propensities” section in the SI, this dispersal or philopatric tendency varies with dominance rank.

We have added: “In each breeding cycle” (L415) to clarify this further.

In response to my query about the reasonableness of the assumption that floaters are in better condition (in the KS treatment) because they don't do any work, the authors have done some additional modeling but I fail to see how that addresses my point. The additional simulations do not touch the feature I was commenting on, and arguably make it stronger (since assuming a positive beta_r -which btw is listed as 0 in Table 1- would make floaters on average be even more stronger than subordinates). It also again confuses me with regard to the previous point, since it implies that now dispersal is also potentially a lifetime event. Is that true?

We are not quite sure where the reviewer gets this idea because we have never assumed a competitive advantage of floaters versus helpers. As stated in the previous revision, floaters can potentially outcompete subordinates of the same age if they attempt to breed without first queuing as a subordinate (step 5 in Figure 1) if subordinates are engaged in work tasks. However, floaters also have higher mortality rates than group members, which makes them have lower age averages. In addition, helpers have the advantage of always competing for an open breeding position in the group, while floaters do not have this preferential access (in Figure S2 we reduce even further the likelihood of a floater to try to compete for a breeding position).

Moreover, in the previous revision (section: “Dominance-dependent dispersal propensities” in the SI) we specifically addressed this concern by adding the possibility that individuals, either floaters or subordinate group members, react to their rank or dominance value to decide whether to disperse (if subordinate) or join a group (if floater). Hence, individuals may choose to disperse when low ranked and then remain on the territory they dispersed to as helpers, OR they may remain as helpers in their natal territory as low ranked individuals and then disperse later when they attain a higher dominance value. The new implementation, therefore, allows individuals to choose when to become floaters or helpers depending on their dominance value. This change to the model affects the relative competitiveness between floaters and helpers, which avoids the assumption that either low- or high-quality individuals are the dispersing phenotype and, instead, allows rank-based dispersal as an emergent trait. As shown in Figure S5, this change had no qualitative impact on the results.

To make this all clearer, we have now added to all of the relevant SI tables a new row with the relative rank of helpers vs floaters. As shown, floaters do not consistently outrank helpers. Rather, which role is most dominant depends on the environment and fitness trade-offs that shape their dispersing and helping decisions.

Some further clarifications: beta_r is a gene that may evolve either positive or negative values, 0 (no reaction norm of dispersal to dominance rank) is the initial value in the simulations before evolution takes place. Therefore, this value may evolve to positive or negative values depending on evolutionary trade-offs. Also, and as clarified in the previous comment, the decision to disperse or not occurs at each breeding cycle, so becoming a floater, for example, is not a lifetime event unless they evolve a fixed strategy (dispersal = 0 or 1). 

Meanwhile, the simplest and most convincing robustness check, which I had suggested last round, is not done: simply reduce the increase in the R of the floater by age relative to subordinates. I suspect this will actually change the results. It seems fairly transparent to me that an average floater in the KS scenario will have R about 15-20% higher than the subordinates (given no defense evolves, y_h=0.1 and H_work evolves to be around 5, and the average lifespan for both floaters and subordinates are in the range of 3.7-2.5 roughly, depending on m). That could be a substantial advantage in competition for breeding spots, depending on how that scramble competition actually works. I asked about this function in the last round (how non-linear is it?) but the authors seem to have neglected to answer.

As we mentioned in the previous comment above, we have now added the relative rank between helpers and floaters to all the relevant SI tables, to provide a better idea of the relative competitiveness of residents versus dispersers for each parameter combination. As seen in Table S1, the competitive advantage of floaters is only marginally in the favor for floaters in the “Only kin selection” implementation. This advantage only becomes more pronounced when individuals can choose whether to disperse or remain philopatric depending on their rank. In this case, the difference in rank between helpers and floaters is driven by the high levels of dispersal, with only a few newborns (low rank) remaining briefly in the natal territory (Table S6). Instead, the high dispersal rates observed under the “Only kin selection” scenario appear to result from the low incentives to remain in the group when direct fitness benefits are absent, unless indirect fitness benefits are substantially increased. This effect is reinforced by the need for task partitioning to occur in an all-or-nothing manner (see the new implementation added to the “Kin selection and the evolution of division of labor” in the Supplementary materials; more details in following comments).

In addition, we specifically chose not to impose this constraint of forcing floaters to be lower rank than helpers because doing so would require strong assumptions on how the floaters rank is determined. These assumptions are unlikely to be universally valid across natural populations (and probably not commonly met in most species) and could vary considerably among species. Therefore, it would add complexity to the model while reducing generalizability.

As stated in the previous revision, no scramble competition takes place, this was an implementation not included in the final version of the manuscript in which age did not have an influence in dominance. Results were equivalent and we decided to remove it for simplicity prior to the original submission, as the model is already very complex in the current stage; we simply forgot to remove it from Table 1, something we explained in the previous round of revisions.

More generally, I find that the assumption (and it is an assumption) floaters are better off than subordinates in a territory to be still questionable. There is no attempt to justify this with any data, and any data I can find points the other way (though typically they compare breeders and floaters, e.g.: https://bioone.org/journals/ardeola/volume-63/issue-1/arla.63.1.2016.rp3/The-Unknown-Life-of-Floaters--The-Hidden-Face-of/10.13157/arla.63.1.2016.rp3.full concludes "the current preliminary consensus is that floaters are 'making the best of a bad job'."). I think if the authors really want to assume that floaters have higher dominance than subordinates, they should justify it. This is driving at least one and possibly most of the key results, since it affects the reproductive value of subordinates (and therefore the costs of helping).

We explicitly addressed this in the previous revision in a long response about resource holding potential (RHP). Once again, we do NOT assume that dispersers are at a competitive advantage to anyone else. Floaters lack access to a territory unless they either disperse into an established group or colonize an unoccupied territory. Therefore, floaters endure higher mortalities due to the lack of access to territories and group living benefits in the model, and are not always able to try to compete for a breeding position.

The literature reports mixed evidence regarding the quality of dispersing individuals, with some studies identifying them as low-quality and others as high-quality, attributing this to them experiencing fewer constraints when dispersing that their counterparts (e.g. Stiver et al. 2007 Molecular Ecology; Torrents‐Ticó, et al. 2018 Journal of Zoology). Additionally, dispersal can provide end-of-queue individuals in their natal group an opportunity to join a queue elsewhere that offers better prospects, outcompeting current group members (Nelson‐Flower et al. 2018 Journal of Animal Ecology). Moreover, in our model floaters do not consistently have lower dominance values or ranks than helpers, and dominance value is often only marginally different.

In short, we previously addressed the concern regarding the relative competitiveness of floaters compared to subordinate group members. To further clarify this point here, we have now included additional data on relative rank in all of the relevant SI tables. We hope that these additions will help alleviate any remaining concerns on this matter.

Regarding division of labor, I think I was not clear so will try again. The authors assume that the group reproduction is 1+H_total/(1+H_total), where H_total is the sum of all the defense and work help, but with the proviso that if one of the totals is higher than "H_max", the average of the two totals (plus k_m, but that's set to a low value, so we can ignore it), it is replaced by that. That means, for example, if total "work" help is 10 and "defense" help is 0, total help is given by 5 (well, 5.1 but will ignore k_m). That's what I meant by "marginal benefit of help is only reduced by a half" last round, since in this scenario, adding 1 to work help would make total help go to 5.5 vs. adding 1 to defense help which would make it go to 6. That is a pretty weak form of modeling "both types of tasks are necessary to successfully produce offspring" as the newly added passage says (which I agree with), since if you were getting no defense by a lot of food, adding more food should plausibly have no effect on your production whatsoever (not just half of adding a little defense). This probably explains why often the "division of labor" condition isn't that different than the no DoL condition.

The model incorporates division of labor as the optimal strategy for maximizing breeder productivity, while penalizing helping efforts that are limited to either work or defense alone. Because the model does not intend to force the evolution of help as an obligatory trait (breeders may still reproduce in the absence of help; k₀ ≠ 0), we assume that the performance of both types of task by the helpers is a non-obligatory trait that complements parental care.

That said, we recognize the reviewer’s concern that the selective forces modeled for division of labor might not be sufficient in the current simulations. To address this, we have now introduced a new implementation, as discussed in the “Kin selection and the evolution of division of labor” section in the SI. In this implementation, division of labor becomes obligatory for breeders to gain a productivity boost from the help of subordinate group members. The new implementation tests whether division of labor can arise solely from kin selection benefits. Under these premises, philopatry and division of labor do emerge through kin selection, but only when there is a tenfold increase in productivity per unit of help compared to the default implementation. Thus, even if such increases are biologically plausible, they are more likely to reflect the magnitudes characteristic of eusocial insects rather than of cooperatively breeding vertebrates (the primary focus of this model). Such extreme requirements for productivity gains and need for coordination further suggest that group augmentation, and not kin selection, is probably the primary driving force particularly in harsh environments. This is now discussed in L210-213.

Reviewer #2 (Public review):

Summary:

This paper formulates an individual-based model to understand the evolution of division of labor in vertebrates. The model considers a population subdivided in groups, each group has a single asexually-reproducing breeder, other group members (subordinates) can perform two types of tasks called "work" or "defense", individuals have different ages, individuals can disperse between groups, each individual has a dominance rank that increases with age, and upon death of the breeder a new breeder is chosen among group members depending on their dominance. "Workers" pay a reproduction cost by having their dominance decreased, and "defenders" pay a survival cost. Every group member receives a survival benefit with increasing group size. There are 6 genetic traits, each controlled by a single locus, that control propensities to help and disperse, and how task choice and dispersal relate to dominance. To study the effect of group augmentation without kin selection, the authors cross-foster individuals to eliminate relatedness. The paper allows for the evolution of the 6 genetic traits under some different parameter values to study the conditions under which division of labour evolves, defined as the occurrence of different subordinates performing "work" and "defense" tasks. The authors envision the model as one of vertebrate division of labor.

