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  1. Feb 2025
    1. Author response:

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

      Public Reviews:

      Reviewer #1 (Public Review):

      The authors studied how hippocampal connectivity gradients across the lifespan, and how these relate to memory function and neurotransmitter distributions. They observed older age with less distinct transitions and observed an association between gradient de-differentiation and cognitive decline.

      This is overall an innovative and interesting study to assess gradient alterations across the lifespan and its associations to cognition.

      The paper is well-written, and the methods appear sound and thoughtful. There are several strengths, including the inclusion of two independent cohorts, the use of gradient mapping and alignment techniques, and an overall sound statistical and analysis framework. There are several areas for potential improvements in the paper, and these are listed below:

      We thank the Reviewer for their positive assessment and summary of our work. We address each of the Reviewer’s comments below, and outline the revisions we have made to the manuscript based on the Reviewer’s suggestions.

      (1) The reported D1 associations appear a bit post-hoc in the current work and I was unclear why the authors specifically focussed on dopamine here, as other transmitter systems are similar present at the level of the hippocampus and implicated in aging.

      Other neurotransmitter systems may indeed be relevant in the context of hippocampal function in aging. In this study, however, we included a specific research question about the DA D1 receptor (D1DR) based on previous research 1) emphasizing the role of DA neuromodulation in maintaining functional network segregation in aging to support cognition (Pedersen et al., 2023), 2) reporting heterogeneous distribution of DA markers across the hippocampus, supporting efficient modulation of distinct behaviors (Dubovyk & ManahanVaughan, 2019; Edelmann & Lessmann, 2018; Gasbarri et al., 1994; Kempadoo et al., 2016), and 3) demonstrating the spatial distribution of D1DRs as varying across neocortex along a unimodal-transmodal gradient (Pedersen et al., 2024). To which degree this variation might be reflected in cortico-hippocampal connectivity, however, remained to be investigated. As such, one of the study’s specific aims was to evaluate the spatial distribution of D1DRs as a molecular correlate of the hippocampus’ functional organization. Importantly, we were interested in mapping associations between individual differences in the organization of connectivity and D1DRs. This was uniquely enabled by utilizing the DyNAMiC sample, as it includes structural and functional MRI data in combination with D1DR PET in the same individuals across the adult lifespan (n=180). However, after observing significant spatial correspondence between functional organization and D1DR expressed by the second hippocampal gradient (G2), we did indeed perform complimentary analyses with group-averaged data of additional dopamine markers (D2DR from a subsample of our participants, as well as DAT and FDOPA from open sources) to test the generalizability of the original finding. Taken together, the original analyses based on subject-level data and complimentary group-level analyses provided support for the interpretation of G2 as a dopaminergic mode.

      We have updated the manuscript to clarify the focus on the D1 receptor and the contribution of including additional DA markers.

      Updated paragraph in the Introduction, pages 5-6:

      “Dopamine (DA) is one of the most important modulators of hippocampus-dependent function(47,48), and influences the brain’s functional architecture through enhancing specificity of neuronal signaling(49). Consistently, there is a DA-dependent aspect of maintained functional network segregation in aging which supports cognition(50). Animal models suggest heterogeneous patterns of DA innervation(51,52) and postsynaptic DA receptors(53), across both transverse and longitudinal hippocampal axes, likely allowing for separation between DA modulation of distinct hippocampus-dependent behaviors(47). Moreover, the human hippocampus has been linked to distinct DA circuits on the basis of long-axis variation in functional connectivity with midbrain and striatal regions(54,55). Taken together with recent findings revealing a unimodal-transmodal organization of the most abundantly expressed DA receptor subtype, D1 (D1DR), across cortex(56), we tested the hypothesis that the organization of hippocampal-neocortical connectivity partly reflects the underlying distribution of hippocampal DA receptors, predicting predominant spatial correspondence for any hippocampal gradient conveying a unimodal-transmodal pattern across cortex.”

      Updated sections in the Results, page 13-14:

      “Our next aim was to investigate to which extent the distribution of hippocampal DA D1 receptors (D1DRs), measured by [<sup>11</sup>C]SCH23390 PET in the DyNAMiC(58) sample, may serve as a molecular correlate of the hippocampus’ functional organization.”

      “Complimentary analyses were then conducted to further evaluate G2 as a dopaminergic hippocampal mode by utilizing additional DA markers at group-level.”

      Moreover, the authors may be aware that multiple PET tracers are somewhat challenged in the mesiotemporal region. Is this the case for the D1 receptor as well? The hippocampus is a small and complex structure, and PET more of a low res technique so one would want to highlight and discuss the limitations of the correlations with PET maps here and/or evaluate whether the analysis adds necessary findings to the study.

      We thank the Reviewer for raising this point. The lower resolution of PET is indeed a relevant aspect to consider when quantifying D1DR availability in the hippocampus, even though previous research indicate high test-retest reliability of [<sup>11</sup>C]SCH23390 PET measurement in this region (Kaller et al., 2017). We have now elaborated on PET limitations in the Discussion of the revised manuscript.

      In our study, we made efforts to reduce potential partial volume effects (PVE) by correcting our PET data, and tested spatial associations between our functional gradients and D1DR maps using trend-surface modelling (TSM), rather than through voxel-wise comparisons. This allowed us to evaluate the spatial correspondence between functional connectivity and D1DRs at a level of spatial trends, estimated using TSM models computed at increasing levels of complexity. The results showed consistent spatial overlap between G2 and D1DRs across these models, that is, across spatial trends described at coarser-to-finer scales. Furthermore, this was replicated across several DA markers with PET and SPECT data from independent samples.

      Taken together, we agree with the Reviewer that the spatial correspondence observed between G2 and hippocampal D1DRs should be interpreted in the context of resolution-related limitations inherent to PET imaging. However, we strongly believe that our DA analyses offer valuable insight to the molecular underpinnings of hippocampal functional organization.

      Updated paragraph in the Discussion, pages 25-26:

      “We discovered that G2, specifically, manifested organizational principles shared among function, behavior, and neuromodulation. Meta-analytical decoding reproduced a unimodalassociative axis across G2 (Figure 3B), and analyses in relation to the distribution of D1DRs – which vary across cortex along a unimodal-transmodal axis(76,77) – demonstrated topographic correspondence both at the level of individual differences and across the group. It should, however, be acknowledged that PET imaging in the hippocampus is associated with resolutionrelated limitations, although previous research indicate high test-retest reliability of [<sup>11</sup>C]SCH23390 PET to quantify D1DR availability in this region(78). As such, mapping the distribution of hippocampal D1DRs at a fine spatial scale remains challenging, and replication of our results in terms of overlap with G2 is needed in independent samples. Here, we evaluated the observed spatial overlap between G2 topography and D1DRs across multiple TSM model orders, showing correspondence between modalities from simple to more complex parameterizations of their spatial properties. Topographic correspondence was additionally observed between G2 and other DA markers from independent datasets (Figure 3B), suggesting that G2 may constitute a mode reflecting a dopaminergic phenotype, which contributes to the currently limited understanding of its biological underpinnings.”

      From my (perhaps somewhat biased) perspective, it might be valuable to instead or in addition look at measures of hippocampal microstructure and how these relate to the functional aging effects. This could be done, if available, using data from the same subjects (eg based on quantitative MRI contrasts and/or structural MRI) and/or using contextualization findings as implemented in eg hippomaps.readthedocs.io

      We thank the Reviewer for this suggestion. We performed additional analyses investigating the spatial overlap between our connectivity gradients and estimates of hippocampal microstructure, computed as the ratio of T1- over T2-weighted (T1w/T2w) images (Glasser & Von Essen, 2011; vos de Wael et al., 2018). Analyses of spatial correspondence then followed the TSM-based method used to test the spatial overlap between functional connectivity gradients and D1DR distribution. Applying TSM to the T1w/T2w image computed for each participant yielded subject-level model parameters describing microstructure topography, which were then entered as predictors of connectivity topography in multivariate GLMs (separate models for each gradient and hemisphere, 6 models in total).

      Analyses revealed that microstructure of the right hippocampus significantly predicted gradient topography of right-hemisphere G1 (F = 1.325, p \= 0.034), while no other links between connectivity gradients and microstructure emerged as significant (F 0.930-1.184, ps 0.7060.079).

      These results, suggesting an association along the anteroposterior axis, deviate from previous findings linking hippocampal microstructure to G3-like, medial-lateral, connectivity organization (vos de Wael et al., 2018). As we believe that comprehensive analyses of our gradients in relation to microstructure across the lifespan would be best addressed in future work, we have not included these analyses of microstructure in the revised manuscript.

      (2) Can the authors clarify why they did not replicate based on cohorts that are more widely used in the community and open access, such as CamCAN and/or HCP-Aging? It might connect their results with other studies if an attempt was made to also show that findings persist in either of these repositories.

      We agree with the Reviewer that replication in samples such as CamCAN and/or HCP-Aging would provide valuable opportunities to connect our findings with those of other studies using those datasets. Here, we included the Betula dataset (Nilsson et al., 2004) as our replication sample, as it was immediately available to us, included a large sample of adults in a comparable age, and a word recall episodic memory task closely aligned with the one included in DyNAMiC. Importantly, leveraging the Betula dataset as our replication sample allows us to link our findings to a wide range of previous studies central to the understanding of neurocognitive aging in general, and hippocampal aging in particular (Nyberg, 2017; Nyberg et al., 2020). Betula is a large longitudinal project that has been tracking individuals since 1988, and is part of the National E-infrastructure for Aging Research (NEAR: www.near-aging.se), through which data from several Swedish studies are made available to both national and international researchers. While we acknowledge the value of extending replication efforts to datasets like CamCAN and HCP-Aging, we emphasize the significant contribution of having replicated our connectivity gradients in the Betula dataset.

      (3) The authors applied TSM and related these parameters to topographic changes in the gradients. I was wondering whether and how such an approach controls for autocorrelation present in both the PET map and gradients. Could the authors clarify?

      The Reviewer raises an important topic in spatial autocorrelation. The TSM approach used to parameterize the topography of the functional gradients and D1DR distribution, and to test the spatial correspondence between modalities, did not include any specific method to control for autocorrelation. Here, we highlight two aspects of our study in relation to this point. First, we demonstrated in the Supplementary information (S. Figure 4) that autocorrelation induced by spatial smoothing likely has limited effects on overall gradient topography and the ability of TSM parameters to capture meaningful inter-individual differences in terms of age. Second, in the case of spatial overlap effects being significantly impacted by autocorrelation, we would expect the association between right-hemisphere G2 and D1DR topography to similarly emerge for G2 in the left hemisphere. The absence of such an association may speak to a limited effect of spatial autocorrelation.

      (4) The TSM approach quantifies the gradients in terms of x/y/z direction in a cartesian coordinate system. Wouldn't a shape intrinsic coordinate system in the hippocampus also be interesting, and perhaps even be more efficient to look at here (see eg DeKraker 2022 eLife or Paquola et al 2020 eLife)?

      This is a very relevant question and we appreciate the Reviewer’s suggestion. We recognize that there may be several benefits associated with adopting a shape-intrinsic coordinate system when characterizing effects in the hippocampus, given its curved/folded anatomy. Approaches like the ones adopted in DeKraker et al., 2022 and Paquola et al., 2020, utilizes geodesic coordinate frameworks to represent the hippocampus in surface space, enabling mapping of connectivity onto the hippocampal surface while respecting its inherent curvature and topology. We anticipate that quantifying gradients within such a framework would especially benefit identification of connectivity change across the hippocampal surface relative to reference points such as subfield boundaries, while minimizing effects of interindividual differences in hippocampal shape and folding. In our study, hippocampal gradients and their associated cortical patterns were computed in volumetric space, with TSM subsequently used to parameterize the change in connectivity along these gradients. This indeed yields a description of connectivity change within a coordinate system less specific to hippocampal anatomy, but may favor generalizability and integration with previous gradient findings within and beyond the hippocampus (e.g., Przeździk et al., 2019; Tian et al., 2020; Katsumi et al., 2023; Navarro-Schröder et al., 2015), as well as connections with broader neuroimaging frameworks through techniques such as meta-analytical decoding. In our view, the different coordinate frameworks offer complimentary insight to hippocampal organization, and while we have opted to not undertake novel analyses to explore our gradients within a geodesic coordinate system for the purposes of this paper, we recognize the importance of such evaluation of our gradients in future analyses. We have made updates to the Discussion in the revised manuscript on this topic (pages 23-24):

      “Greater anatomical specificity, with more precise characterization of connectivity in relation to subfield boundaries while minimizing effects of inter-individual differences in hippocampal shape and folding, might be achieved by adopting techniques implementing a geodesic coordinate system to represent effects within the hippocampus(68,69).”

      Reviewer #2 (Public Review):

      Summary:

      This paper derives the first three functional gradients in the left and right hippocampus across two datasets. These gradient maps are then compared to dopamine receptor maps obtained with PET, associated with age, and linked to memory. Results reveal links between dopamine maps and gradient 2, age with gradients 1 and 2, and memory performance.

      Strengths:

      This paper investigates how hippocampal gradients relate to aging, memory, and dopamine receptors, which are interesting and important questions. A strength of the paper is that some of the findings were replicated in a separate sample.

      Weaknesses:

      The paper would benefit from added clarification on the number of models/comparisons for each test. Furthermore, it would be helpful to clarify whether or not multiple comparison correction was performed and - if so - what type or - if not - to provide a justification. The manuscript would furthermore benefit from code sharing and clarifying which results did/did not replicate.

      We thank the Reviewer for their positive assessment and suggestions regarding further clarifications. We have addressed the Reviewer’s comments in a point-by-point manner under the “Recommendations for the authors” section.

      Reviewer #3 (Public Review):

      Summary:

      In this study, the authors analyzed the complex functional organization of the hippocampus using two separate adult lifespan datasets. They investigated how individual variations in the detailed connectivity patterns within the hippocampus relate to behavioral and molecular traits. The findings confirm three overlapping hippocampal gradients and reveal that each is linked to established functional patterns in the cortex, the arrangement of dopamine receptors within the hippocampus, and differences in memory abilities among individuals. By employing multivariate data analysis techniques, they identified older adults who display a hippocampal gradient pattern resembling that of younger individuals and exhibit better memory performance compared to their age-matched peers. This underscores the behavioral importance of maintaining a specific functional organization within the hippocampus as people age.

      Strengths:

      The evidence supporting the conclusions is overall compelling, based on a unique dataset, rich set of carefully unpacked results, and an in-depth data analysis. Possible confounds are carefully considered and ruled out.

      Weaknesses:

      No major weaknesses. The transparency of the statistical analyses could be improved by explicitly (1) stating what tests and corrections (if any) were performed, and (2) justifying the elected statistical approaches. Further, some of the findings related to the DA markers are borderline statistically significant and therefore perhaps less compelling but they line up nicely with results obtained using experimental animals and I expect the small effect sizes to be largely related to the quality and specificity of the PET data rather than the derived functional connectivity gradients.

      We thank the Reviewer for the thoughtful summary and positive assessment of our work. To increase transparency of the statistical analyses, we have in the revised manuscript added information regarding statistical tests and corrections for multiple comparisons. In the Results, p-values were reported at an uncorrected statistical threshold, and we have in the revised manuscript included the corresponding p-values adjusted for multiple comparisons using the Benjamini-Hochberg method to control the false discovery rate (FDR). Finally, in the revised manuscript, we have now elaborated on the potential limitations of our PET analyses and we include the updated paragraph below.

      Addition made to the Results section, page 13:

      “Individual maps of D1DR binding potential (BP) were also submitted to TSM, yielding a set of spatial model parameters describing the topographic characteristics of hippocampal D1DR distribution for each participant. D1DR parameters were subsequently used as predictors of gradient parameters in one multivariate GLM per gradient (in total 6 GLMs, controlled for age, sex, and mean FD). Results are reported with p-values at an uncorrected statistical threshold and p-values after adjustment for multiple comparisons using the Benjamini-Hochberg method to control the false discovery rate (FDR).”

      Addition made to the Results section, page 15:

      “Effects of age on gradient topography were assessed using multivariate GLMs including age as the predictor and gradient TSM parameters as dependent variables (controlling for sex and mean frame-wise displacement; FD). One model was fitted per gradient and hemisphere, each model including all TSM parameters belonging to a gradient (in total, 6 GLMs).”

      Addition made to the Results section, page 17:

      “Models were assessed separately for left and right hemispheres, across the full sample and within age groups, yielding eight hierarchical models in total. Results are reported with p-values at an uncorrected statistical threshold and p-values after FDR adjustment.”

      Updated paragraph in the Discussion, pages 25-26:

      “We discovered that G2, specifically, manifested organizational principles shared among function, behavior, and neuromodulation. Meta-analytical decoding reproduced a unimodalassociative axis across G2 (Figure 3B), and analyses in relation to the distribution of D1DRs – which vary across cortex along a unimodal-transmodal axis(76,77) – demonstrated topographic correspondence both at the level of individual differences and across the group. It should, however, be acknowledged that PET imaging in the hippocampus is associated with resolutionrelated limitations, although previous research indicate high test-retest reliability of [<sup>11</sup>C]SCH23390 PET to quantify D1DR availability in this region(78). As such, mapping the distribution of hippocampal D1DRs at a fine spatial scale remains challenging, and replication of our results in terms of overlap with G2 is needed in independent samples. Here, we evaluated the observed spatial overlap between G2 topography and D1DRs across multiple TSM model orders, showing correspondence between modalities from simple to more complex parameterizations of their spatial properties. Topographic correspondence was additionally observed between G2 and other DA markers from independent datasets (Figure 3B), suggesting that G2 may constitute a mode reflecting a dopaminergic phenotype, which contributes to the currently limited understanding of its biological underpinnings.”

      Recommendations for the authors:

      Reviewer #1 (Recommendations For The Authors):

      Please see the comments in the public review.

      We thank the Reviewer for their comments and recommendations, and have addressed them in the “Public review” section.

      Reviewer #2 (Recommendations For The Authors):

      (1) All statistical analyses are based on linear regressions using trend surface modeling (TSM) parameters that parameterize gradients at the subject level. These models resulted in 9 parameters for gradient 1 and 12 parameters each for gradients 2 and 3. The text states that 'Effects of age on gradient topography was assessed using multivariate GLMs including age as the predictor and gradient TSM parameters as dependent variables (controlling for sex and mean frame-wise displacement; FD)'. Please clarify whether these GLMs were fitted separately for each TSM parameter (i.e., 9+12+12=33 models for both left and right = 66 total models) or on the overall model?

      We appreciate the Reviewer’s request for clarification on this matter. These GLMs were fitted on the overall TSM model, that is, through one GLM per gradient (3) and hemisphere (2), each one including all TSM parameters belonging to a gradient (in total, 6 GLMs).

      In the revised manuscript, we have added more details to the Results section, page 15: “Effects of age on gradient topography were assessed using multivariate GLMs including age as the predictor and gradient TSM parameters as dependent variables (controlling for sex and mean frame-wise displacement; FD). One model was fitted per gradient and hemisphere, each model including all TSM parameters belonging to a gradient (in total, 6 GLMs).”

      (2) Similarly, for memory it appears that multiple models were performed (left and right, young, middle-aged, old, whole groups). Please clarify whether and how multiple comparison correction was performed in this case.

      In the revised manuscript, we have now specified the number of analyses conducted in relation to memory performance. We have also clarified that p-values were reported at an uncorrected statistical threshold, and we have in the revised manuscript included the corresponding p-values adjusted for multiple comparisons using the Benjamini-Hochberg method to control the FDR.

      Updated section in the Results, page 17:

      “Models were assessed separately for left and right hemispheres, across the full sample and within age groups, yielding eight hierarchical models in total. Results are reported with p-values at an uncorrected statistical threshold and p-values after FDR adjustment.”

      (3) Although I applaud the authors for their replication efforts, the results do not appear to replicate well. For example, memory was linked to gradient 2 in the whole group but to gradient 1 in the young group. Furthermore, dopamine was linked to gradient 2 in the right but not the left hemisphere. Although the overall group-level gradients were very stable between the two datasets, it is not clear whether the age findings replicated and the memory subgroup findings only replicated at trend level for memory and only partially replicated at the TSM parameter level.

      We thank the Reviewer for highlighting the inclusion of a replication dataset as a strength of our study, and we appreciate the recommendation to clarify to which extent results replicated. We provide a response to the Reviewer’s points below, and specify the revisions made to the manuscript in relation to this topic.

      The main aim of our study was to characterize the topographic organization of functional hippocampal-neocortical connectivity within the hippocampus across the adult lifespan, as previous studies have limited their focus to younger adults. Given the lack of previous studies for comparison, together with our identification of a novel secondary long-axis connectivity gradient (G2) taking precedence over the previously established medial-lateral G3, we included the Betula sample (Nilsson et al., 2004) for the purpose of replication. There was a high level of consistency between our main dataset and our replication dataset, with gradients 1-3 in left and right hemispheres identified in both samples.

      Further use of the replication dataset, beyond the identification of the connectivity gradients, was originally not planned. As such, not all subsequent analyses in the main dataset were conducted in the replication dataset. However, we found it critical to evaluate the observation that older individuals who maintained a youth-like gradient topography also exhibited higher levels of memory performance in an independent sample. This was possible given that the replication dataset included a comparable number of participants in similar ages and a word recall episodic memory task corresponding well to the one used in DyNAMiC. Overall, we conclude that these analyses replicated well across samples. Firstly, topography of lefthemisphere G1 informed the classification of older adults into youth-like and aged subgroups in both samples. Furthermore, in both samples, we observed that the older subgroups identified based on G1 topography also exhibited the youth-like vs. aged pattern in G2 topography. This pattern was, however, evident also in G3 only in the main sample, possibly suggesting a limited contribution of G3 topography in determining overall functional profiles in older age. In terms of the behavioral relevance of maintaining youth-like gradient topography in older age, we observed effects on word recall performance in both samples; although the Reviewer correctly points out that, the difference between subgroups was significant at trend-level (p = 0.058) in the replication dataset. While this indeed underscores the importance of replication efforts in additional samples, we argue that the pattern observed in our replication dataset is overall consistent with, and conveys effects in the expected direction based on, the original observations in our main dataset.

      In revising the manuscript, we have performed additional analyses for replication purposes in terms of memory. Originally, we observed a significant association between G2 topography and episodic memory across the main sample. However, this effect did not remain significant after FDR adjustment for multiple comparisons. To evaluate this association further, we conducted a corresponding hierarchical multiple regression analysis in the replication dataset, which supported a role of G2 in memory (Adj. R<sup>2</sup> = 0.368, ΔR<sup>2</sup> = 0.081, F= 1.992, p = 0.028). Together, these analyses suggest that inter-individual differences in episodic memory performance may in part be explained by the spatial characteristics of G2 across the adult lifespan, although increased statistical power in relation to the large number of TSM parameters included in the hierarchical regression models may be needed to explore this association in smaller, age-stratified, groups. Relatedly, it is worth mentioning that higher levels of memory performance in older age were linked to the maintenance of youth-like G2 topography in both our main and replication datasets.

      In parallel, topographic parameters of G1 predicted memory performance in the younger adults, which successfully replicates TSM-based results previously reported in Przeździk et al., 2019. Although similar associations were not evident within the other age groups, a link between G1 topography and memory was demonstrated in older age based on a) the identification of individuals maintaining a youth-like G1 profile and higher levels of memory, within which b) memory performance was, as in young adults, significantly predicted by G1 topography.

      The spatial correspondence between G2 topography and distribution of hippocampal D1DRs was lateralized to the right, and as the Reviewer points out, as such did not replicate across hemispheres. To which extent replication across hemispheres should be expected in this case is, however, difficult to determine. Lateralization and/or hemispheric asymmetry is commonly observed in numerous hippocampal features, from the molecular level to its functional involvement in behavior (Nematis et al., 2023; Persson & Söderlund, 2015), including various dopaminergic markers tested in the animal literature (Afonso et al., 1993; Sadeghi et al., 2017). Yet, potential differences between hemispheres in D1DR availability and the spatial distribution of receptors along hippocampal axes remain less studied in humans. More data is therefore needed to determine the nature of this right-hemisphere lateralization.

      In sum, we argue that our results show a good level of replication across independent datasets and across analyses in our main dataset. Whereas this study did not attempt replication of all analyses conducted in the main dataset, it has through replication across independent samples provided support for its main findings – the organization of hippocampal-neocortical connectivity along three main hippocampal gradients across the adult lifespan, and the gradient topography-based identification of older individuals maintaining a youth-like hippocampal organization in older age.

      The revised manuscript includes edits made to incorporate the new analyses and clarifications of observations in relation to memory.

      In the Results, page 17:

      “Observing that the association between G2 and memory did not remain significant after FDR adjustment, we performed the same analysis in our replication dataset, which also included episodic memory testing. Consistent with the observation in our main dataset, G2 significantly predicted memory performance (Adj. R<sup>2</sup> = 0.368, ΔR<sup>2</sup> = 0.081, F= 1.992, p = 0.028) over and above covariates and topography of G1. Here, the analysis also showed that G1 topography predicted performance across the sample (Adj. R<sup>2</sup> = 0.325, ΔR<sup>2</sup> = 0.112, F= 3.431, p < 0.001).”

      In the Discussion, page 26:

      “Results linked both G1 and G2 to episodic memory, suggesting complimentary contributions of these two overlapping long-axis modes. Considered together, analyses in the main and replication datasets indicated a role of G2 topography in memory across the adult lifespan, independent of age. A similar association with G1 was only evident across the entire sample in the replication dataset, whereas results in the main sample seemed to emphasize a role of youthlike G1 topography in memory performance. In line with previous research, memory was successfully predicted by G1 topography in young adults(30), and similarly predicted by G1 in older adults exhibiting a youth-like functional profile.”

      (4) Please share the data and code and add a description of data and code availability in the manuscript.

      We have now made our code available, and added a statement on data and code availability in the revised manuscript.

      On page 37: “Data from the DyNAMiC study are not publicly available. Access to the original data may be shared upon request from the Principal investigator, Dr. Alireza Salami. The Matlab, R, and FSL codes used for analyses included in this study are openly available at https://github.com/kristinnordin/hcgradients. Computation of gradients was done using the freely available toolbox ConGrads: https://github.com/koenhaak/congrads.”

      Reviewer #3 (Recommendations For The Authors):

      Please see the comments in the public review.

      We thank the Reviewer for their comments and recommendations, and have addressed them in the “Public review” section.

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      Paquola, C., Benkarim, O., DeKraker, J., Larivière, S., Frässle, S., Royer, J., Tavakol, S.,

      Valk, S., Bernasconi, A., Bernasconi, N., Khan, A., Evans, A. C., Razi, A., Smallwood, J., & Bernhardt, B. C. (2020). Convergence of cortical types and functional motifs in the human mesiotemporal lobe. eLife, 9, e60673. https://doi.org/10.7554/eLife.60673

      Pedersen, R., Johansson, J., Nordin, K., Rieckmann, A., Wåhlin, A., Nyberg, L., Bäckman, L., & Salami, A. (2024). Dopamine D1-Receptor Organization Contributes to Functional Brain Architecture. Journal of Neuroscience, 44(11). https://doi.org/10.1523/JNEUROSCI.0621-23.2024

      Pedersen, R., Johansson, J., & Salami, A. (2023). Dopamine D1-signaling modulates maintenance of functional network segregation in aging. Aging Brain, 3, 100079. https://doi.org/10.1016/j.nbas.2023.100079

      Persson, J., & Söderlund, H. (2015). Hippocampal hemispheric and long-axis differentiation of stimulus content during episodic memory encoding and retrieval: An activation likelihood estimation meta-analysis. Hippocampus, 25(12), 1614–1631. https://doi.org/10.1002/hipo.22482

      Przeździk, I., Faber, M., Fernández, G., Beckmann, C. F., & Haak, K. V. (2019). The functional organisation of the hippocampus along its long axis is gradual and predicts recollection. Cortex, 119, 324–335. https://doi.org/10.1016/j.cortex.2019.04.015

      Sadeghi, L., Rizvanov, A. A., Salafutdinov, I. I., Dabirmanesh, B., Sayyah, M., Fathollahi, Y., & Khajeh, K. (2017). Hippocampal asymmetry: Differences in the left and right hippocampus proteome in the rat model of temporal lobe epilepsy. Journal of Proteomics, 154, 22–29. https://doi.org/10.1016/j.jprot.2016.11.023

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    1. Les points suivants résument les informations importantes du live de l'Académie de Grenoble sur l'orientation post-bac, en mettant l'accent sur les conseils et les ressources disponibles pour les étudiants:

      • Rôle des Centres d'Information et d'Orientation (CIO) Les CIO sont des lieux d'accueil ouverts à tous les publics, y compris les jeunes scolarisés et les adultes, offrant un accompagnement personnalisé dans la réflexion sur l'orientation. Ils mettent à disposition de la documentation de l'ONISEP et du CIDJ, accessible en consultation libre ou en prêt.

      • Psychologues de l'Éducation Nationale (PsyEN) Les PsyEN, anciennement conseillers d'orientation psychologues, sont des psychologues formés au système éducatif et à l'orientation. Ils offrent un accompagnement bienveillant et sans jugement, aident à identifier les points forts, les centres d'intérêt, et les compétences des étudiants, et informent sur les différentes voies de formation et les secteurs professionnels.

      • Importance de la connaissance de soi et de l'information Il est essentiel de bien se connaître (qualités, compétences, centres d'intérêt) et de s'informer sur les formations et les métiers. Les PsyEN peuvent aider à travers des questionnaires d'intérêt, mais soulignent que ces outils ne sont pas des solutions miracles.

      • Parcoursup comme ressource d'information Parcoursup est une mine d'or d'informations sur les formations : présentation, apprentissage, matières enseignées, critères d'analyse des candidatures, taux de bacheliers recrutés, etc.. Il est possible de consulter Parcoursup même sans l'utiliser pour postuler, et ce dès la seconde.

      • Journées portes ouvertes Les journées portes ouvertes sont des occasions importantes pour se familiariser avec les lieux, discuter avec les étudiants et les professeurs, et poser des questions. Les dates de ces événements sont disponibles sur Parcoursup et les sites des CIO.

      • Ne pas rester seul et oser demander de l'aide Il est important de ne pas rester isolé face aux questions d'orientation et de solliciter l'aide des PsyEN, que ce soit au CIO ou dans les établissements scolaires. Les parents peuvent également prendre rendez-vous.

      • Droit à l'erreur et réorientation L'orientation est un processus qui peut nécessiter du temps, et il est normal de changer d'avis ou de se tromper de voie. La réorientation est possible, et il existe des passerelles entre les formations. Se tromper une fois peut même renforcer la motivation à mieux s'informer par la suite.

      • Ressources en ligne Plusieurs ressources en ligne sont disponibles, telles que la plateforme Avenir, le site de l'ONISEP (avec ONISEP TV), et le site Côté Formation pour la région Auvergne-Rhône-Alpes.

      • Dépasser les idées reçues et les conseils Il est important de tenir compte des conseils de l'entourage, mais la décision finale revient à l'étudiant. Il ne faut pas s'autocensurer ni prendre pour argent comptant les idées reçues sur les formations et les métiers.

      • Spécialités au lycée et orientation Le choix des spécialités au lycée peut être important pour certaines formations scientifiques, mais il existe des solutions de remise à niveau pour les étudiants qui souhaitent se réorienter vers ces domaines. Il ne faut pas se mettre trop de pression sur ce choix et se renseigner sur les attendus des formations visées.