The main conclusion of the paper is that group augmentation is the primary factor causing the evolution of vertebrate division of labor, rather than kin selection. This conclusion is drawn because, for the parameter values considered, when the benefit of group augmentation is set to zero, no division of labor evolves and all subordinates perform "work" tasks but no "defense" tasks.

Strengths:

The model incorporates various biologically realistic details, including the possibility to evolve age polytheism where individuals switch from "work" to "defence" tasks as they age or vice versa, as well as the possibility of comparing the action of group augmentation alone with that of kin selection alone.

Weaknesses:

The model and its analysis is limited, which makes the results insufficient to reach the main conclusion that group augmentation and not kin selection is the primary cause of the evolution of vertebrate division of labor. There are several reasons.

First, the model strongly restricts the possibility that kin selection is relevant. The two tasks considered essentially differ only by whether they are costly for reproduction or survival. "Work" tasks are those costly for reproduction and "defense" tasks are those costly for survival. The two tasks provide the same benefits for reproduction (eqs. 4, 5) and survival (through group augmentation, eq. 3.1). So, whether one, the other, or both tasks evolve presumably only depends on which task is less costly, not really on which benefits it provides. As the two tasks give the same benefits, there is no possibility that the two tasks act synergistically, where performing one task increases a benefit (e.g., increasing someone's survival) that is going to be compounded by someone else performing the other task (e.g., increasing that someone's reproduction). So, there is very little scope for kin selection to cause the evolution of labour in this model. Note synergy between tasks is not something unusual in division of labour models, but is in fact a basic element in them, so excluding it from the start in the model and then making general claims about division of labour is unwarranted. I made this same point in my first review, although phrased differently, but it was left unaddressed.

The scope of this paper was to study division of labor in cooperatively breeding species with fertile workers, in which help is exclusively directed towards breeders to enhance offspring production (i.e., alloparental care), as we stated in the previous review. Therefore, in this context, helpers may only obtain fitness benefits directly or indirectly by increasing the productivity of the breeders. This benefit is maximized when division of labor occurs between group members as there is a higher return for the least amount of effort per capita. Our focus is in line with previous work in most other social animals, including eusocial insects and humans, which emphasizes how division of labor maximizes group productivity. This is not to suggest that the model does not favor synergy, as engaging in two distinct tasks enhances the breeders' productivity more than if group members were to perform only one type of alloparental care task. We have expanded on the need for division of labor by making the performance of each type of task a requirement to boost the breeders productivity, see more details in a following comment.

Second, the parameter space is very little explored. This is generally an issue when trying to make general claims from an individual-based model where only a very narrow parameter region has been explored of a necessarily particular model. However, in this paper, the issue is more evident. As in this model the two tasks ultimately only differ by their costs, the parameter values specifying their costs should be varied to determine their effects. Instead, the model sets a very low survival cost for work (yh=0.1) and a very high survival cost for defense (xh=3), the latter of which can be compensated by the benefit of group augmentation (xn=3). Some very limited variation of xh and xn is explored, always for very high values, effectively making defense unevolvable except if there is group augmentation. Hence, as I stated in my previous review, a more extensive parameter exploration addressing this should be included, but this has not been done. Consequently, the main conclusion that "division of labor" needs group augmentation is essentially enforced by the limited parameter exploration, in addition to the first reason above.

We systematically explored the parameter landscape and report in the body of the paper only those ranges that lead to changes in the reaction norms of interest (other ranges are explored in the SI). When looking into the relative magnitude of cost of work and defense tasks, it is important to note that cost values are not directly comparable because they affect different traits. However, the ranges of values capture changes in the reaction norms that lead to rank-depending task specialization.

To illustrate this more clearly, we have added a new section in the SI (Variation in the cost of work tasks instead of defense tasks section) showing variation in y_h, which highlights how individuals trade off the relative costs of different tasks. As shown, the results remain consistent with everything we showed previously: a higher cost of work (high y_h) shifts investment toward defense tasks, while a higher cost of defense (high x_h) shifts investment toward work tasks.

Importantly, additional parameter values were already included in the SI of the previous revision, specifically to favor the evolution of division of labor under only kin selection. Basically, division of labor under only kin selection does happen, but only under conditions that are very restrictive, as discussed in the “Kin selection and the evolution of division of labor” section in the SI. We have tried to make this point clearer now (see comments to previous reviewer above, and to this reviewer right below).

Third, what is called "division of labor" here is an overinterpretation. When the two tasks evolve, what exists in the model is some individuals that do reproduction-costly tasks (so-called "work") and survival-costly tasks (so-called "defense"). However, there are really no two tasks that are being completed, in the sense that completing both tasks (e.g., work and defense) is not necessary to achieve a goal (e.g., reproduction). In this model there is only one task (reproduction, equation 4,5) to which both "tasks" contribute equally and so one task doesn't need to be completed if the other task compensates for it. So, this model does not actually consider division of labor.

Although it is true that we did not make the evolution of help obligatory and, therefore, did not impose division of labor by definition, the assumptions of the model nonetheless create conditions that favor the emergence of division of labor. This is evident when comparing the equilibria between scenarios where division of labor was favored versus not favored (Figure 2 triangles vs circles).

That said, we acknowledge the reviewer’s concern that the selective forces modeled in our simulations may not, on their own, be sufficient to drive the evolution of division of labor under only kin selection. Therefore, we have now added a section where we restrict the evolution of help to instances in which division of labor is necessary to have an impact on the dominant breeder productivity. Under this scenario, we do find division of labor (as well as philopatry) evolving under only kin selection. However, this behavior only evolves when help highly increases the breeders’ productivity (by a factor of 10 what is needed for the evolution of division of labor under group augmentation). Therefore, group augmentation still appears to be the primary driver of division of labor, while kin selection facilitates it and may, under certain restrictive circumstances, also promote division of labor independently (discussed in L210-213).

Reviewer #1 (Recommendations for the authors):

I really think you should do the simulations where floaters do not come out ahead by floating. That will likely change the result, but if it doesn't, you will have a more robust finding. If it does, then you will have understood the problem better.

As we outlined in the previous round of revisions, implementing this change would be challenging without substantially increasing model complexity and reducing its general applicability, as it would require strong assumptions that could heavily influence dispersal decisions. For instance, by how much should helpers outcompete floaters? Would a floater be less competitive than a helper regardless of age, or only if age is equal? If competitiveness depends on equal age, what is the impact of performing work tasks given that workers always outcompete immigrants? Conversely, if floaters are less competitive regardless of age, is it realistic that a young individual would outcompete all immigrants? If a disperser finds a group immediately after dispersal versus floating for a while, is the dominance value reduced less (as would happen to individuals doing prospections before dispersal)? 

Clearly it is not as simple as the referee suggests because there are many scenarios that would need to be considered and many assumptions made in doing this. As we explained to the points above, we think our treatment of floaters is consistent with the definition of floaters in the literature, and our model takes a general approach without making too many assumptions.

Reviewer #2 (Recommendations for the authors):

The paper's presentation is still unclear. A few instances include the following. It is unclear what is plotted in the vertical axes of Figure 2, which is T but T is a function of age t, so this T is presumably being plotted at a specific t but which one it is not said.

The values graphed are the averages of the phenotypically expressed tasks, not the reaction norms per se. We have now rewritten the the axis to “Expressed task allocation T (0 = work, 1 = defense)” to increase clarity across the manuscript.

The section titled "The need for division of labor" in the methods is still very unclear.

We have rephased this whole section to improve clarity.

Author response:

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

Reviewer #1 (Public review):

Nielsen et al have identified a new disease mechanism underlying hypoplastic left heart syndrome due to variants in ribosomal protein genes that lead to impaired cardiomyocyte proliferation. This detailed study starts with an elegant screen in stemcell-derived cardiomyocytes and whole genome sequencing of human patients and extends to careful functional analysis of RP gene variants in fly and fish models. Striking phenotypic rescue is seen by modulating known regulators of proliferation, including the p53 and Hippo pathways. Additional experiments suggest that the cell type specificity of the variants in these ubiquitously expressed genes may result from genetic interactions with cardiac transcription factors. This work positions RPs as important regulators of cardiomyocyte proliferation and differentiation involved in the etiology of HLHS, although the downstream mechanisms are unclear.

We thank Reviewer 1 for the thoughtful assessment of our manuscript. Our point-bypoint responses to the recommendations are provided (Reviewer 1, “Recommendations for the authors”).

Reviewer #2 (Public review):

Tanja Nielsen et al. present a novel strategy for the identification of candidate genes in Congenital Heart Disease (CHD). Their methodology, which is based on comprehensive experiments across cell models, Drosophila and zebrafish models, represents an innovative, refreshing and very useful set of tools for the identification of disease genes, in a field which are struggling with exactly this problem. The authors have applied their methodology to investigate the pathomechanisms of Hypoplastic Left Heart Syndrome (HLHS) - a severe and rare subphenotype in the large spectrum of CHD malformations. Their data convincingly implicates ribosomal proteins (RPs) in growth and proliferation defects of cardiomyocytes, a mechanism which is suspected to be associated with HLHS.

By whole genome sequencing analysis of a small cohort of trios (25 HLHS patients and their parents), the authors investigated a possible association between RP encoding genes and HLHS. Although the possible association between defective RPs and HLHS needs to be verified, the results suggest a novel disease mechanism in HLHS, which is a potentially substantial advance in our understanding of HLHS and CHD. The conclusions of the paper are based on solid experimental evidence from appropriate high- to medium-throughput models, while additional genetic results from an independent patient cohort are needed to verify an association between RP encoding genes and HLHS in patients.

We thank Reviewer 2 for the thoughtful assessment of our manuscript. Our point-by-point responses to the recommendations are provided (Reviewer 2, “Recommendations for the authors”).

Reviewer #1 (Recommendations for the authors): 

(1) Despite an interesting surveillance model, the disease-causing mechanisms directly downstream of the RP variants remain unclear. Can the authors provide any evidence for abnormal ribosomes or defects in translation in cells harboring such variants? The possibility that reduced translation of cardiac transcription factors such as TBX5 and NKX2-5 may contribute to the functional interactions observed should be considered. How do the authors consider that the RP variants are affecting transcript levels as observed in the study?

Our model implies that cell cycle arrest does not require abnormal ribosomes or translational defects but instead relies on the sensing of RP levels or mutations as a fitness-sensing mechanism that activates TP53/CDKN1A-dependent arrest. Supporting this framework, we observed no significant changes in TBX5 or NKX2-5 expression (data not shown), but rather an upregulation of CDKN1A levels upon RP KD.

(2) The authors suggest that a nucleolar stress program is activated in cells harboring RP gene variants. Can they provide additional evidence for this beyond p53 activation? 

We added additional data to support nucleolar stress (Suppl. Fig. 6) and text (lines 52635):

To determine whether cardiac KD of RpS15Aa causes nucleolar stress in the Drosophila heart, we stained larval hearts for Fibrillarin, a marker for nucleoli and nucleolar integrity.  We found that RpS15Aa KD causes expansion of nucleolar Fibrillarin staining in cardiomyocyte, which is a hallmark of nucleolar stress (Suppl. Fig. 6A-C). As a control, we also performed cardiac KD of Nopp140, which is known to cause nucleolar stress upon loss-of-function. We found a similar expansion of Fibrillarin staining in larval cardiomyocyte nuclei (Suppl. Fig. 6C,D). This suggests that RpS15Aa KD indeed causes nucleolar stress in the Drosophila heart, that likely contributes to the dramatic heart loss in adults.

Other recommendations: 

(3) Concerning the cell type specificity, in the proliferation screen, were similar effects seen on the actinin negative as actinin positive EdU+ cells? It would be helpful to refer to the fibroblast result shown in Supplementary Figure 1C in the results section. 

As suggested by reviewer #1, we have added a reference to Supplementary Fig. 1C, D and noted that RP knockdown exerts a non–CM-specific effect on proliferation.

(4) The authors refer to HLHS patients with atrial septal defects and reduced right ventricular ejection fraction. Please clarify the specificity of the new findings to HLHS versus other forms of CHD, as implied in several places in the manuscript, including the abstract.

This study focused on a cohort of 25 HLHS proband-parent trios selected for poor clinical outcome, including restrictive atrial septal defect and reduced right ventricular ejection fraction.  We have revised the following sentence  in response to the Reviewer’s comment (lines 567-571): “While our study highlights the potential of this approach for gene prioritization, additional research is needed to directly demonstrate the functional consequence of the identified genetic variants, verify an association between RP encoding genes and HLHS in other patient cohorts with and without poor outcome, and determine if RP variants have a broader role in CHD susceptibility.

(5) The multi-model approach taken by the authors is clearly a good system for characterizing disease-causing variants. Did the authors score for cardiomyocyte proliferation or the time of phenotypic onset in the zebrafish model? 

We used an antibody against phosphohistone 3 to identify proliferating cells and DAPI to identify all cardiac cells in control injected, rps15a morphants, and rps15a crispants. We found that  cell numbers and proliferating cells were significantly reduced at 24 and 48 hpf. By 72 hpf cardiac cell proliferation is greatly diminished even in controls, where proliferation typically declines. 

Reduced ventricular cardiomyocyte numbers could potentially result from impaired addition of LTPB3-expressing progenitors. In experiments where altered cardiac rhythm is observed, please comment on the possible links to proliferation.

Heart function data showed that heart period (R-R interval) was unaffected in morphants and crispants at 72 hpf where we also observed significant reductions in cell numbers. This suggests that the bradycardia observed in the rps15a + nkx2.5 or tbx5a double KD (Sup. Fig. 5D & E) was not due to the reduction in cell numbers alone. 

Author response image 1.

Finally, the use of the mouse to model HLHS in potential follow-up studies should be discussed. 

We have added a mouse model comment to the discussion (lines 571-74): “In conclusion, we propose that the approach outlined in this study provides a novel framework for rapidly prioritizing candidate genes and systematically testing them, individually or in combination, using a CRISPR/Cas9 genome-editing strategy in mouse embryos (PMID: 28794185)”.

(6) When the authors scored proliferation in cells from the proband in family 75H, did they validate that RPS15A expression is reduced, consistent with a regulatory region defect? 

Good point. We examined RPS15A expression in these cells and found no significant reduction in gene expression in day 25 cardiomyocytes (data not shown). One possible explanation is that this variant may regulate RPS15A expression in a stage-specific manner during differentiation or under additional stress conditions.

(7) Minor point. Typo on line 494: comma should be placed after KD, not before.

Thank you, this has now been corrected (new line 490)

Reviewer #2 (Recommendations for the authors):  

(1) The authors are invited to revise the part of the manuscript that describes the genetic analysis and provide a more balanced discussion of the WGS data, with a conclusion that aligns with the strength of the human genetic data. 

We disagree with reviewer #2’s assessment. The goal of our study is not to apply a classical genetic approach to establish variant pathogenicity, but rather to employ a multidisciplinary framework to prioritize candidate genes and variants and to examine their roles in heart development using model systems. In this context, genetic analysis serves primarily as a filtering tool rather than as a means of definitively establishing causality.

(2) The genetic analysis of patients does not appear to provide strong evidence for an association between RP gene variants and HLHS. More information regarding methodology and the identified variants is needed. 

HLHS is widely recognized as an oligogenic and heterogeneous genetic disease in which traditional genetic analyses have consistently failed to prioritize any specific gene class as reviewer#2 is pointing out. Therefore, relying solely on genetic analysis is unlikely to yield strong evidence for association with a given gene class. This limitation provides the rationale for our multidisciplinary gene prioritization strategy, which leverages model systems to interrogate candidate gene function. Ultimately, definitive validation of this approach will require studies in relevant in vivo models to establish causality within the context of a four-chambered heart (see also Discussion).

In Table S2, it would be appropriate to provide information on sequence, MAF, and CADD. Please note the source of MAF% (GnomAD version?, which population?).  

As summarized in Figure 2A, the 292 genes from the families with the 25 proband with poor outcome displayed in Supplemental Table 2 fulfilled a comprehensive candidate gene prioritization algorithm based on the variant, gene, inheritance, and enrichment, which required all of the following: 1) variants identified by whole genome sequencing with minor allele frequency <1%; 2) missense, loss-of-function, canonical splice, or promoter variants; 3) upper quartile fetal heart expression; and 4)De novo or recessive inheritance. Unbiased network analysis of these 292 genes, which are displayed in Supplemental Table 2 for completeness, identified statistically significant enrichment of ribosomal proteins. The details about MAF, CADD score, and sequence highlighted by the Reviewer are provided for the RP genes in Table 1, which are central to the focus and findings of the manuscript.    

It would also be helpful for the reader if genome coordinates (e.g., 16-11851493-G-A for RSL1D1 p.A7V) were provided for each variant in both Table 1 and S2.

Genome coordinates have been added to Table 1.

(3) The dataset from the hPSC-CM screen could be of high value for the community. It would be appropriate if the complete dataset were made available in a usable format. 

The dataset from the hPSC-CM screen has been added to the manuscript as Supp Table 1

(4) The "rare predicted-damaging promoter variant in RPS15A" (c.-95G>A) does not appear so rare. Considering the MAF of 0,00662, the frequency of heterozygous carriers of this variant is 1 out of 76 individuals in the general population. Thus, considering the frequency of HLHS in the population (2-3 out of 10,000) and the small size of family 75H, the data do not appear to indicate any association between this particular variant and HLHS. The variants in Table 1 also appear to have relatively mild effects on the gene product, judging from the MAF and CADD scores. The authors are invited to discuss why they find these variants disease-causing in HLHS. 

Our study design is based on the widely held premise that HLHS is an oligogenic disorder. Our multi-model systems platform centered on comprehensive filtering of coding and regulatory variants identified by whole genome sequencing of HLHS probands to identify candidate genes associated with susceptibility to this rare developmental phenotype. 75H proved to be a high-value family for generating a relatively short list of candidate genes for left-sided CHD. Given the rarity of both left-sided CHD and the RPS15A variant identified in the HLHS proband and his 5th degree relative, with a frequency consistent with a risk allele for an oligogenic disorder, we made the reasonable assumption that this was a bona fide genotype-phenotype association rather than a chance occurrence. Moreover, incomplete penetrance and variable expression is consistent with a genetically complex basis of disease whereby the shared variant is risk-conferring and acts in conjunction with additional genetic, epigenetic, and/or environmental factors that lead to a left-sided CHD phenotype. In sum, we do not claim these variants are definitively disease causing, but rather potentially contributing risk factors.

(5) Information is lacking on how clustering of RP genes was demonstrated using STRING (with P-values that support the conclusions). What is meant by "when the highest stringency filter was applied"? Does this refer to the STRING interaction score or something else? The authors could also explain which genes were used to search STRING (e.g., all 292 candidate genes) and provide information on the STRING interaction score used in the analysis, the number of nodes and edges in the network.