    1. Voici un sommaire avec horodatage basé sur la transcription de la vidéo "🍭 Le sucre, entre plaisir et ravages" :

      • Présentation du café des sciences [00:00:01-00:00:03]
      • Introduction des spécialistes du sucre [00:00:03-00:00:27]
        • Emelyine Leroux, maître de conférence en biochimie alimentaire à l'université de Rennes.
        • David Lailler, directeur de recherche en neurosciences comportementale et nutrition à l'Institut Numecan à Rennes.
      • Annonce de la possibilité de poser des questions par le public [00:00:27-00:00:35]
      • Introduction au sujet du sucre [00:00:35-00:00:54]
        • Le sucre est partout et sous différentes formes.
      • Définition biochimique des sucres [00:00:54-00:01:07]
        • Il faudrait parler "des sucres" et non "du sucre" car c'est une grande famille, les glucides. Le sucre classique est le saccharose [00:00:54, 00:00:55].
      • Oses simples [00:00:55-00:01:07]
        • Petites molécules comme le glucose et le fructose [00:00:55, 00:00:56].
      • Dioloides [00:01:07-00:01:10]
        • Association de deux oses simples [00:01:07].
        • Exemples: saccharose (fructose + glucose), lactose (glucose + galactose) [00:01:07, 00:01:08].
      • Différences entre les sucres [00:01:10-00:01:13]
        • Le lactose est moins sucré que le saccharose [00:01:10].
      • Molécules complexes [00:01:13-00:01:18]
        • L'amidon, composé de chaînes de glucose, est plus difficile à absorber [00:01:13, 00:01:17].
      • Goût sucré et analyse sensorielle [00:01:18-00:01:28]
        • Le saccharose est la référence pour le goût sucré [00:01:18, 00:01:19].
        • L'analyse sensorielle utilise des panels de consommateurs pour évaluer le goût [00:01:21-00:01:27].
      • Pouvoir sucrant des différents sucres [00:01:28-00:01:30]
        • Le lactose a un pouvoir sucrant de 0,3 par rapport au saccharose (1) [00:01:28, 00:01:29].
      • Faux sucres [00:01:30-00:01:40]
        • Dérivés de sucres ou molécules sans lien avec les sucres (ex: aspartame) [00:01:30-00:01:34].
        • Pouvoir sucrant parfois 600 fois supérieur au saccharose [00:01:34-00:01:36].
        • Dilution nécessaire pour une consommation acceptable [00:01:37-00:01:40].
      • Importance du sucre pour la vie [00:01:40-00:01:42]
        • Le glucose est notre carburant [00:01:40, 00:01:41].
      • Sources de glucose [00:01:41-00:01:48]
        • Amidon, transformation des lipides [00:01:41-00:01:42].
        • Le corps peut fabriquer le glucose nécessaire [00:01:42].
        • Les glucides devraient représenter 40 à 50% de l'alimentation [00:01:45-00:01:48].
      • Stockage du glucose [00:01:48-00:01:52]
        • Glycogène (équivalent de l'amidon chez les plantes) [00:01:48, 00:01:49].
        • Réserve de glucides dégradée après consommation du glucose circulant [00:01:50-00:01:52].
      • Perception du goût sucré [00:01:52-00:01:55]
        • L'amidon n'est pas sucré, mais la mastication prolongée permet aux enzymes de la salive de le dégrader en glucose, libérant ainsi le goût sucré [00:01:52-00:01:55].
      • Coup de fatigue et hypoglycémie [00:01:55-00:02:00]
        • En cas d'hypoglycémie, la consommation de sucres rapides peut apporter de l'énergie à court terme [00:01:55-00:01:59].
      • Risques de la consommation de sucres rapides [00:02:00-00:02:18]
        • Pic de glycémie suivi d'une sécrétion d'insuline pour réguler le taux de glucose [00:02:00-00:02:06].
        • Risque d'hypoglycémie paradoxale [00:02:07-00:02:08].
      • Prévention des carences en sucre [00:02:08-00:02:12]
        • Privilégier les glucides complexes pour une libération progressive du sucre dans le sang [00:02:08-00:02:11].
      • Effet de la matrice alimentaire [00:02:12-00:02:22]
        • L'effet sur la glycémie dépend de la matrice alimentaire et de la présence de fibres qui ralentissent l'absorption du sucre [00:02:12-00:02:17].
        • Un fruit entier aura un effet différent d'un jus de fruit [00:02:13].
        • La mastication et la digestion ralentissent également l'absorption du sucre [00:02:17-00:02:18].
      • Rôle de la satiété [00:02:19-00:02:22]
        • Les aliments solides et peu transformés favorisent la satiété grâce à des signaux cérébraux [00:02:19-00:02:22].
      • Différents types de jus d'orange [00:02:22-00:02:24]
        • Pur jus, jus à base de concentré, nectars (bas de gamme, coupés avec de l'eau sucrée) [00:02:22-00:02:24].
      • Sucres ajoutés dans les produits industriels [00:02:24-00:02:27]
        • Les aliments transformés contiennent souvent des sirops de glucose-fructose issus de l'industrialisation [00:02:24-00:02:26].
      • Risques associés aux sirops de glucose-fructose [00:02:26-00:02:27]
        • Augmentation du risque de maladies cardiovasculaires et de syndrome métabolique [00:02:26].
      • Évolution de la consommation de sucre [00:02:27-00:02:37]
        • Consommation stable ces dernières années, mais très faible avant 1850 car le sucre était un produit de luxe [00:02:27-00:02:32].
        • Aujourd'hui, consommation mondiale autour de 20 kg par an par habitant, environ 35 kg en France [00:02:33].
        • L'OMS recommande un maximum de 50g par jour, voire 25g [00:02:33-00:02:34].
      • Impact du sucre sur le cerveau [00:02:37-00:02:42]
        • Le glucose est le carburant de base des neurones [00:02:37-00:02:39].
        • Le cerveau est informé de la consommation de sucre par des récepteurs intestinaux et des hormones [00:02:40-00:02:42].
      • Détection et intégration des signaux liés à la consommation de sucre [00:02:42-00:02:51]
        • Le cerveau intègre des informations exteroceptives (goût, odeur, vision) et interoceptives (sensations intestinales) [00:02:42-00:02:50].
        • L'image sensorielle de l'aliment se forme et influence le comportement alimentaire [00:02:50-00:02:51].
      • Chiffres de la consommation de sucre [00:02:51-00:02:57]
        • La consommation mondiale est d'environ 20 kg par an et par habitant [00:02:52-00:02:53]. En France, elle est d'environ 35 kg [00:02:53].
        • L'OMS recommande un maximum de 50 g par jour et par personne, idéalement 25 g [00:02:53-00:02:55].
      • Types de sucres pris en compte dans les chiffres [00:02:57-00:03:01]
        • Le sucre en poudre ou en morceaux (saccharose), ajouté aux aliments ou présent dans les produits industriels [00:02:57-00:03:00].
      • Expérience d'arrêt du sucre et migraines [00:03:01-00:03:08]
        • L'arrêt brutal du sucre peut provoquer des symptômes de sevrage et des migraines [00:03:01-00:03:06].
      • Addiction au sucre et plasticité cérébrale [00:03:06-00:03:15]
        • Le cerveau s'habitue à une certaine concentration de sucre, et une modification soudaine peut provoquer des réactions [00:03:06-00:03:08].
        • Hyperglycémie et hypoglycémie peuvent causer des maux de tête [00:03:08-00:03:11].
        • Le corps réagit mal à un afflux soudain de sucre après une période d'abstinence [00:03:11-00:03:15].
      • Différence entre sucre raffiné et non raffiné [00:03:15-00:03:26]
        • La différence réside principalement dans les minéraux accompagnant le sucre [00:03:15-00:03:20].
        • La charge en saccharose reste similaire [00:03:19].
        • Le sucre roux peut avoir un côté plus aromatique [00:03:20].
      • Raffinage des aliments [00:03:26-00:03:31]
        • Le raffinage simplifie la matrice alimentaire, ce qui est moins bon pour la santé [00:03:26-00:03:29].
      • Pain blanc vs pain complet [00:03:29-00:03:34]
        • Le pain blanc a un index glycémique plus élevé que le pain complet [00:03:29-00:03:31].
        • Un aliment non raffiné est préférable [00:03:32].
      • Cuisson des pâtes [00:03:34-00:03:37]
        • Les pâtes "al dente" ont un index glycémique plus bas que les pâtes trop cuites [00:03:34-00:03:37].
      • Culpabilisation et information [00:03:37-00:03:45]
        • Il est important de distinguer culpabilisation et information [00:03:37-00:03:40].
        • Les participants à la conférence sont probablement déjà plus conscients et ont une consommation plus raisonnable [00:03:40-00:03:42].
      • Évolution de la consommation et pathologies [00:03:45-00:03:49]
        • L'obésité et le diabète ont émergé avec l'industrialisation de l'alimentation [00:03:45-00:03:47].
      • Transitions alimentaires [00:03:49-00:03:55]
        • Le feu, l'agriculture, les grandes cités et l'industrialisation [00:03:49-00:03:54].
        • La quatrième transition (industrialisation) a conduit à l'émergence de pathologies [00:03:54-00:03:55].
      • Responsabilité individuelle et sociétale [00:03:55-00:04:04]
        • Il est difficile de changer les habitudes alimentaires, surtout celles prises dans l'enfance [00:03:55-00:03:58].
        • Il ne faut pas culpabiliser les gens, mais il y a une part de responsabilité individuelle [00:03:58-00:04:01].
        • La société, le système de production alimentaire et l'éducation ont également un rôle important [00:04:01-00:04:04].
      • Les 1000 premiers jours de la vie [00:04:04-00:04:13]
        • Période capitale pour la mise en place des conduites alimentaires et du métabolisme [00:04:04-00:04:09].
        • L'alimentation de la mère et du père influence la santé future de l'enfant [00:04:09-00:04:13].
      • Conclusion sur la responsabilité [00:04:13-00:04:17]
        • Tout ne repose pas sur les épaules de la personne malade, mais elle a un rôle à jouer dans la modification de ses comportements [00:04:13-00:04:17].
      • Plaisir du sucre [00:04:17-00:04:23]
        • Le sucre est réconfortant et associé à des moments positifs [00:04:17-00:04:23].
      • Préférence innée pour le sucre [00:04:23-00:04:27]
        • Les nourrissons préfèrent spontanément le goût sucré, lié à un besoin de survie [00:04:23-00:04:25].
        • Attirance naturelle pour le sucre, source d'énergie rapide [00:04:25-00:04:27].
      • Conseils pour réduire sa consommation de sucre [00:04:27-00:04:33]
        • Réduire les boissons sucrées [00:04:27-00:04:29].
        • Cuisiner soi-même pour contrôler les quantités de sucre [00:04:29-00:04:33].
      • Addiction au sucre [00:04:33-00:04:35]
        • Plus on mange sucré, plus on a besoin de sucre [00:04:33-00:04:35].
      • Adaptation et contre-adaptation [00:04:35-00:04:40]
        • Le cerveau a gardé son appétence innée pour le sucre, ce qui devient problématique dans un environnement où le sucre est omniprésent [00:04:35-00:04:40].
      • Activation des centres de récompense [00:04:40-00:04:45]
        • Le sucre active les centres de la récompense dans le cerveau, comme d'autres sources de plaisir [00:04:40-00:04:43].
      • Addiction au sucre comparée à la cocaïne [00:04:45-00:04:49]
        • Des expériences sur des rongeurs ont montré que le sucre peut être plus addictif que la cocaïne [00:04:45-00:04:49].
      • Lien entre sucre et mémoire [00:04:59-00:05:02]
        • Le cerveau a besoin de sucre pour fonctionner correctement [00:04:59-00:05:02].
        • L'hypoglycémie peut altérer les capacités cognitives [00:05:01].
      • Effets de l'hyperglycémie sur les capacités cognitives [00:05:02-00:05:07]
        • Des pics d'hyperglycémie réguliers peuvent avoir des effets négatifs sur les capacités cognitives [00:05:02-00:05:07].
      • Peut-on se passer de sucre raffiné ? [00:05:07-00:05:12]
        • Oui, nos ancêtres n'en consommaient presque pas [00:05:07-00:05:09].
        • Le corps est capable de produire du glucose à partir d'autres sources [00:05:09-00:05:12].
      • Faux sucres [00:05:12-00:05:13]
        • Effets potentiels sur la santé et addiction [00:05:12-00:05:13].
      • Polyols [00:05:13-00:05:16]
        • Dérivés d'oses, peu caloriques, non cariogènes [00:05:13-00:05:15].
        • Risque d'effet laxatif en cas de consommation excessive [00:05:15-00:05:16].
      • Aspartame et sucralose [00:05:16-00:05:24]
        • L'aspartame est décrié (suspicions de potentiel cancérogène) [00:05:16-00:05:19].
        • Le sucralose est du saccharose modifié avec du chlore (pouvoir sucrant 600 fois supérieur) [00:05:19-00:05:24].
      • Stevia [00:05:24-00:05:25]
        • Édulcorant intense naturel [00:05:24-00:05:25].
      • Effets des édulcorants sur la perception du goût [00:05:25-00:05:31]
        • Perturbation de l'intégration des signaux, le cerveau peut ne plus associer le goût sucré aux calories [00:05:25-00:05:29].
        • Déséquilibre de la régulation de la faim et de la satiété [00:05:29-00:05:31].
      • Fructose et sirop d'agave [00:05:31-00:05:33]
        • Fructose pas forcément mauvais en soi (présent dans les fruits), mais attention aux sirops de glucose-fructose industriels [00:05:31-00:05:32].
      • Pathologies hépatiques non alcooliques [00:05:33-00:05:37]
        • Liées à une alimentation déséquilibrée et à une consommation excessive de sucre [00:05:33-00:05:37].
      • Sirop d'agave [00:05:37-00:05:39]
        • Alternative végétale, mais à consommer avec modération [00:05:37-00:05:39].
      • Cholestérol et sucre [00:05:39-00:05:45]
        • Pas de lien direct, mais une alimentation globalement déséquilibrée (riche en sucre et en graisses saturées) favorise les troubles métaboliques [00:05:39-00:05:42].
      • Triglycérides [00:05:45-00:05:48]
        • Le sucre peut être stocké sous forme de triglycérides [00:05:45-00:05:48].
      • Sucre et excitation chez les enfants [00:05:48-00:05:51]
        • Une hyperglycémie peut avoir un effet excitant [00:05:48-00:05:51].
      • Effet réconfortant du sucre [00:05:51-00:05:55]
        • Peut calmer les enfants stressés en activant les circuits du plaisir [00:05:51-00:05:54].
      • Ordre de consommation des aliments (sucré/salé) [00:05:55-00:05:58]
        • L'important est de privilégier une matrice alimentaire complexe pour ralentir l'absorption des nutriments [00:05:55-00:05:58].
      • Jeûne [00:05:58-00:06:01]
      • Jeûne intermittent [00:06:01-00:06:09]

        • Alternance de périodes de jeûne et de périodes d'alimentation [00:06:01-00:06:04].
        • Risque d'hypoglycémie et de baisse des capacités cognitives [00:06:04-00:06:07].
        • Être vigilant face aux recommandations et études contradictoires [00:06:07-00:06:09].
        • Sucre et cancer [00:06:09-00:06:17]
        • Les cellules cancéreuses consomment du sucre, mais il ne faut pas affamer le corps entier [00:06:09-00:06:13].
        • Les cellules cancéreuses consomment plus de glucose que les cellules saines [00:06:13-00:06:17].
        • Addiction [00:06:17-00:06:22]
        • La consommation de sucre peut être plus addictive que la cocaïne chez les rongeurs [00:06:17-00:06:22].
        • Addiction alimentaire [00:06:22-00:06:25]
        • Concept polémique, non reconnu dans les classifications des troubles mentaux [00:06:22-00:06:25].
        • Critères d'addiction [00:06:25-00:06:32]
        • Craving, perte de contrôle, tolérance, abandon d'autres plaisirs, consommation malgré les effets négatifs, symptômes de sevrage [00:06:25-00:06:32].
        • Addiction à l'aliment ou au fait de manger ? [00:06:32-00:06:35]
        • Les rongeurs développent une addiction au sucre, mais pas au gras [00:06:32-00:06:35].
        • Addiction chez l'humain [00:06:35-00:06:38]
        • Pas d'addiction au sucre démontrée chez l'humain [00:06:35-00:06:38].
        • Troubles liés à l'usage alimentaire [00:06:38-00:06:42]
        • Aliments transformés, sucrés, salés [00:06:38-00:06:42].
        • Échelle d'évaluation de l'addiction alimentaire [00:06:42-00:06:47]
        • Yell Food Addiction Scale (YFAS), questionnaire clinique avec un score permettant de déterminer le niveau d'addiction [00:06:42-00:06:47].
        • Prise en charge de l'addiction alimentaire [00:06:47-00:06:52]
        • Études cliniques et développement de nouvelles thérapies [00:06:47-00:06:52].
        • Addiction alimentaire et obésité [00:06:52-00:06:55]
        • L'addiction alimentaire n'est pas exclusive aux personnes obèses, elle touche environ 20% de la population générale [00:06:52-00:06:55].
        • Index glycémique (IG) [00:06:55-00:07:04]

        • Score attribué aux aliments en fonction de leur capacité à élever la glycémie [00:06:55-00:06:59].

        • Le glucose pur sert de référence (IG = 100) [00:06:59].
        • Glycémie sanguine [00:07:04-00:07:08]

        • Taux de sucre dans le sang, mesuré par une prise de sang [00:07:04-00:07:08].

        • Pics de glycémie [00:07:08-00:07:14]

        • La consommation de glucose entraîne un pic de glycémie, suivi d'une sécrétion d'insuline pour réguler le taux de sucre [00:07:08-00:07:11].

        • Une matrice alimentaire complexe ralentit l'absorption du sucre et atténue le pic de glycémie [00:07:11-00:07:14].
    1. Voici un sommaire avec horodatage basé sur la transcription de la vidéo "🌪️ La santé mentale des adolescents" :

      • Présentation du café des sciences [00:00:00-00:00:24]
      • Introduction du sujet : la santé mentale des adolescents [00:00:24-00:00:44]
      • Définition de l'adolescence [00:00:44-00:02:26]
        • L'adolescence est un concept social plus que physiologique.
        • Début de l'adolescence : marqué par la puberté et les hormones.
        • Fin de l'adolescence : difficile à définir, liée à l'indépendance.
        • Période de transformation sociale, psychologique et physique.
      • Chiffres clés sur la santé mentale des adolescents [00:02:26-00:02:53]
        • Plus d'un élève sur 10 présente un risque important de dépression en France.
        • 24% des lycéens déclarent des pensées suicidaires au cours des 12 derniers mois.
      • Présentation de l'intervenante, Emmanuel godau [00:02:53-00:03:26]
        • Médecin de santé publique et enseignante-chercheuse.
        • Réalise des enquêtes auprès des collégiens et lycéens.
      • Méthodologie des enquêtes sur la santé des adolescents [00:03:26-00:04:57]
        • Période de la vie où l'on meurt et est malade le moins.
        • Questionnaires auto-administrés en classe, anonymes et confidentiels.
        • Les adolescents sont les mieux placés pour parler de leur santé.
        • On croit ce que disent les adolescents dans les enquêtes.
      • Dégradation de la santé mentale des adolescents depuis la pandémie [00:04:57-00:05:37]
        • Dégradation observée dans plusieurs pays, surtout chez les filles de 15 ans.
      • Différences de genre et hypothèses explicatives [00:05:37-00:07:38]
        • Différences importantes entre filles et garçons au collège.
        • Hypothèses : éducation différenciée, puberté vécue différemment, stress scolaire.
        • Les filles sont plus stressées par le travail scolaire.
      • Avance des filles et impact sur leur bien-être [00:07:38-00:08:04]
        • Maturation physique et psychique plus précoce chez les filles.
        • Les filles commencent à donner des indicateurs négatifs de leur santé mentale plus tôt.
      • Impact des émotions négatives et du contexte anxiogène [00:08:04-00:08:27]
        • Les filles ressentent plus fortement les émotions négatives.
        • Construction culturelle du ressenti et de l'expression des émotions.
      • Distinction entre santé mentale et troubles [00:08:27-00:09:21]
        • La santé mentale est un continuum.
        • Les troubles sont des pathologies psychiatriques.
      • Témoignage sur le vécu au collège et l'impact des réseaux sociaux [00:09:21-00:11:16]
        • Pression au collège pour être comme les autres.
        • Le lycée : plus de liberté et d'acceptation.
      • Facteurs de dégradation de la santé mentale pendant la pandémie [00:11:16-00:12:53]
        • Confinement, contexte géopolitique, crise climatique, réseaux sociaux, #MeToo.
        • Les adolescents ont moins fumé et bu pendant le confinement.
      • Facteurs biologiques et rôle des hormones [00:12:53-00:13:35]
        • Impact des hormones sur l'humeur, syndrome prémenstruel.
      • Contexte politique et pression scolaire [00:13:35-00:14:24]
        • Pression de Parcoursup.
        • Classes surchargées.
      • Chronobiologie et rythme scolaire [00:14:24-00:14:52]
        • Décalage de la chronobiologie à l'adolescence.
        • Difficulté à se coucher tôt et à se lever tôt.
      • Évolution de la perception de la souffrance des adolescents [00:14:52-00:15:58]
        • La souffrance a toujours existé, mais elle est aujourd'hui plus écoutée et prise en compte.
        • Déstigmatisation de la santé mentale.
      • Omniprésence des préoccupations sur la vie sexuelle [00:15:58-00:17:34]
        • Âge du premier rapport sexuel : pas d'augmentation, voire diminution.
        • Accès à la pornographie et rôle de l'éducation à la vie affective et sexuelle.
      • Influence des réseaux sociaux sur la santé mentale [00:17:34-00:19:06]
        • Ne pas diaboliser les réseaux sociaux.
        • Pression de l'image, mais aussi opportunités d'interaction et de soutien.
        • Rôle des adultes pour accompagner et aider à identifier les bonnes ressources.
      • Liens entre santé mentale et santé physique [00:19:06-00:19:44]
        • Les deux sont liés et s'influencent mutuellement.
      • Comment changer la perception de la santé mentale ? [00:19:44-00:21:08]
        • Définition de la santé par l'OMS : physique et mentale.
        • Santé mentale : grande cause nationale.
        • En parler à toute occasion, déstigmatiser.
      • Différences de symptômes entre garçons et filles [00:21:08-00:22:00]
        • Les filles ont des symptômes internalisés, les garçons externalisés (moins vrai aujourd'hui).
        • Conditionnement culturel sur l'expression du mal-être.
      • Surconsommation des réseaux sociaux et risque de dépression [00:22:00-00:23:00]
        • Question de la dose et des algorithmes.
        • Rester sur la ligne de crête et ne pas interdire.
      • Signes à détecter et comment réagir face à un adolescent qui va mal [00:23:00-00:24:06]
        • Changement de comportement, dégradation des résultats scolaires.
        • En parler, écouter, donner le message qu'il y a un adulte de confiance.
        • Former les adultes au premier secours en santé mentale.
      • Différences liées à l'identité de genre et à l'orientation sexuelle [00:24:06-00:25:54]
        • Difficulté à se positionner sur le sexe et impact sur la santé mentale.
        • Les élèves attirés par le même sexe ou les deux vont globalement plus mal.
      • Rôle de l'argent et des industriels derrière les réseaux sociaux [00:25:54-00:26:40]
      • Terminants commerciaux de la santé et vulnérabilité des adolescents [00:26:40-00:26:56]
      • Conclusion et prochain rendez-vous [00:26:56-00:27:12]
    1. Voici un document de synthèse pour un briefing sur la filière STI2D, basé sur la transcription de la vidéo "Les rendez-vous de la techno : Promotion de la série STi2D".

      Présentation générale

      La filière STI2D (Sciences et Technologies de l'Industrie et du Développement Durable) est un baccalauréat technologique destiné aux élèves intéressés par les sciences, la technologie, l'ingénierie et le développement durable. Cette filière met l'accent sur l'apprentissage par projet, le travail en équipe et la recherche de solutions à des problèmes concrets.

      Accès à la filière

      • Les élèves de 3e doivent obtenir un avis favorable pour passer en seconde générale et technologique.
      • En fin de seconde, ils doivent obtenir un avis favorable du conseil de classe pour être admis en première technologique STI2D.

      À qui s'adresse cette filière ?

      Le bac STI2D s'adresse aux élèves qui:

      • Aiment les sciences et la technologie.
      • Sont intéressés par le métier d'ingénieur et l'industrie en général.
      • Aiment travailler en groupe et sur des projets.
      • Aiment trouver des solutions à des problèmes.
      • Sont curieux et inventifs.

      Compétences développées

      Les élèves de STI2D développent des compétences solides en:

      • Sciences et technologies.
      • Ingénierie.
      • Mathématiques.
      • Créativité, approche design et innovation.

      Secteurs d'activité

      Après un bac STI2D, les élèves peuvent s'orienter vers des secteurs d'activité variés tels que:

      • Bâtiment, travaux publics, architecture.
      • Construction navale, maintenance, matériaux.
      • Énergies renouvelables.
      • Audiovisuel, informatique, télécommunications, numérique.
      • Recherche et développement.

      Poursuites d'études

      Les principales voies d'études après un bac STI2D sont:

      • BTS (Brevet de Technicien Supérieur) : formations de 2 ans, spécialisées et professionnalisantes. Plusieurs BTS sont proposés au lycée Louis-Vincent, comme CPI (Conception des Processus de Réalisation de Produits), CRSA (Conception et Réalisation de Systèmes Automatiques), CIRA (Contrôle Industriel et Régulation Automatique), électrotechnique (lycée Cormontaigne), FED fluides énergies domotique (Talange) et environnement nucléaire (Thionville).
      • BUT (Bachelor Universitaire de Technologie) : formations de 3 ans, plus généralistes que les BTS, avec une insertion professionnelle ou une poursuite d'études possibles.
      • Classes préparatoires aux grandes écoles (CPGE) :
        • TSI (Technologie et Sciences Industrielles) : prépare aux écoles d'ingénieurs. Cette formation est notamment proposée au lycée Louis-Vincent.
        • ATS (Adaptation Technicien Supérieur) : Prépare les étudiants de BTS à intégrer une école d'ingénieur.
      • Écoles d'ingénieurs : accessibles après une prépa ou directement après le bac pour certaines écoles.
      • Licences universitaires : dans les domaines des sciences, de l'informatique, des mathématiques, etc., avec possibilité de poursuivre en master.

      Spécialités en terminale

      En classe de terminale, les élèves de STI2D doivent choisir une spécialité parmi les quatre suivantes:

      • Architecture et Construction (AC) : Étude de l'architecture et de la construction.
      • Énergie et Environnement (EE) : Étude des systèmes énergétiques et des enjeux environnementaux.
      • Systèmes d'Information et Numérique (SIN) : Étude des systèmes informatiques et numériques.
      • Innovation Technologique et Éco-conception (ITEC) : Étude de l'innovation et de la conception de produits respectueux de l'environnement.

      Enseignement et projets

      • L'enseignement en STI2D combine des cours théoriques, des travaux dirigés (TD) et des travaux pratiques (TP).
      • Les élèves réalisent des projets concrets, souvent en groupe, qui leur permettent de mettre en application leurs connaissances et de développer leurs compétences. Par exemple, ils peuvent concevoir un robot suiveur de ligne, un système de gestion d'énergie, ou un générateur d'électricité pour ferme-portes.

      Atelier de Fabrication (FabLab)

      Les lycées proposant la filière STI2D disposent souvent d'un FabLab, un espace équipé de machines de fabrication numérique (découpe laser, imprimantes 3D, etc.). Les élèves peuvent y prototyper et réaliser leurs projets.

      Témoignages

      • Des élèves apprécient la filière STI2D pour la possibilité de mettre en pratique les connaissances acquises en cours.
      • Les projets réalisés en STI2D permettent de développer l'esprit d'équipe, la créativité et l'autonomie.

      Ce document de synthèse donne un aperçu global de la filière STI2D. Pour plus d'informations, il est conseillé de consulter les ressources mentionnées dans la vidéo, telles que le site de l'Onisep et le diaporama interactif de la région Grand Est. Il est également recommandé de contacter les professeurs principaux et les psychologues de l'éducation nationale.

    1. “Muchas personas consideran que afirmar que el pensamiento tiene una estructura lógica normativa es falso, pues piensan que el pensamiento es algo propio de cada persona.”(Pag 92) No entiendo a que se refiere exactamente.

      Interesante. El pensamiento no es solo algo individual, el persamiento como lo vimos tiene que ver con lo subjetivo, intersubjetivo y objetivo. Hay que diferenciar individualidad, identidad, subjetivididad, sujeto, etc.

    1. Reviewer #1 (Public review):

      Summary:

      In this manuscript submitted by Qi et al., the authors study the RNA methylation mechanism by the METTL3-METTL14 complex. This complex catalyzes the major epitranscriptome methylation mark of nuclear RNA, including mRNA and lncRNAs. They catalyze the transfer of methyl group from SAM to convert the N6 of adenosine in RNA to m6A. Mutations in this complex have been associated with several diseases, such as type 2 diabetes and several types of cancer. The primary focus of this study was to understand the post-catalytic state of the METTL3-14 bound to a structural mimic of a reaction product known as N6-methyladenosine monophosphate (m6A) using X-ray crystallography. The authors show that the m6A occupies a novel pocket at the interface of the METTL3-14 complex and identified that residues interacting with m6A are mutated in several cancers. Furthermore, the authors demonstrate that the mutations lead to a significant loss in catalytic activity, alter RNA binding, and hinder the proper positioning of the substrate adenine in the active site. Lastly, the authors perform supervised molecular dynamics simulations to understand the effect of the mutations on the interaction network with m6A. The evidence for this study is good, with the combination of X-ray, functional assays, and molecular dynamics justifying their overall conclusions. This structure is significant as it provides new insights into the structural determinants of known cancer-associated mutations of this important class of enzymes. However, some issues need to be addressed.

      Strengths:

      (1) The X-ray structure is well determined, and the density map has the quality to observe all the interactions of the METTL3-14 complex with m6A.

      (2) The structure reveals a novel 'cryptic pocket' in the complex that is 16 Å away from the SAM binding site. It is a functional m6A-sensor, illustrating a mechanism where the complex switches its functionality from an m6A writer to a reader.

      (3) The structure illustrates that the residues forming cryptic pockets are found in multiple Cancer-associated mutations and are well conserved across several organisms.

      (4) The functional assays (methyl transferase, RNA binding, kinetic, and SPR assays) provide a complete picture of the effect of the mutations on the activity of the METTL3-14 complex.

      (5) Molecular dynamics simulations were done to understand the impact of the mutations on the pocket structure and its dynamics and support the X-ray structure findings.

      Weaknesses:

      (1) Although the X-ray structure is well determined, the statistics are a bit troubling, particularly the Ramachandran, Sidechain and RSRZ outliers. It is well above the average for structures at that resolution. Maybe the use of alternative software such as ISOLDE may be adequate to improve those parameters.

      (2) The authors should expand their discussion as to why the affinity for the product is higher than the substrate and the implications on the mechanism.

      (3) The SPR profiles of the association kinetics look to have several minor association-dissociation events occurring. Multiple binding sites? Authors should provide an explanation for such behavior. Also, what is the structural explanation of the difference in binding modes between the wt vs. mutant (one vs. two-state binding modes)?

      (4) In materials and methods, it shows the data in Figure 2a was fitted to a Michaelis-Menten equation, however, the Y axis shows Normalized methylation and not initial rates. The authors should elaborate on their approach. In addition, more than three initial velocity rate points per protein are needed to fit a Michaelis-Menten curve confidently. Additionally, where can the Michaelis-Menten parameters be found?

    1. how observations help improve model/understanding Observations of these carbon-water couplings have been made from the leaf scale to the ecosystem and global scales. Depending on the scale, research has focused on different aspects, such as the coupling between photosynthesis and transpiration, ecosystem water use efficiency’s control over changes in gross primary productivity, and the links between atmospheric CO₂ growth rates and terrestrial water storage. Despite these efforts, there remain clear discrepancies in modeling the processes linking the water and carbon cycles, resulting in mismatches with observations and uncertainties in past and future simulations. model data integration and their challenges The models representing vegetation-water-carbon interactions vary significantly in process formulation, complexity, and parameterization, often dictated by the spatial scale of model development and application. As the spatial scale increases, model complexity typically decreases, requiring simplification that introduces empirical model parameters, often under-constrained. The rapid growth of satellite Earth observations, observational networks, and observation-based data presents unprecedented opportunities to enhance the representation and understanding of key carbon-water cycle processes in models. However, many terrestrial biogeochemical models remain too rigid for flexible model structures and too demanding for model-data-integration experiments that leverage observational data constraints.