To determine whether certain gene networks were over-represented, two online bioinformatics tools were used. First, genes were inputted into STRING (Author response table 2 below) to investigate experimental and predicted protein-protein and genetic interactions. Clustering of ribosomal protein genes was demonstrated when applying the highest stringency filter. Next, genes were analyzed for potential enrichment of genes by ontology classification using PANTHER .Applying Fisher’s exact test and false discovery rate corrections, ribosomal proteins were the most enriched class when compared to the reference proteome, including data annotated by molecular function (4.84-fold, p=0.02), protein class (6.45-fold, p=0.00001), and cellular component (9.50fold, p=0.001). A majority of the identified RP candidate genes harbored variants that fit a recessive inheritance disease model.

Author response image 2.

Synthèse du Débat : Le Genre Précède-t-il le Sexe ?

Résumé Exécutif

Ce document de synthèse analyse le débat contradictoire portant sur l'affirmation « Le genre précède le sexe », opposant Lou Girard (position affirmative) et Franck Ramus (position négative).

Le débat met en lumière une divergence fondamentale entre deux cadres d'analyse :

• l'un, issu des études de genre et de la sociologie, postule que les structures sociales (le genre) façonnent la conceptualisation scientifique de la biologie (le sexe) ;

• l'autre, ancré dans la biologie évolutionniste, soutient que les réalités biologiques (le sexe) constituent le substrat sur lequel se développent les constructions culturelles (le genre).

Lou Girard, s'appuyant sur les travaux de Christine Delphy et Thomas Laqueur, argue que la notion de sexe binaire est une construction scientifique récente (XVIIIe siècle), historiquement contingente et influencée par le système patriarcal qu'elle visait à justifier.

Pour Girard, le genre, en tant que système social hiérarchique, est donc premier.

Franck Ramus contre-argumente sur trois niveaux : ontologique (le phénomène biologique du sexe existe depuis un milliard d'années), développemental (un individu est sexué dès la conception, bien avant l'influence du genre) et évolutionniste (les différences de stratégies reproductives entre mâles et femelles expliquent l'émergence de rôles de genre récurrents dans les sociétés humaines).

La divergence principale ne réside pas seulement dans la conclusion, mais dans l'épistémologie :

quel poids accorder aux preuves issues de la sociologie historique par rapport à celles de la biologie évolutionniste ?

Le débat révèle que même lorsque les deux intervenants partagent des sources communes, leurs cadres interprétatifs radicalement différents les mènent à des conclusions opposées, notamment sur la nature binaire du sexe et la validité des reconstructions historiques des concepts scientifiques.

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1. Contexte et Cadre du Débat

Le débat a été organisé dans un format de "débat constructif" visant à clarifier les points d'accord et de désaccord plutôt qu'à déterminer un vainqueur.

Les deux intervenants ont été invités à défendre des positions opposées sur la proposition "Le genre précède le sexe".

• Position Affirmative ("Oui") : Défendue par Lou Girard.

• Position Négative ("Non") : Défendue par Franck Ramus.

Le format incluait des phases distinctes :

• une prise de position initiale, une session de clarification pour assurer la compréhension mutuelle,

• une phase de "personne de fer" où chaque intervenant reformulait la position de l'autre de manière charitable,

• et des discussions sur les racines des convictions, les limites des approches respectives,

• et enfin les points de convergence et de divergence.

2. Position Affirmative (Lou Girard) : Le Genre comme Principe Organisateur

La position de Lou Girard s'ancre dans le champ pluridisciplinaire des études sur le genre (sociologie, philosophie, études féministes).

Son argument central est que notre compréhension du "sexe" biologique est une construction sociale façonnée par le système de genre préexistant.

Origine et Définitions Clés

• Source de l'affirmation : La sociologue Christine Delphy.

• Définition du Genre : Un "système bicatégorisé (hommes/femmes) et hiérarchisé" où les femmes sont subordonnées aux hommes, notamment par l'exploitation de leur travail domestique et reproductif (patriarcat).

• Définition du Sexe : Il ne s'agit pas des organes génitaux, mais du concept de sexe tel qu'utilisé en biologie, c'est-à-dire la "distinction antagoniste entre les mâles et les femelles".

L'Argument Principal : Une Construction Sociale du Sexe Biologique

L'affirmation "Le genre précède le sexe" signifie que le concept scientifique du sexe biologique a été construit épistémologiquement sur les bases du patriarcat.

Il s'agit d'une "justification scientifique d'un système social".

La science n'a pas découvert le sexe binaire dans un vide neutre ; elle a formalisé une catégorie qui servait à rationaliser une organisation sociale déjà en place.

Preuves Historiques (Thomas Laqueur)

Girard s'appuie fortement sur les travaux de l'historien Thomas Laqueur (La fabrique du sexe) pour démontrer que la conception binaire du sexe est une idée récente.

• Avant le XVIIIe siècle : Le sexe n'était pas conçu comme deux catégories distinctes.

◦ Antiquité : Un modèle à "sexe unique" prévalait, où les organes féminins étaient vus comme une version invertie des organes masculins.  

◦ Moyen Âge : Le sexe était perçu comme un continuum basé sur la "chaleur vitale", les hommes représentant le plus haut degré de cette chaleur.

• À partir du XVIIIe siècle : Le modèle binaire s'impose, coïncidant avec une volonté de naturaliser les rôles sociaux.

Implications et Continuité du Biais Patriarcal

Le modèle binaire, une fois établi, a eu des conséquences concrètes, servant d'outil de normalisation sociale.

• Personnes intersexes : Plutôt que de remettre en question le modèle binaire face à des cas qui ne s'y conforment pas, la médecine a historiquement "mutilé" les personnes intersexes pour les faire correspondre à l'une des deux catégories.

• Homosexuels et personnes trans : Leur existence contrevenant au modèle biomédical, ils ont été psychiatrisés et internés.

• Biais actuel : Ce biais patriarcal continue, selon Girard, d'influencer la recherche scientifique, qui tend à justifier inconsciemment les normes patriarcales plutôt qu'à décrire les faits de manière neutre.

3. Position Négative (Franck Ramus) : Le Sexe comme Prérequis Biologique

La position de Franck Ramus repose sur une distinction claire entre le phénomène biologique du sexe et le concept humain de sexe.

Il soutient que le sexe, en tant que réalité biologique fondamentale, précède et influence l'émergence des constructions sociales comme le genre.

Définition Fondamentale du Sexe

• Le Sexe comme Stratégie Reproductive : Ramus définit le sexe à son niveau le plus fondamental, stabilisé en biologie, comme la distinction entre deux types sexuels dans la reproduction sexuée anisogame :

◦ Femelles : Porteurs de gros gamètes (ovocytes).    ◦ Mâles : Porteurs de petits gamètes (spermatozoïdes).

• Cette définition est primordiale, et les autres aspects (génétiques, hormonaux) en découlent.

L'Argument Principal : Trois Niveaux d'Analyse

Ramus défend que le sexe précède le genre à trois échelles distinctes :

1. Niveau Ontologique : Le phénomène du sexe existe dans la nature depuis environ un milliard d'années, bien avant l'apparition de l'humanité, du patriarcat ou de la conceptualisation humaine du sexe.

2. Niveau Développemental (Individuel) : Un individu possède un sexe dès la conception (chromosomes sexuels).

L'influence du genre et des représentations sociales n'intervient qu'après la naissance. Pour le fœtus, le sexe précède donc clairement le genre.

3. Niveau Évolutionniste (Espèce) : Le genre, en tant que phénomène social, n'émerge pas de rien.

Il se développe sur la base de prédispositions biologiques issues de l'évolution.

Le Modèle Évolutionniste : De l'Anisogamie à la Domination Masculine

Ramus propose une explication évolutionniste à l'origine des rôles de genre.

• Investissement Parental Différentiel : L'anisogamie (différence de taille des gamètes) entraîne un investissement reproductif initial plus élevé pour les femelles.

Cela les incite à investir davantage dans la survie de la progéniture (gestation, allaitement, élevage).

L'investissement des mâles peut rester minimal.

• Conséquences Comportementales :

◦ Les mâles sont en compétition pour l'accès aux femelles, ce qui sélectionne des traits comme l'agressivité, la taille et la force.  

◦ Les femelles, ayant plus à perdre, sont plus sélectives dans le choix de leurs partenaires.

• Origine de la Domination Masculine : La sélection pour une plus grande taille et force chez les mâles (pour la compétition inter-mâles) a pour "effet secondaire" de les rendre physiquement plus forts que les femelles, rendant ainsi la domination masculine possible.

• Division du Travail : Les contraintes reproductives (grossesse, allaitement) rendent les femelles plus sédentaires, tandis que les mâles sont plus mobiles.

Cela favorise une "répartition relativement naturelle des rôles et des tâches", que l'on retrouve dans de multiples cultures.

Ramus précise que ce n'est pas une justification morale, mais une explication causale.

4. Points de Divergence Fondamentaux

Le débat a cristallisé plusieurs points de désaccord profonds, qui sont moins factuels qu'épistémologiques.

Primauté de la Nature vs. la Culture

C'est l'opposition centrale du débat.

• Pour Girard : La culture précède la nature. Les systèmes sociaux (genre) déterminent la manière dont nous conceptualisons et même percevons la réalité biologique (sexe).

• Pour Ramus : La nature précède la culture. Les prédispositions biologiques humaines constituent le socle sur lequel les cultures se développent.

La Binarité du Sexe : Concept vs. Réalité Biologique

• Pour Ramus : Le sexe, défini par la stratégie reproductive (production de deux types de gamètes), est fondamentalement binaire.

• Pour Girard : Le sexe biologique n'est pas binaire. Cette vision est le produit d'un modèle social imposé à une réalité plus complexe (comme en témoignent les personnes intersexes).

L'Interprétation des Preuves Historiques et Scientifiques

Le cas de Thomas Laqueur est emblématique de cette divergence.

• Girard accepte les conclusions de Laqueur comme une preuve historique valide que la conception binaire du sexe est une construction récente.

• Ramus exprime son "incrédulité" face à cette affirmation, la trouvant contre-intuitive.

Il a du mal à imaginer qu'avant le XVIIIe siècle, les humains n'avaient pas conscience de l'existence de deux sexes.

Pour lui, le critère d'arbitrage serait le consensus scientifique parmi les historiens, pas la thèse d'un seul auteur.

Poids Épistémologique des Disciplines et des Données

Initialement présentée comme une opposition entre sociologie (Girard) et biologie (Ramus), la divergence est plus subtile.

• Girard accorde une grande valeur aux analyses des études de genre pour déconstruire les biais inhérents à la production du savoir scientifique.

• Ramus ne rejette pas les sciences humaines et sociales, mais se dit "non convaincu" par certains arguments et données spécifiques issus des études de genre, qu'il confronte à des données issues de la biologie ou de la psychologie.

Le débat a montré que même en lisant les mêmes auteurs (ex: Anne Fausto-Sterling), ils en tirent des conclusions radicalement opposées, révélant des cadres d'analyse irréconciliables.

5. Racines des Positions et Limites Reconnues

Parcours et Motivations Personnelles

• Franck Ramus : Son intérêt pour le sujet provient de ses recherches en sciences cognitives, où il a observé de manière répétée et non sollicitée des différences entre sexes (prévalence de l'autisme, dyslexie, développement du langage, neuroanatomie), le poussant à en chercher les origines.

• Lou Girard : Sa position est façonnée par son expérience de femme transgenre.

La confrontation au sexisme et à la transphobie l'a conduite à s'intéresser au féminisme, puis aux études de genre, dont elle a adopté le cadre d'analyse matérialiste comme étant le plus pertinent pour comprendre la société.

Limites et Incertitudes Avouées

• Franck Ramus : Admet que l'approche évolutionniste est une "inférence à la meilleure explication" et qu'il ne peut apporter de "preuves irréfutables" pour chaque détail de ce récit historique.

Sa force réside dans sa cohérence et son pouvoir explicatif global.

• Lou Girard : Reconnaît ses limites personnelles en tant que non-experte diplômée, ce qui pourrait limiter sa compréhension des théories qu'elle expose.

Elle admet également la possibilité de faiblesses épistémologiques dans l'approche des études de genre elle-même, ainsi que l'existence de limites qu'elle ne perçoit pas.

6. Points de Convergence Identifiés

Malgré les divergences profondes, quelques points d'accord ont été établis :

• L'existence du patriarcat en tant que système social qui désavantage les femmes.

• La préexistence de phénomènes biologiques ("nature") avant l'émergence de la culture humaine.

• Le fait que les individus sont biologiquement sexués avant d'être socialisés.

• Un désaccord commun sur la validité du premier modèle des "cinq sexes" d'Anne Fausto-Sterling, bien que leur analyse de l'évolution de son travail diverge par la suite.

Author response:

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

Reviewer #1 (Public review): 

“The study analyzes the gastric fluid DNA content identified as a potential biomarker for human gastric cancer. However, the study lacks overall logicality, and several key issues require improvement and clarification. In the opinion of this reviewer, some major revisions are needed:” 

(1) “This manuscript lacks a comparison of gastric cancer patients' stages with PN and N+PD patients, especially T0-T2 patients.”

We are grateful for this astute remark. A comparison of gfDNA concentration among the diagnostic groups indicates a trend of increasing values as the diagnosis progresses toward malignancy. The observed values for the diagnostic groups are as follows:

Author response table 1.

The chart below presents the statistical analyses of the same diagnostic/tumor-stage groups (One-Way ANOVA followed by Tukey’s multiple comparison tests). It shows that gastric fluid gfDNA concentrations gradually increase with malignant progression. We observed that the initial tumor stages (T0 to T2) exhibit intermediate gfDNA levels, which in this group is significantly lower than in advanced disease (p = 0.0036), but not statistically different from non-neoplastic disease (p = 0.74).

Author response image 1.

(2) “The comparison between gastric cancer stages seems only to reveal the difference between T3 patients and early-stage gastric cancer patients, which raises doubts about the authenticity of the previous differences between gastric cancer patients and normal patients, whether it is only due to the higher number of T3 patients.”

We appreciate the attention to detail regarding the numbers analyzed in the manuscript. Importantly, the results are meaningful because the number of subjects in each group is comparable (T0-T2, N = 65; T3, N = 91; T4, N = 63). The mean gastric fluid gfDNA values (ng/µL) increase with disease stage (T0-T2: 15.12; T3-T4: 30.75), and both are higher than the mean gfDNA values observed in non-neoplastic disease (10.81 ng/µL for N+PD and 10.10 ng/µL for PN). These subject numbers in each diagnostic group accurately reflect real-world data from a tertiary cancer center.

(3) “The prognosis evaluation is too simplistic, only considering staging factors, without taking into account other factors such as tumor pathology and the time from onset to tumor detection.”

Histopathological analyses were performed throughout the study not only for the initial diagnosis of tissue biopsies, but also for the classification of Lauren’s subtypes, tumor staging, and the assessment of the presence and extent of immune cell infiltrates. Regarding the time of disease onset, this variable is inherently unknown--by definition--at the time of a diagnostic EGD. While the prognosis definition is indeed straightforward, we believe that a simple, cost-effective, and practical approach is advantageous for patients across diverse clinical settings and is more likely to be effectively integrated into routine EGD practice.

(4) “The comparison between gfDNA and conventional pathological examination methods should be mentioned, reflecting advantages such as accuracy and patient comfort. “

We wish to reinforce that EGD, along with conventional histopathology, remains the gold standard for gastric cancer evaluation. EGD under sedation is routinely performed for diagnosis, and the collection of gastric fluids for gfDNA evaluation does not affect patient comfort. Thus, while gfDNA analysis was evidently not intended as a diagnostic EGD and biopsy replacement, it may provide added prognostic value to this exam.

(5) “There are many questions in the figures and tables. Please match the Title, Figure legends, Footnote, Alphabetic order, etc. “

We are grateful for these comments and apologize for the clerical oversight. All figures, tables, titles and figure legends have now been double-checked.

(6) “The overall logicality of the manuscript is not rigorous enough, with few discussion factors, and cannot represent the conclusions drawn. “

We assume that the unusual wording remark regarding “overall logicality” pertains to the rationale and/or reasoning of this investigational study. Our working hypothesis was that during neoplastic disease progression, tumor cells continuously proliferate and, depending on various factors, attract immune cell infiltrates. Consequently, both tumor cells and immune cells (as well as tumor-derived DNA) are released into the fluids surrounding the tumor at its various locations, including blood, urine, saliva, gastric fluids, and others. Thus, increases in DNA levels within some of these fluids have been documented and are clinically meaningful. The concurrent observation of elevated gastric fluid gfDNA levels and immune cell infiltration supports the hypothesis that increased gfDNA—which may originate not only from tumor cells but also from immune cells—could be associated with better prognosis, as suggested by this study of a large real-world patient cohort.

In summary, we thank Reviewer #1 for his time and effort in a constructive critique of our work.

Reviewer #2 (Public review):

Summary: 

“The authors investigated whether the total DNA concentration in gastric fluid (gfDNA), collected via routine esophagogastroduodenoscopy (EGD), could serve as a diagnostic and prognostic biomarker for gastric cancer. In a large patient cohort (initial n=1,056; analyzed n=941), they found that gfDNA levels were significantly higher in gastric cancer patients compared to non-cancer, gastritis, and precancerous lesion groups. Unexpectedly, higher gfDNA concentrations were also significantly associated with better survival prognosis and positively correlated with immune cell infiltration. The authors proposed that gfDNA may reflect both tumor burden and immune activity, potentially serving as a cost-effective and convenient liquid biopsy tool to assist in gastric cancer diagnosis, staging, and follow-up.”

Strengths: 

“This study is supported by a robust sample size (n=941) with clear patient classification, enabling reliable statistical analysis. It employs a simple, low-threshold method for measuring total gfDNA, making it suitable for large-scale clinical use. Clinical confounders, including age, sex, BMI, gastric fluid pH, and PPI use, were systematically controlled. The findings demonstrate both diagnostic and prognostic value of gfDNA, as its concentration can help distinguish gastric cancer patients and correlates with tumor progression and survival. Additionally, preliminary mechanistic data reveal a significant association between elevated gfDNA levels and increased immune cell infiltration in tumors (p=0.001).”

Reviewer #2 has conceptually grasped the overall rationale of the study quite well, and we are grateful for their assessment and comprehensive summary of our findings.

Weaknesses: 

(1) “The study has several notable weaknesses. The association between high gfDNA levels and better survival contradicts conventional expectations and raises concerns about the biological interpretation of the findings.“

We agree that this would be the case if the gfDNA was derived solely from tumor cells. However, the findings presented here suggest that a fraction of this DNA would be indeed derived from infiltrating immune cells. The precise determination of the origin of this increased gfDNA remains to be achieved in future follow-up studies, and these are planned to be evaluated soon, by applying DNA- and RNA-sequencing methodologies and deconvolution analyses.

(2) “The diagnostic performance of gfDNA alone was only moderate, and the study did not explore potential improvements through combination with established biomarkers. Methodological limitations include a lack of control for pre-analytical variables, the absence of longitudinal data, and imbalanced group sizes, which may affect the robustness and generalizability of the results.“

Reviewer #2 is correct that this investigational study was not designed to assess the diagnostic potential of gfDNA. Instead, its primary contribution is to provide useful prognostic information. In this regard, we have not yet explored combining gfDNA with other clinically well-established diagnostic biomarkers. We do acknowledge this current limitation as a logical follow-up that must be investigated in the near future.