      References: Reichstein, M., Camps-Valls, G., Stevens, B., Jung, M., Denzler, J., Carvalhais, N., and Prabhat: Deep learning and process understanding for data-driven Earth system science, Nature, 566, 195-204, 10.1038/s41586-019-0912-1, 2019. Reichstein, M., Mahecha, M. D., Ciais, P., Seneviratne, S. I., Blyth, E. M., Carvalhais, N., and Luo, Y.: Elk–testing climate–carbon cycle models: a case for pattern–oriented system analysis, iLEAPS Newsletter, 11, 14-21, 2011. Williams, M., Richardson, A. D., Reichstein, M., Stoy, P. C., Peylin, P., Verbeeck, H., Carvalhais, N., Jung, M., Hollinger, D. Y., Kattge, J., Leuning, R., Luo, Y., Tomelleri, E., Trudinger, C. M., and Wang, Y. P.: Improving land surface models with FLUXNET data, Biogeosciences, 6, 1341-1359, 10.5194/bg-6-1341-2009, 2009. Reichstein, M. and Beer, C.: Soil respiration across scales: The importance of a model-data integration framework for data interpretation, Journal of Plant Nutrition and Soil Science, 171, 344-354, 10.1002/jpln.200700075, 2008.

    1. why it is false.

      (a) False → Removing a collinear predictor does influence other coefficients. (b) True → A 1-unit increase in 𝑥 1 x 1 ​ increases 𝑦 y by 2.5. (c) True → A 1-unit increase in 𝑥 1 x 1 ​ increases 𝑦 y by 5.7.

    1. 与YUV关系

      是的,你的理解是准确的!YUV本质上与CIELAB有相似之处,它们都基于人眼对亮度的高度敏感性,并且都将亮度和色度信息分开处理,旨在提高效率并适应人眼的感知方式。

      1. YUV与CIELAB的相似性

      在YUV格式中,Y分量代表亮度(即图像的明暗程度),而UV分量表示色度(即颜色的色相和饱和度)。这与CIELAB的设计非常相似,后者通过L*表示亮度,a*b*表示色度(红绿和蓝黄轴上的色差)。

      为什么YUV与CIELAB类似?

      • 人眼对亮度更敏感:人眼对亮度(或明暗)变化的敏感度要远高于对色度(色相和饱和度)的敏感度。这是因为我们对图像的细节和对比度的识别依赖于亮度信息,而对颜色的变化则不那么敏感。这一特性使得在视频和图像处理中,分离亮度和色度可以有效减少信息冗余,提高数据压缩的效率。

      在YUV中,Y分量表示亮度,这个分量在人眼视觉感知中占据主导地位;UV分量则表示色度,它们的变化对视觉感知的影响较小。因此,在视频压缩和传输中,UV分量通常可以进行下采样(比如4:2:0采样),从而减少数据量,而不显著影响图像的视觉质量。

      • 色度分量压缩:由于人眼对色度(如红绿、蓝黄的变化)不如对亮度敏感,YUV格式将色度分量压缩,并且在许多视频编码格式中,色度分量的分辨率通常低于亮度分量。这类似于CIELAB中,色度部分(a*和b*)与亮度(L*)是分开的,并且色度部分的变化对视觉的影响相对较小。

      2. YUV与CIELAB的区别

      尽管YUV和CIELAB有相似的结构,它们的主要区别在于它们的设计目标和应用领域:

      • CIELAB是一个设备无关的色彩空间,旨在提供一种统一的标准来描述人眼对颜色的感知,并且可以用于不同设备之间的颜色转换。它并不依赖于任何特定的设备或显示技术。

      • YUV是为视频编码和传输而设计的,它特别考虑了数据压缩的需求,并且通过色度的下采样来减少冗余信息。它主要用于视频信号的传输和存储,尤其是高效压缩(例如,H.264、MPEG标准)中的应用。

      3. 总结

      • YUVCIELAB都基于人眼对亮度的敏感性,因此它们都通过分离亮度和色度信息来提高效率。YUV通过将Y作为亮度分量,UV作为色度分量,实现了色度的压缩,而CIELAB通过L*表示亮度,a*b*表示色度,确保颜色的感知与人眼一致。

      • YUV的目标高效的视频压缩和传输,而CIELAB的目标是确保设备无关的颜色表示跨设备色彩管理,以确保不同设备(如显示器、打印机)之间的颜色一致性。

      因此,YUV和CIELAB的设计理念相似,但它们各自的应用场景和功能有所不同,YUV更侧重于视频数据的传输效率,而CIELAB侧重于提供一个统一的色彩标准,用于色彩管理和设备之间的颜色转换。

    1. Esto significa que cuando nosencontramos con un período largo, con incisos también extensos, nuestra memoria sesobrecarga, no puede retener todas las palabras y perdemos el hilo de la prosa

      Concuerdo totalmente, ya que me pasa que cuando hay muchas palabras complicadas o parráfos extensos pierdo el hilo de la lectura o de la idea principal del párrafo, por ello concuerdo que en la escritura a veces lo simple y claro es lo mejor.

    2. ¡Vaya tostones!

      El autor intenta una "conversación" con el lector y empatizar con el para que asimile mejor la información que se le esta dando. Utiliza para ello un lenguaje coloquial y cómico.

    3. —¡y el tema y el tono del texto no tienendesperdicio!—

      Este tipo de exclamativas me parecen útiles para motivar al lector a perstarle atención a los detalles que menciona el autor.

    Annotators

    1. Author response:

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

      Recommendations for the authors:

      Reviewer #1 (Recommendations For The Authors):

      Abstract

      I don't think you need the first two sentences of the abstract. This is not a grant and your results are exciting enough to justify a full basic science-based approach.

      We fully understand this perspective.  However, we prefer to introduce the work in the broader context of sleep medicine.  This manuscript is part of our long-standing efforts to develop cavefish as a model for sleep disorders and we believe this provides important context.

      Last sentence of the abstract: the subject is missing. "That have developed..." who has developed?

      Thank you. We have corrected this error, the sentence now reads “...these findings suggest that cavefish have developed resilience to sleep loss...”

      Introduction

      First paragraph. Worth explaining in a sentence what is the link between DNA damage and ROS.

      We now state ‘Further, chronic sleep loss results in elevated reactive oxygen species (ROS), a known mediatior of DNA damage, in the gut and/or brain that contribute to mortality in Drosophila and mice [11,16].’

      "A. mexicanus exists as blind cave populations and an extant surface population that are interfertile". This needs rephrasing. As it is, it sounds like the surface population is infertile.

      We have rephrased for clarity; the line now reads: “while the surface and cave populations are geographically isolated, they remain interfertile and capable of hybridization in nature as well as laboratory settings”.

      "Further, the evolved differences in DNA repair genes, including links between mechanisms regulating sleep, light responsiveness, and DNA repair across all three cave populations studied to date [27,29]" This sentence is incomplete.

      We have corrected the phrasing, which now reads “...evolved differences in DNA repair genes have been identified across all three cave populations studied to date, including links between mechanisms regulating sleep, light responsiveness, and DNA repair”:

      Figure 1

      I recommend improving the legibility of the figure copying some of the information provided in the legend directly within the figure itself.

      A, B: label in the panel itself what is blue and what is green.

      Thank you, we have made this change.

      C: Make it clear in the figure itself that you are measuring yH2AX. Also, probably you have enough room in the figure to avoid abbreviations for Rhomb, mes, and tele. It may also help if you could add a little cartoon that explains what those three brain regions are.

      We have added text to the y axis indicating that yH2AX fluorescence is being measured, and replaced the abbreviations with eh full names of the regions.

      G: again, explain that DHE is being measured here. And perhaps pick a different colour choice to highlight the difference from C?

      We have added clarifiaction to the y-axis of the figure, but have retained the color scheme for consistency; in all surface-cave comparisons in the manuscript, gray is used for surface fish and red for cavefish.

      In the text: I would recommend adding some quantitative reminder of what is the difference in sleep amount between the two species (cave vs surface).

      We have added the following to highlight the magnitude of the difference in sleep: “Strikingly, cavefish sleep as little as 1-2 hours per day, in contrast to their surface counterparts, which sleep as much as 6-10 hours a day”

      "Together, these findings fortify the notion that cellular stress is elevated in the gut of cavefish relative to surface fish." Were the two populations fed the same diet and raised in the same lab conditions? If this is pinpointed to sleep amount, it's worth ruling out possible confounding factors.

      We have added a sentence to the results underlining this point: “Prior to imaging, both surface and cavefish had been reared in a temperature-controlled incubator, and relied solely on their yolk sac for nutrients; so, differences in gut ROS cannot be attributed to differences in rearing or feeding conditions.”

      Figure 2

      Spell out, somewhere in the figure itself, that the 30s and 60s refer to UV treatment protocols.

      We have added X-axis titles to clarify this in Fig 2 and supp. Fig 1.

      It would be worth providing a cartoon of the experimental setup that shows for instance what time of the day UV was given (it's only specified in the text) and which subsequent sleep period was selected for comparisons.

      We have added arrows to all sleep plots indicating the time of UV treatment, and brackets indicating the time period used for statistical comparisons, as well as text in the figure legends indicating this.

      Figure 3

      A. I don't think this is needed, to be honest, and if you want to keep it, it needs a better legend.

      We have edited the figure legend to increase clarity.

      B. I would make it clear in the figure that this refers to transcriptomics analysis. Perhaps you could change the order and show C, D, and then B.

      We have added text to the figure legend and the results text to more explicitly state that the PCA plot is of transcriptional response. We have however retained the original figure order, as well feel this figure is important to establish that both populations have strong, but distinct responses to the UV treatment.

      Figure 4

      A. Spell it out in the figure itself that you're staining for CPD.

      Thank you, we have made this change.

      B. You are using the same colour combination you had in Figure 1 but for yet another pairing. This is a bit confusing.

      Thank you for bringing this to our attention.  We have added descriptions of the colors in the figure legend.

      Discussion

      "Beyond the Pachón cavefish population, all three other cavefish populations have been found to have reduced sleep (Cite)." Citation missing here.

      Thank you.  We have now clarified this sentence and included a citation.

      Reviewer #2 (Recommendations For The Authors):

      Consideration of Environmental Conditions:

      Evaluate whether the lab conditions, which may more closely resemble surface environments, could influence the observed increase in neuronal DNA damage and gut ROS levels in cavefish. Adjusting these conditions or discussing their potential impact in the manuscript would strengthen the findings.

      We are very excited about these experiments.  We have a paper that will be submitted to BioRxiv this week where we record wild-caught fish, as well as fish in caves.  The conclusion is that sleep loss is present in both populations.  This field work took over 10 years to come together and still lacks the power of the lab based assays.  Nevertheless, we can conclusively say that the phenotypes we have observed for the last ~15 years in the lab are present in a natural setting.  We have included a statement about the need for future work to test these findings in a natural setting.

      Alternative Stressors:

      Given that cavefish are albino and blind (to my knowledge), consider using alternative sources of genotoxic stress beyond UV-induced damage. This could include chemical agents or other forms of environmental stress to provide a more comprehensive assessment of DDR.

      We agree and are enthusiastic about looking more generally at stress.  We note that we have previously found that cavefish rebound following sleep deprivation (McGaugh et al, 2020) suggesting that they are responsive to sleep disruption.  This will be a major research focus area moving forward.

      Broader Stress Responses:

      Investigate whether other forms of stress, such as dietary changes or temperature fluctuations, elicit similar differences in sleep patterns and DDR responses. This could provide additional insights into the robustness of the observed phenomena.

      We fully agree.  This will be the primary focus of this research area moving forward. We hypothesize that cavefish are generally less responsive to their environment.  Unpublished data reveals that temperature stress, circadian changes, and aging (presented here) to little to impact gene expression in surface fish.  We would like to test the hypothesis that transcriptional stability of cavefish contributes to their longevity.

      Potential Protective Mechanisms:

      Discuss the possibility that lower levels of gamma-H2AX in cavefish might be protective, as DDR can lead to cellular senescence or cancer. This perspective could add depth to the interpretation of the results.

      This was the hypothesis underlying this manuscript.  However, we found elevated levels of gamma-H2AX.  We believe there may be additional protective mechanisms that have evolved in cavefish, but cannot identify them to date.  Our hope is future functional studies by our group, as well as other groups’ access to this published work, may help address these questions.

      Strengthening the Sleep-DNA Damage Link:

      Further experiments are needed to directly link sleep differences to the observed variations in DNA damage and DDR. This could involve manipulating sleep patterns in surface fish and cavefish to observe corresponding changes in DNA repair mechanisms.

      We agree.  We have referenced work that conclusively showed this relationship in zebrafish. Our current methods for limiting sleep involves shaking, and this has too many confounds.  We are working on developing genetic tools, and applying the gentle rocking methods used previously in zebrafish to address these questions.

      Clarification of Causal Directionality:

      Address the potential that sleep patterns and DDR responses may both be downstream effects of a common cause or independent adaptations to the cave environment. Clarifying this in the manuscript would provide a more nuanced understanding of the evolutionary adaptations.

      Thank you for this suggestion.  We have now added a paragraph describing how these experiments (and the ones described above) are necessary for understanding the relationship between sleep and DDR.

      Clarification and Presentation:

      Fix the many typos, and improve the clarity of the figures and their legends to ensure they are easily interpretable. Additional context in the discussion section would help readers understand the significance and potential implications of the findings.

      Thank you, we have now included this.

      Reviewer #3 (Recommendations For The Authors):

      There are a number of suggestions that I have made in the public review, but there are a few things that I would like to add here.

      The methods section is missing many important details, for instance, the intensity of the illumination used in the UV exposure in larvae is not reported but is vital for the interpretation/replication of these experiments. In general, this section should be redone with a greater effort to include all important information. Similarly, the figure legends could be greatly improved, with important details like n-number and definition of significance thresholds defined (e.g. see Figures 1, C, and G.)

      We have added greater detail to the methods section to specify the spectral peak and power output of the bulbs used.

      There are a number of passages in the manuscript that do not make sense, which suggests that a future version of record should be carefully proofread. I know that this can be a case of reading multiple versions of a manuscript so many times that one doesn't really see it anymore, but, for example, phrases like "To differentiate between these two possibilities" are confusing to the reader when there has been no introduction of alternate possibilities.

      Thank you for this comment.  We have fixed this mistake and proofread the manuscript.

      Additionally, there are multiple examples of errors in citations/references. A few examples are below:

      "Further, chronic sleep loss results in elevated reactive oxygen species (ROS) in the gut and/or brain that contribute to mortality in Drosophila and mice [11, 16]". Reference 16 does not include mice at all, and reference 11 is Vaccaro et al. 2020, where Drosophila mortality is assessed, but mouse mortality is not.

      We have added the appropriate citations and revised this sentence.

      References 13 and 15 are the same.

      Thank you, we have fixed.

      References 24 and 26 are the same.

      Thank you, we have fixed.

      Public Reviews:

      Reviewer #1 (Publc Review):

      Summary:

      Lloyd et al employ an evolutionary comparative approach to study how sleep deprivation affects DNA damage repair in Astyanax mexicanus, using the cave vs surface species evolution as a playground. The work shows, convincingly, that the cavefish population has evolved an impaired DNA damage response both following sleep deprivation or a classical paradigm of DNA damage (UV).

      Strengths:

      The study employs a thorough multidisciplinary approach. The experiments are well conducted and generally well presented.

      Weaknesses:

      Having a second experimental mean to induce DNA damage would strengthen and generalise the findings.

      Overall, the study represents a very important addition to the field. The model employed underlines once more the importance of using an evolutionary approach to study sleep and provides context and caveats to statements that perhaps were taken a bit too much for granted before. At the same time, the paper manages to have an extremely constructive approach, presenting the platform as a clear useful tool to explore the molecular aspects behind sleep and cellular damage in general. The discussion is fair, highlighting the strengths and weaknesses of the work and its implications.

      We fully agree with this assessment.  We are currently performing experiments to test the effects of additional DNA damaging agents.  We hope to extend these studies beyond DNA-damage agents to look more generally at how animals respond to stress including ROS, sleep deprivation, and high temperature.  This will be a major direction of the laboratory moving forward.

      The manuscript investigates the relationship between sleep, DNA damage, and aging in the Mexican cavefish (Astyanax mexicanus), a species that exhibits significant differences in sleep patterns between surface-dwelling and cave-dwelling populations. The authors aim to understand whether these evolved sleep differences influence the DNA damage response (DDR) and oxidative stress levels in the brain and gut of the fish.

      Summary of the Study:

      The primary objective of the study is to determine if the reduced sleep observed in cave-dwelling populations is associated with increased DNA damage and altered DDR. The authors compared levels of DNA damage markers and oxidative stress in the brains and guts of surface and cavefish. They also analyzed the transcriptional response to UV-induced DNA damage and evaluated the DDR in embryonic fibroblast cell lines derived from both populations.

      Strengths of the Study:

      Comparative Approach:

      The study leverages the unique evolutionary divergence between surface and cave populations of A. mexicanus to explore fundamental biological questions about sleep and DNA repair.

      Multifaceted Methodology:

      The authors employ a variety of methods, including immunohistochemistry, RNA sequencing, and in vitro cell line experiments, providing a comprehensive examination of DDR and oxidative stress.

      Interesting Findings:

      The study presents intriguing results showing elevated DNA damage markers in cavefish brains and increased oxidative stress in cavefish guts, alongside a reduced transcriptional response to UV-induced DNA damage.

      Weaknesses of the Study:

      Link to Sleep Physiology:

      The evidence connecting the observed differences in DNA damage and DDR directly to sleep physiology is not convincingly established. While the study shows distinct DDR patterns, it does not robustly demonstrate that these are a direct result of sleep differences.

      We agree with this assessment.  We are currently working to apply tools developed in zebrafish to examine the physiology of sleep.  While this is important, and our results our promising, we will note that functional analysis of sleep physiology in fish has been limited to zebrafish.  We hope future studies will allow us to integrate approaches that examine the physiology of sleep.

      Causal Directionality:

      The study fails to establish a clear causal relationship between sleep and DNA damage. It is possible that both sleep patterns and DDR responses are downstream effects of a common cause or independent adaptations to the cave environment.

      We agree, however, we note that this could be the case for all animals in which sleep has been linked to DNA damage.  We believe the most likely explanation for Astyanax and other animals studied, is that sleep is that sleep and DDR are downstream/interface with the sleep homeostat.

      Environmental Considerations:

      The lab conditions may not fully replicate the natural environments of the cavefish, potentially influencing the results. The impact of these conditions on the study's findings needs further consideration.

      This is correct. We have considered this carefully.  After nearly a decade of effort,  we have completed analysis of sleep in the wild.  These will be uploaded to BioRxiv within the next week.

      Photoreactivity in Albino Fish:

      The use of UV-induced DNA damage as a primary stressor may not be entirely appropriate for albino, blind cavefish. Alternative sources of genotoxic stress should be explored to validate the findings.

      We have addressed this above.  Future work will examine additional stressors. Both fish are transparent at 6dpf and so it is unlikely that albinism impacts the amount of UV that reaches the brain.

      Assessment of the Study's Achievements:

      The authors partially achieve their aims by demonstrating differences in DNA damage and DDR between surface and cavefish. However, the results do not conclusively support the claim that these differences are driven by or directly related to the evolved sleep patterns in cavefish. The study's primary claims are only partially supported by the data.

      Impact and Utility:

      The findings contribute valuable insights into the relationship between sleep and DNA repair mechanisms, highlighting potential areas of resilience to DNA damage in cavefish. While the direct link to sleep physiology remains unsubstantiated, the study's data and methods will be useful to researchers investigating evolutionary biology, stress resilience, and the molecular basis of sleep.

      Reviewer #3 (Public Review):

      Lloyd, Xia, et al. utilised the existence of surface-dwelling and cave-dwelling morphs of Astyanax mexicanus to explore a proposed link between DNA damage, aging, and the evolution of sleep. Key to this exploration is the behavioural and physiological differences between cavefish and surface fish, with cavefish having been previously shown to have low levels of sleep behaviour, along with metabolic alterations (for example chronically elevated blood glucose levels) in comparison to fish from surface populations. Sleep deprivation, metabolic dysfunction, and DNA damage are thought to be linked and to contribute to aging processes. Given that cavefish seem to show no apparent health consequences of low sleep levels, the authors suggest that they have evolved resilience to sleep loss. Furthermore, as extended wake and loss of sleep are associated with increased rates of damage to DNA (mainly double-strand breaks) and sleep is linked to repair of damaged DNA, the authors propose that changes in DNA damage and repair might underlie the reduced need for sleep in the cavefish morphs relative to their surface-dwelling conspecifics.

      To fulfill their aim of exploring links between DNA damage, aging, and the evolution of sleep, the authors employ methods that are largely appropriate, and comparison of cavefish and surface fish morphs from the same species certainly provides a lens by which cellular, physiological and behavioural adaptations can be interrogated. Fluorescence and immunofluorescence are used to measure gut reactive oxygen species and markers of DNA damage and repair processes in the different fish morphs, and measurements of gene expression and protein levels are appropriately used. However, although the sleep tracking and quantification employed are quite well established, issues with the experimental design relate to attempts to link induced DNA damage to sleep regulation (outlined below). Moreover, although the methods used are appropriate for the study of the questions at hand, there are issues with the interpretation of the data and with these results being over-interpreted as evidence to support the paper's conclusions.

      This study shows that a marker of DNA repair molecular machinery that is recruited to DNA double-strand breaks (γH2AX) is elevated in brain cells of the cavefish relative to the surface fish and that reactive oxygen species are higher in most areas of the digestive tract of the cavefish than in that of the surface fish. As sleep deprivation has been previously linked to increases in both these parameters in other organisms (both vertebrates and invertebrates), their elevation in the cavefish morph is taken to indicate that the cavefish show signs of the physiological effects of chronic sleep deprivation.

      It has been suggested that induction of DNA damage can directly drive sleep behaviour, with a notable study describing both the induction of DNA damage and an increase in sleep/immobility in zebrafish (Danio rerio) larvae by exposure to UV radiation (Zada et al. 2021 doi:10.1016/j.molcel.2021.10.026). In the present study, an increase in sleep/immobility is induced in surface fish larvae by exposure to UV light, but there is no effect on behaviour in cavefish larvae. This finding is interpreted as representing a loss of a sleep-promoting response to DNA damage in the cavefish morph. However, induction of DNA damage is not measured in this experiment, so it is not certain if similar levels of DNA damage are induced in each group of intact larvae, nor how the amount of damage induced compares to the pre-existing levels of DNA damage in the cavefish versus the surface fish larvae. In both this study with A. mexicanus surface morphs and the previous experiments from Zada et al. in zebrafish, observed increases in immobility following UV radiation exposure are interpreted as following from UV-induced DNA damage. However, in interpreting these experiments it is important to note that the cavefish morphs are eyeless and blind. Intense UV radiation is aversive to fish, and it has previously been shown in zebrafish larvae that (at least some) behavioural responses to UV exposure depend on the presence of an intact retina and UV-sensitive cone photoreceptors (Guggiana-Nilo and Engert, 2016, doi:10.3389/fnbeh.2016.00160). It is premature to conclude that the lack of behavioural response to UV exposure in the cavefish is due to a different response to DNA damage, as their lack of eyes will likely inhibit a response to the UV stimulus.

      We believe that in A. mexicanus, like in zebrafish, it is highly unlikely that the effects of UV are mediated through visual processing. Even if this were the case, the timeframe of UV activation is very short compared to the time-scale of sleep measurements so this is unlikely to be a confound.

      Indeed, were the equivalent zebrafish experiment from Zada et al. to be repeated with mutant larvae fish lacking the retinal basis for UV detection it might be found that in this case too, the effects of UV on behaviour are dependent on visual function. Such a finding should prompt a reappraisal of the interpretation that UV exposure's effects on fish sleep/locomotor behaviour are mediated by DNA damage.

      We prefer not to comment on Zada et al, as that is a separate manuscript.

      An additional note, relating to both Lloyd, Xia, et al., and Zada et al., is that though increases in immobility are induced following UV exposure, in neither study have assays of sensory responsiveness been performed during this period. As a decrease in sensory responsiveness is a key behavioural criterion for defining sleep, it is, therefore, unclear that this post-UV behaviour is genuinely increased sleep as opposed to a stress-linked suppression of locomotion due to the intensely aversive UV stimulus.

      We understand this concern and are working on improved methodology for measuring sleep.  However, behavioral measurements are the standard for almost every manuscript that has studied sleep in zebrafish, flies, and worms to date. 

      The effects of UV exposure, in terms of causing damage to DNA, inducing DNA damage response and repair mechanisms, and in causing broader changes in gene expression are assessed in both surface and cavefish larvae, as well as in cell lines derived from these different morphs. Differences in the suite of DNA damage response mechanisms that are upregulated are shown to exist between surface fish and cavefish larvae, though at least some of this difference is likely to be due to differences in gene expression that may exist even without UV exposure (this is discussed further below).

      UV exposure induced DNA damage (as measured by levels of cyclobutene pyrimidine dimers) to a similar degree in cell lines derived from both surface fish and cave fish. However, γH2AX shows increased expression only in cells from the surface fish, suggesting induction of an increased DNA repair response in these surface morphs, corroborated by their cells' increased ability to repair damaged DNA constructs experimentally introduced to the cells in a subsequent experiment. This "host cell reactivation assay" is a very interesting assay for measuring DNA repair in cell lines, but the power of this approach might be enhanced by introducing these DNA constructs into larval neurons in vivo (perhaps by electroporation) and by tracking DNA repair in living animals. Indeed, in such a preparation, the relationship between DNA repair and sleep/wake state could be assayed.

      Comparing gene expression in tissues from young (here 1 year) and older (here 7-8 years) fish from both cavefish and surface fish morphs, the authors found that there are significant differences in the transcriptional profiles in brain and gut between young and old surface fish, but that for cavefish being 1 year old versus being 7-8 years old did not have a major effect on transcriptional profile. The authors take this as suggesting that there is a reduced transcriptional change occurring during aging and that the transcriptome of the cavefish is resistant to age-linked changes. This seems to be only one of the equally plausible interpretations of the results; it could also be the case that alterations in metabolic cellular and molecular mechanisms, and particularly in responses to DNA damage, in the cavefish mean that these fish adopt their "aged" transcriptome within the first year of life.

      This is indeed true.  However, one could also interpret this as a lack of aging.  If the profile does not change over time, the difference seems largely semantic.

      A major weakness of the study in its current form is the absence of sleep deprivation experiments to assay the effects of sleep loss on the cellular and molecular parameters in question. Without such experiments, the supposed link of sleep to the molecular, cellular, and "aging" phenotypes remains tenuous. Although the argument might be made that the cavefish represent a naturally "sleep-deprived" population, the cavefish in this study are not sleep-deprived, rather they are adapted to a condition of reduced sleep relative to fish from surface populations. Comparing the effects of depriving fish from each morph on markers of DNA damage and repair, gut reactive oxygen species, and gene expression will be necessary to solidify any proposed link of these phenotypes to sleep.

      We agree this would be beneficial.  We note that relatively few papers have sleep deprived fish.  While we done have this before in A. mexicanus the assay is less than ideal and likely induces generalizable stress.  We are working on adapting more recently developed methods in zebrafish.

      A second important aspect that limits the interpretability and impact of this study is the absence of information about circadian variations in the parameters measured. A relationship between circadian phase, light exposure, and DNA damage/repair mechanisms is known to exist in A. mexicanus and other teleosts, and differences exist between the cave and surface morphs in their phenomena (Beale et al. 2013, doi: 10.1038/ncomms3769). Although the present study mentions that their experiments do not align with these previous findings, they do not perform the appropriate experiments to determine if such a misalignment is genuine. Specifically, Beale et al. 2013 showed that white light exposure drove enhanced expression of DNA repair genes (including cpdp which is prominent in the current study) in both surface fish and cavefish morphs, but that the magnitude of this change was less in the cave fish because they maintained an elevated expression of these genes in the dark, whereas the darkness suppressed the expression of these genes in the surface fish. If such a phenomenon is present in the setting of the current study, this would likely be a significant confound for the UV-induced gene expression experiments in intact larvae, and undermine the interpretation of the results derived from these experiments: as samples are collected 90 minutes after the dark-light transition (ZT 1.5) it would be expected that both cavefish and surface fish larvae should have a clear induction of DNA repair genes (including cpdp) regardless of 90s of UV exposure. The data in Supplementary Figure 3 is not sufficient to discount this potentially serious confound, as for larvae there is only gene expression data for time points from ZT2 to ZT 14, with all of these time points being in the light phase and not capturing any dynamics that would occur at the most important timepoints from ZT0-ZT1.5, in the relevant period after dark-light transition. Indeed, an appropriate control for this experiment would involve frequent sampling at least across 48 hours to assess light-linked and developmentally-related changes in gene expression that would occur in 5-6dpf larvae of each morph independently of the exposure to UV.

      We agree that this would be useful, however, frequent sampling is not feasible given the experiments presented here and the challenges of working with an emerging model.

      On a broader point, given the effects of both circadian rhythm and lighting conditions that are thought to exist in A. mexicanus (e.g. Beale et al. 2013) experiments involving measurements of DNA damage and repair, gene expression, and reactive oxygen species, etc. at multiple times across >1 24 hour cycle, in both light-dark and constant illumination conditions (e.g. constant dark) would be needed to substantiate the authors' interpretation that their findings indicate consistently altered levels of these parameters in the cavefish relative to the surface fish. Most of the data in this study is taken at only single time points.

      Again, see comment above.  The goal was to identify whether there are differences in DNA Damage response between A. mexcicanus. Extending on this to examine interactions with the circadian system could be a useful path to pursue in the future.

      On a broader point, given the effects of both circadian rhythm and lighting conditions that are thought to exist in A. mexicanus (e.g. Beale et al. 2013) experiments involving measurements of DNA damage and repair, gene expression, and reactive oxygen species, etc. at multiple times across >1 24 hour cycle, in both light-dark and constant illumination conditions (e.g. constant dark) would be needed to substantiate the authors' interpretation that their findings indicate consistently altered levels of these parameters in the cavefish relative to the surface fish. Most of the data in this study is taken at only single time points.

      In summary, the authors show that there are differences in gene expression, activity of DNA damage response and repair pathways, response to UV radiation, and gut reactive oxygen species between the Pachón cavefish morph and the surface morph of Astyanax mexicanus. However, the data presented does not make the precise nature of these differences very clear, and the interpretation of the results appears to be overly strong. Furthermore, the evidence of a link between these morph-specific differences and sleep is unconvincing.

      In summary, the authors show that there are differences in gene expression, activity of DNA damage response and repair pathways, response to UV radiation, and gut reactive oxygen species between the Pachón cavefish morph and the surface morph of Astyanax mexicanus. However, the data presented does not make the precise nature of these differences very clear, and the interpretation of the results appears to be overly strong. Furthermore, the evidence of a link between these morph-specific differences and sleep is unconvincing.