Moreover, we collected a substantial number of pre-analytical variables within the limitations of a study involving over 1,000 subjects. Longitudinal samples and data were not analyzed here, as our aim was to evaluate prognostic value at diagnosis. Although the groups are imbalanced, this accurately reflects the real-world population of a large endoscopy center within a dedicated cancer facility. Subjects were invited to participate and enter the study before sedation for the diagnostic EGD procedure; thus, samples were collected prospectively from all consenting individuals.

Finally, to maintain a large, unbiased cohort, we did not attempt to balance the groups, allowing analysis of samples and data from all patients with compatible diagnoses (please see Results: Patient groups and diagnoses).

(3) “Additionally, key methodological details were insufficiently reported, and the ROC analysis lacked comprehensive performance metrics, limiting the study's clinical applicability.“

We are grateful for this useful suggestion. In the current version, each ROC curve (Supplementary Figures 1A and 1B) now includes the top 10 gfDNA thresholds, along with their corresponding sensitivity and specificity values (please see Suppl. Table 1). The thresholds are ordered from-best-to-worst based on the classic Youden’s J statistic, as follows:

Youden Index = specificity + sensitivity – 1 [Youden WJ. Index for rating diagnostic tests. Cancer 3:32-35, 1950. PMID: 15405679]. We have made an effort to provide all the key methodological details requested, but we would be glad to add further information upon specific request.

Reviewer #1 (Recommendations for the authors):

The authors should pay attention to ensuring uniformity in the format of all cited references, such as the number of authors for each reference, the journal names, publication years, volume numbers, and page number formats, to the best extent possible. 

Thank you for pointing this inconsistency. All cited references have now been revisited and adjusted properly. We apologize for this clerical oversight.

Reviewer #2 (Recommendations for the authors):

(1) “High gfDNA levels were surprisingly linked to better survival, which conflicts with the conventional understanding of cfDNA as a tumor burden marker. Was any qualitative analysis performed to distinguish DNA derived from immune cells versus tumor cells?“

Tumor-derived DNA is certainly present in gfDNA, as our group has unequivocally demonstrated in a previous publication [Pizzi M. P., et al. (2019) Identification of DNA mutations in gastric washes from gastric adenocarcinoma patients: Possible implications for liquid biopsies and patient follow-up Int J Cancer 145:1090–1097. DOI: 10.1002/ijc.32114]. However, in the present manuscript, our data suggest that gfDNA may also contain DNA derived from infiltrating immune cells. This may also be the case for other malignancies, and qualitative deconvolution studies could provide more informative information. To achieve this, DNA sequencing and RNA-Seq analyses may offer relevant evidence. Our study should be viewed as an original and preliminary analysis that may encourage such quantitative and qualitative studies in biofluids from cancer patients. Currently, this is a simple approach (which might be its essential beauty), but we hope to investigate this aspect further in future studies.

(2) “The ROC curve AUC was 0.66, indicating only moderate discrimination ability. Did the authors consider combining gfDNA with markers such as CEA or CA19-9 to improve diagnostic accuracy?“

This is indeed a logical idea, which shall certainly be explored in planned follow-up studies.

(3) “DNA concentration could be influenced by non-biological factors, including gastric fluid pH, sampling location, time delay, or freeze-thaw cycles. Were these operational variables assessed for their effect on data stability?“

We appreciate the rigor of the evaluation. Yes, information regarding gastric fluid pH was collected. All samples were collected from the stomach during EGD procedure. Samples were divided in aliquots and were thawed only once. This information is now provided in the updated manuscript text.

(4) “This cross-sectional study lacks data on gfDNA changes over time, limiting conclusions on its utility for monitoring treatment response or predicting recurrence.“

Again, temporal evaluation is another excellent point, and it will be the subject of future analyses. In this exploratory study, samples were collected at diagnosis, at a single point. We have not obtained serial samples, as participants received appropriate therapy soon following diagnosis.

(5) The normal endoscopy group included only 10 patients, the precancerous lesion group 99 patients, while the gastritis group had 596 patients. Such uneven sample sizes may affect statistical reliability and generalizability. Has weighted analysis or optimized sampling been considered for future studies?“

Yes, in future studies this analysis will be considered, probably by employing stratified random sampling with relevant patient attributes recorded.

(6) “The SciScore was only 2 points, indicating that key methodological details such as inclusion/exclusion criteria, randomization, sex variables, and power calculation were not clearly described. It is recommended that these basic research elements be supplemented in the Methods section. “

This was an exploratory research, the first of its kind, to evaluate prognostic potential of gfDNA in the context of gastric cancer. Patients were not included if they did not sign the informed consent or excluded if they withdrew after consenting. Other exclusion criteria included diagnoses of conditions such as previous gastrectomy or esophagectomy, or the presence of non-gastric malignancies. Randomization and power analyses were not applicable, as no prior data were available regarding gfDNA concentration values or its diagnostic/prognostic potential. All subjects, regardless of sex, were invited to participate without discrimination or selection.

(7) “Although a ROC curve was provided in the supplementary materials (Supplementary Figure 1), only the curve and AUC value were shown without sensitivity, specificity, predictive values, or cutoff thresholds. The authors are advised to provide a full ROC performance assessment to strengthen the study's clinical relevance.

These data are now given alongside the ROC curves in the Supplementary Information section, specifically in Supplementary Figure 1 and in the newly added Supplementary Table 1.

We thank Reviewer #2 for an insightful and positive overall assessment of our work.

L'Idéologie et l'Esprit Critique : Synthèse du Débat

Résumé Exécutif

Ce document synthétise les arguments et les conclusions du débat sur la compatibilité entre l'idéologie et l'esprit critique, opposant Gwen Pallarès (position positive) et Pascal Wagner-Egger (position négative).

Gwen Pallarès soutient que l'idéologie est non seulement compatible mais souvent un prérequis et un moteur pour l'esprit critique, arguant que tout individu possède une idéologie qui structure sa pensée et motive sa curiosité.

Pascal Wagner-Egger défend la position selon laquelle l'idéologie est fondamentalement un obstacle à la pensée critique et à la démarche scientifique, un ensemble de préconceptions qu'il faut activement chercher à minimiser en s'appuyant sur des données empiriques.

Malgré leurs positions de départ opposées, un consensus significatif a émergé sur plusieurs points.

Les deux intervenants s'accordent sur l'existence d'un "point de bascule" ou d'un "saut qualitatif" où l'idéologie devient incompatible avec l'esprit critique, notamment dans les cas de fanatisme, de radicalisation ou lorsque les croyances fondamentales liées à l'identité sont menacées.

Ils reconnaissent également que l'idéologie peut agir comme une puissante "motivation épistémique", incitant à la recherche et à l'analyse.

La divergence principale réside dans la nature de cette relation.

Pour Pascal, la motivation induite par l'idéologie est une arme à double tranchant qui exige une vigilance épistémique accrue pour contrer les biais.

Pour Gwen, cette motivation est un moteur fondamental, et la volonté de se placer dans une position "centriste" pour éviter les biais est elle-même une position idéologique.

Cette différence de perspective trouve sa source dans des divergences épistémologiques plus profondes sur la nature des sciences, la construction des données et la porosité entre les domaines scientifique et politique.

1. Introduction au Débat

Le débat, animé par Peter Barret, a pour objectif d'explorer la question "L’idéologie est-elle compatible avec l’esprit critique ?" dans un format visant à être constructif et à clarifier les positions plutôt qu'à encourager la contre-argumentation.

Les deux intervenants sont :

• Gwen Pallarès : Maîtresse de conférence en didactique des sciences à l'Université de Reims Champagne-Ardenne, défendant la position positive.

• Pascal Wagner-Egger : Psychologue social à l'Université de Fribourg, défendant la position négative.

2. Définitions Clés

Les intervenants se sont accordés sur les définitions suivantes pour encadrer le débat.

Terme

Définition de Gwen Pallarès (Psychologie Sociale)

Définition de Pascal Wagner-Egger (Larousse)

Idéologie

Un système d'attitudes, de croyances et de stéréotypes qui coordonne les actions des institutions et des individus.

Ce système vise notamment à justifier ou à critiquer les hiérarchies sociales existantes (ex: féminisme vs. masculinisme).

Un système d'idées générales constituant un corps de doctrine philosophique et politique à la base d'un comportement individuel ou collectif (ex: idéologie marxiste, nationaliste).

Esprit Critique : Défini par Gwen Pallarès comme un ensemble de compétences (analyse, évaluation d'arguments et d'informations) et de dispositions (humilité intellectuelle, curiosité, réflexivité).

Cet ensemble est orienté vers la prise de décision raisonnée ("Qu'est-ce qu'il convient de croire ou de faire ?") et s'opérationnalise souvent par une argumentation de bonne qualité.

3. Positions Initiales

3.1. Position de Gwen Pallarès (Positive) : L'Idéologie comme Prérequis Compatible

L'argument central de Gwen Pallarès repose sur l'universalité de l'idéologie :

• Tout le monde a une idéologie : La pensée de chaque individu est structurée par des systèmes de croyances, d'attitudes et de stéréotypes.

Refuser cela serait nier une réalité fondamentale du fonctionnement humain.

• L'incompatibilité rendrait l'esprit critique impossible : Si l'idéologie était incompatible avec l'esprit critique, et puisque tout le monde a une idéologie, alors personne ne pourrait avoir d'esprit critique.