    1. Voici une checklist pour vérifier la bonne pratique de l'utilisation des sanctions comme outil éducatif, en tenant compte des informations fournies par la source:

      • Avant la sanction :

        • Vérifier si le comportement est lié à un trouble spécifique de l'élève. Il est essentiel de prendre en compte les difficultés propres à chaque élève afin de ne pas sanctionner un comportement qui est directement relié à un trouble.
        • S'assurer que les règles sont claires et connues de tous. Les règles doivent être déterminées au préalable et expliquées au groupe.
        • Privilégier la sanction à la punition. La sanction est une réponse à une transgression basée sur des règles connues, tandis que la punition est une réaction émotionnelle et subjective.
        • Avoir un entretien avec l'élève. Un entretien avec l’élève est un préalable indispensable afin que celui-ci comprenne les objectifs, ce que l’on attend de lui pour la suite.
      • Pendant l'annonce de la sanction :

        • Annoncer la sanction de manière neutre. L’annonce doit être faite de façon neutre pour tous les élèves, sans tonalité affective ni émotionnelle.
        • Différer l'annonce si nécessaire, surtout pour les élèves ayant des difficultés d'expression comportementale (DEC). On peut proposer simplement à l’élève de rester à la fin de la classe pour échanger sur son comportement et lui énoncer la sanction.
        • Dans les situations graves, impliquer l'équipe éducative et éventuellement les parents. La sanction devra être prononcée par un autre enseignant, ce qui montre la communication et la cohésion de l’équipe éducative, et augmente l’autorité des adultes.
      • Types de sanctions :

        • Éviter les sanctions qui confrontent directement l'élève à sa difficulté. Par exemple, ne pas faire copier des lignes à un élève dysgraphique.
        • Privilégier les sanctions réparatrices. Certains établissements proposent des travaux civiques comme nettoyer la classe.
        • Envisager l'exclusion temporaire de classe avec prudence. L'exclusion temporaire de classe peut être envisagée s’il n’y a pas de risque d’escalade et si cela a été travaillé en amont avec le jeune et l’équipe éducative.
        • Manier l'exclusion temporaire de l'établissement avec précaution. Tant que possible il est nécessaire de maintenir le jeune sur l’établissement scolaire, et un système d’exclusion-inclusion est à privilégier.
      • Après la sanction :

        • Inscrire la sanction dans un projet de remédiation. Rendre l’élève acteur du suivi de son comportement par des dispositifs qui pourront varier d’un établissement à l’autre permettra, si sanction il y a, de davantage faire sens.
        • Mettre en place un tutorat si nécessaire. Des entretiens réguliers sont prévus pour permettre de faire le point, constater les avancées et les objectifs à atteindre.
        • Travailler en collaboration avec les familles. Travailler avec les familles va conditionner la réussite et l’intérêt des sanctions posées par l’établissement scolaire.
      • Suivi et ajustement :

        • Évaluer l'efficacité de la sanction. Chez les élèves n’ayant pas de difficulté spécifique leur application se révèlera simple, avec des résultats rapides.
        • Ajuster les réactions aux difficultés. Pour un certain nombre de difficultés, l’élève peut garder un contrôle même si c’est plus difficile pour lui que pour les autres élèves.
        • Être prêt à renégocier les règles si nécessaire. Des espaces de (re)négociation et de retour sur l’action permettent de faire évoluer les règles de la classe en fonction des besoins identifiés par le groupe classe ou l’enseignant.

      En suivant cette checklist, il est possible de mettre en place des sanctions qui soient à la fois justes, efficaces et adaptées aux besoins spécifiques de chaque élève, tout en maintenant un environnement d'apprentissage positif et respectueux.

    1. Voici une checklist de bonnes pratiques pour gérer la discipline en classe, basée sur les sources fournies:

      • Établir un cadre clair et stable Définir clairement les règles, les possibilités et les interdits. S'assurer que le cadre est stable, impartial et basé sur le respect réciproque.
      • Impliquer les élèves dans la définition des règles Discuter des règles de vie en collectivité avec les élèves pour qu'ils se les approprient.
      • Privilégier les sanctions éducatives Viser à faire grandir l'enfant, à lui faire comprendre les fondements de la vie en société et à l'aider à y trouver sa place. Transformer un acte perturbateur en moment de civisme.
      • Associer sanctions et dialogue Accompagner la sanction de parole pour réexpliquer les règles et le motif de la sanction.
      • Individualiser les sanctions Tenir compte du degré de responsabilité de l'élève, de son âge, de son implication et de ses antécédents. Adapter les sanctions aux profils différents des élèves.
      • Respecter les principes juridiques S'assurer que les sanctions respectent les principes de légalité, du contradictoire, de proportionnalité et d'individualisation.
      • Valoriser les comportements adéquats Encourager et valoriser les comportements adaptés à l'activité scolaire (calme, attention, soin, entraide, respect d'autrui). Mettre en place des actions valorisantes et encourageantes.
      • Utiliser des outils de valorisation Mettre en place des outils comme un tableau de comportement évolutif et un cahier de réussite pour valoriser les élèves.
      • Être attentif aux signaux : être attentif aux signaux faibles qui peuvent dégénérer.
      • Proportionnalité et cohérence : Adapter la sanction à la gravité de la faute.
      • Réagir aux comportements : Réagir rapidement aux incivilités, aux injustices, aux brimades, au non-respect des règles, etc..
      • Bannir les sanctions dégradantes ou humiliantes Éviter tout châtiment corporel ou traitement humiliant.
      • Former une communauté éducative : Impliquer tous les acteurs de l'école, les parents, les services sociaux et de santé, etc.

      En mettant en œuvre ces bonnes pratiques, il est possible de créer un environnement d'apprentissage positif et respectueux, où les élèves se sentent valorisés et responsabilisés.

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

      Learn more at Review Commons


      Reply to the reviewers

      Manuscript number: RC-2024-02767

      Corresponding author(s): Kazuaki Maruyama

      1. General Statements

      Response to Reviewer #1:

      We sincerely appreciate your thoughtful review of our manuscript. Our primary objective is to elucidate the pathogenic mechanisms underlying congenital low-flow vascular malformations, thereby informing the development of novel therapeutic strategies. We recognize that, given the dual nature of our study encompassing both fundamental and clinical science, the presentation may have appeared somewhat convoluted. In response, we have revised the manuscript to clarify these points and have reformatted the text corresponding to your comments—originally presented as a single continuous block—into defined, numbered sections to enhance readability.

      Response to Reviewer #2:

      We are deeply grateful for the time and effort you have dedicated to reviewing our manuscript despite your busy schedule. Your comments have been particularly insightful, especially regarding the section on the preclinical mouse model. In light of your suggestions, we have conducted additional experiments and revised the manuscript accordingly. We trust that these modifications address your concerns and contribute to the overall improvement of our work.

      The revised sections have been highlighted in red in the text.

      2. Point-by-point description of the revisions

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

      The authors investigate the pathogenesis of congenital vascular malformations by overexpressing the Pik3caH1047R mutation under the R26 locus in different cell populations and developmental stages using various Cre and CreERT2 lines, including endothelial-specific and different mesoderm precursor lines. The authors provide a thorough characterization of the vascular malformation phenotypes across models. Specifically, they claim that expressing Pik3caH1047R in the cardiopharyngeal mesoderm (CPM) precursors results in vascular abnormalities localized to the head and neck region of the embryo. The study also includes scRNAseq data analyses, including from previously published data and new data generated by the authors. Trajectory inference analysis of a previous scRNA-seq dataset revealed that Isl1+ mesodermal cells can differentiate into ETV2+ cells, directly giving rise to Prox1+ lymphatic endothelial cell progenitors, bypassing the venous stage. Single-cell RNA sequencing of their CPM model and other in vitro datasets show that Pik3caH1047R upregulates VEGF-A via HIF-1α-mediated hypoxia signaling, findings further corroborated in human samples. Finally, preclinical studies in adult mice confirm that pharmacological inhibition of HIF-1α and VEGF-A reduces the number and size of mutant vessels.

      Major comments

      1. While the study provides a nice characterization of Pik3caH1047R-derived vascular phenotypes induce by expressing this mutation in different cells, the main message of the study is unclear. What is the main question that the authors want to address with this manuscript?

      Response:

      Our main message is as follows:

      1. __ Elucidation of pathogenesis based on developmental cellular origins:__ This study focuses on using embryonic models to elucidate the mechanism by which the Pik3caH1047R mutation induces low-flow vascular malformations. Specifically, we demonstrate that expression of Pik3caH1047R in cells derived from the cardiopharyngeal mesoderm (CPM) induces vascular abnormalities that are confined to the head and neck region. Furthermore, vascular malformations originating from another cell type—for example, Pax3+ cells—are confined to the lower body. This suggests that the embryonic origin of endothelial cells may determine the anatomical location of vascular malformations, with important implications for clinical severity and treatment strategies.

      Molecular ____s____i____gnaling pathways and targeted therapeutic approaches:

      Through single-cell RNA sequencing, we have identified hypoxia signaling—particularly via HIF-1α and VEGF-A—as central to the pathogenesis of these malformations. Moreover, preclinical mouse model experiments demonstrate that pharmacological inhibition of HIF-1α and VEGF-A significantly reduces lesion formation, supporting the potential of targeting these pathways as a novel therapeutic strategy.

      In summary, our main message is that by elucidating the developmental and molecular mechanisms underlying Pik3caH1047R-driven low-flow vascular malformations—especially the pivotal role of hypoxia signaling via HIF-1α/VEGF-A—we provide a strong rationale for novel therapeutic strategies aimed at these challenging conditions

      To further clarify these points, we have revised the manuscript by incorporating additional experiments and reorganizing the text into clearly defined sections.

      The precursor type form where these lesions appear, that venous and lymphatic malformations emerge independently, when and where this phenotype appear?

      Response:

      In Tie2-Cre; R26R-Pik3caH1047R mutant embryos, no prominent phenotype was observed at E9.5 or E11.5. Vascular (venous) malformations are evident from E12.5, whereas lymphatic malformations become prominent from E13.5. We propose that the emergence of the lymphatic phenotype after E13.5 is due to the fact that lymphatic vessels, particularly in the upper body, begin forming a luminal structure mainly from E13.5 onward(Maruyama et al, 2022) . For further details, please refer to the explanation provided in Question 6.

      To address this, we have newly included Supplemental Figure 2 and revised the Results section as follows:

      Whereas clear phenotypes were evident at E12.5 and E13.5, no pronounced external abnormalities were observed at E9.5 or E11.5 (Supplemental Figure 2A–B). Similarly, histological examination revealed no significant differences in the short-axis diameter of the PECAM+ CV or in the number of Prox1+ LECs surrounding the CV between control and mutant embryos at E11.5 (Supplemental Figure 2C–F). We also assessed Tie2-Cre; R26R-Pik3caH1047R mutant embryos at E14.0 from five pregnant mice. Only two embryos were alive at this stage, and both showed severe edema and hemorrhaging, indicating they were nearly moribund. These observations suggest that the critical point for survival of these mutant embryos lies between E13.5 and E14.0 (Supplemental Figure 2G). (Page 5, lines 157–165)

      The manuscript needs some work to make the sections more cohesive and to structure better the main findings and the rationale for choosing the models. Authors should explain better when and where the pathogenic phenotypes refer to blood and/or lymphatic malformations. From the quantifications provided in Figure 1, Pik3caH1047R leads to different phenotypes in blood and lymphatic vessels. These are larger diameters with no difference in the number of blood vessels (are you quantifying all pecam1 positive? Vein, arteries, capillaries?), and an increase in the number of lymphatics vessels. Please clarify and discuss.

      Response:

      We interpreted this as a question regarding which vessels were quantified. The answer to this question is provided in Question 4.

      Which vessel types are considered for the quantifications shown in Fig. 1I, M, Q? All Pecam1+ vessels, including lymphatic, vein, capillaries and arteries or which ones? Provide clarifications.

      __Response: __

      Vessel types were characterized based on anatomical and histological features. For the anatomical details, we referred to The Atlas of Mouse Development by M.H. Kaufman.

      This aspect is described in the Methods section, as follows:

      Veins and arteries were classified based on anatomical criteria. Vessels demonstrating continuity with a clearly identifiable vein (e.g., the anterior cardinal vein) in serial sections were defined as veins. In contrast, the aorta and pulmonary artery, each exhibiting a distinct wall structure indicative of a direct connection to the heart, were designated as arteries. Lymphatic vessels were identified based on the combined expression of Prox1, VEGFR3, and PECAM, along with the developmental stage, morphology, and anatomical location as described in our previous studies (Maruyama et al, 2019, 2022, 2021) . PECAM+ vessels that lacked a definitive wall structure, did not express lymphatic markers, or did not exhibit clearly identifiable continuity necessary for classification as veins or capillaries were collectively designated as blood vessels or vasculatures. (Page 16, lines 530-539)

      Regarding Figure 1I:

      In the tongue and mandible, the facial vein—which branches from the anterior cardinal vein—is dilated, and its continuity with the venous system is confirmed. In contrast, Figure 1J shows the number of PECAM+ vasculatures; however, for smaller vessels, continuity is not always demonstrable, so these are designated as vasculatures according to the criteria.

      Regarding Figures 1M and N:

      In the liver, the dilated vessels are classified as veins because they exhibit continuity with the inferior vena cava. Even in the control group, the central veins tend to have relatively large diameters. Therefore, we compared the average area and quantified the number of abnormal central veins—defined as those contiguous with a vein and exceeding a specified area.

      Regarding Figures 1Q and R:

      Cerebral vessels are classified as veins due to their continuity with the common cardinal and jugular veins. However, as these vessels extend into the periphery, this continuity becomes less distinct, and they are consequently designated as blood vessels lacking Prox1 expression.

      The authors propose that the CPM model results in localized head and neck vascular malformations. However, I am not convinced. The images supporting the neck defects are evident, but it is unclear whether there are phenotypes in the head.

      Response:

      Perhaps the discrepancy arises from a terminological issue. According to the WHO Classification of Tumours, commonly used in clinical settings, the term "Head and Neck" refers to the facial and cervical regions (including the oral cavity, larynx, pharynx, salivary glands, nasal cavity, etc.) and excludes the central nervous system. The inclusion of the brain in Figure 1O-R may have led to some confusion. We included the brain because cerebral cavernous malformations are classified as venous malformations, and thus serve as an example of common sites for venous malformations in humans. To clarify this point, we have made slight revisions to the first part of the Introduction, as follows:

      They frequently manifest in the head and neck region—here defined as the orofacial and cervical areas, excluding the brain. (Page2, lines 52-53)

      Why are half of the experiments with the Tie2-Cre model conducted at E12.5 (e.g., validation of recombination, signaling, proliferation) and the others at E13.5? It becomes confusing for the reader why the authors start the results section with E13.5 and then study E12.5.

      Response:

      This is also related to the previous question (Question 4). We decided to include extensive anatomical information in a single figure. In Supplemental Figure 1, sagittal sections at E12.5 were used so that the pulmonary artery, aorta, and dilated common cardinal vein could be visualized within one sample. This allowed us to demonstrate that the Pik3caH1047R mutation does not affect arteries by contrasting them with the dilated veins. At E13.5, in addition to the dilation observed at E12.5, the common cardinal vein becomes markedly dilated and compresses the surrounding structures. Capturing both veins and arteries simultaneously would require multiple images, which could potentially confuse the reader. Moreover, lymphatic and other organ phenotypes (e.g., in the liver) are more prominent at E13.5. Therefore, we selectively employed both E12.5 and E13.5 stages to suit our specific objectives.

      The quantifications provided do not clarify what the "n" represents or how many embryos or litters were analyzed. 

      Response:

      Thank you for your feedback. We have now incorporated the sample size (n) directly into the graphs and figure legends.

      Blasio et al. (2018), Hare et al (2015) reported that Pik3caH1047R with Tie2-Cre embryos die before E10.5. How do the authors explain the increase in survival here? Were embryos at E13.5alive? What was the Mendelian ratio observed by the authors? Please provide this information and discuss this point.

      Response:

      Two types of Tie2-Cre lines are widely used worldwide. The mouse line employed by Blasio et al. (2018) differs from that used in our study (their manuscript did not specify whether the background was B6 or a mixed strain). In contrast, although Hare et al. (2015) used the same mouse line as we did, they maintained a C57BL/6 background. We selected a mixed background of B6 and ICR, as we believe that a heterogeneous genetic background more accurately reflects the diversity of human pathology. We examined five pregnant females, which yielded approximately 30 embryos from five pregnant mice, of which only two survived until E14.0. Based on these observations, we consider E13.5 to be the appropriate survival limit (see Supplemental Figure 2G for additional details). In our breeding strategy, mice in the Tie2-Cre or Tie2-Cre; R26R-eYFP line were maintained as heterozygotes for Tie2-Cre and homozygotes for R26R-eYFP, whereas those carrying the R26R-Pik3caH1047R allele were homozygous. This approach produced control(Cre (-)) and heterozygous offspring in an expected 1:1 ratio at all examined stages: E9.5 (mutant n = 4, control n = 4 from two pregnant females), E11.5 (mutant n = 8, control n = 8 from two pregnant females), E12.5 (mutant n = 4, control n = 4 from two pregnant females), and E13.5 (mutant n = 5, control n = 5 from two pregnant females), with no deviation from the anticipated Mendelian ratio.

      Regarding this point, we have described it in the Results section as follows:

      Whereas clear phenotypes were evident at E12.5 and E13.5, no pronounced external abnormalities were observed at E9.5 or E11.5 (Supplemental Figure 2A–B). Similarly, histological examination revealed no significant differences in the short-axis diameter of the PECAM+ CV or in the number of Prox1+ LECs surrounding the CV between control and mutant embryos at E11.5 (Supplemental Figure 2C–F). We also assessed Tie2-Cre; R26R-Pik3caH1047R mutant embryos at E14.0 from five pregnant mice. Only two embryos were alive at this stage, and both showed severe edema and hemorrhaging, indicating they were nearly moribund. These observations suggest that the critical point for survival of these mutant embryos lies between E13.5 and E14.0 (Supplemental Figure 2G). (Page 5, lines 157-165)

      Please explain the rationale for using the Cdh5-CreERT2. It is likely due to the lethality observed with Tie2Cre, but this was not mentioned.

      Response:

      Thank you very much for your comment. As mentioned above, nearly all Tie2‐Cre;Pik3caH1047R embryos fail to survive past E14.0.

      The lethality observed with Tie2‐Cre mice is described as follows:

      We also assessed Tie2-Cre; R26R-Pik3caH1047R mutant embryos at E14.0 from five pregnant mice. Only two embryos were alive at this stage, and both showed severe edema and hemorrhaging, indicating they were nearly moribund. These observations suggest that the critical point for survival of these mutant embryos lies between E13.5 and E14.0 (Supplemental Figure 2G). (Page 5, lines 161-165)

      The rationale for using CDH5-CreERT2 mice is described as follows:

      To investigate whether the resulting human disease subtype (e.g., lesions confined to the head and neck region) is determined by the specific embryonic stage at which Pik3caH1047R is expressed, we crossed tamoxifen-inducible, pan-endothelial CDH5-CreERT2 mice with R26R-Pik3caH1047R mice and analyzed the embryos at E16.5 or E17.5. (Page 5, lines 169-172)

      Why were tamoxifen injections done at various time points (E9.5, E12.5, E15.5)? Please clarify the reasoning behind administering tamoxifen at these specific times. Explaining the rationale will help the reader follow the experimental design more easily. Additionally, including an initial diagram summarizing all the strategies to guide the reader from the beginning would be helpful.

      Response:

      Martinez‐Corral et al. (Nat. Commun., 2020) focused on lymphatic malformations, arguing that the timing of tamoxifen administration during the embryonic period determines the anatomical features of these lesions. They stated, “The majority of lesions appeared as large isolated cysts that were localized mainly to the cervical, and less frequently to the sacral region of the skin (Figure 2)”. Although not stated definitively, their data suggest that early embryonic tamoxifen administration results in the formation of large‐caliber lymphatic vessels with region‐specific distribution in the cervical skin (Figure 2C, Supplemental Figure 2). This description likely reflects an intention to model human vascular malformations, implying that the anatomical characteristics of these malformations are influenced by the developmental stage at which the Pik3caH1047R somatic mutation occurs.

      Inspired by these findings, we conducted experiments to determine whether altering the timing of tamoxifen administration would yield region-specific anatomical patterns in vascular malformation development. However, our results indicate that changing the timing of tamoxifen administration does not lead to an anatomical bias similar to that observed in human vascular malformations. Instead, we propose that the embryological cellular origin plays a more significant role in the formation of these human pathologies.

      Regarding this section, we have slightly revised the introductory part of the Figure 2 explanation as follows:

      To investigate whether the resulting human disease subtype (e.g., lesions confined to the head and neck region) is determined by the specific embryonic stage at which Pik3caH1047R is expressed, we crossed tamoxifen-inducible, pan-endothelial CDH5-CreERT2 mice with R26R-Pik3caH1047R mice and analyzed the embryos at E16.5 or E17.5. (Page 5, lines 169-172)

      Additionally, we have added a schematic diagram of the tamoxifen administration schedule at the beginning of Figure 2 and Supplemental Figure 3.

      Why do you use the Isl1-Cre constitutive line (instead of the CreERT2)? The former does not allow control of the timing of recombination (targeting specifically your population of interest) and loses the ability to trace the mutant cell behaviors over time. Is the constitutive expression of Pik3caH1047R in Isl1+ cells lethal at any embryonic time, or do the animals survive into adulthood? When you later use the Isl1-CreERT2 line, why do you induce recombination specifically at E8.5? It would be helpful for the reader to have an explanation for this choice, along with a reference to your previous paper.

      Response:

      Thank you for your comments. We did attempt the same experiments using Isl1-CreERT2 under various conditions. However, administering tamoxifen earlier than E8.5 invariably caused embryonic lethality, likely due to both Pik3ca activity and tamoxifen toxicity, leaving no embryos for analysis. In our previous study, repeated attempts from E6.5 to E16.5 resulted in only two surviving embryos (Maruyama et al., eLife, 2022, Supplemental Figure 3). We also failed to recover any live embryos with tamoxifen administration at E7.5.

      Even reducing the tamoxifen dose to one-fifth did not succeed when given before E8.5. Although E8.5 administration was feasible, the observed phenotype remained mild, and no phenotype was detected at E9.5, E11.5, E12.5, or later stages. These findings align with our earlier observations that moving tamoxifen injection from E8.5 to E9.5 markedly diminishes the Isl1+ contribution to the endothelial lineage.

      Furthermore, Supplemental Figure 5____ and 6 suggest that a decrease in Isl1 mRNA, which occurs as early as E8.0–E8.25, triggers the shift toward endothelial differentiation. Considering these data and the mild phenotype at E8.5, earlier administration would be ideal for impacting Isl1+ cell fate. However, technical constraints prevented us from doing so, leading us to utilize the constitutive Isl1-Cre line instead.

      This section was already included in the Discussion; however, for clarity, we have revised it as follows:

      Given that Isl1 expression disappears at a very early stage and contributes to endothelial differentiation, experiments using Isl1-Cre or Isl1-CreERT2 mice cannot clearly distinguish between LMs, VMs, and capillary malformations, In other words, Isl1+ cells likely label a common progenitor population for multiple endothelial subtypes. Consequently, the diverse vascular malformations in the head and neck—including mixed venous-lymphatic and capillary malformations, as well as the macro- and microcystic subtypes of LMs—cannot be fully accounted for by this study alone. (Page 13, lines 419-425)

      What is the purpose of using this battery of CreERT2 lines (for example, the Myf5-CreERT2)?

      Response:

      The head and neck mesoderm arises primarily from the cardiopharyngeal mesoderm and the cranial paraxial mesoderm. Myf5-CreERT2 labels the cranial paraxial mesoderm in the facial region, which gives rise to facial skeletal muscles. Stone et al. (Dev Cell, 2019) reported that a subset of this lineage contributes to head and neck lymphatic vessels, whereas our study (Maruyama et al., eLife, 2022) found no such contribution—an ongoing point of debate. Nevertheless, expressing Pik3caH1047R in this lineage did not induce any vascular malformations.

      Pax3-CreERT2 mice label Pax3____⁺ paraxial mesoderm (including cranial paraxial mesoderm), which reportedly contributes to the common cardinal vein and subsequently forms trunk lymphatics (Stone & Stainier, 2019; Lupu et al, 2022) . When Pik3caH1047R was expressed in Pax3⁺ cells, we observed abnormal vasculature in the lower trunk and around the vertebrae, consistent with that report.

      Synthesizing these observations with our results from Isl1-Cre, Isl1-CreERT2, and Mef2c-AHF-Cre lines, we propose that Pik3caH1047R mutations within the cardiopharyngeal mesoderm underlie the clinically significant vascular malformations seen in the head and neck region.

      We have also incorporated the following explanation into the main text.

      Regarding the Pax3-CreERT2:

      The head and neck mesoderm arises primarily from the cardiopharyngeal mesoderm and the cranial paraxial mesoderm. In Pax3-CreERT2; R26R-Pik3caH1047R embryos, Pax3+ paraxial mesoderm (including cranial paraxial mesoderm) is labeled; this lineage reportedly contributes to the common cardinal vein and subsequently forms trunk lymphatics(Lupu et al, 2022), (Page 8, lines 247-250)

      Regarding the Myf5-CreERT2;

      In Myf5-CreERT2; R26R-tdTomato mice—which label the cranial paraxial mesoderm, particularly muscle satellite cells—crossed with R26R-Pik3caH1047R, tamoxifen was administered to pregnant mice at E9.5. (Page 8, lines 255-257)

      I find the scRNAseq data in Fig S4 and S5 results very interesting, although I am unsure how they fit with the rest of the story. In principle, a subset of Isl1+ cardiopharyngeal mesoderm (CPM) derivatives into lymphatic endothelial cells was already demonstrated in a previous publication from the group. What is the novelty and purpose here?

      Response:

      This also addresses Question 11. Our aim in using the Isl1⁺ lineage was to determine the extent of analysis possible with this experimental system. Through reanalysis, we found that the downregulation of Isl1 triggers a switch toward endothelial cell differentiation, with this cell fate decision occurring at a very early embryonic stage. Consequently, our single‐cell analysis supports the conclusion that, regardless of the Isl1-CreERT2 line used or the timing of tamoxifen administration, it is challenging to precisely recapitulate the fine clinical phenotypes observed in humans (e.g., lymphatic or venous malformations) with this experimental system. We believe that this single‐cell analysis provides a theoretical basis for the notion that our Isl1-Cre-based developmental model can only generate a mixed phenotype of vascular and lymphatic malformations.

      This section is explained in a similar manner in the revised Discussion for Question 11 as follows:

      Given that Isl1 expression disappears at a very early stage and contributes to endothelial differentiation, experiments using Isl1-Cre or Isl1-CreERT2 mice cannot clearly distinguish between LMs, VMs, and capillary malformations, In other words, Isl1+ cells likely label a common progenitor population for multiple endothelial subtypes. Consequently, the diverse vascular malformations in the head and neck—including mixed venous-lymphatic and capillary malformations, as well as the macro- and microcystic subtypes of LMs—cannot be fully accounted for by this study alone. (Page 13, lines 419-425)

      Why in Fig. 4 ECs were not subclustered for further analysis (as in Fig. S4,5)? This is a missed opportunity to understand the pathogenic phenotypes.

      Response:

      Thank you for your question. We performed sub-clustering analysis, particularly focusing on why no phenotype is observed in arteries, as we believed this approach could provide molecular-level insights. Accordingly, we conducted the analysis presented in Figure 1 for Reviewer 1.





      Figure legends for Figure ____1 ____for Reviewer 1. The number of endothelial cells was insufficient, making subclustering ineffective.

      (Figure for Reviewer 1A, B) Left: UMAP plot showing color-coded clusters (0–3). Subcluster analysis of the Endothelium (Cluster 1) from Fig. 4B. Right: UMAP plot color-coded by condition. (Figure for Reviewer 1C) Heatmap showing the average gene expression of marker genes for each cluster by condition. After cluster annotation, subclusters 0, 1, 2, and 3 were defined as Vein, Capillary, Artery, and Lymphatics, respectively. (Figure for Reviewer 1D) Cell type proportions. (Figure for Reviewer 1E) Number of differentially expressed genes (DEGs) in each sucluster of the PIK3CAH1047R group relative to Control. (Figure for Reviewer 1F) Comparison of enrichment analysis between EC subclusters from scRNA-seq. The bar graph shows the top 20 significantly altered Hallmark gene sets in EC subclusters from scRNA-seq using ssGSEA (escape R package). Red bars represent significantly upregulated Hallmark gene sets in mutants (FDR Initially, we performed sub-clustering on endothelial cells; however, this resulted in a considerably reduced number of cells per sub-cluster, especially in control group (Figure for Reviewer 1A, B). In the control group, there were only approximately 149 endothelial cells in total, and dividing these into four clusters led to very few cells per cluster, thereby introducing statistical instability. Although arterial endothelial cells were relatively well defined by their high expression of Hey1 and Hey2 and lower levels of Nr2f2 and Aplnr, the boundaries between venous, capillary, and lymphatic endothelial cells were less distinct. In particular, defining lymphatic endothelial cells solely by Prox1 expression yielded a very small population; even after incorporating additional lymphatic markers such as Flt4 and Lyve1, it remained challenging to clearly separate the venous, capillary, and lymphatic populations (Figure for Reviewer 1C). Consequently, the proportion of lymphatic endothelial cells was markedly low, and discrepancies with the histological findings further reduced our confidence in this dataset (Figure for Reviewer 1D, E). Moreover, the number of differentially expressed genes (DEGs) increased with the number of cells, and the results of the enrichment analysis as well as the volcano plot were nearly identical to those shown in Figure 4 (Figure for Reviewer 1F, G). In other words, the subclustering process itself had limitations, resulting in the overall outcome being dominated by the most abundant venous cluster.

      It is possible that these limitations in sub-clustering are due to the relatively small number of endothelial cells. Nonetheless, a major strength of our single-cell analysis is its ability to compare various cell types derived from Isl1+ lineages, not just endothelial cells. Therefore, the relative scarcity of endothelial cells represents a limitation of this experimental system. For these reasons, we decided to omit this figure from the final version of the manuscript.

      This point is described in the Discussion section as follows:

      Additionally, we performed endothelial subclustering to explore potential differences in gene expression among arterial, venous, capillary, and lymphatic endothelium. However, in the control embryos, the number of endothelial cells was too low to yield reliable data (data not shown). (Page 13, lines 434-437)

      Hypoxia and glycolysis signatures are not specific to mutant ECs. Do the authors have an explanation for this? It is well known that PI3K overactivation increases glycolysis; please acknowledge this.

      __Response: __

      Thank you for your important comment. We have now incorporated a discussion, along with relevant references, on the section addressing that PI3K overactivation increases glycolysis into the Discussion section as follows:

      It is well known that overactivation of PI3K enhances glycolysis(Hu et al, 2016) . In our study, the elevated expression of glycolytic enzymes, including Ldha, suggests a shift toward aerobic glycolysis, consistent with the Warburg effect. (Page 13, lines447-450)

      Do you have an explanation for the expression of VEGFA by lymphatic mutant cells?

      __Response: __

      VEGF-A acts on VEGFR2 expressed on LECs, thereby promoting their proliferation and migration(Hong et al, 2004; Dellinger & Brekken, 2011) .To clarify this point, we have revised the text accordingly and added additional references as follows:

      We focused on Vegf-a, a key regulator of ECs proliferation and a downstream target of Hif-1α. Vegf-a likely drives both cell-autonomous and non-cell-autonomous effects on blood ECs , as well as LECs(Hong et al, 2004; Dellinger & Brekken, 2011). (Page 13, lines 445-447)

      Likewise, why mesenchymal cells traced from the Islt1-Cre decreased upon expression of Pik3caH1047R?

      Response: When comparing the mesenchyme cluster with other mesoderm-derived cells, we observed a marked downregulation of signaling pathways—notably those involved in inhibiting EMT, such as TGF-β, Wnt/βcatenin, and MYC target genes (Supplemental Figure 7B). Many of these pathways are associated with decreased epithelial-to-mesenchymal transition(Xu et al, 2009; Singh et al, 2012; Larue & Bellacosa, 2005; Yu et al, 2015), which could explain the reduction in the number of mesenchymal cells. However, PI3K activation is generally considered to promote EMT, which is at odds with previous studies.

      On the other hand, several investigations—including those using ES cells—suggest that PI3K activation could suppress TGF-β signaling via SMAD2/3(Yu et al, 2015) , and in some undifferentiated cell contexts, it may also inhibit the Wnt/β-catenin pathway via Smad2/3(Singh et al, 2012) . These multifaceted roles of PI3K could be particularly important during embryonic development(Larue & Bellacosa, 2005).