• L'esprit critique est un spectre : Tout le monde possède des compétences minimales d'analyse et d'argumentation, même si leur application peut être biaisée (ex: biais de confirmation où l'on critique plus durement les informations qui contredisent nos croyances).

• Limite de la compatibilité : Elle concède que les formes extrêmes d'idéologie (radicalisation, emprise sectaire, fanatisme) sont, elles, incompatibles avec l'esprit critique car elles poussent à une acceptation acritique des informations.

3.2. Position de Pascal Wagner-Egger (Négative) : L'Idéologie comme Obstacle à la Science

Pascal Wagner-Egger ancre sa position dans l'histoire des sciences et la psychologie sociale :

• La science s'est construite contre l'idéologie : Il cite l'exemple de la science luttant contre l'idéologie religieuse, qu'il qualifie de "régime totalitaire".

• La "méthode idéologique" : Elle postule que la vérité est contenue dans un texte fondateur (la Bible, Le Capital) et que toute observation doit s'y conformer. C'est l'inverse de la méthode scientifique.

• L'ennemi intérieur et extérieur : L'idéologie est un obstacle institutionnel (externe) mais aussi un obstacle interne aux chercheurs eux-mêmes.

Il cite Gaston Bachelard et ses "obstacles épistémologiques" (opinion, connaissance générale) comme précurseurs de la notion de biais cognitifs.

• Le rôle des données empiriques : La méthode scientifique est le principal outil pour limiter les effets de nos idéologies et tester nos préconceptions contre la réalité.

Il cite des études montrant plus de dogmatisme et de complotisme aux extrêmes politiques.

4. Racine des Convictions : Les Parcours Académiques

Les positions des deux débatteurs sont fortement influencées par leurs expériences personnelles et académiques.

• Pascal Wagner-Egger : Son parcours l'a mené des sciences "dures" vers les sciences sociales.

Il a été frappé par ce qu'il a perçu comme des positions idéologiques dogmatiques chez certains collègues, notamment le rejet des méthodes quantitatives qualifiées d'"impérialisme anglo-saxon".

Cette expérience a forgé sa conviction que l'idéologie peut nuire à la recherche de la vérité scientifique et qu'il faut s'en prémunir.

• Gwen Pallarès : Son parcours est inverse, des mathématiques vers la didactique des sciences.

L'étude approfondie des controverses socio-scientifiques (IA, genre, écologie) pour sa thèse l'a progressivement politisée.

Son engagement politique est devenu un moteur pour produire une recherche scientifique plus rigoureuse et utile socialement, notamment pour l'éducation.

Pour elle, l'idéologie n'est pas un obstacle à la rigueur, mais ce qui la motive.

5. Analyse de la Convergence et de la Divergence

Le débat a révélé un terrain d'entente plus large qu'attendu, tout en précisant la nature des désaccords.

5.1. Points de Convergence Fondamentaux

1. Le "Point de Bascule" : Les deux intervenants s'accordent sur le fait qu'il existe un seuil où l'idéologie devient incompatible avec l'esprit critique.

Ce seuil est atteint dans les cas de fanatisme, de radicalisation, ou lorsque des croyances fondamentales liées à l'identité de la personne sont menacées, rendant le dialogue et la remise en question impossibles.

2. La Motivation Épistémique : Il est admis par les deux parties que l'idéologie est un puissant moteur.

Un engagement idéologique (ex: écologiste, féministe) peut stimuler la curiosité intellectuelle, la recherche d'informations et la volonté d'analyser des arguments, qui sont des dispositions centrales de l'esprit critique.

3. L'Universalité de l'Idéologie : Les deux débatteurs partagent le postulat que chaque individu, y compris les scientifiques, possède une ou plusieurs idéologies qui structurent sa vision du monde.

5.2. Points de Divergence Clés

La principale divergence ne porte pas tant sur la compatibilité en soi, mais sur la nature de la relation entre idéologie et esprit critique.

Point de Divergence

Position de Pascal Wagner-Egger

Position de Gwen Pallarès

Nature du lien

Une arme à double tranchant : L'idéologie motive, mais elle biaise simultanément.

Il est donc crucial d'exercer une vigilance épistémique accrue et de chercher à minimiser l'influence de ses propres idéologies, notamment en les confrontant aux données empiriques.

Un moteur fondamental : L'idéologie est le moteur principal de la recherche et de l'engagement critique. Tenter de l'annuler est illusoire.

La posture qui consiste à se vouloir "au centre" pour être moins biaisé est elle-même une idéologie ("biais du juste milieu").

Épistémologie sous-jacente

Plus proche de l'empirisme et du rationalisme critique (citant Popper et se revendiquant de Lakatos).

Les données, bien que partiellement construites, permettent par triangulation de s'approcher d'une réalité indépendante de la méthode.

Plus proche du constructivisme et du pragmatisme. Les données sont fondamentalement construites par la méthodologie, qui est elle-même issue de cadres théoriques.

La distinction entre science et politique est plus poreuse.

Rapport Science / Politique

Vise à maintenir une distinction claire. Dans le domaine scientifique, les données doivent primer sur les préconceptions. Dans le domaine politique, l'idéologie et le militantisme sont utiles et nécessaires.

La distinction est moins nette. Le travail scientifique est intrinsèquement lié à des enjeux de société et peut être motivé par un engagement politique, cet engagement pouvant être un gage de rigueur pour rendre la science utile.

bonjour, j'ai une preoccupation et elle est la suivante : je suis aller dans cette section de code pen j'ai constaté que les valeurs attribuées à l'attribu class ne sont declarées de la meme maniére dans le css. et j'aimerai comprendre pourquoi s'il vous plait.

aussi j'aimerai comprendre si Box apres l'espace dans le HTML lors de l'affectation des valeurs à l'attribut veut tout simplement dire que l'on aura des valeurs geometriques comme par exemple un carré et donc c'est la raison pour laquelle il ne figure pas dans le CSS. merci d'avance pour votre orientation

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Manuscript number: RC -2025-03175

Corresponding author(s): Gernot Längst

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

Summary: Holzinger et al. present a new automated pipeline, nucDetective, designed to provide accurate nucleosome positioning, fuzziness, and regularity from MNase-seq data. The pipeline is structured around two main workflows-Profiler and Inspector-and can also be applied to time-series datasets. To demonstrate its utility, the authors re-analyzed a Plasmodium falciparum MNase-seq time-series dataset (Kensche et al., 2016), aiming to show that nucDetective can reliably characterize nucleosomes in challenging AT-rich genomes. By integrating additional datasets (ATAC-seq, RNA-seq, ChIP-seq), they argue that the nucleosome positioning results from their pipeline have biological relevance.

Major Comments:

Despite being a useful pipeline, the authors draw conclusions directly from the pipeline's output without integrating necessary quality controls. Some claims either contradict existing literature or rely on misinterpretation or insufficient statistical support. In some instances, the pipeline output does not align with known aspects of Plasmodium biology. I outline below the key concerns and suggested improvements to strengthen the manuscript and validate the pipeline:

Clarification of +1 Nucleosome Positioning in P. falciparum The authors should acknowledge that +1 nucleosomes have been previously reported in P. falciparum. For example, Kensche et al. (2016) used MNase-seq to map ~2,278 TSSs (based on enriched 5′-end RNA data) and found that the +1 nucleosome is positioned directly over the TSS in most genes:

"Analysis of 2278 start sites uncovered positioning of a +1 nucleosome right over the TSS in almost all analysed regions" (Figure 3A).

They also described a nucleosome-depleted region (NDR) upstream of the TSS, which varies in size, while the +1 nucleosome frequently overlaps the TSS. The authors should nuance their claims accordingly. Nevertheless, I do agree that the +1 positioning in P. falciparum may be fuzzier as compared to yeast or mammals. Moreover, the correlation between +1 nucleosome occupancy and gene expression is often weak and that several genes show similar nucleosome profiles regardless of expression level. This raises my question: did the authors observe any of these patterns in their new data?

We appreciate the reviewer’s insightful comment and agree that +1 nucleosomes and nucleosome depleted promoter regions have been previously reported in P. falciparum, notably by the Bartfai and Le Roch groups, including Kensche et al. (PMID: 26578577). Our study advances this understanding by providing, for the first time, a comprehensive view of the entirety of a canonical eukaryotic promoter architecture in P. falciparum—encompassing the NDR, the well-positioned +1 nucleosome, and the downstream phased nucleosome array. This downstream nucleosome array structure has not been characterized before, as prior studies noted a “lack of downstream nucleosomal arrays” (PMID: 26578577) or “relatively random” nucleosome organization within gene bodies (PMID: 24885191). We have revised the manuscript to more clearly acknowledge previous work and highlight our contributions. The changes we applied in the manuscript are highlighted in yellow and shown as well below.

In the Abstract L26-L230: Contrary to the current view of irregular chromatin, we demonstrate for the first time regular phased nucleosome arrays downstream of TSSs, which, together with the established +1 nucleosome and upstream nucleosome-depleted region, reveal a complete canonical eukaryotic promoter architecture in Pf.

Introduction L156-L159: For example, we identify a phased nucleosome array downstream of the TSS. Together with a well-positioned +1 nucleosome and an upstream nucleosome-free region. These findings support a promoter architecture in Pf that resembles classical eukaryotic promoters (Bunnik et al. 2014, Kensche et al. 2016).

Results L181-L183: These new Pf nucleosome maps reveal a nucleosome organisation at transcription start sites (TSS) reminiscent of the general eukaryotic chromatin structure, featuring a reported well-positioned +1 nucleosome , an upstream nucleosome-free region (NFR, Bunnik et al. 2014, Kensche et al. 2016), and shown for the first time in Pf, a phased nucleosome array downstream of the TSS.