      Understanding how mesenchymal cell changes under PI3K activation affect endothelial cells is an important issue that requires further study. Accordingly, we have added these points to the Discussion section as follows:

      In our data, the mesenchymal cell population was decreased, and within this cluster, pathways typically promoting epithelial mesenchymal tansition (EMT) (e.g., TGF-β, Wnt, and MYC target genes) were downregulated (Supplemental Figure 7B). Although PI3K activation is generally thought to enhance EMT, several studies in undifferentiated cells have reported that PI3K can suppress these signals via SMAD2/3(Singh et al, 2012; Yu et al, 2015) . Elucidating how these changes in the mesenchyme contribute to vascular malformation pathogenesis remains an important avenue for future research. (Page 13, lines 437-444)

      Authors need to characterize the preclinical model before conducting any preclinical study. No controls are provided, including wild-type mice and phenotypes, before starting the treatment (day 4).

      Response:

      Thank you very much for your comment. We have now added new images illustrating skin under three conditions: untreated skin at Day 7, skin from Cre-negative animals that received tamoxifen, and skin from Cre-positive animals examined 4 days after tamoxifen administration. Additionally, we have included the corresponding statistical data for these skin samples (Figure 6C–E).

      Why did the authors not use their developmental model of head and neck malformation model for preclinical studies? This would be much more coherent with the first part of the manuscript. Also, how many animals were treated and quantified for the different conditions?

      Response:

      We have now indicated the number of animals (n) used under each condition directly on the graphs for clarity. As for why we did not use the Isl1-Cre model, we observed that—similar to the Tie2-Cre line—all Isl1-Cre mutant embryos died between E13.5 and E14.0 (indeed, none survived beyond E14.0; see our newly added Figure 3N). Consequently, we could not perform any postnatal treatment experiments. Moreover, as previously noted, the Isl-CreERT2 line has an extremely narrow developmental window for vascular malformation formation, making it less suitable as a general model.

      Although we considered potential in utero or maternal interventions (e.g., direct uterine injection or placental transfer), these approaches demand extensive technical optimization and remain an area for future investigation. From a clinical standpoint, postnatal therapy meets a more immediate need: while vascular malformations are congenital, they often enlarge over time(Ryu et al, 2023) , becoming more apparent and more likely to require treatment.

      In this study, because embryonic Pik3caH1047R expression was lethal before birth, we generated and treated postnatal cutaneous vascular malformations instead. Although this model does not strictly recapitulate the embryonic disease state, previous studies assessing drug efficacy have similarly employed postnatal tamoxifen-inducible mouse models(Martinez-Corral et al, 2020) , lending validity to this approach. Moreover, because lesions typically become evident later in life rather than in utero, this method more closely aligns with clinical reality and may be more readily translated into practice.

      Minor Comments

      References in the introduction need to be revised. Specifically, how authors reached the stats on head and neck vascular malformations needs to be clarified. For instance, one of the cited papers refers to all types of vascular malformation, while the other focuses exclusively on lymphatic malformations with PIK3CA mutations. Moreover, in the latter, the groups are divided into orofacial and neck and body categories. How do authors substrate the information from the neck and head here?

      Response:

      We have clarified our definition of the “head and neck” region early in the Introduction and separated the discussion on anatomical localization from that on PIK3CA genetics. Additionally, we removed the percentage data of localization to avoid potential confusion with the genetic aspects.

      In Japan, lymphatic and other vascular malformations of the head and neck typically require complex, multidisciplinary management. Consequently, these conditions are officially designated as “intractable diseases,” and the government provides financial assistance for their treatment. Although most of the information is available only in Japanese, we refer reviewers to the following websites for details on head and neck vascular malformations:

      https://www.nanbyou.or.jp/entry/4893 https://www.nanbyou.or.jp/entry/4631 https://www.nanbyou.or.jp/entry/4758.

      (Please read with English translator, e.g., Google chrome translator)

      We are not aware of a comparable system in other countries. However, it is well recognized that vascular malformations frequently occur in the head and neck region(Nair, 2018; Alsuwailem et al, 2020; Sadick et al, 2017), as evidenced by over 250 PubMed hits when searching for “vascular malformation” and “head and neck.

      Incorporating this comment, we have revised the early part of the Introduction as follows:

      They frequently manifest in the head and neck region—here defined as the orofacial and cervical areas, excluding the brain (Zenner et al, 2019; Lee & Chung, 2018; Nair, 2018; Alsuwailem et al, 2020). (Page 2, lines 52-53)

      Also, in line 79, I need clarification on ref 24 about fibrosis.

      __Response: __

      Thank you very much for pointing out the error. We have corrected the placement of the reference accordingly.

      Include references: Studies in mice have shown that p110α is essential for normal blood and lymphatic vessel development. Please clarify and correct. 

      __Response: __

      Thank you very much. We have now added the references(Graupera et al, 2008; Gupta et al, 2007; Stanczuk et al, 2015).

      Please define PIP2 and PIP3

      __Response: __

      Thank you very much for your comment. We have now added the following definitions to the Introduction:

      PIP2: Phosphatidylinositol 4,5-bisphosphate

      PIP3: Phosphatidylinositol 3,4,5-trisphosphate


      Why is Prox1 showing positivity in erythrocytes in Figure 1?

      Response:

      We used paraffin-embedded sections to preserve tissue morphology. Although we applied a reagent to suppress autofluorescence, some spillover from excitation around 488 nm was unavoidable. Moreover, in the mutant mice, blood remained within the abnormal vessels rather than being completely flushed out, which further increased the autofluorescence. Despite our efforts to mitigate this, some residual autofluorescence persisted. Consequently, we also employed DAB-based staining to confirm the specificity of Prox1 labeling in other Figures.

      Regarding Figure 1, I suggest organizing the quantifications in the same order to facilitate phenotype comparisons. For example, I, J vs. Q, R. What is the difference between M and N?

      Response:

      To facilitate the comparison between Figures 1I, J and 1Q, R, we have swapped Figures 1Q and R. Regarding Figures 1M and N, these panels represent the average cross-sectional area of an enlarged malformed vessel and the number of vessels exceeding a defined size, respectively. Although some central veins appeared slightly enlarged in the control group, the liver exhibits both a significant dilation of malformed vessels and an increased number of such vessels.

      Add the reference of the Bulk RNseq data.

      __Response: __

      We have added the following references: (Jauhiainen et al, 2023)

      Mark in the Fig. 4F that the volcano plots are from cluster one of the scRNASeq (this is explained in text and legend, but when you go to the figure, it isn't very clear).

      __Response: __

      We have added the label “Cluster 1: Volcano Plot (genes associated with hypoxia/glycolysis)” to

      Figure 4F.

      Please label Figure 6D/E with the proper labels.

      __Response: __

      We have provided appropriate labels for Figure 6.

      In Fig. 6, it is mentioned that vacuoles are from the tamoxifen injection, how do you know? Do you also see them if you add oil alone (without tamoxifen) or tamoxifen in a WT background?

      __Response: __

      In Figure 6C, we have included both the image at Day 4 and the condition of Cre(–) animals 7 days after tamoxifen injection.

      **Referees cross-commenting**

      I complete agree with referee #2 regarding the preclinical studies. Bevacizumab, does not neutralize murine VEGFA. This is a major issue.

      __Response: __

      As noted in the Reviewer #2 section, there appears to be some effect on mouse vasculature (Lin et al, 2022). However, given the ongoing debate regarding this issue, we performed additional experiments using a neutralizing antibody against mouse VEGF-A (clone 2G11). This antibody has been shown to suppress the proliferation of mouse vascular endothelial cells in vivo, for example(Mashima et al, 2021; Wuest & Carr, 2010). Our results demonstrate that it more sharply suppresses the proliferation of malformed vasculatures (both blood and lymphatic vessels) than bevacizumab. Based on these additional experiments, we revised the figures and updated them as Figure 6.

      Reviewer #1 (Significance (Required)):

      This study addresses a timely and relevant question: the origins, onset and progression of congenital vascular malformations, a field with limited understanding. The work is novel in its approach, employing complex embryonic models that aim to mimic the disease in its native context. By focusing on the effects of Pik3caH1047R mutations in cardiopharyngeal mesoderm-derived endothelial cells, it sheds light on how these mutations drive phenotypic outcomes through specific pathways, such as HIF-1α and VEGF-A signaling, while also identifying potential therapeutic targets. A strong aspect of the study is the use of embryonic models, which enables the investigation of disease onset in a context that closely resembles the in vivo environment. This is particularly valuable for congenital disorders, where native developmental cues are an integral aspect of disease progression. The study also integrates advanced techniques, including single-cell RNA sequencing, to dissect the cellular and molecular responses induced by the Pik3caH1047R mutation. Moreover, from a translational perspective, it provides novel therapeutic strategies for these diseases. Limitations of the study are (1) unclarity of the main question authors try to address, and main conclusions dereived thereof; (2) the different parts of the manuscripts are not well connected, not clear the rationale; (3) scRNAseq analysis is underdeveloped; (4) characterization of the preclinical model is not provided.

      Audience:

      The findings presented here interest specialized audiences within developmental biology, vascular biology, and congenital disease research fields, and clinicians by providing new therapies to treat vascular anomalies. Moreover, the study's integration of single-cell and in vivo models could inspire further research in other contexts where understanding clonal behavior and signaling pathways is critical.

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

      This paper focuses on vascular malformations driven by PI3K mutation, with particular interest on the vascular defects localized at head and neck anatomical sites. The authors exploit the H1047R mutant which has been largely demonstrated to induce both vascular and lymphatic malformation. To limit the effect of H1047R to tissues originated from cardiopharinegal mesoderm, PI3caH1047R mice were crossed with mice expressing Cre under the control of the promoter of Ils1 , a transcription factor that contributes to the development of cardiopharinegal mesoderm-derived tissues. By comparing the embryo phenotype of this model with that observed by inducing at different times of development the expression of PI3caH1047R, the authors conclude that Isl-Cre; PI3caH1047R; R26R-eYFP model recapitulates better the anatomical features of human vascular malformations and in particular those localized at head and neck. In my opinion the new proposed model represents a significant progress to study human vascular malformations. Furthermore, scRNA seq analysis has allowed to propose a mechanism focused on the role of HIF and VEGFA. The authors provides partial evidences that HIF and VEGFA inhibitors halt the development of vascular malformation in VeCAdCre; Pik3caH1047 mice. This experiment is characterized by a conceptual mistake because bevacizumab does not recognize murine VEGFA (see for instance 10.1073/pnas.0611492104; 10.1167/iovs.07-1175. This error dampens my enthusiasm

      CRITICISM

      1. Fig 1A. E13.5 corresponds to the early phase of vascular remodelling. Which is the phenotype at earliest stages (e.g. 9.5 or 10.5)

      Response:

      Thank you very much for your comment. We have created new Supplemental Figure 2, which demonstrates that no obvious phenotype is observed in mutant embryos at E9.5 and E11.5, and that the survival limit of these mutant embryos is around E13.5 to E14.0.

      In response to Reviewer 1’s question, previous study(Hare et al, 2015) have shown that on a B6 background, this mouse model exhibits an earlier onset of phenotype, resulting in early lethality. However we selected a mixed background of B6 and ICR, as we believe that a heterogeneous genetic background more accurately reflects the diversity of human pathology. We examined five pregnant females, which yielded approximately 30 embryos, of which only two survived until E14.0. Based on these observations, we consider E13.5 to E14.0 to be the appropriate survival limit (see Supplemental Figure 2G for additional details).

      We have described this in the Results section as follows:

      Whereas clear phenotypes were evident at E12.5 and E13.5, no pronounced external abnormalities were observed at E9.5 or E11.5 (Supplemental Figure 2A–B). Similarly, histological examination revealed no significant differences in the short-axis diameter of the PECAM+ CV or in the number of Prox1+ LECs surrounding the CV between control and mutant embryos at E11.5 (Supplemental Figure 2C–F). We also assessed Tie2-Cre; R26R-Pik3caH1047R mutant embryos at E14.0 from five pregnant mice. Only two embryos were alive at this stage, and both showed severe edema and hemorrhaging, indicating they were nearly moribund. These observations suggest that the critical point for survival of these mutant embryos lies between E13.5 and E14.0 (Supplemental Figure 2G). (Page 5, lines 157-165)

      Fig 1,2,3. The analysis of VEGFR2 expression is required. This request is important for the paradigmatic and non-overlapping role of this receptor in early and late vascular development. Furthermore ,these data better clarify the mechanism suggested by the experiments reported in fig 5 (VEGFA and HIF expression)

      __Response: __

      Thank you very much for your comment. For each mouse presented in Figures 1, 2, and 3, we performed VEGFR2 immunostaining on serial sections corresponding to each figure and created a new Supplemental Figure 9. VEGFR2 was broadly expressed in both vascular and lymphatic endothelial cells in control and mutant embryos.

      We have described this in the Results section as follows:

      Furthermore, to verify whether VEGF‐A can act via VEGFR2, we performed VEGFR2 immunostaining on several mouse models: Tie2‐Cre; R26R‐Pik3caH1047R embryos (E13.5, corresponding to Figure 1), CDH5‐CreERT2; R26R‐Pik3caH1047R embryos (tamoxifen administered at E9.5 and analyzed at E16.5, corresponding to Figure 2), and Isl1‐Cre; R26R‐Pik3caH1047R embryos (E11.5 and E13.5, corresponding to Figure 3). In all cases, both control and mutant embryos exhibited widespread VEGFR2 expression in blood and lymphatic vessels at early and late developmental stages (Supplemental Figure 9A-R’). These findings suggest that Pik3caH1047R may act in an autocrine manner, at least in part via the VEGF‐A/VEGFR2 axis in endothelial cells, potentially explaining the observed phenotype. (Page 11, lines352-361)

      As done in Fig 1,2 and 3, data quantification by morphometric analysis is also required for results reported in supplemental figure 3

      __Response: __

      Thank you for your comment. We have now added additional statistics and graphs for clarity, which are presented as Supplemental Figure 4.

      Lines 166-174. I suppose that the reported observations were done at E16.5. What happens later? It's crucial to sustain the statement at lines 187-190

      Response:

      At E9.5 and E12.5, we reduced the tamoxifen dose to one-fifth of the standard dose. After collecting embryos from approximately 10 pregnant females, we were only able to obtain three embryos at these stages. When tamoxifen was administered at E15.5, three embryos were obtained from two litters. In most cases, miscarriages occurred by E16.5, making further observation difficult. We focused on the time point around E16.5 because it is generally believed that the basic distribution of the lymphatic system throughout the body is established around this stage (Srinivasan et al, 2007; Maruyama et al, 2022).

      A similar experiment has been reported using T-CreERT2 to induce mosaic expression of Pik3caH1047R in the mesoderm, which resulted in subcutaneous venous malformations in mice at P1–P5 (Castillo et al, 2016). However, that study did not report whether the mice survived normally after birth. In fact, regarding the survival rate, the authors stated, “Our observations on the lethality and vascular defects in MosMes-Pik3caH1047R (T-CreERT2;R26R-Pik3caH1047R) embryos are similar to the previously reported phenotypes of ubiquitous or EC-specific expression of Pik3caH1047R in the developing embryo (Hare et al, 2015),” suggesting a high mortality rate when Pik3caH1047R is expressed using Tie2-Cre. Moreover, according to Hare et al., analysis of 250 Tie2-Cre; R26R-Pik3caH1047R embryos revealed that all were lethal by E11.5. Thus, considering our results in conjunction with those from previous studies, it appears that expression of Pik3caH1047R in the mesoderm or endothelial cells during embryonic development results in the death of most embryos before birth.

      We have supplemented the Results section with the following details:

      Since the standard tamoxifen dose (125 mg/kg body weight) leads to miscarriage or embryonic death within 1–2 days, we diluted it to one-fifth of the original concentration. (Pages 5-6, lines 175-177)

      scRNAseq was performed at E13.5 (Fig 4). It's mandatory to perform the same analysis at E16.5, which corresponds to the phenotypic analysis shown in fig 3. This experiment is required to understand how hypoxia and glycolysis genes changes along the development of the vascular malformation.

      __Response: __

      Thank you very much for your comment. First, regarding the experiments using Isl1‐Cre, we would like to clarify that the survival aspect was not adequately addressed. Our Isl1‐Cre embryos die between E13.5 and E14.0, which makes it practically impossible to perform single‐cell analysis beyond this stage (please refer to the newly added Figure 4N). Similarly, for experiments using CDH5‐CreERT2, the limited number of embryos obtained renders further analysis extremely challenging. Additionally, we have supplemented the Results section with the following description:

      These Isl1-Cre; R26R-Pik3caH1047R mutant embryos likely died from facial hemorrhaging between E13.5 and E14.0 (Figure 3N). (Page 7, lines 236-237)

      Further analysis at later embryonic stages proved challenging. Consequently, we aimed to investigate the effects of Pik3caH1047R on endothelial cells by comparing gene expression at E10.5 with that at E13.5. We performed single‐cell RNA sequencing on E10.5 embryos from both the control (Isl1-Cre; R26R-eYFP) and mutant (Isl1-Cre; R26R-eYFP; R26R-Pik3caH1047R) embryos. Unfortunately, the quality of both datasets was insufficient for reliable analysis. In the control sample, only 40.3% of reads were assigned to cell‐associated barcodes—substantially below the ideal threshold of >70%—with an estimated 790 cells and a median of 598 genes per cell. Similarly, in the mutant sample, only 37.0% of reads were associated with cells, despite an estimated cell count of 7,326 and a median of only 526 genes per cell. These metrics indicate that both datasets were severely compromised by high levels of ambient RNA or by a significant number of cells with low RNA content, precluding robust downstream analysis. This may be due to the fact that immature cells are particularly susceptible to damage incurred during FACS sorting and transportation to the analysis facility. Moreover, the relatively low number of control endothelial cells at E13.5 led us to conclude that performing similar experiments at earlier stages would be difficult. Despite our best efforts, we acknowledge this as a limitation of the present study.

      Lines 326-343. In this section the authors provide pharmacological evidences that HIF and VEGFa are involved in vascular malformation caused by H1047R . However , I'm surprised of efficacy of bevacizumab, which neutralizes human but not murine VEGFA. Genetech has developed B20 mAb that specifically neutralizes murine VEGFA. So the data shown require a. clarification by the authors and the experiments must be done with the appropriate reagent. Furthermore, which is the pharmacokynetics of these compounds topically applied?

      Response:

      Thank you very much for your comment. There are reports that bevacizumab exerts an in vivo inhibitory effect on neovascularization mediated by mouse Vegf-A (Lin et al, 2022). However, given the contentious nature of this issue, we conducted additional experiments. Due to the requirement for an MTA to obtain B20 mAb from Genentech—and considering the time constraints during revision—we opted to use a neutralizing antibody against mouse VEGF-A (clone 2G11) instead. This antibody has been shown to suppress the proliferation of mouse vascular endothelial cells in vivo (Mashima et al, 2021; Wuest & Carr, 2010) .

      The dosing regimen for 2G11 was determined based on previous studies (Surve et al, 2024; Churchill et al, 2022). Moreover, an example of effective local administration is provided in (Nagao et al, 2017). Since this product is an antibody drug, it is metabolized and does not function as a prodrug. Although the precise half-life of 2G11 is unknown, rat IgG2a antibodies generally have a circulating half-life of approximately 7–10 days in rats. However, when administered to mice, the half-life is often significantly reduced due to interspecies differences in neonatal Fc receptor (FcRn) binding affinity, with estimates in murine models typically around 2–4 days(Abdiche et al, 2015; Medesan et al, 1998) . However, in our model the injection is subcutaneous—almost equivalent to an intradermal injection (Figure 6B, C). Because this method is expected to provide a more sustained, slow-release effect (similar to the tuberculin reaction), the half-life should be longer than that achieved with intravenous administration. Consequently, we believe that sufficient efficacy is maintained in this model.

      Regarding LW-6:

      LW-6 is a small molecule that, due to its hydrophobic nature, is believed to freely cross cell membranes. Once inside the cell, it facilitates the degradation of HIF-1α, leading to reduced expression of its downstream targets (Lee et al, 2010). Although its half-life is estimated to be around 30 minutes, the active metabolites may exert sustained secondary effects (Lee et al, 2021). When administered intravenously, peak blood concentrations are reached within 5 minutes, making Cmax a critical parameter due to the rapid onset of action. In our experiments, we based the dosing regimen on previous studies (Lee et al, 2010; Song et al, 2016; Xu et al, 2022, 2024). While those studies administered doses comparable to or twice as high as ours via intravenous, intraperitoneal, or oral routes, our experimental design—in which a single dose was administered on Day 4 and samples were collected on Day 7—necessitated a single-dose protocol.

      Regarding Rapamycin:

      Several studies have demonstrated that local administration yields anti-inflammatory effects (Takayama et al, 2014; Tyler et al, 2011). Similar outcomes have been observed in vascular malformations (Boscolo et al, 2015; Martinez-Corral et al, 2020). Although the half-life of rapamycin is estimated to be approximately 6 hours following intravenous administration, it may be even shorter (Comas et al, 2012; Popovich et al, 2014).

      In light of these comments, we have revised Figure 6. Furthermore, the Results section pertaining to Figure 6 has been updated as follows:

      Hif-1α and Vegf-A inhibitors suppress the progression of vascular malformations.

      We next examined whether administering Hif-1α and Vegf-A inhibitors could effectively treat vascular malformations. Tamoxifen was administered to 3–4-week-old CDH5-CreERT2;R26R-Pik3caH1047R mice to induce mutations in the dorsal skin. Anti-VEGF-A, a Vegf-A neutralizing antibody; LW6, a Hif-1α inhibitor; and rapamycin, an mTOR inhibitor, were topically applied, and their effects were analyzed (Figure 6A). Both anti-VEGF-A and LW6 reduced the visible swelling in the dorsal skin, whereas the difference between the drug-treated and control groups was less pronounced with rapamycin (Figure 6B). In tamoxifen-treated Cre(–) mice, inflammatory cell infiltration and fibrosis were observed from the dermis to the subcutaneous tissue; however, there were no changes in the number of PECAM⁺ vasculatures or VEGFR3⁺ lymphatic vessels, including their enlarged forms, compared to the untreated control (Figure 6C–E). In contrast, tamoxifen administration to CDH5-CreERT2;R26R-Pik3caH1047R mice resulted in an increase in these vascular structures by day 4 (Figure 6C–E). At day 7, comparing mice with or without treatment using anti-VEGF-A, LW6, or rapamycin, the number of PECAM⁺ vasculatures was reduced in the treated groups; however, in the rapamycin group, the number of enlarged PECAM⁺ vasculatures did not differ from that in the untreated group (Figure 6F–M). Similarly, for VEGFR3⁺ lymphatic vessels, both anti-VEGF-A and LW6 induced a reduction, whereas rapamycin did not produce a statistically significant decrease (Figure 6N–U). (Page 11, lines 363-381)

      **Referees cross-commenting**

      The issues raised by refereee #1 related to the phenotype analysis are right. In my opinion the Isl model here proposed well mimic human pathology evenf the vascular damage at. head is not so evident

      Response:

      Perhaps the discrepancy arises from a terminological issue. According to the WHO Classification of Tumours, commonly used in clinical settings, the term "Head and Neck" refers to the facial and cervical regions (including the oral cavity, larynx, pharynx, salivary glands, nasal cavity, etc.) and excludes the central nervous system. The inclusion of the brain in Figure 1O-R may have led to some confusion. We included the brain because cerebral cavernous malformations are classified as venous malformations, and thus serve as an example of common sites for venous malformations in humans.

      To clarify this point, we have made slight revisions to the first part of the Introduction, as follows:

      They frequently manifest in the head and neck region—here defined as the orofacial and cervical areas, excluding the brain. (Page2, lines 52-53)

      Reviewer #2 (Significance (Required)):

      General assessment

      STRENGTH : a new mouse model seems to well recapitulate human vascular malformation. Possible key molecules have been identified

      WEAKNESS. The pharmacological approach to support the role of VEGFA e HIF is not appropriate

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    1. “Wife and child of destitute Ozark Mountains family.” Ben Shahn, Arkansas, October 1935.

      Esta fotografía tiene mucha profundidad. Se puede ver en los ojos de la madre que ella es muy seria, tratando de sobrevivir y dar toda su vida a sus hijos.

    2. With this reissue, I hope that a whole new generation sees these pictures within the context of the times and realizes the importance of a strong image and the impact it can have in helping people. I want people to continue making pictures with such power

      ¡Sí! Eso es lo que estaba diciendo y pensando.

    3. It was tough times. Fortunately, these pictures made a difference.

      Es una locura cuántas fotos pueden marcar la diferencia. ahora los ves todos los días y en todas partes, pero en aquel entonces eran muy limitados y unas pocas fotos podían hacer mucho.

    4. She said that something drew her to a certain tent in the camp, where she found a lady with her seven children. The family had sold the tires on their car to buy food, which was running low. They were essentially stuck.

      Varias cosas me llamaron la atención de esta historia y estas imágenes. Primero, hay tantos viviendo allí sin comida y qué triste es, y segundo, ¿por qué la madre tendría siete hijos en tiempos tan escasos? Sé que el control de la natalidad no era una gran opción entonces, pero ¿vivían cómodamente antes?

    5. At the height of the Great Depression, scores of US citizens found themselves homeless and without work.

      Esta foto realmente resalta la emoción de la situación. Mostrando a una madre soltera con dos hijos pequeños y sin hogar. Lo que más me llama la atención es la mirada en sus ojos y la desesperación.

    6. rural hom

      Esta fotografía me llamo la atención por la sencilles de la casa o el lugar en donde están, es un lugar bastante básico que parece tener solo lo necesario, esto me llamo la atención porque hoy en día a la gente le importa mucho como se presenta su casa, las decoraciones las ediciones caras y lo que otra gente piense se su hogar.

    7. movie theater,”

      Esta fotografía me llamo la atención por la manera en que la gente está vestida solo para ver una película. es muy sorprendente pensar que antes ir a una película se significaba salir muy elegantes y bien vestidos, cuando ahora en día ver una película se considera algo bastante casual.

    8. Family of migrant packinghouse workers in their quarters, Homestead, Florida.” Marion Post Wolcott, February 1939.

      La moda circula, pero algunas cosas son para siempre, como los overalls. Estos niños los trajeron en 1939, mis papás los usaban en 1970 y yo todavía los uso en 2025. Me pregunto de qué tipo de material están hechos: mezclilla, algodón, poliéster?

    9. “In front of the movie theater,” Russell Lee, Chicago, April 1941.

      Esta imagen parece ser de una película antigua. La manera de vestir tan formal, el filtro blanco y negro, y las entradas de los niños siendo solo 10 centavos es muy interesante. Es loco que esta foto se haya tomado hace casi 100 años.

    10. Today, it’s ironic to think that California is worried about migrants coming in from Mexico, when in the 1930s they were trying to stop migrants from coming in from Nebraska!

      La historia siempre se repite, desafortunadamente. Los migrantes son una parte crucial de nuestro país y economía. Sin ellos, no puedo imaginar cómo será la vida. Espero que las personas tengan una mente más abierta y que se den cuenta de que solo quieren proveer una vida mejor para ellos y sus familias, especialmente en este período de nuestra política.

    11. In the convict camp in Greene County, Georgia,” Jack Delano, May 1941. {"adType":"ex","adPos":"promo5-wide","wid":224,"size":[[970,90],[970,250],[728,90],"fluid"],"viewability":"high","platform":"desktop","zone1":"bfnews","renderLookahead":"x0.25"}

      Parecen tan contentos en el campo de convictos. Bailando, cantando, aplaudiendo, tocando la guitarra, parecen más optimistas que en las otras fotos donde no están encarcelados. Estoy curiosa de qué hicieron para llegar a esta situación del campamento y cómo todavía tienen una perspectiva tan brillante a pesar de la situación?

    12. the FSA brought together what was easily the finest group of photographers ever assembled in the United States, if not the world.{"adType":"ex","adPos":"promo2-wide","wid":221,"size":[[970,90],[970,250],[728,90],"fluid"],"viewability":"high","platform":"desktop","zone1":"bfnews","renderLookahead":"x0.25"}

      Qué fascinante que ensamblaron el mejor grupo de fotógrafos para este proyecto. Imagino que muchos eran de ciudades y no estaban muy afectados por las sequías, pero esta misión les enseñó un nuevo punto de vista en la vida que llevaron con ellos el resto de sus vidas.

    1. Data set 1:

      I’d reorganize these data into a horizontal bar graph although a vertical bar graph would also work. x-axis units = “Percentage of students “satisfied” or “very satisfied”” with tick marks from 0 to 100. y-axis units = “Major", with a unique bar for each of the four majors given in the table.

      Title: “Percentage of Students Satisfied With Their Major”.

    2. Data set 2:

      Since this data set compares two groups across time (12 months), I’d reformulate this information into a line graph with one line for Detroit and another line for Austin. The lines would need different colors and/or patterns (i.e., one solid line, one dashed). Both or the latter option is more accessible to color-blinded individuals and is hence recommended. The legend would contain a line segment for each of the two groups followed by “Detroit” or “Austin”. The x-axis units = “Month” with tick-marks for Sept through Aug. The y-axis units = “Energy used (average kilowatts per hour)” with tick marks from 0 to ~ 40-50 (depending on the intended audience / any conventional technicalities for reporting energy usage).

      Title = “Monthly Energy Usage in Detroit and Austin Buildings”

    3. Data set 3:

      Since multiple groups are being compared without regard to time, I would use a cluster bar graph with beef, meat, and poultry clustered together under two categories labeled underneath the x-axis: “Average calories” and “Average sodium content (in mg)”. The y-axis = “Average content” with tick marks from 0 to 500. The sample size can be in parentheses after each Type in the legend. Title = “Average Calories and Sodium Content of Different Hotdog Meat”

    4. Data set 4:

      Since two groups are compared with repect to time, I would reformulate this data set into a line graph with one line for “Revolution helmet” and another for “Standard helmet”. Each line would have a unique color and pattern labeled in a legend. The x-axis label = “Interval (weeks)” with tick marks for 1, 2, 3, and 4. The y-axis label = “Return to Play Rates (%)” with tick marks from 0 to 100.

      Title of the graph = “Return to Play Rates After Experiencing a Concussion When Wearing a Revolution vs. Standard Helmet”

    5. Figure 3.11a.

      The y-axis could be rescaled so it goes to 5 to make it clearer what “1 = poor; 5 = excellent” in the caption refers to. Additionally, rescaling the y-axis would make both the control and experimental groups more accurately look less effective than they currently do.

    6. Data set 4:

      I would use a horizontal bar chart for this. However, I would stack the colors on top of each other to make it more readable. An alpha value of 0.5 would make this easier to read. Title: Revolution vs Standard Hemet for Concussion Downtime x = percentages y = interval color = helmet labs( x = Percentage of Players Recovered y = Time to Recover color = Helmet Type )

    7. Data set 3:

      I would use another line graph for this. The number of brands analyzed isn't that important and can be put in a caption. Title: Average Calories and Average of Sodium in Common Hotdog Meats x = calories y = sodium color = type label( x = "Average Amount of Calories" y = "Average Amount of Sodium (mg)" color = "Hotdog meat type" ) Caption: Based on analysis of 20 beef hotdog brands. 17 meat hotdogs brands, and 17 poultry hotdogs brands.

    8. Data set 2:

      I would use a line graph as it shows a trend over time. Title: Energy used in a building in Detroit and Austin x = time y = energy color = location label( x = "Month" y = "Energy used (averaged kilowatts / hour) ) A legend should be created with color.

    9. Data set 1:

      I would use a bar chart. Title: Figure 1: Percentage of Students Satisfaction with their Majors x = major y = percent label( x = "Major" y = "Percent of students "satisfied" or "very satisfied" )

    10. Figure 3: Levelized costs (in dollars per megawatt hours of electricity) of five different power plants.

      I think this would be a good title for a graph. It seems detailed and introduces the y and x axis

    11. Figure 2 shows the average test scores of male and female students.

      Average test scores should once again be an x or y axis label instead of a caption.

    12. Figure 1: Average GPA (on 4-point scale) by hours studied per week of students at University X. Figure 2 shows the average test scores of male and female students. Figure 3: Levelized costs (in dollars per megawatt hours of electricity) of five different power plants. Table 1: Memory usage of five different web browsers with one tab open. Figure 4: Harley-Davidson is the most popular motorcycle. Table 2: Popularity of the top-selling motorcycle brands among registered motorcycle owners in Pittsburgh. Harley-Davidson is the most popular.