Discussion L414-L419: Previous analyses of Pf chromatin have identified +1 nucleosomes and NFRs (Bunnik et al 2014, Kensche et al. 2016). Here we extend this understanding by demonstrating phased nucleosome array structures throughout the genome. This finding provides evidence for a spatial regulation of nucleosome positioning in Pf, challenging the notion that nucleosome positioning is relatively random in gene bodies (Bunnik et al. 2014, Kensche et al. 2016). Consequently our results contribute to the understanding that Pf exhibits a typical eukaryotic chromatin structure, including well-defined nucleosome positioning at the TSS and regularly spaced nucleosome arrays (Schones et al. 2008; Yuan et al. 2005).

Regarding the reviewer’s question on +1 nucleosome dynamics. Our data agrees with the reviewer and other studies (e.g. PMID: 31694866), that the +1 nucleosome position is robust and does not correlate with gene expression strength. In the manuscript we show that dynamic nucleosomes are preferentially detected at the –1 nucleosome position (Figure 2C). In line with that we show that the +1 nucleosome position does not markedly change during transcription initiation of a subset of late transcribed genes (Figure 5A). However, we observe an opening of the NDR and within the gene body increased fuzziness and decreased nucleosome array regularity (Figure S4A). To illustrate the relationship between the +1 nucleosome positioning and expression strength, we have included a heatmap showing nucleosome occupancy at the TSS, ordered according to expression strength (NEW Figure S4C):

We included a sentence describing the relationship of +1 nucleosome position with gene expression in L257-L258: Furthermore, the +1 nucleosome positioning is unaffected by the strength of gene expression (Figure S2C).

__ Lack of Quality Control in the Pipeline __

The authors claim (lines 152-153) that QC is performed at every stage, but this is not supported by the implementation. On the GitHub page (GitHub - uschwartz/nucDetective), QC steps are only marked at the Profiler stage using standard tools (FastQC, MultiQC). The Inspector stage, which is crucial for validating nucleosome detection, lacks QC entirely. The authors should implement additional steps to assess the quality of nucleosome calls. For example, how are false positives managed? ROC curves should be used to evaluate true positive vs. false positive rates when defining dynamic nucleosomes. How sequencing biases are adressed?

The workflow overview chart on GitHub was not properly color coded. Therefore, we changed the graphics and highlighted the QC steps in the overview charts accordingly:

Based on our long standing expertise of analysing MNase-seq data (PMID: 38959309, PMID: 37641864, PMID: 30496478, PMID: 25608606), the best quality metrics to assess the performance of the challenging MNase experiment are the fragment size distributions revealing the typical nucleosomal DNA lengths and the TSS plots showing a positioned +1 nucleosome and regularly phased nucleosome arrays downstream of the +1 nucleosome. Additionally, visual inspection of the nucleosome profiles in a genome browser is advisable. We make those quality metrics easily available in the nucDetective Profiler workflow (Insertsize Histogram, TSS plot and provide nucleosome profile bigwig files). Furthermore, the PC and correlation analysis based on the nucleosome occupancy in the inspector workflow allows to evaluate replicate reproducibility or integrity of time series data, as shown for data evaluated in this manuscript.

The inspector workflow uses the well-established DANPOS toolkit to call nucleosome positions. Based on our experience, this step is particularly robust and well-established in the DANPOS toolkit (PMID: 23193179), so there is no need to reinvent it. Nevertheless, appropriate pre-processing of the data as done in the nucDetective pipeline is crucial to obtain highly resolved nucleosome positions. Using the final nucleosome profiles (bigwig) and the nucleosome reference positions (bed) as output of the Inspector workflow allows visual inspection of the called nucleosomes in a genome viewer. Furthermore, to avoid using false positive nucleosome positions for dynamic nucleosome analysis, we take only the 20% best positioned nucleosomes of each sample, as determined by the fuzziness score.

We understand the value of a gold standard of dynamic nucleosomes to test performance using ROC curves. However, we are not aware that such a gold standard exists in the nucleosome analysis field, especially not when using multi-sample settings, such as time series data. One alternative would be to use simulated data; however, this has several limitations:

• __Lack of biological complexity: __simulated data often fails to capture the full complexity of biological systems including the heterogeneity, variability, and subtle dependencies present in real-world data. Simplifications and omissions in simulation models can result in test datasets that are more tractable but less realistic, causing software to appear robust or accurate under idealized conditions, while underperforming on actual experimental data.
• __Risks of Overfitting: __Software may be tuned to perform well on simulated datasets leading to overfitting and falsely inflated performance metrics. This undermines the predictive or diagnostic value of the results for real biological data

• Poor Model Fidelity and Hidden Assumptions: The authenticity of simulated data is bounded by the fidelity of the underlying models. If those models are inaccurate or make untested assumptions, the generated data may not reflect real experimental or clinical scenarios. This can mask software shortcomings or bias validation toward specific, perhaps irrelevant, scenarios. Therefore, we decided to validate the performance of the pipeline in the biological context of the analyzed data:

• PCA analysis of the individual nucleosome features shows a cyclic structure as expected for the IDC (Fig. 1D-G).

• Nucleosome occupancy changes anti-correlate with chromatin accessibility (Fig. 3B) as expected.
• Dynamic nucleosome features correlate with expression changes (Fig. 5C) We are aware that MNase-seq experiments might have sequence bias caused by the enzyme's endonuclease sequence preference (PMID: 30496478). However, the main aim of the nucDetective pipeline is to identify dynamic nucleosome features genome wide. Therefore, we are comparing the nucleosome features across multiple samples to find the positions in the genome with the highest variability. Comparisons are performed between the same nucleosome positions at the same genomic sites across multiple conditions, so the sequence context is constant and does not confound the analysis. This is like the differential expression analysis of RNA-seq data, where the gene counts are not normalized by gene length. Introducing a sequence normalization step might distort and bias the results of dynamic nucleosomes.

We included a paragraph describing the limitations to the discussion (L447-457):

Depending on the degree of MNase digestion, preferentially nucleosomes from GC rich regions are revealed in MNase-seq experiments (Schwartz et al. 2019). However, no sequence or gDNA normalisation step was included in the nucDetective pipeline. To identify dynamic nucleosomes, comparisons are performed between the same nucleosome positions at the same genomic sites across multiple samples. Hence, the sequence context is constant and does not confound the analysis. Introducing a sequence normalization step might even distort and bias the results. Nevertheless, it is highly advisable to use low MNase concentrations in chromatin digestions to reduce the sequence bias in nucleosome extractions. This turned out to be a crucial condition to obtain a homogenous nucleosome distribution in the AT-rich intergenic regions of eukaryotic genomes and especially in the AT-rich genome of Pf (Schwartz et al. 2019, Kensche et al. 2016).

__ Use of Mono-nucleosomes Only __

The authors re-analyze the Kensche et al. (2016) dataset using only mono-nucleosomes and claim improved nucleosome profiles, including identification of tandem arrays previously unreported in P. falciparum. Two key issues arise: 1. Is the apparent improvement due simply to focusing on mono-nucleosomes (as implied in lines 342-346)?

The default setting in nucDetective is to use fragment sizes of 140 – 200 bp, which corresponds to the main mono-nucleosome fraction in standard MNase-seq experiments. However, the correct selection of fragment sizes may vary depending on the organism and the variations in MNase-seq protocols. Therefore, the pipeline offers the option of changing the cutoff parameter (--minLen; --maxLen), accordingly. Kensche et al thoroughly tested and established the best parameters for the data set. We agree with their selected parameters and used the same cutoffs (75-175 bp) in this manuscript. For this particular data set, the fragment size selection is not the reason why we obtain a better resolution. MNase-seq analysis is a multistep process which is optimized in the nucDetective pipeline. Differences in the analysis to Kensche et al are at the pre-processing stage and alignment step:

Kensche et al. : “Paired-end reads were clipped to 72 bp and all data was mapped with BWA sample (Version 0.6.2-r126)”

nucDetective:

• Trimming using TrimGalore --paired -q 10 --stringency 2
• Mapping using bowtie2 --very-sensitive –dovetail --no-discordant
• MAPQ >= 20 filtering of aligned read-pairs (samtools). The manuscript text L379 was changed to

This is achieved using MNase-seq optimized alignment settings, and proper selection of the fragment sizes corresponding to mono-nucleosomal DNA to obtain high resolution nucleosome profiles.

How does the pipeline perform with di- or tri-nucleosomes, which are also biologically relevant (Kensche et al., 2016 and others)? Furthermore, the limitation to mono-nucleosomes is only mentioned in the methods, not in the results or discussion, which could mislead readers.

The pipeline is optimized for mono-nucleosome analysis. However, the cutoffs for fragment size selection can be adjusted to analyse other fragment populations in MNase-seq data (--minLen; --maxLen). For example we know from previous studies that the settings in the pipeline could be used for sub-nucleosome analysis as well (PMID: 38959309). Di- or Tri-nucleosome analysis we have not explicitly tested. However, in a previous study (PMID: 30496478) we observed that the inherited MNase sequence bias is more pronounced in di-nucleosomes, which are preferentially isolated from GC-rich regions. This is in line with the depletion of di-nucleosomes in AT-rich intergenic regions in Pf, as was already described by Kensche et al.

Changes to the manuscript text: We included a paragraph describing the limitations to the discussion (L428-434):

The nucDetective pipeline has been optimized for the analysis of mono-nucleosomes. However, the selection of fragment sizes can be adjusted manually, enabling the pipeline to be used for other nucleosome categories. The pipeline is suitable to map and annotate sub-nucleosomal particles (

__ Reference Nucleosome Numbers __

The authors identify 49,999 reference nucleosome positions. How does this compare to previous analyses of similar datasets? This should be explicitly addressed.