      This caption is too long and encapsulates two main points. Average GPA should be on the x or y axis. The hours studied per week at Uni X should be a title.

    13. E. Shorter women receive more unsolicited emails than taller women do (especially women above 5'10"), while the opposite trend is true for men.

      1

      Violates: - 1. The y-axis is different from women and men. - 2. Arguably for women and especially for men, the number of unsolicited emails vs. height is negligible or at least a far smaller difference than the line graph implies. - 3. The demographic (i.e., locale, profession, race) of participant pool is unclear other than the distinction between women and men. - 4. Given #2, the conclusions from the data are exaggerated.

    1. Voici un document de synthèse pour un briefing, basé sur les informations tirées de la source fournie concernant la délinquance des mineurs :

      • Travaux parlementaires du Sénat Le Sénat a pour missions principales le vote de la loi, le contrôle du Gouvernement et l'évaluation des politiques publiques. Les travaux parlementaires incluent divers rapports et documents de travail, notamment des rapports de commission d'enquête.

      • Commission d'enquête sur la délinquance des mineurs Un rapport de commission d'enquête sur la délinquance des mineurs a été déposé le 27 juin 2002. Jean-Pierre Schosteck était le président de cette commission et Jean-Claude Carle, le rapporteur.

      • Auditions et thèmes abordés La commission a mené des auditions de différents experts et acteurs concernés par la délinquance des mineurs :

        • Sociologues Sebastian Roché a récapitulé ce qu'est la délinquance des mineurs et a tenté de décrire les explications du phénomène. Laurent Mucchielli a parlé de l'état actuel de confusion du débat sur l'insécurité.

        • Cadre législatif et son adaptation Sebastian Roché a souligné l'ampleur des dysfonctionnements des administrations publiques et leur inadaptation aux réalités actuelles, mais il ne pense pas que la solution réside dans de nouveaux textes de loi. Alain Bauer a déclaré qu'il est toujours stupéfait du débat concernant l'ordonnance de 1945, car, selon lui, cette ordonnance, ainsi que celle de 1958, permettent de répondre à presque tous les problèmes. Denis Salas a estimé que les réponses législatives existent et qu'il est erroné de penser que l'ordonnance de 1945 n'a pas été modifiée depuis sa promulgation. D'autres estiment qu'il convient d'abroger l'ordonnance de 1945.

        • Acteurs et structures de prévention Sebastian Roché a souligné le manque de coordination entre les différentes structures de prévention.

        • Chiffres et statistiques Bruno Aubusson de Cavarlay a analysé en profondeur les chiffres relatifs à la délinquance juvénile. Il a rappelé quelques principes de bon usage des statistiques du ministère de l'intérieur, en soulignant qu'elles fournissent une mesure de l'activité des services de police bien plus qu'une mesure de la délinquance réelle. Philippe Lutz a indiqué qu'en Seine-Saint-Denis, 26 % des personnes mises en cause étaient mineures.

        • Politiques de prévention Alain Bauer a souligné le caractère flou, voire inexistant et dépourvu de moyens, de la prévention en France.

        • Ordonnance de 1945 Plusieurs intervenants ont commenté l'ordonnance de 1945 relative à l'enfance délinquante.

        • Victimes L'accompagnement des victimes tout au long de la procédure est mal assuré.

        • Centres éducatifs Il serait temps d'avancer sur une évaluation raisonnable de l'ordre de grandeur des besoins en matière de centres éducatifs renforcés.

        • Responsabilité parentale Lorsque les familles se montrent défaillantes, voire complices, les textes permettent déjà de les sanctionner.

        • Liens entre acteurs L'organisation de la synergie entre les acteurs doit se faire dans la durée, de manière permanente, pour créer l'habitude de travailler en commun.

        • Mesures et cohérence Il y a un problème de cohérence dans la réponse des adultes et il faut que tout le monde travaille de concert sur des cas concrets.

        • Signalements André Tanti s'est dit tout à fait favorable au signalement, voire à la plainte.

        • Tutelle des allocations familiales Plusieurs intervenants ont évoqué la mise sous tutelle des allocations familiales.

        • Rôle de l'école L'école doit transmettre le savoir-être et le comportement.

        • Comparution immédiate Certains proposent d'étendre la comparution immédiate aux mineurs.

        • Spécificités locales Dominique Buchert a présenté la situation à Strasbourg, notamment dans le quartier du Neuhof.

        • Mixité culturelle Nécessité d'introduire la mixité culturelle.

        • Cannabis Jean-Luc Saladin a souligné les effets du cannabis sur le cerveau des jeunes.

        • Sport Guillaume Marignan a souligné les vertus éducatives du sport.

      Ce document de synthèse offre un aperçu des discussions et des thèmes clés abordés lors des auditions de la commission d'enquête sur la délinquance des mineurs.

    1. Voici un sommaire de l'entretien avec Gabrielle Légeret, fondatrice de l'association "De l'or dans les mains", avec les horodatages:

      • Vrai ou faux sur la perception des métiers manuels [0:00]:
        • L'orientation vers les filières professionnelles est souvent perçue comme un échec [0:00].
        • L'intelligence manuelle est moins valorisée que l'intelligence intellectuelle [0:06].
        • Les jeunes ne s'intéressent plus aux métiers de l'artisanat (Faux) [0:11].
        • L'artisanat et les métiers manuels sont incompatibles avec l'innovation (Faux) [0:17].
        • Les métiers manuels sont associés à un manque d'intelligence [0:24].
        • La valorisation des métiers manuels pourrait réduire le chômage [0:31].
        • Les jeunes préfèrent les carrières intellectuelles aux manuelles (Faux) [0:37].
        • Les métiers manuels peuvent offrir des salaires compétitifs [0:42].
        • Les compétences manuelles sont de plus en plus demandées [0:48].
      • Interventions de "De l'or dans les mains" dans les collèges [1:07]:
        • Le mépris pour les métiers manuels est ancré dans la société et l'éducation [1:09-1:18].
        • Comment redonner leurs lettres de noblesse aux métiers manuels? [1:35]
        • Présentation du programme phare "Je découvre les métiers manuels" [2:04].
        • Programme de 15h en classe de 5ème [2:10].
        • Création de liens entre les programmes scolaires et la pratique artisanale [2:19-2:26].
        • Intervention d'artisans dans les classes pour fabriquer des objets liés au programme scolaire [2:41-2:50].
        • Découverte de neuf métiers à travers la pratique artisanale [3:18-3:27].
        • Les élèves découvrent qu'ils ont des pouvoirs avec leurs mains et réussissent à l'école [3:55-4:02].
        • Les interventions changent le regard des enseignants sur leurs élèves [4:02-4:11].
        • Les élèves en difficulté peuvent se retrouver en situation de réussite [4:11].
        • Un bon élève peut découvrir la menuiserie comme option [5:14-5:25].
        • Le système scolaire n'est adapté qu'à une certaine typologie de cerveaux [5:25-5:43].
        • Importance de la manipulation et de l'expérimentation pour l'apprentissage [5:43-5:52].
        • L'intelligence du geste sollicite aussi l'intellect [6:14-6:19].
        • Il faut apprendre aux enfants à connecter leur corps à ce qu'ils vivent [6:27-6:41].
      • Reconnecter la tête et les mains [7:00]:
        • L'opposition entre la tête et les mains fait beaucoup de mal et place des jeunes en situation d'échec scolaire [7:17-7:23].
        • Nécessité de replacer l'intelligence manuelle au cœur du projet de société [7:32-7:39].
        • Importance d'une culture matérielle face aux logiques de consommation [7:39-7:58].
        • Faire des études supérieures n'assure plus une bonne place dans la société [8:10-8:16].
        • Les métiers manuels peuvent répondre à la transition écologique et retisser du lien social [8:42-9:01].
        • Les métiers de demain sont des métiers de la transition écologique et manuels [9:15-9:35].
        • Importance de révéler les talents manuels des jeunes à équivalence avec les talents intellectuels [9:42-9:58].
        • La pratique manuelle doit irriguer les savoirs fondamentaux [10:20-10:41].
        • Importance de donner aux élèves un réel choix concernant ce qu'ils ont envie d'expérimenter [11:53-12:13].
        • Les jeunes ne vont pas vers les métiers manuels par manque d'information et de valorisation de leurs compétences manuelles [12:21-12:50].
      • Expliquer l’opposition [13:23]:
        • Les origines de la dévalorisation des métiers manuels sont complexes et typiquement françaises [13:23-13:39].
        • En Suisse et en Allemagne, l'orientation vers une filière professionnelle est moins dévalorisée et les passerelles sont plus intenses [13:39-14:13].
        • Décisions politiques en France de délocaliser les industries et de devenir une société de services [14:22-14:34].
        • Les métiers manuels étaient souvent pénibles [14:34-14:47].
        • Il y a aussi des enjeux de pénibilité dans les métiers de bureau [14:53-15:06].
        • Importance de sortir de la pénibilité dans les entreprises et d'avoir des salaires décents [15:16-15:53].
        • La France place les enfants en situation de compétitivité et de comparaison dès le plus jeune âge [16:29-16:45].
        • Sur un chantier, il y a une responsabilité collective face au métier et à la société [16:45-17:38].
        • Importance de faire des projets collectifs à l'école [17:55-18:13].
        • Une société qui ne regarde pas de façon condescendante les métiers va mieux [18:19-18:41].
        • Beaucoup de jeunes se retrouvent à 40 ans à vouloir se reconvertir vers des métiers manuels [18:41-19:13].
        • Un salarié sur trois dans les entreprises du patrimoine vivant est en reconversion [19:20-19:29].
        • Il ne faut pas attendre d'avoir prouvé quelque chose à la société pour choisir un métier manuel [20:05-20:19].
      • Changer la donne [20:27]:
        • Réintégrer la pratique manuelle à l'école [20:48-20:53].
        • L'objectif est de replacer l'intervention manuelle dans le système éducatif au profit des jeunes [21:19-21:40].
        • Favoriser le développement de soi et la réussite académique [21:40].
        • S'adosser aux politiques publiques éducatives existantes et venir au service des enseignants et des élèves [22:33-22:48].
        • Changer le regard des pouvoirs publics, de l'opinion publique, des parents, des enfants, des enseignants [22:57-23:02].
        • Mobiliser l'ensemble de la société sur cette question [23:29-23:36].
      • Pour un autre futur [23:47]:
        • Comment imaginer un système où l'orientation soit au service de la réalisation des citoyens? [23:47-23:58]
        • Il faut désacraliser l'orientation et permettre de se tromper et de recommencer [24:06-24:19].
        • Valoriser et évaluer d'autres compétences que les compétences académiques [24:42-24:50].
        • Faire rentrer plus le monde du travail et de l'entreprise au collège [25:23-25:32].
        • L'orientation a besoin d'être vécue [26:02].
        • Mettre en pratique des expériences, des mini-entreprises [26:22-26:32].
        • Faire en sorte que l'école soit plus ancrée sur son territoire et réponde à des projets d'intérêt général [26:38-26:50].
        • Travailler au bien commun [27:24-27:33].
        • Revaloriser le métier d'enseignant [28:00-28:05].
        • Permettre et donner du temps et des moyens aux enseignants d'innover [29:10-29:19].
        • Faire rentrer des artisans au sein du collège et faire travailler des artisans et des enseignants ensemble [30:12-30:34].
        • Le monde de l'entreprise aurait tout intérêt à mieux venir pénétrer nos classes [30:45-30:50].
        • Importance des partenariats avec l'Onisep et sa plateforme Parcours Avenir [31:34-31:43].
        • Il y a des choses à changer et des prises de conscience [32:08-32:26].
        • Le corps ne doit pas être utilisé qu'en sport [33:10-33:19].
        • Importance de l'agencement des salles et de la pratique dans les cours [33:19-33:33].
        • Apprendre aux enfants comment fonctionne leur cerveau et les encourager à développer des méthodes d'apprentissage qui mobilisent le corps [34:33-34:46].
        • Une société qui est en mouvement dans son corps est une société qui est bien dans son corps [35:09-35:15].
        • Importance d'une éducation à la nourriture [35:21-35:36].
        • Retrouver un regard global sur ce qu'est la vie et ce dont on a besoin pour créer des individus en santé mentale et physique [36:13-36:31].
        • Recréer un rapport holistique au monde qui nous entoure [36:59-37:05].
        • Donner aux enfants les clés pour s'ancrer dans la société et avoir le pouvoir d'agir [38:06-38:14].
        • Faire l'expérience du sensible et redonner la place au sensible [39:16-39:22].
    1. Briefing Document : Auditions Commission des Affaires Culturelles - 25 Septembre 2024 Objet : Examen des auditions diverses de la Commission des Affaires Culturelles, axées sur les politiques de jeunesse, la vie associative et les événements culturels, notamment la cérémonie d'ouverture des Jeux Olympiques.

      Date : 2024-10-27

      Sources : vidéo "Commission des affaires culturelles : Auditions diverses - Mercredi 25 septembre 2024

      Thèmes Principaux et Idées Clés

      Politiques de Jeunesse et Vie Associative :

      Vision Silotée et Interministérielle : La nécessité d'une vision plus transversale des politiques de jeunesse est soulignée, avec une critique de l'approche actuelle "silotée". " On a une vision extrêmement, je n'aime pas ce mot mais c'est le mot qui convient le mieux, silotée de la jeunesse. On a des politiques de jeunesse dans les collectivités, des politiques de jeunesse au ministère de l'éducation nationale, des politiques de jeunesse dans le sport etc, etc."

      Accès à l'Autonomie : Discussion sur les politiques favorisant l'autonomie des jeunes, avec une référence à des recommandations issues de contributions externes. Difficultés du Secteur Associatif Local : Préoccupation concernant la " tragération du secteur associatif local" due à la généralisation des marchés publics et des appels à projets. Bien que les appels à projets puissent soutenir l'innovation, ils peuvent aussi créer des difficultés.

      Dispositifs existants : Le document mentionne un dispositif créé en 2022 pour les jeunes de 16 à 25 ans (jusqu'à 30 ans pour les personnes handicapées), offrant un stage, une formation ou un service civique et une allocation. "Ce dispositif a rencontré un réel succès, plus de 702 000 jeunes en ont en effet bénéficié entre mars 2022 et avril 2024."

      Engagement des Jeunes : Il n'y a pas de crise de l'engagement des jeunes mais une nécessité de mieux le reconnaître et le valoriser. Le SNU (Service National Universel) est questionné dans sa capacité à favoriser cet engagement.

      SNU (Service National Universel) :Le document relate des inquiétudes concernant le SNU. "Son coût est une dérive estimée entre 3,5 et 5 milliards d'euros, et jusqu'à 10 milliards en intégrant les coûts pour les autres acteurs, tels que les collectivités territoriales." Nécessité de mieux calibrer le rôle des services de l'État et d'améliorer la gouvernance. "Sur le pilotage, sur le besoin de mieux calibrer le rôle des services de l'Etat, sur le côté gouvernance"

      Articulation complexe avec d'autres outils d'engagement comme les services civiques. "sur l'articulation qu'il va devoir falloir penser entre le SNU et les autres outils d'engagement pour justement éviter cette logique concurrentielle qui peut y avoir avec les services civiques,"

      Difficulté à définir le SNU : "Enfin, sur les jeunes, la problématique qu'on identifie aussi aujourd'hui, c'est qu'on ne sait pas trop ce que c'est ce SNU, on le définit toujours par la négative, ce n'est ni la colonie, l'école, ni le militaire."

      Il y a des points d'alerte concernant le pilotage, le rôle des services de l'Etat, la gouvernance, et l'articulation avec les outils existants.

      Culture et Jeux Olympiques :

      Cérémonie d'Ouverture : La cérémonie d'ouverture est reconnue comme un outil d'expression d'une identité et de cohésion, valorisant le patrimoine et l'histoire de la France. On loue le fait que les cérémonies étaient "loin des clichés touristiques mais avec tout ce qui nous ressemble et nous rassemble, cet esprit français impertinent, un peu submersif."

      Réussite Artistique et Culturelle des Jeux : Les Jeux Olympiques ont permis de mettre en avant l'universalisme et de lutter contre les fractures sociales. "Et cela, on le doit en partie au bilan artistique et culturel des Jeux."

      Diversité et Inclusion : L'importance de la diversité et de l'inclusion, y compris l'accessibilité à la culture pour les personnes handicapées, est soulignée.

      Critiques et Polémiques : Les polémiques autour de la cérémonie d'ouverture, notamment, sont mentionnées.

      Lien Culture et Politique: Le document affirme que "La culture et l'art sont politiques par essence, dans ce qu'ils racontent, dans les récits et imaginaires qu'ils mettent en scène."

      Critiques envers les choix musicaux : Des interrogations ont été émises sur les choix musicaux, notamment "Le relative silence de voix française bien établie, tels que les Souchon, Wulzik, Allogero, Benabar, Badiou, Pravi."

      Théâtre et Culture Vivante :

      Importance du Théâtre Public : Le théâtre public est vu comme un outil essentiel pour la société, permettant de retrouver une forme de discernement et de développer un esprit critique.

      Lien entre Culture et Pouvoir : L'histoire montre que tous les grands pouvoirs se sont appuyés sur la culture.

      Accès à la Culture : Importance de rendre la culture accessible à tous, en allant au public et en proposant des tarifs abordables.

      Héritage Culturel : La nécessité de préserver et de développer les lieux culturels existants est mise en avant.

      Points d'Attention

      SNU : Les interrogations autour du SNU, de son coût, de sa définition et de sa place par rapport aux autres dispositifs d'engagement nécessitent un suivi attentif.

      Financement des associations locales : La "tragération" du secteur associatif est un sujet préoccupant.

      Vision transversale des politiques de jeunesse : L'approche "silotée" actuelle risque de limiter l'efficacité des actions menées.

      Garantir l'accès à la culture : S'assurer que la culture est accessible à tous, y compris aux personnes handicapées et aux populations rurales.

      Conclusion

      Les auditions de la Commission des Affaires Culturelles mettent en lumière des enjeux importants concernant les politiques de jeunesse, la vie associative et la culture.

      Elles soulignent la nécessité d'une approche transversale, d'un soutien accru au secteur associatif local, et d'une réflexion approfondie sur le rôle de la culture dans la société, notamment dans la construction d'un esprit critique et d'une identité collective.

    1. Voici les points saillants concernant la lutte contre la dépendance numérique chez les enfants, d'après le compte rendu de la Commission des affaires culturelles et de l'éducation du 27 novembre 2024:

      • Audition et Examen La commission a entendu M. Jean-Karl Deschamps et Mme Bernadette Groison sur le rapport "Réussite à l'école, réussite de l'école". Ensuite, elle a examiné une proposition de résolution européenne visant à lutter contre les addictions numériques chez les enfants.

      • Proposition de résolution européenne (PPRE) La PPRE vise à définir une position publique et à inviter l'exécutif à la défendre au niveau européen, car l'Union européenne est en mesure d'imposer une réglementation protectrice et juridiquement contraignante aux géants du net. L'objectif est que l'Assemblée nationale s'associe à l'initiative du Parlement européen, qui a adopté une résolution sur la conception addictive des services en ligne et la protection des consommateurs.

      • Constat et chiffres clés

        • Les parents sont souvent démunis face à l'usage massif et précoce du numérique par leurs enfants.
        • En 2021, 82 % des enfants de 10 à 14 ans vont régulièrement sur internet sans leurs parents, et 95 % des 15 à 17 ans font de même.
        • La première inscription sur un réseau social intervient en moyenne vers 8 ans et demi, et plus de la moitié des enfants de 10 à 14 ans y sont présents.
        • Les jeunes de 16 à 24 ans passent en moyenne plus de sept heures par jour sur internet.
        • Un enfant ou un jeune sur quatre a une utilisation problématique de son smartphone, indiquant une dépendance.
        • En France, un enfant sur dix souffre de troubles liés à l'utilisation excessive des écrans.
        • 22 % des adolescents présentent des symptômes d'addiction numérique.
      • Causes et mécanismes de l'addiction

        • Le cerveau recherche des satisfactions simples et rapides, ce que les écrans peuvent facilement provoquer par la libération de dopamine.
        • Les services numériques sont conçus intentionnellement pour capter l'attention et créer une dépendance, avec des caractéristiques comme le défilement infini, la lecture automatique, la restriction temporelle de contenus (syndrome Fomo), et les techniques de gratification sociale (bouton "j'aime").
        • Ces techniques de manipulation visent à maximiser la durée d'utilisation et les revenus des entreprises de la tech.
      • Actions et réglementations existantes

        • L'Union européenne a adopté le règlement sur les services numériques (DSA) en octobre 2022, mais il ne traite pas directement de l'addiction aux écrans.
        • Le Parlement européen a adopté une résolution sur la conception addictive des services en ligne.
      • Propositions et objectifs

        • Interdire les caractéristiques de conception addictive des services en ligne à l'échelon européen.
        • Donner à l'utilisateur la possibilité de désactiver facilement les paramètres addictifs dès l'inscription à un service en ligne.
        • Consacrer un droit numérique à ne pas être dérangé.
        • Imposer aux fournisseurs de services en ligne une conception éthique des caractéristiques techniques.
        • Mener des actions combinant régulation, éducation et sensibilisation.
      • Arguments en faveur de la PPRE

        • Nécessité d'une action collective, urgente et coordonnée au niveau européen.
        • La régulation de l'usage des réseaux sociaux par les mineurs ne peut reposer uniquement sur les parents.
        • Importance de la régulation, de la prévention et de la sensibilisation.
        • La France doit peser de tout son poids pour une réglementation protectrice de la santé des mineurs.
      • Amendements proposés

        • Souligner le rôle majeur de l'éducation dans la prévention des risques liés au numérique.
        • Financer prioritairement les recherches publiques indépendantes sur les addictions numériques.
        • Prendre en compte l'usage des écrans individuels dans les établissements scolaires.
        • Insérer une charte parentale sur le numérique dans le carnet de santé.
        • Instaurer une obligation de formation des enseignants aux dangers du numérique.
        • Interdire l'exposition aux écrans des enfants de moins de 3 ans.
    1. Ce document est le procès-verbal d'une réunion de la Commission des affaires culturelles, axée sur deux sujets principaux.

      Premièrement, il aborde la réussite scolaire et les moyens de lutter contre les inégalités sociales dans l'éducation, en mettant en avant des propositions pour favoriser la mixité sociale et adapter les objectifs de l'école aux besoins contemporains.

      Deuxièmement, il traite de la lutte contre les addictions numériques chez les enfants, examinant une proposition de résolution européenne visant à rendre les plateformes numériques moins addictives et à protéger les jeunes utilisateurs.

      La réunion comprend des discussions, des amendements et des votes sur ces propositions, soulignant un effort concerté pour améliorer le système éducatif et protéger les enfants des dangers du numérique.

      Voici un sommaire minuté des principaux points abordés dans la source, organisés chronologiquement :

      • 00:13-03:24 : Répétition de la phrase : "Le gouvernement est en train de dépasser les conditions de la situation".

      • 10:27-11:13 : Remerciements pour la présentation d'un rapport publié en juin 2024, jugé riche et original, appelant à refonder la conception de l'école.

      • 11:13-12:00 : Proposition de loi visant à garantir la mixité sociale au sein des établissements scolaires, notamment par la réduction des dotations en cas de non-respect des contrats d'objectifs et de moyens.

      • 12:00-12:28 : Questions sur l'application pratique de l'indice de position sociale comme indicateur de mixité et sur les préconisations en matière de formation initiale des enseignants.

      • 12:41-13:29 : Évocation des territoires ultramarins et de leurs situations particulièrement dégradées, avec une question sur les mesures à prendre pour adapter les politiques éducatives à ces réalités.

      • 14:53-15:03 : Présentation des résultats des travaux du Conseil économique, social et environnemental (CSE) sur le dossier intitulé "Réussir à l'école réussite de l'école".

      • 15:03-15:20 : Information sur la méthodologie du travail, basée sur une cinquantaine d'auditions et d'entretiens.

      • 16:16-16:21 : Mention d'un vote à l'unanimité (moins une voix contre) sur le rapport du Conseil économique, social et environnemental.

      • 16:56-17:05 : Présentation du rapport et avis du Conseil économique, social et environnemental (CESE), contenant 20 préconisations sur l'école.

      • 17:05-17:23 : Définition du terme "école" recouvrant le périmètre de l'obligation scolaire, annulée en 2015 par le Conseil économique.

      • 21:57-22:17 : Proposition de généraliser des dispositifs existants et de redéfinir la carte scolaire et celle de l'éducation prioritaire afin d'agir contre la concentration.

      • 24:01-25:00 : Constat que l'école est traversée par les problématiques de la société et soumise à des injonctions contradictoires.

      • 25:56-26:24 : Axe de préconisation consacré à l'amélioration des conditions de scolarisation des élèves.

      • 27:53-28:11 : Mention d'un plan spécifique d'urgence pour les territoires ultramarins, détaillé en annexe.

      • 29:31-29:41 : Remerciements et présentation de l'échange entre l'Assemblée nationale et le Conseil économique, social et environnemental.

      • 30:02-30:21 : Le groupe rassemblement national estime que le système éducatif doit être repensé et que son organisation actuelle est un problème.

      • 33:17-33:27 : Ce rapport constitue une base solide pour une éducation inclusive et performante.

      • 33:44-34:15 : Salutation du CSE pour son rapport et son attention à la question des métiers de l'Éducation.

      • 35:19-35:41 : Satisfaction de voir reprise la préconisation des contrats d'objectifs et de moyens.

      • 35:41-35:52 : Question sur les actions envisagées par le CESE dans les semaines et mois à venir.

      • 36:06-36:21 : Satisfaction de constater que les constats politiques sont partagés par l'expertise de la société civile.

      • 37:05-38:26 : Question sur la pertinence de faire coïncider la géographie de l'éducation prioritaire avec celle des quartiers prioritaires de la politique de la ville (QPV) et l'utilisation de l'IPS.

      • 45:47-46:23 : Question sur l'opposition entre la réussite des élèves et la méritocratie.

      • 49:56-50:30 : Question sur les moyens de faire du lycée professionnel une option à part entière.

      • 52:56-54:05 : Réflexion sur les objectifs de la société en matière d'éducation et sur l'évolution de ces objectifs au fil du temps.

      • 59:29-1:00:02 : Discussion sur la mixité et la question de l'enseignement privé.

      • 1:00:40-1:01:14 : Explication du fonctionnement des contrats avec l'enseignement privé et de la conditionnalité à une redevabilité financière.

      • 1:01:50-1:02:34 : Explication de la position du CESE sur la non-superposition de la carte de l'éducation prioritaire avec les QPV.

      • 1:06:30-1:07:01 : Invitation à une prochaine réunion au CESEU le 28 janvier.

      • 1:09:24-1:09:41 : Préoccupation concernant la méritocratie et l'idéalisme républicain.

      • 1:09:53-1:10:04 : Question sur les moyens de garantir l'égalité des chances et de rétablir la confiance dans le système éducatif.

      • 1:10:04-1:10:22 : Question sur la place des collectivités territoriales dans le processus de décision concernant l'éducation.

      • 1:11:05-1:11:34 : Constat que les communes sont peu associées aux décisions malgré leur engagement financier et leur responsabilité dans le cadre éducatif.

      • 1:11:34-1:11:51 : Proposition de faire évoluer la gouvernance du premier degré et les rôles de chacun dans la communauté éducative.

      • 1:13:49-1:14:35 : Question sur les effets de l'interdiction des téléphones portables dans les écoles et collèges, et sur la généralisation de la pause numérique.

      • 1:15:21-1:15:52 : Question sur les préconisations pour prévenir et lutter contre le décrochage scolaire.

      • 1:16:30-1:17:01 : Question sur la nécessité de remplacer le Conseil supérieur des programmes (CSP).

      • 1:18:41-1:19:21 : Question sur la prise en compte des caractéristiques des territoires ruraux dans la définition des critères d'évaluation.

      • 1:19:57-1:20:13 : Question sur la reconsidération sociale du métier d'enseignant.

      • 1:20:13-1:20:28 : Absence d'évocation de deux événements majeurs bouleversant l'institution scolaire dans le rapport.

      • 1:26:10-1:26:29 : Question sur le bilan des réunions favorisant la souscription de projets au sein du Fonds d'innovation pédagogique.

      • 1:26:29-1:27:04 : Question sur l'impact du dédoublement des classes depuis 2017.

      • 1:29:04-1:29:26 : Question sur l'organisation des relations avec les collectivités territoriales pour favoriser la réussite des élèves.

      • 1:29:26-1:30:15 : Question sur la formation à l'éducation à la sexualité et à la vie affective, et sur la réponse aux familles anti-choix.

      • 1:31:08-1:31:19 : Nécessité de ne pas se désintéresser de certains sujets même s'ils ne sont pas écrits dans le rapport.

      • 1:31:19-1:31:45 : Précision sur la question des observatoires.

      • 1:31:45-1:32:06 : Importance de repenser les finalités et le système éducatif, et d'avoir une instance prenant en compte la diversité des approches.

      • 1:32:34-1:32:50 : Le décrochage n'est pas traité précisément mais l'importance des conditions de scolarisation est soulignée.

      • 1:38:20-1:39:10 : La titularisation des enseignants devrait se faire au niveau master.

      • 1:39:10-1:39:31 : Difficultés et différences entre le premier et le second degré, sujet des contractuels à réouvrir.

      • 1:39:48-1:40:05 : L'IPS est un indicateur fiable mais d'autres indicateurs peuvent être utilisés.

      • 1:40:29-1:40:59 : D'autres outils et pistes sont à explorer, notamment en milieu rural.

      • 1:42:13-1:43:25 : Nécessité de changer de paradigme pour favoriser la mixité.

      • 1:44:40-1:45:42 : L'uniforme n'a pas été retenu car il semble plus important de traiter les causes que les stigmates.

      • 1:51:17-1:51:22 : Reprise de la séance.

      • 1:51:22-1:51:34 : Examen de la proposition de résolution européenne visant à lutter contre les addictions numériques chez les enfants.

      • 1:55:17-1:56:07 : Présentation de la proposition de résolution européenne visant à lutter contre les addictions numériques chez les enfants.

      • 1:56:07-1:57:22 : Importance que l'Assemblée nationale se saisisse de ce sujet grave, porteur d'enjeux de santé publique et d'éducation.

      • 1:57:22-1:57:57 : Souhait que la proposition de résolution européenne soit débattue en séance.

      • 1:57:57-1:58:26 : Statistiques alarmantes sur le temps passé sur internet par les jeunes et sur l'utilisation problématique des smartphones.

      • 2:02:06-2:02:34 : Les écrans entraînent une libération rapide de dopamine.

      • 2:03:46-2:04:21 : Exemples de caractéristiques addictives des services en ligne : défilement infini, lecture automatique, restriction temporale de contenu (FOMO).

      • 2:05:16-2:07:22 : L'Union Européenne a adopté des règlements sur les marchés et les services numériques pour protéger les enfants.

      • 2:07:22-2:09:15 : Nécessité d'agir de manière efficace à l'échelon européen pour interdire les caractéristiques addictives des services en ligne.

      • 2:09:15-2:10:03 : Invitation à adopter largement la proposition de résolution européenne.

      • 2:10:03-2:10:27 : Appropriation du texte par la Commission des Affaires Européennes.

      • 2:10:54-2:11:20 : Nécessité de légiférer pour protéger les plus jeunes et d'avoir des sanctions adaptées.

      • 2:13:27-2:14:05 : Définition de solutions concrètes face à cette problématique, combinant régulation, éducation et sensibilisation.

      • 2:14:47-2:15:52 : Importance de limiter le temps d'écran et l'accès aux outils et matériels.

      • 2:15:55-2:16:36 : Rappel des statistiques sur le temps passé devant les écrans par les enfants de différents âges.

      • 2:16:53-2:18:29 : Parallèle avec le tabagisme et la nécessité de prendre des mesures pour protéger les enfants des dangers du numérique.

      • 2:23:56-2:24:44 : Condamnation du groupe Meta pour non-respect des règles de la concurrence et prise de conscience des comportements addictifs générés chez les jeunes.

      • 2:24:53-2:26:18 : Nécessité d'une réglementation forte et protectrice de la santé des mineurs, et soutien à la proposition de résolution européenne.

      • 2:28:19-2:29:01 : Importance de faire du numérique une opportunité et non une menace pour la santé mentale des enfants.

      • 2:29:12-2:30:44 : Les recommandations de la proposition de résolution européenne fournissent des pistes concrètes et pertinentes, notamment le droit numérique à ne pas être dérangé.

      • 2:30:44-2:31:12 : Soutien à la proposition de résolution européenne et remerciements aux rapporteurs.

      • 2:35:57-2:37:57 : Importance de se concentrer sur ce qui est proposé dans la proposition de résolution européenne et d'agir auprès de la Commission européenne.

      • 2:37:57-2:38:13 : Il ne s'agit pas de proposer des éléments sur le droit français, mais d'agir sur le droit européen.

      • 2:38:13-2:39:46 : Nécessité de trouver la juste mesure entre régulation et contrainte face aux plateformes numériques.

      • 2:44:57-2:45:55 : La technologie aliénante a été complètement domestiquée, y compris pour les enfants.

      • 2:45:55-2:47:17 : Nécessité d'un accompagnement et de la création de contenus pédagogiques pour accompagner les politiques publiques.

      • 2:48:03-2:49:15 : Intérêt pour des dispositifs législatifs mis en place dans d'autres pays européens et pour une réflexion sur un cadre précis concernant la publicité.

      • 2:49:15-2:49:44 : Remerciements pour la qualité des travaux menés sur l'addiction aux écrans des enfants.

      • 2:49:44-2:50:10 : Question sur les actions concrètes à mettre en place pour harmoniser les législations européennes et assurer un équilibre entre la régulation des plateformes et le respect des droits numériques des mineurs.

      • 2:51:26-2:52:14 : Préoccupation concernant l'algorithme en entonnoir et le contrôle de l'accès aux contenus inadaptés.

      • 2:52:14-2:52:35 : La responsabilité du numérique doit se discuter à l'échelon européen.

      • 2:52:35-2:53:02 : Un rapport a été rendu sur "enfants et écrans à la recherche du temps perdu", avec des préconisations pouvant être intégrées dans une proposition de loi nationale.

      • 2:53:56-2:55:33 : Le niveau européen permet d'être plus efficace, et cela demande des initiatives nouvelles à prendre, y compris en tant que parlementaires au niveau national.

      • 2:55:33-2:56:53 : Il faut trouver les leviers pour la taxation et la publicité.

      • 2:56:53-2:58:03 : Il faut choisir le bon niveau pour agir, en tenant compte du cadre du droit européen.

      • 2:58:03-2:59:29 : La proposition de résolution européenne est une indication de la France au sein du parlement européen.

      • 3:00:12-3:02:00 : Il est préférable de retirer certains amendements car ils relèvent du niveau national et non européen.

      • 3:06:18-3:09:38 : Discussion sur des amendements concernant la priorisation des acteurs publics dans la recherche sur l'impact des écrans, l'usage des écrans individuels dans les établissements scolaires, et l'inscription de données relatives à l'exposition aux écrans dans le carnet de santé.

      • 3:11:06-3:14:19 : Explication des rapporteurs sur la nécessité de se concentrer sur le droit européen dans le cadre de cette proposition de résolution.

      • 3:15:53-3:17:02 : Insistance sur le retrait des amendements ne relevant pas du cadre européen.

      • 3:17:09-3:18:25 : Explication sur la possibilité de sous-amender les amendements pour un passage en séance.

      • 3:18:25-3:18:31 : Avis des rapporteurs sur l'amendement AC5.

      • 3:18:31-3:20:14 : Les rapporteurs ont la volonté de travailler sur quelque chose au niveau national.

      • 3:20:14-3:21:11 : L'échelle européenne est la plus appropriée pour agir sur ces questions.

      • 3:21:11-3:21:36 : Rappel que ce qui est du réglementaire ne relève pas du droit européen.

      • 3:21:58-3:23:23 : Discussion sur l'interdiction de l'exposition aux écrans des enfants de moins de 3 ans.

      • 3:26:48-3:27:25 : Fin de l'étude des amendements et désignation de rapporteurs sur la proposition de loi relative à la réforme de l'audiovisuel public.

    2. Je peux fournir un résumé des principaux sujets et arguments présentés dans les sources, afin de faciliter la préparation de réunions d'information.

      • Mixité sociale à l'école Un conférencier du Conseil économique, social et environnemental (CESE) remet en question la mixité sociale à l'école et la redéfinition de la carte scolaire, y compris l'utilisation du financement public des établissements privés comme levier. Il est proposé de réduire les dotations en cas de non-respect des contrats d'objectifs et de moyens, en utilisant le critère social comme indicateur de mixité. L'enseignement privé scolarise davantage d'élèves d'origine favorisée, une tendance à la hausse. Il est suggéré d'établir des contrats d'objectifs triennaux avec les établissements privés, avec une révision des financements si les objectifs ne sont pas atteints.

      • Formation des enseignants Les sources soulignent l'importance de la formation initiale et continue des enseignants. Un conférencier s'interroge sur les préconisations en matière de formation initiale, étant donné le projet de réforme annoncé. La formation continue est également mise en avant, avec la nécessité de la préserver au sein du temps scolaire. L'amélioration de la formation des enseignants est jugée nécessaire, tant au niveau initial que continu.

      • Inégalités sociales et système éducatif Le système éducatif français est décrit comme un accélérateur d'inégalités sociales. Un conférencier critique le modèle méritocratique, opposant la méritocratie à la réussite de tous les élèves. L'école est présentée comme un projet de société où les enjeux sont collectifs, nécessitant l'association de tous les acteurs. L'OCDE souligne que la France reste l'un des pays où l'acquisition de compétences à 15 ans est fortement influencée par le milieu social.

      • Crise d'attractivité du métier d'enseignant Les sources notent une crise du métier d'enseignant et un manque d'attractivité. La reconnaissance et la revalorisation du métier sont préconisées, en s'inspirant de modèles comme celui de la Finlande.

      • Adaptation aux réalités territoriales Les territoires ultramarins présentent des situations particulièrement dégradées, nécessitant des mesures adaptées. Il est souligné la nécessité d'adapter les politiques éducatives aux réalités locales et aux besoins des territoires. La coordination entre les collectivités territoriales est essentielle pour favoriser la mixité sociale et adapter les mesures aux spécificités locales.

      • Débat démocratique sur l'école Plusieurs intervenants appellent à un débat démocratique sur les finalités de l'école, impliquant l'ensemble de la communauté éducative. La nécessité de redéfinir les objectifs de l'école est soulignée, afin de garantir une éducation adaptée aux besoins contemporains.

      • Démocratie scolaire et rôle des élèves Il est proposé de renforcer la démocratie scolaire en prenant en compte la parole et le rôle des élèves, afin de conforter leur apprentissage de la citoyenneté. L'apprentissage des droits par les enfants, et pas seulement de leurs droits civiques, est également mis en avant.

      • Réussite scolaire et bien-être des élèves L'importance d'un climat scolaire serein est soulignée, avec un accent sur la question du harcèlement, du bien-être et de la santé des élèves et des personnels.

      • Addiction numérique chez les enfants

        • Une proposition de résolution européenne vise à lutter contre l'addiction numérique chez les enfants, décrite comme un problème grave de santé publique.
        • Les chiffres montrent une utilisation massive et précoce des écrans par les enfants, avec des conséquences sur leur développement cognitif et émotionnel.
        • Les plateformes numériques sont accusées d'utiliser des caractéristiques de conception addictives pour capter l'attention des utilisateurs et créer une dépendance.
        • Il est proposé d'interdire les caractéristiques de conception addictive aux services en ligne et de consacrer un droit numérique à ne pas être dérangé.
        • Plusieurs intervenants soulignent la nécessité d'une action collective et coordonnée au niveau européen pour protéger les enfants.
        • L'importance de la responsabilisation, de la prévention et de l'éducation à une utilisation responsable du numérique est mise en avant.
        • Des mesures restrictives pour les mineurs, garantissant leur liberté et leur libre arbitre, sont également proposées.
        • Le rôle de la lecture et de l'écriture est souligné comme une alternative aux écrans.
    1. Pour un document de synthèse pour un briefing, en utilisant les sources à disposition et notre conversation, on peut tirer les informations suivantes du webinaire sur l'habitat privé dans les quartiers prioritaires:

      • Contexte du webinaire: Le webinaire avait pour but de mieux faire connaître les spécificités de l’habitat privé dans les quartiers prioritaires. L'étude présentée fait suite à une première étude menée en 2018 et a été commanditée par la NCT et la direction générale de l’aménagement du logement et de la nature.

      • Objectifs de l'étude: L'étude visait à calculer des indicateurs sur la situation du parc privé dans les quartiers prioritaires, en s'appuyant sur trois sources de données. Elle couvre désormais les départements et régions d'outre-mer et apporte des indicateurs sur les quartiers de veille active, permettant d'apprécier l'évolution des indicateurs dans le temps.

      • Méthodologie:

        • Sources de données: L'étude s'appuie sur les fichiers fonciers (base de données d'origine fiscale), la base de données DB3S relative aux transactions foncières immobilières, et la base de données Fidéli de l'INSEE, qui permet d'étudier l'occupation des logements.
        • Périmètre d'analyse: Les indicateurs sont calculés à l'échelle précise des quartiers, en comparant la situation du quartier à celle de son unité urbaine de référence. Le parc privé est défini comme l'ensemble des logements qui ne sont pas locatifs sociaux.
        • Segmentation du parc: Les logements sont segmentés en logements vacants, logements locatifs et logements occupés par des propriétaires occupants.
      • Résultats clés:

        • Diversité des quartiers: Il existe une grande diversité de taille des quartiers, avec une hausse du nombre de logements entre 2016 et 2021.
        • Parc social: La spécificité du parc social est importante dans ces quartiers, avec un pourcentage de 64% en 2021, contre 26% dans les espaces de référence. La part du logement social diminue légèrement entre 2016 et 2021.
        • Locatif: Le segment locatif représente environ 44% du parc privé dans ces quartiers, contre 31% dans les espaces de référence.
        • Typologie des logements: Les logements collectifs sont fortement dominants, avec 9 logements sur 10 de type collectif.
        • Ancienneté: Les logements sociaux datent majoritairement des années 60, tandis qu'il y a une sur-représentation des logements datant d'avant 49 pour le parc privé des QPV.
        • Vacance: Le taux de vacance de longue durée est en hausse sur l'ensemble des segments et touche davantage les QPV que les espaces de référence, particulièrement pour le parc privé.
        • Niveau de vie: Le niveau de vie des ménages est plus faible dans les QPV, notamment sur le parc social.
        • Sur-occupation: La sur-occupation du parc privé est sur-représentée par rapport à l'espace de référence.
        • Marché immobilier: Le décrochage du marché immobilier est en moyenne de 25% entre le quartier et son espace de référence.
      • Outils et ressources:

        • Outil cartographique: Un outil cartographique a été créé pour permettre aux acteurs locaux de se saisir des résultats de l'étude.
        • Fichier d'indicateurs: Un fichier d'indicateurs propres à chaque quartier est accessible en téléchargement.
        • Typologies de QPV: Deux typologies de QPV ont été développées pour rendre compte de la diversité des quartiers.
      • Veille active: Les quartiers en veille active sont en moyenne plus petits, avec un taux de logement social plus faible, un locatif du parc privé moins important, un habitat collectif moins fréquent, une vacance de longue durée moins présente et une pauvreté moins forte.

      • Accès aux données et à la cartographie: Les données sont relativement homogènes sur le territoire national, malgré quelques exceptions. La cartographie est accessible via un lien, et il est possible qu'un compte soit nécessaire pour y accéder.

      Ce briefing permettra de donner un aperçu des principaux résultats de l'étude et des outils mis à disposition des acteurs locaux pour mieux comprendre les enjeux de l'habitat privé dans les quartiers prioritaires.

    1. Ce document de synthèse porte sur le témoignage de Mike, un père célibataire élevant seul sa fille de 7 ans, Karma, après le décès de sa femme des suites d'un cancer.

      • Situation initiale : Mike s'est retrouvé seul avec sa fille de 3 ans après le décès de sa femme. Il a dû faire face à un deuil et à la nécessité de s'organiser en tant que parent solo.

      • Défis rencontrés :

        • Organisation et éducation : Mike a dû jongler avec l'organisation quotidienne, l'éducation de sa fille et les tâches ménagères. Il a anticipé les premières fois importantes comme la rentrée scolaire et les relations amoureuses.
        • Pression sociale : Il avait l'impression d'être constamment observé et jugé.
        • Difficultés financières : Mike aInitialement bien gagné sa vie, mais il a eu tendance à dépenser excessivement pour compenser le manque. Il a reconnu la complexité de trouver un emploi compatible avec sa situation de parent isolé.
        • Équilibre personnel : Trouver un équilibre entre sa vie de parent et sa vie d'homme a été difficile, surtout face à une situation inattendue à 30 ans.
      • Solutions et adaptations :

        • Routine et organisation : Mike a mis en place une routine quotidienne pour stabiliser sa vie et celle de sa fille.
        • Soutien familial : La famille de sa femme est très présente, aidant avec Karma les week-ends et pendant les vacances. Ses proches l'ont aussi soutenu dès le début.
        • Changement de carrière : Il a quitté son emploi dans le commerce pour devenir artiste digital, ce qui lui permet de travailler à domicile et de s'occuper de sa fille.
        • Gestion des émotions : Il communique ouvertement avec sa fille au sujet de sa mère et n'évite pas le sujet.
      • Préoccupations et avenir :

        • Dangers extérieurs : Mike craint les dangers extérieurs tels que le harcèlement scolaire ou les mauvaises rencontres.
        • Relations futures : Il est ouvert à l'idée de rencontrer quelqu'un, mais sans dépendance affective. Il se demande comment l'arrivée d'une nouvelle figure féminine pourrait affecter Karma.
      • Points positifs :

        • Relation avec sa fille : La relation entre Mike et Karma est remplie de bonheur et de joie. Karma est souriante et bien entourée.
        • Ouverture et communication : Mike aborde tous les sujets avec sa fille, y compris ceux liés à la féminité et aux origines. Il encourage le dialogue et l'absence de tabous.
        • Présence maternelle : Karma se souvient de sa maman et lui parle quotidiennement. Mike entretient la mémoire de sa femme pour qu'elle reste présente dans leurs vies.
    1. At the time, rumours had been circulating that the public-health agency, which tracks disease outbreaks and makes its data publicly available, would start removing pages from its website, in response to executive orders issued by President Donald Trump directing government departments to take down public information on gender and diversity.

      Rumours.. AKA misinformation

      The fact is that data will not be removed from the CDC website.


      Implying Intent Behind the Data Removals:

      The article suggests that data is being removed due to executive orders from President Trump, particularly related to "gender and diversity." However, the Trump administration has not explicitly stated it would remove such data. The article frames these removals as intentional and politically motivated without providing concrete evidence or official statements, implying a political agenda when the true reasons for the removals remain unclear. Language Choices:

      The term “willy-nilly” describes the removal of pages, portraying the action as reckless or careless. This informal language casts the actions in a negative light without offering justification for the removals. Similarly, describing researchers’ actions as “scrambling” implies a crisis, which could exaggerate the situation. Framing of Researchers’ Actions:

      The article highlights researchers working to back up and preserve data, presenting their efforts as central to the story. This framing focuses on their actions without exploring any broader context or complexities, giving the impression of a singular focus on their role. Lack of Diverse Perspectives:

      The article mainly features voices critical of the data removals, primarily scientists and attorneys advocating for public access. This selective representation limits a full understanding of the situation, reinforcing a biased narrative without exploring other angles.

    1. 146151156PositionwildtypeO5O5O5O5O5U5U5U5U5U5O10O10O10O10O10U10U10U10U10U10VariantF F K S A M P E G Y V Q E R T I F F K D D G N Y K T R A E V K F E G D T L V N R I E L K G I D F K E D G N I L G H K L E Y N Y N S H N V Y I M A D K Q K N G I KVI WYIV VS S VG E VVE T VWY IM W RE M RW161166171176181186191196201206211216221226231236PositionwildtypeO5O5O5O5O5U5U5U5U5U5O10O10O10O10O10U10U10U10U10U10VariantV N F K I R H N I E D G S V Q L A D H Y Q Q N T P I G D G P V L L P D N H Y L S T Q S A L S K D P N E K R D H M V L L E F V T A A G I T H G M D E L Y KR L Y WR L YR L Y T RL W HR L Y R FI K RR W C FR V RV R KM W MR L Y N T GWR L Y R F HL Y T R HL N T GW FR L Y T W HM E Y R M C I L RR V F W W QR V M Q M WV F W S N QK M F R RFigure S20. The 20 engineered GFP variants.

      It's interesting to see how many mutant positions these variants share, even across observed versus unobserved, as well as the preference for later residues in the primary sequence. I'm curious if the model is learning something more significant about the features driving activity, particularly if this pattern holds true for the other ten datasets.

    1. Author response:

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

      Public Reviews:

      Reviewer #1 (Public review):

      Summary:

      In this work, Harpring et al. investigated divisome assembly in Chlamydia trachomatis serovar L2 (Ct), an obligate intracellular bacterium that lacks FtsZ, the canonical master regulator of bacterial cell division. They find that divisome assembly is initiated by the protein FtsK in Ct by showing that it forms discrete foci at the septum and future division sites. Additionally, knocking down ftsK prevents divisome assembly and inhibits cell division, further supporting their hypothesis that FtsK regulates divisome assembly. Finally, they show that MreB is one of the last chlamydial divisome proteins to arrive at the site of division and is necessary for the formation of septal peptidoglycan rings but does not act as a scaffold for division assembly as previously proposed.

      Strengths:

      The authors use microscopy to clearly show that FtsK forms foci both at the septum as well as at the base of the progenitor cell where the next septum will form. They also show that the Ct proteins PBP2, PBP3, MreC, and MreB localize to these same sites suggesting they are involved in the divisome complex.

      Using CRISPRi the authors knock down ftsK and find that most cells are no longer able to divide and that PBP2 and PBP3 no longer localized to sites of division suggesting that FtsK is responsible for initiating divisome assembly. They also performed a knockdown of pbp2 using the same approach and found that this also mostly inhibited cell division. Additionally, FtsK was still able to localize in this strain, however PBP3 did not, suggesting that FtsK acts upstream of PBP2 in the divisome assembly process while PBP2 is responsible for the localization of PBP3.

      The authors also find that performing a knockdown of ftsK also prevents new PG synthesis further supporting the idea that FtsK regulates divisome assembly. They also find that inhibiting MreB filament formation using A22 results in diffuse PG, suggesting that MreB filament formation is necessary for proper PG synthesis to drive cell division.

      Overall the authors propose a new hypothesis for divisome assembly in an organism that lacks FtsZ and use a combination of microscopy and genetics to support their model that is rigorous and convincing. The finding that FtsK, rather than a cytoskeletal or "scaffolding" protein is the first division protein to localize to the incipient division site is unexpected and opens up a host of questions about its regulation. The findings will progress our understanding of how cell division is accomplished in bacteria with non-canonical cell wall structure and/or that lack FtsZ.

      Weaknesses:

      No major weaknesses were noted in the data supporting the main conclusions. However, there was a claim of novelty in showing that multiple divisome complexes can drive cell wall synthesis simultaneously that was not well-supported (i.e. this has been shown previously in other organisms). In addition, there were minor weaknesses in data presentation that do not substantially impact interpretation (e.g. presenting the number of cells rather than the percentage of the population when quantifying phenotypes and showing partial western blots instead of total western blots).

      We agree with the weaknesses identified by the reviewer. We removed the statements in the Results and Discussion that multiple independent divisome complexes can simultaneously direct PG synthesis. We presented the data in Figs. 3-5 as % of the cells in the population, and complete western blots are shown in Supp. Fig. S1.

      Reviewer #2 (Public review):

      Summary:

      Chlamydial cell division is a peculiar event, whose mechanism was mysterious for many years. C. trachomatis division was shown to be polar and involve a minimal divisome machinery composed of both homologues of divisome and elongasome components, in the absence of an homologue of the classical division organizer FtsZ. In this paper, Harpring et al., show that FtsK is required at an early stage of the chlamydial divisome formation.

      Strengths:

      The manuscript is well-written and the results are convincing. Quantification of divisome component localization is well performed, number of replicas and number of cells assessed are sufficient to get convincing data. The use of a CRISPRi approach to knock down some divisome components is an asset and allows a mechanistic understanding of the hierarchy of divisome components.

      Weaknesses:

      The authors did not analyse the role of all potential chlamydial divisome components and did not show how FtsK may initiate the positioning of the divisome. Their conclusion that FtsK initiates the assembly of the divisome is an overinterpretation and is not backed by the data. However, data show convincingly that FtsK, if perhaps not the initiator of chlamydial division, is definitely an early and essential component of the chlamydial divisome.

      The following statement has been included in the Discussion (pg. 16 of the revised manuscript)  “Although we focused our study on a subset of the divisome and elongasome proteins that Chlamydia expresses (bolded in Fig. 6G), our results support our conclusion that chlamydial budding is dependent upon a hybrid divisome complex and that FtsK is required for the assembly of this hybrid divisome. At this time, we cannot rule out that other proteins act upstream of FtsK to initiate divisome assembly in this obligate intracellular bacterial pathogen.”

      We will soon be submitting another manuscript that addresses how FtsK specifies the site of divisome assembly. This work is too extensive to be included in this manuscript.

      Reviewer #3 (Public review):

      Summary:

      The obligate intracellular bacterium Chlamydia trachomatis (Ct) divides by binary fission. It lacks FtsZ, but still has many other proteins that regulate the synthesis of septal peptidoglycan, including FtsW and FtsI (PBP3) as well as divisome proteins that recruit and activate them, such as FtsK and FtsQLB. Interestingly, MreB is also required for the division of Ct cells, perhaps by polymerizing to form an FtsZ-like scaffold. Here, Harpring et al. show that MreB does not act early in division and instead is recruited to a protein complex that includes FtsK and PBP2/PBP3. This indicates that Ct cell division is organized by a chimera between conserved divisome and elongasome proteins. Their work also shows convincingly that FtsK is the earliest known step of divisome activity, potentially nucleating the divisome as a single protein complex at the future division site. This is reminiscent of the activity of FtsZ, yet fundamentally different.

      Strengths:

      The study is very well written and presented, and the data are convincing and rigorous. The data underlying the proposed localization dependency order of the various proteins for cell division is well justified by several different approaches using small molecule inhibitors, knockdowns, and fluorescent protein fusions. The proposed dependency pathway of divisome assembly is consistent with the data and with a novel mechanism for MreB in septum synthesis in Ct.

      Weaknesses:

      The paper could be improved by including more information about FtsK, the "focus" of this study. For example, if FtsK really is the FtsZ-like nucleator of the Ct divisome, how is the Ct FtsK different sequence-wise or structurally from FtsK of, e.g. E. coli? Is the N-terminal part of FtsK sufficient for cell division in Ct like it is in E. coli, or is the DNA translocase also involved in focus formation or localization? Addressing those questions would put the proposed initiator role of FtsK in Ct in a better context and make the conclusions more attractive to a wider readership.

      We will be submitting another manuscript soon that details the conserved domain organization of FtsK from different bacteria, and the role of the various domains of chlamydial FtsK (including the N-terminus and the C-terminal translocase domain) in directing its localization in dividing Chlamydia. We have added text to the discussion (pg. 16 of the revised manuscript) that describes the sequence homology of chlamydial FtsK to FtsK from E. coli.

      Another weakness is that the title of the paper implies that FtsK alone initiates divisome assembly. However, the data indicate only that FtsK is important at an early stage of divisome assembly, not that it is THE initiator. I suggest modifying the title to account for this--perhaps "FtsK is required to initiate....".

      We agree with the reviewer and modified the title to “FtsK is Critical for the Assembly of the Unique Divisome Complex of the FtsZ-less Chlamydia trachomatis”. We have also modified the text throughout to indicate that FtsK is required for the assembly of the hybrid divisome of Chlamydia

      Recommendations for the authors:

      Reviewer #1 (Recommendations for the authors):

      Suggestions for improvement (mostly minor):

      (1) For several of the graphs, the authors plot the number of cells with a given phenotype on the y-axis, but then describe percentages of cells in the text. It would make it clearer if all the graphs had the percentage of cells on the y-axis instead.

      We have modified the figures to indicate the percentage of cells on the y-axis with a given phenotype.

      (2) In Figures 3, 4, and 5 the authors show separate graphs for plus/minus drug or inducer. These should be on the same graph as they are directly comparing these two different conditions. Having them on separate graphs makes it less clear whether these differences are significant between the two conditions

      We modified Fig. 4 to show +/- inducer in ftsk and pbp2 knockdown strains in the same graph.  Regarding Figures 3 and 5, we believe the figures in the original submission effectively demonstrate the +/- drug conditions, so these figures remain unchanged in the revised manuscript.

      (3) In Figure 2 the authors show microscopy of the colocalization of FtsK with several other divisome proteins from Ct. Quantification of the colocalization of FtsK with these other proteins would provide a more holistic understanding of their colocalization and help further support their argument that FtsK initiates the assembly of the divisome.

      Supp. Fig. S4A of the revised manuscript contains images showing the colocalization of FtsK with the fusions at the septum and the base of dividing cells, and the colocalization of FtsK with the fusions that are only at the base of dividing cells. Supp. Fig. S4B quantified the percentage of dividing cells where FtsK overlaps the localization of each of the fusions at the septum, at the septum and the base, and at the base alone.

      (4) In Figure 6 the authors mention that the PG ring was at a slight angle relative to the MOMP-stained septum. What is the significance of this? The authors mention it several times but do not explain its relevance to divisome assembly. It is not really evident in the images presented.

      We mention in the discussion pgs. 17-18 of the revised manuscript that “The relevance of the angled orientation of PG and MreC rings relative to the MOMP-stained septum in division intermediates is unclear. However, it appears to be a conserved feature of the cell division process and may arise because the divisome proteins are often positioned slightly above or below the plane of the MOMP-stained septum. The positioning of divisome proteins above or below the septum is indicated in Figs. 1 and 2.

      We included cartoons in Fig. 6C of the revised manuscript to assist the reader in visualizing the angled orientation of the PG ring relative to the MOMP-stained septum.

      (5) In line 270 the authors claim that "these are the first data in any system to suggest that septal PG synthesis/modification is simultaneously directed by multiple independent divisome complexes." However, their experiments do not demonstrate that multiple divisome complexes are active at the same time. They show that multiple foci of FtsK etc. are present at sites where PG synthesis has occurred, but that does not necessarily mean that each focus/complex was actively synthesizing PG at the same time. Moreover, similar approaches were used to support a claim that septal PG synthesis is directed by multiple discrete divisome complexes previously (e.g. in Figure 1 of Bisson-Filho et al. 2017 (PMID: 28209898) in Bacillus subtilis and in Perez et al 2021 (PMID: 33269494) in Streptococcus pneumoniae). This claim is not central to the main conclusions of the study and could just be removed.

      This statement has been removed from the Results and the Discussion.

      (6) In Figure 6B the authors see three distinct FtsK foci. Why is this the only place in the manuscript where they see three foci? They mentioned previously that they saw foci at the septum and at the base of the progenitor mother cell, but why are there three foci here?

      The vast majority of dividing cells displayed one foci at the septum and/or the base.  Representative images were chosen that reflected the localization profiles observed in the majority of cells. While we observed cells with  multiple foci, as shown in Figure 6C, these cells were relatively rare   (~2% of cells for all the divisome proteins in 3 independent experiments).  Since  the number of cells with multiple foci were relatively rare, we chose to group these cells with the cells that had single foci at the septum, the septum and base, or base alone categories in the quantification shown in Fig. 2C. This is stated in the legend of Fig. 2 of the revised manuscript.

      (7) The Discussion section is lacking a couple of things that would put the data in a broader context. Can the authors speculate on how FtsK knows how to find the division site? I.e. what might be upstream of FtsK localization? Additionally, the authors do not talk about the FtsK sequence or domains at any point in the paper. Does Ct FtsK have a similar sequence/structure to FtsKs from other bacteria? Are there any differences in sequence/structure that might tell us about its function in Ct?

      We will be submitting another manuscript soon that examines how the site of assembly of the divisome is defined in dividing Chlamydia. This manuscript will also define the localization of the different sub-domains of chlamydial FtsK during cell division.  For this manuscript, we added a paragraph in the Discussion (pg. 16 of the revised manuscript) that states the domain organization is conserved in FtsK proteins from different bacteria. This paragraph includes information regarding the % sequence identity of the C-terminus and the N-terminus of chlamydial FtsK when compared to E. coli FtsK.

      (8) For Supplementary Figure S1B-C. The authors should show the full blots rather than just the single band of the protein of interest to show that the antibodies are specific. Additionally, the authors should include a loading control to show that they loaded the same amount of protein for each sample.

      We have included the full blots in Supp. Fig. S1 of the revised manuscript. We do not see the need for including a loading control for these blots because we are not making arguments about the relative level of the proteins that were assayed. We only use the blots to show that the fusion proteins are primarily a single species of the predicted molecular mass.

      (9) In Supplementary Figure S4A the authors use RT-qPCR to measure ftsK and pbp2 transcript levels. Since they have antibodies against these proteins, they should also include Western blots to show that the proteins are not being produced when targeted using CRISPRi.

      We have included data in Supp. Fig. S5E of the resubmission that indicates foci of FtsK and PBP2 could not be detected following the knockdown of ftsk and pbp2. We feel that these data support our conclusion that the induced expression of dCas12 in the the ftsk and pbp2 knockdown strains results in the downregulation of the endogenous FtsK and PBP2 polypeptides.

      (10) In lines 261-262 the authors say that "PG organization was the same or differed at the septum." What is the PG organization being compared to? Same or different from what?

      We agree with the reviewer that the text in lines 261-262 in the original submission was confusing.  The text has been modified.

      (11) Lines 201-215 the authors refer to Supplementary Figure S3 throughout this section, but they should refer to Supplementary Figure S4.

      This has been corrected.

      Reviewer #2 (Recommendations for the authors):

      I am not convinced that this paper shows that FtsK initiates the assembly of the divisome since the authors did not analyse the role and localization of all other chlamydial divisome components. Out of the ten homologues of divisome and elongasome components encoded by C. trachomatis genome, only five are investigated in this study. There is no explanation about how these five were chosen.

      We state on pg. 16 of the revised manuscript that “Although we focused our study on a subset of the divisome and elongasome proteins that Chlamydia expresses (bolded in Fig. 6G), our results support our conclusion that chlamydial budding is dependent upon a hybrid divisome complex and that FtsK is required for the assembly of this hybrid divisome. At this time, we cannot rule out that other proteins act upstream of FtsK to initiate divisome assembly in this obligate intracellular bacterial pathogen.

      Results convincingly indicate that FtsK is an early divisome component, but proofs are lacking to indicate that it initiates the divisome formation. Indeed, the authors do not show how FtsK would be the first protein to selectively accumulate at a given location to initiate the divisome formation. For this reason, the model they propose at the end of their study is not backed by sufficient data, to my opinion.

      We agree with the reviewer that our data does not show that FtsK initiates divisome assembly. The title of the manuscript has been modified to “FtsK is Critical for the Assembly of the Unique Divisome Complex of the FtsZ-less Chlamydia trachomatis” and the text throughout has been modified to indicate that FtsK is the first protein we assayed that associates with nascent divisomes at the base of dividing cells. We will soon be submitting another manuscript that details how FtsK is recruited to a specific site to initiate nascent divisome assembly, This work is too extensive to be included in this manuscript.

      There are also discrepancies in the number of cells analysed to quantify the localization of divisome components, ranging from 50 to 250 cells. The authors could better explain why there are such variations.

      There were differences in the number of cells analyzed in the various experiments, but in every instance the effect of inhibitors (A22 and mecillinam) or ftsk and pbp2 knockdown on divisome assembly was statistically significant.

      There are a few mistakes in the text regarding figure numbering (Figure S4 is mentioned as S3 in the text). Figures 5B and D are not specifically cited.

      These mistakes have been corrected in the revised manuscript.

      Line 261-262: the sentence starting "Our imaging analysis.." is not clear to me.

      We agree with the reviewer that the text in lines 261-262 was confusing.  The text has been modified (pg. 14 of the revised manuscript).

      Line 270-271: there are insufficient proofs to say that there are multiple independent divisome complexes. This is in my opinion an overinterpretation of the data, since there is no proof that these complexes are independent.

      This statement has been removed from the text.

      A few details are lacking in the figure legends:

      Figure 2C: when was the expression of the different mCherry and 6xHis constructs induced?

      The onset and length of the induction of the fusions have been included in the legend of Fig. 2.

      Bars are sometimes mentioned as uM and should be um. Bars sizes, number of replicates, and/or meaning of the error bars are lacking in legends of Figures S2, S3, and S4

      This has been corrected in the revised manuscript.

      The consistency of Figures could be improved between Figures 3A, 4A, B, and 5A. The results of treated cells could be always shown as dark grey. It would help the reader.

      We have used consistent coloring in Figs. 3-5 to indicate the treated cells.

      Reviewer #3 (Recommendations for the authors):

      (1) Lines 113-118: do Ct cells increase in size as they get closer to starting division? If so, could a pseudo-time course (demograph) be done to bolster the evidence that the base foci formed mainly in predivisional cells and not newborn cells? This evidence might be more convincing than the data in Figures 1F and G.

      Chlamydial cells in the population were heterogeneous in size at the timepoint we are studying. This observation is consistent with previous reports in the literature (Liechti et al.,2021). While we agree that a pseudo-time course could potentially bolster the evidence about when FtsK foci appear, we believe our current analysis sufficiently demonstrates that basal foci of FtsK appear prior to the appearance of new buds at the base of dividing cells.

      (2) Figure 3E: It looks like MreC localization to foci doesn't strictly require MreB polymerization. Is this known for E. coli or other species?

      To our knowledge, MreC assembly into a filament has not been shown to be dependent upon MreB in other bacteria.  In Caulobacter crescentus, MreC forms a helical structure that is not dependent upon MreB or MreB filament formation (Dye et al., 2005. PNAS; Divakaruni et al., 2005. PNAS).

      (3) Figure 5E: why is nearly half of PBP2 and PBP3 still localized to foci at the membrane even after treatment with mecillinam? This suggests, as the authors mention, that mecillinam reduces the efficiency of localization to the divisome but does not eliminate it. Any ideas why?

      At this time, we do not know why inhibiting the catalytic activity of PBP2 with mecillinam does not fully prevent the association of PBP2 with the chlamydial divisome. We have included a statement in the Results (pg. 13 of the revised manuscript) that inhibiting the catalytic activity of PBP2 prevents it from efficiently associating with or maintaining its association with polarized divisome complexes.

      (4) Line 262-263: This sentence is confusing-please rephrase. The same as what? Differed from what?

      We agree with the reviewer. The wording in lines 262-263 of the original submission has been modified.  

      (5) Lines 265-267 and Figure 6: Adding cartoon schematics might help readers visualize cell orientations in Fig. 6 (especially 6B).

      Cartoons have been added to Fig. 6C (Fig. 6B in the original submission) to orient the reader.

      (6) Line 294-298: Do the authors think that the residual 5-10% of PG foci after FtsK knockdown is due to the ability of residual FtsK to organize divisomes?

      We show that knockdown of FtsK is not complete, and while we cannot be certain, it is likely, that the PG foci detected in FtsK knockdown cells is due to the ability of the residual FtsK to organize divisomes that direct PG synthesis.

      (7) Do the authors have any evidence that FtsK foci are mobile like treadmilling FtsZ?

      We have not performed real-time imaging studies, and we currently have no evidence that FtsK foci are mobile.

      (8) FtsK foci here are reminiscent of mobile foci formed by the FtsK-like SpoIIIE at the Bacillus subtilis sporulation septum. This might be a good idea to mention in the Discussion. Is it possible that Ct FtsK is also involved in coordinating chromosome partitioning through the developing septum? (That is another reason why it would be useful to know if the translocase domain was dispensable for localization/activity).

      We are currently preparing another manuscript that documents the contribution of the various domains of FtsK to its localization profile and whether the division defect in ftsk knockdown cells can be suppressed by specific subdomains of FtsK. This manuscript not only will include these data, it will also include experiments that address how the site of polarized budding is defined. In the revised manuscript, we have included a description of how the domain organization of chlamydial FtsK is similar to E. coli FtsK (pg. 16 of revision). Chlamydial FtsK also has a similar domain organization as SpoIIIE from B. subtilis. The C-terminal catalytic domain of SpoIIIE is 45% identical to chlamydial FtsK. The N-terminus of SpoIIIE is predicted to encode 4 transmembrane spanning helices, like chlamydial FtsK. However, the N-terminus of SpoIIIE shares no sequence homology with the N-terminus of chlamydial FtsK.  We have not included the similar domain organization of SpoIIIE and chlamydial FtsK in the revised manuscript.

      (9) It seems that FtsK foci localize to a particular spot opposite from the active septum, although how this spot is specified is not clear. Is there any geometric clue for FtsK's localization like there is for Min-specified FtsZ localization?

      As mentioned above, we are currently preparing another manuscript that documents our efforts to understand how the site of polarized budding is defined.  This analysis is too extensive to include in this study.

      (10) As mentioned in the Summary, do the authors know whether the N-terminal membrane binding part of FtsK (FtsKn) sufficient for localization/divisome assembly in Ct as it is in other species? Oullette et al. 2012 showed that FtsKn could interact with MreB in BACTH.

      We are currently preparing another manuscript that documents the contribution of the various domains of FtsK to its localization profile.

      (11) The previous BACTH result with MreB and FtsKn implies that this interaction is direct, yet the current data suggest that this is not the case. Can the authors comment on this? Is this due to bridging effects inherent in the BACTH system?

      We have not presented any data to indicate that FtsK and MreB do not interact. We have only shown that FtsK localization is not dependent upon MreB filament formation (Fig. 3).

      (12) The FtsZ-independent role of FtsK in nucleating the divisome suggests that Ct FtsK may differ from other FtsKs structurally - can this be explored, perhaps with AlphaFold 3?

      As mentioned above, we have included a paragraph in the discussion of the revised manuscript (pg. 16 of the revised manuscript) that states the domain organization of chlamydial FtsK is similar to E.coli FtsK. This conserved domain organization is evident when we view the structures of the proteins using Alphafold.

      (13) Typo on line 559: should be HeLa.

      This has been corrected.

    1. For Nieto de Herrera, it was clear that jazz, the dance, was the creation of a single individual,Oscar Duryea, a well-known dancer, choreographer, and dance instructor in New York at thetime, who had contributed in 1914 to the standardization and popularization of the steps forthe foxtrot and who apparently invented a new dance in 1916: the ‘two-two’.47 Thus, the intri-cate set of dancing instructions that Nieto de Herrera presented in Spanish to her readers inHavana came from a set of instructions published previously in English by Duryea. All thingsconsidered, following the steps seems to make evident that jazz dance – as prescribed byDuryea and amplified by Nieto de Herrera – was bounded within the stylistic realm of foxtrot,tango, waltz, one-step, two-step, and other ‘society dances’ of the 1910s. While the specificityand sophistication of the instructions reminds us, in a way, of much older society dances –such as the contradance – they also point to social parameters defined by Victorian and bour-geois sensibilities:Walk slowly during three steps in the line of direction, starting with the left foot. Stopon the third step, balancing the weight of the body [bending?] over the left foot;count long ‘one, two, three,’ which should account for six [beats] in the music.Quickly bring the right foot next to the left foot, put the left foot forward and changethe step so that it falls over the right foot. Count two and four. Repeat the last twosteps starting with the right foot and stopping over the right foot when giving thethird step forward and count two. Advance with the left foot and stop for an instant.(1). Cross the right foot behind the left foot, closing suddenly to find the step of the45 ‘[E]l jazz, elegantísimo baile que está haciendo furor en los salones de alta sociedad neoyorkinos. El jazz es el últimoderivado del Fox Trot, más animado, más entusiasta y más elegante que el primero, pues su ritmo hace lucir, en toda suamplitud, la gracia y la gentileza de los bailadores.’ El Universal, 17 April 1918, 8. The ‘Dancing casino’ parties,announced as ‘recepciones’ and ‘bailes de etiqueta’ were to take place at the business’s salons, located at ‘Casa No.12 de la calle San Juan de Letrán’.46 See Wald, How the Beatles Destroyed Rock ’n’ Roll, 49–59.47 ‘Oscar Duryea,’ in Sonny Watson’s Street Swing website: www.streetswing.com/histmai2/d2durya1.htm (accessed 13August 2022); The Northeastern Reporter, vol. 90, 4 January–22 March 1910, 1019, 1141.362 Ospina Romero The Dawn of the Jazz Age in the Caribbean

      As a dancer who was trained in ballet, modern, jazz, afro Caribbean, hip hop, African and many more styles for about 15 years, to learn more about the origin of jazz is quite interesting in a way because there is a known saying of " Ballet is the foundation of all dance" and for a while I have believed that because ballet is what we all started in, it's the most popular etc. But when I read this, it makes me wonder how untrue that is. It's a series of genres hat can form one new style of dance it's no always ballet or on its own. I wonder what caused ballet dance to take the title. (And for a dancer who has recently left high school for dance and forgot most of the dance terms the bottom is really helpful)

    1. Reviewer #1 (Public review):

      The results of these experiments support a modest but important conclusion: If sub-optimal methods are used to collect retrospective reports, such as simple yes/no questions, inattentional blindness (IB) rates may be overestimated by up to ~8%.

      (1) In experiment 1, data from 374 subjects were included in the analysis. As shown in figure 2b, 267 subjects reported noticing the critical stimulus and 107 subjects reported not noticing it. This translates to a 29% IB rate if we were to only consider the "did you notice anything unusual Y/N" question. As reported in the results text (and figure 2c), when asked to report the location of the critical stimulus (left/right), 63.6% of the "non-noticer" group answered correctly. In other words, 68 subjects were correct about the location while 39 subjects were incorrect. Importantly, because the location judgment was a 2-alternative-forced-choice, the assumption was that if 50% (or at least not statistically different than 50%) of the subjects answered the location question correctly, everyone was purely guessing. Therefore, we can estimate that ~39 of the subjects who answered correctly were simply guessing (because 39 guessed incorrectly), leaving 29 subjects from the non-noticer group who were correct on the 2AFC above and beyond the pure guess rate. If these 29 subjects are moved from the non-noticer to the noticer group, the corrected rate of IB for Experiment 1 is 20.86% instead of the original 28.61% rate that would have been obtained if only the Y/N question was used. In other words, relying only on the "Y/N did you notice anything" question led to an overestimate of IB rates by 7.75% in Experiment 1.

      In the revised version of their manuscript, the authors provided the data that was missing from the original submission, which allows this same exercise to be carried out on the other 4 experiments. Using the same logic as above, i.e., calculating the pure-guess rate on the 2AFC, moving the number of subjects above this pure-guess rate to the non-noticer group, and then re-calculating a "corrected IB rate", the other experiments demonstrate the following:

      Experiment 2: IB rates were overestimated by 4.74% (original IB rate based only on Y/N question = 27.73%; corrected IB rate that includes the 2AFC = 22.99%)

      Experiment 3: IB rates were overestimated by 3.58% (original IB rate = 30.85%; corrected IB rate = 27.27%)

      Experiment 4: IB rates were overestimated by ~8.19% (original IB rate = 57.32%; corrected IB rate for color* = 39.71%, corrected IB rate for shape = 52.61%, corrected IB rate for location = 55.07%)

      Experiment 5: IB rates were overestimated by ~1.44% (original IB rate = 28.99%; corrected IB rate for color = 27.56%, corrected IB rate for shape = 26.43%, corrected IB rate for location = 28.65%)

      *note: the highest overestimate of IB rates was from Experiment 4, color condition, but the authors admitted that there was a problem with 2AFC color guessing bias in this version of the experiment which was a main motivation for running experiment 5 which corrected for this bias.

      Taken as a whole, this data clearly demonstrates that even with a conservative approach to analyzing the combination of Y/N and 2AFC data, inattentional blindness was evident in a sizeable portion of the subject populations. An important (albeit modest) overestimate of IB rates was demonstrated by incorporating these improved methods.

      (2) One of the strongest pieces of evidence presented in this paper was the single data point in Figure 3e showing that in Experiment 3, even the super subject group that rated their non-noticing as "highly confident" had a d' score significantly above zero. Asking for confidence ratings is certainly an improvement over simple Y/N questions about noticing, and if this result were to hold, it could provide a key challenge to IB. However, this result can most likely be explained by measurement error.

      In their revised paper, the authors reported data that was missing from their original submission: the confidence ratings on the 2AFC judgments that followed the initial Y/N question. The most striking indication that this data is likely due to measurement error comes from the number of subjects who indicated that they were highly confident that they didn't notice anything on the critical trial, but then when asked to guess the location of the stimulus, indicated that they were highly confident that the stimulus was on the left (or right). There were 18 subjects (8.82% of the high-confidence non-noticer group) who responded this way. To most readers, this combination of responses (high confidence in correctly judging a stimulus feature that one is highly confident in having not seen at all) indicates that a portion of subjects misunderstood the confidence scales (or just didn't read the questions carefully or made mistakes in their responses, which is common for experiments conducted online).

      In the authors' rebuttal to the first round of peer review, they wrote, "it is perfectly rationally coherent to be very confident that one didn't see anything but also very confident that if there was anything to be seen, it was on the left." I respectfully disagree that such a combination of responses is rationally coherent. The more parsimonious interpretation is that a measurement error occurred, and it's questionable whether we should trust any responses from these 18 subjects.

      In their rebuttal, the authors go on to note that 14 of the 18 subjects who rated their 2AFC with high confidence were correct in their location judgment. If these 14 subjects were removed from analysis (which seems like a reasonable analysis choice, given their contradictory responses), d' for the high-confidence non-noticer group would most likely fall to chance levels. In other words, we would see a data pattern similar to that plotted in Figure 3e, but with the first data point on the left moving down to zero d'. This corrected Figure 3e would then provide a very nice evidence-based justification for including confidence ratings along with Y/N questions in future inattentional blindness studies.

      (3) In most (if not all) IB experiments in the literature, a partial attention and/or full attention trial is administered after the critical trial. These control trials are very important for validating IB on the critical trial, as they must show that, when attended, the critical stimuli are very easy to see. If a subject cannot detect the critical stimulus on the control trial, one cannot conclude that they were inattentionally blind on the critical trial, e.g., perhaps the stimulus was just too difficult to see (e.g., too weak, too brief, too far in the periphery, too crowded by distractor stimuli, etc.), or perhaps they weren't paying enough attention overall or failed to follow instructions. In the aggregate data, rates of noticing the stimuli should increase substantially from the critical trial to the control trials. If noticing rates are equivalent on the critical and control trials, one cannot conclude that attention was manipulated in the first place.

      In their rebuttal to the first round of peer review, the authors provided weak justification for not including such a control condition. They cite one paper that argues such control conditions are often used to exclude subjects from analysis (those who fail to notice the stimulus on the control trial are either removed from analysis or replaced with new subjects) and such exclusions/replacements can lead to underestimations of inattentional blindness rates. However, the inclusion of a partial or full attention condition as a control does not necessitate the extra step of excluding or replacing subjects. In the broadest sense, such a control condition simply validates the attention manipulation, i.e., one can easily compare the percent of subjects who answered "yes" or who got the 2AFC judgment correct during the critical trial versus the control trial. The subsequent choice about exclusion/replacement is separate, and researchers can always report the data with and without such exclusions/replacements to remain more neutral on this practice.

      If anyone were to follow-up on this study, I highly recommend including a partial or full attention control condition, especially given the online nature of data collection. It's important to know the percent of online subjects who answer yes and who get the 2AFC question correct when the critical stimulus is attended, because that is the baseline (in this case, the "ceiling level" of performance) to which the IB rates on the critical trial can be compared.

    2. Author response:

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

      Responses to Reviewer #1:

      We thank the reviewer for these additional comments, and more generally for their extensive engagement with our work, which is greatly appreciated. Here, we respond to the three points in their latest review in turn.

      The results of these experiments support a modest but important conclusion: If sub-optimal methods are used to collect retrospective reports, such as simple yes/no questions, inattentional blindness (IB) rates may be overestimated by up to ~8%.

      It is true, of course, that we think the field has overstated the extent of IB, and we appreciate the reviewer characterizing our results as important along these lines. Nevertheless, we respectfully disagree with the framing and interpretation the reviewer attaches to them. As explained in our previous response, we think this interpretation — and the associated calculations of IB overestimation ‘rates’ — perpetuates a binary approach to perception and awareness which we regard as mistaken.

      A graded approach to IB and visual awareness 

      Our sense is that many theorists interested in IB have conceived of perception and awareness as ‘all or nothing’: You either see a perfectly clear gorilla right in front of you, or you see nothing at all. This is implicit in the reviewer’s characterization of our results as simply indicating that fewer subjects fail to see the critical stimulus than previously assumed. To think that way is precisely to assume the orthodox binary position about perception, i.e., that any given subject can neatly be categorized into one of two boxes, saw or didn’t see.

      Our perspective is different. We think there can be degraded forms of perception and awareness that fall neatly into neither of the categories “saw the stimulus perfectly clearly” or “saw nothing at all”. On this graded conception, the question is not: “What proportion of subjects saw the stimulus?” but: “What is the sensitivity of subjects to the stimulus?” This is why we prefer signal detection measures like d′ over % noticing and % correct. This powerful framework has been successful in essentially every domain to which it has been applied, and we think perception and visual awareness are no exception. We understand that the reviewer may not think the same way about this foundational issue, but since part of our goal is to promote a graded approach to perception, we are keen to highlight our disagreement here and so resist the reviewer’s interpretation of our results (even to the extent that it is a positive one!).

      Finally, we note that given this perspective, we are correspondingly inclined to reject many of the summary figures following below in Point (1) by the reviewer. These calculations (given in terms of % noticing and not noticing) make sense on the binary conception of awareness, but not on the SDT-based approach we favor. We say more about this below. 

      (1) In experiment 1, data from 374 subjects were included in the analysis. As shown in figure 2b, 267 subjects reported noticing the critical stimulus and 107 subjects reported not noticing it. This translates to a 29% IB rate if we were to only consider the "did you notice anything unusual Y/N" question. As reported in the results text (and figure 2c), when asked to report the location of the critical stimulus (left/right), 63.6% of the "non-noticer" group answered correctly. In other words, 68 subjects were correct about the location while 39 subjects were incorrect. Importantly, because the location judgment was a 2-alternative-forced-choice, the assumption was that if 50% (or at least not statistically different than 50%) of the subjects answered the location question correctly, everyone was purely guessing. Therefore, we can estimate that ~39 of the subjects who answered correctly were simply guessing (because 39 guessed incorrectly), leaving 29 subjects from the nonnoticer group who were correct on the 2AFC above and beyond the pure guess rate. If these 29 subjects are moved from the non-noticer to the noticer group, the corrected rate of IB for Experiment 1 is 20.86% instead of the original 28.61% rate that would have been obtained if only the Y/N question was used. In other words, relying only on the "Y/N did you notice anything" question led to an overestimate of IB rates by 7.75% in Experiment 1.

      In the revised version of their manuscript, the authors provided the data that was missing from the original submission, which allows this same exercise to be carried out on the other 4 experiments.  

      (To briefly interject: All of these data were provided in our public archive since our original submission and remain available at https://osf.io/fcrhu. The difference now is only that they are included in the manuscript itself.)

      Using the same logic as above, i.e., calculating the pure-guess rate on the 2AFC, moving the number of subjects above this pure-guess rate to the non-noticer group, and then re-calculating a "corrected IB rate", the other experiments demonstrate the following:

      Experiment 2: IB rates were overestimated by 4.74% (original IB rate based only on Y/N question = 27.73%; corrected IB rate that includes the 2AFC = 22.99%)

      Experiment 3: IB rates were overestimated by 3.58% (original IB rate = 30.85%; corrected IB rate = 27.27%)

      Experiment 4: IB rates were overestimated by ~8.19% (original IB rate = 57.32%; corrected IB rate for color* = 39.71%, corrected IB rate for shape = 52.61%, corrected IB rate for location = 55.07%)

      Experiment 5: IB rates were overestimated by ~1.44% (original IB rate = 28.99%; corrected IB rate for color = 27.56%, corrected IB rate for shape = 26.43%, corrected IB rate for location = 28.65%)

      *note: the highest overestimate of IB rates was from Experiment 4, color condition, but the authors admitted that there was a problem with 2AFC color guessing bias in this version of the experiment which was a main motivation for running experiment 5 which corrected for this bias.

      Taken as a whole, this data clearly demonstrates that even with a conservative approach to analyzing the combination of Y/N and 2AFC data, inattentional blindness was evident in a sizeable portion of the subject populations. An important (albeit modest) overestimate of IB rates was demonstrated by incorporating these improved methods.

      We appreciate the work the reviewer has put into making these calculations. However, as noted above, such calculations implicitly reflect the binary approach to perception and awareness that we reject. 

      Consider how we’d think about the single subject case where the task is 2afc detection of a low contrast stimulus in noise. Suppose that this subject achieves 70% correct. One way of thinking about this is that the subject fully and clearly sees the stimulus on 40% of trials (achieving 100% correct on those) and guesses completely blindly on the other 60% (achieving 50% correct on those) for a total of 40% + 30% = 70% overall. However, this is essentially a ‘high threshold’ approach to the problem, in contrast to an SDT approach. On an SDT approach — an approach with tremendous evidential support — on every trial the subject receives samples from probabilistic distributions corresponding to each interval (one noise and one signal + noise) and determines which is higher according to the 2afc decision rule. Thus, across trials, they have access to differentially graded information about the stimulus. Moreover, on some trials they may have significant information from the stimulus (perhaps, well above their single interval detection criterion) but still decide incorrectly because of high noise from the other spatial interval. From this perspective, there is no nonarbitrary way of saying whether the subject saw/did not see on a given trial. Instead, we must characterize the subject’s overall sensitivity to the stimulus/its visibility to them in terms of a parameter such as d′ (here, ~ 0.7).

      We take the same attitude to the subjects in our experiments (and specifically to our ‘super subject’). Instead of calculating the proportion of subjects who saw or failed to see the stimulus (with some characterized as aware and some as unaware), we think the best way to characterize our results is that, across subjects (and so trials also), there was differential graded access to information from the stimulus, and this is best represented in terms of the group-level sensitivity parameter d′. This is why we frame our results as demonstrating that subjects traditionally considered inattentionally blind exhibit significant residual visual sensitivity to the critical stimulus.

      (2) One of the strongest pieces of evidence presented in this paper was the single data point in Figure 3e showing that in Experiment 3, even the super subject group that rated their non-noticing as "highly confident" had a d' score significantly above zero. Asking for confidence ratings is certainly an improvement over simple Y/N questions about noticing, and if this result were to hold, it could provide a key challenge to IB. However, this result can most likely be explained by measurement error.

      In their revised paper, the authors reported data that was missing from their original submission: the confidence ratings on the 2AFC judgments that followed the initial Y/N question. The most striking indication that this data is likely due to measurement error comes from the number of subjects who indicated that they were highly confident that they didn't notice anything on the critical trial, but then when asked to guess the location of the stimulus, indicated that they were highly confident that the stimulus was on the left (or right). There were 18 subjects (8.82% of the high-confidence non-noticer group) who responded this way. To most readers, this combination of responses (high confidence in correctly judging a stimulus feature that one is highly confident in having not seen at all) indicates that a portion of subjects misunderstood the confidence scales (or just didn't read the questions carefully or made mistakes in their responses, which is common for experiments conducted online).

      In the authors' rebuttal to the first round of peer review, they wrote, "it is perfectly rationally coherent to be very confident that one didn't see anything but also very confident that if there was anything to be seen, it was on the left." I respectfully disagree that such a combination of responses is rationally coherent. The more parsimonious interpretation is that a measurement error occurred, and it's questionable whether we should trust any responses from these 18 subjects.

      In their rebuttal, the authors go on to note that 14 of the 18 subjects who rated their 2AFC with high confidence were correct in their location judgment. If these 14 subjects were removed from analysis (which seems like a reasonable analysis choice, given their contradictory responses), d' for the high-confidence non-noticer group would most likely fall to chance levels. In other words, we would see a data pattern similar to that plotted in Figure 3e, but with the first data point on the left moving down to zero d'. This corrected Figure 3e would then provide a very nice evidence-based justification for including confidence ratings along with Y/N questions in future inattentional blindness studies.

      We appreciate the reviewer’s highlighting of this particular piece of evidence as amongst our strongest. (At the same time, we must resist its characterization as a “single data point”: it derives from a large pre-registered experiment involving some 7,000 subjects total, with over 200 subjects in the relevant bin — both figures being far larger than a typical IB experiment.) We also appreciate their raising the issue of measurement error.

      Specifically, the reviewer contends that our finding that even highly confident non-noticers exhibit significant sensitivity is “most likely … explained by measurement error” due to subjects mistakenly inverting our confidence scale in giving their response. In our original reply, we gave two reasons for thinking this quite unlikely; the reviewer has not addressed these in this revised review. First, we explicitly labeled our confidence scale (with 0 labeled as ‘Not at all confident’ and 3 as ‘Highly confident’) so that subjects would be very unlikely simply to invert the scale. This is especially so as it is very counterintuitive to treat “0” as reflecting high confidence. More importantly, however, we reasoned that any measurement error due to inverting or misconstruing the confidence scale should be symmetric. That is: If subjects are liable to invert the confidence scale, they should do so just as often when they answer “yes” as when they answer “no” – after all the very same scale is being used in both cases. This allows us to explore evidence of measurement error in relation to the large number of high-confidence “yes” subjects (N = 2677), thus providing a robust indicator as to whether subjects are generally liable to misconstrue the confidence scale. Looking at the number of such high confidence noticers who subsequently respond to the 2afc question with low confidence (a pattern which might, though need not, suggest measurement error), we found that the number was tiny. Only 28/2677 (1.05%) of high-confidence noticers subsequently gave the lowest level of confidence on the 2afc question, and only 63/2677 (2.35%) subjects gave either of the two lower levels of confidence. For these reasons, we consider any measurement error due to misunderstanding the confidence scale to be extremely minimal.

      The reviewer is correct to note that 18/204 (9%) subjects reported both being highly confident that they didn't notice anything and highly confident in their 2afc judgment, although only 14/18 were correct in this judgment. Should we exclude these 14? Perhaps if we agree with the reviewer that such a pattern of responses is not “rationally coherent” and so must reflect a misconstrual of the scale. But such a pattern is in fact perfectly and straightforwardly intelligible. Specifically, in a 2afc task, two stimuli can individually fall well below a subject’s single interval detection criterion — leading to a high confidence judgment that nothing was presented in either interval. Quite consistent with this, the lefthand stimulus may produce a signal that is much higher than the right-hand stimulus — leading to a high confidence forced-choice judgment that, if something was presented, it was on the left. (By analogy, consider how a radiologist could look at a scan and say the following: “We’re 95% confident there’s no tumor. But even on the 5% chance that there is, our tests completely rule out that it’s a malignant one, so don’t worry.”) 

      (3) In most (if not all) IB experiments in the literature, a partial attention and/or full attention trial is administered after the critical trial. These control trials are very important for validating IB on the critical trial, as they must show that, when attended, the critical stimuli are very easy to see. If a subject cannot detect the critical stimulus on the control trial, one cannot conclude that they were inattentionally blind on the critical trial, e.g., perhaps the stimulus was just too difficult to see (e.g., too weak, too brief, too far in the periphery, too crowded by distractor stimuli, etc.), or perhaps they weren't paying enough attention overall or failed to follow instructions. In the aggregate data, rates of noticing the stimuli should increase substantially from the critical trial to the control trials. If noticing rates are equivalent on the critical and control trials, one cannot conclude that attention was manipulated in the first place.

      In their rebuttal to the first round of peer review, the authors provided weak justification for not including such a control condition. They cite one paper that argues such control conditions are often used to exclude subjects from analysis (those who fail to notice the stimulus on the control trial are either removed from analysis or replaced with new subjects) and such exclusions/replacements can lead to underestimations of inattentional blindness rates. However, the inclusion of a partial or full attention condition as a control does not necessitate the extra step of excluding or replacing subjects. In the broadest sense, such a control condition simply validates the attention manipulation, i.e., one can easily compare the percent of subjects who answered "yes" or who got the 2AFC judgment correct during the critical trial versus the control trial. The subsequent choice about exclusion/replacement is separate, and researchers can always report the data with and without such exclusions/replacements to remain more neutral on this practice.

      If anyone were to follow-up on this study, I highly recommend including a partial or full attention control condition, especially given the online nature of data collection. It's important to know the percent of online subjects who answer yes and who get the 2AFC question correct when the critical stimulus is attended, because that is the baseline (in this case, the "ceiling level" of performance) to which the IB rates on the critical trial can be compared.

      We agree with the reviewer that future studies could benefit from including a partial or full attention condition. They are surely right that we might learn something additional from such conditions. 

      Where we differ from the reviewer is in thinking of these conditions as “controls” appropriate to our research question. This is why we offered the justification we did in our earlier response. When these conditions are used as controls, they are used to exclude subjects in ways that serve to inflate the biases we are concerned with in our work. For our question, the absence of these conditions does not impact the significance of the findings, since such conditions are designed to answer a question which is not the one at the heart of our paper. Our key claim is that subjects who deny noticing an unexpected stimulus in a standard inattentional blindness paradigm nonetheless exhibit significant residual sensitivity (as well as a conservative bias in their response to the noticing question); the presence or absence of partial- or full-attention conditions is orthogonal to that question.

      Moreover, we note that our tasks were precisely chosen to be classic tasks widely used in the literature to manipulate attention. Thus, by common consensus in the field, they are effective means to soak up attention, and have in effect been tested in partial- and full-attention control settings in a huge number of studies. Second, we think it very doubtful that subjects in a full-attention trial would not overwhelmingly have detected our critical stimuli. The reviewer worries that they might have been “too weak, too brief, too far in the periphery, too crowded by distractor stimuli, etc.” But consider E5 where the stimulus was a highly salient orange or green shape, present on the screen for 5 seconds. The reviewer also suggests that subjects in the full-attention control might not have detected the stimulus because they “weren't paying enough attention overall”. But evidently if they weren’t paying attention even in the full-attention trial this would be reason for thinking that there was inattentional blindness even in this condition (a point made by White et al. 2018) and certainly not a reason for thinking there was not an attentional effect in the critical trial. Lastly, the reviewer suggests that a full-attention condition would have helped ensure that subjects were following instructions. But we ensured this already by (as per our pre-registration) excluding subjects who performed poorly in the relevant primary tasks.

      Thus, both in principle and in practice, we do not see the absence of such conditions as impacting the interpretation of our findings, even as we agree that future work posing a different research question could certainly learn something from including such conditions.

      Responses to Reviewer #2:

      We note that this report is unchanged from an earlier round of review, and not a response to our significantly revised manuscript. We believe our latest version fully addresses all the issues which the reviewer originally raised. The interested reader can see our original response below. We again thank the reviewer for their previous report which was extremely helpful.

      —-

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

      eLife Assessment

      This study presents valuable findings to the field interested in inattentional blindness (IB), reporting that participants indicating no awareness of unexpected stimuli through yes/no questions, still show above-chance sensitivity to specific properties of these stimuli through follow-up forced-choice questions (e.g., its color). The results suggest that this is because participants are conservative and biased to report not noticing in IB. The authors conclude that these results provide evidence for residual perceptual awareness of inattentionally blind stimuli and that therefore these findings cast doubt on the claim that awareness requires attention. Although the samples are large and the analysis protocol novel, the evidence supporting this interpretation is still incomplete, because effect sizes are rather small, the experimental design could be improved and alternative explanations have not been ruled out.

      We are encouraged to hear that eLife found our work “valuable”. We also understand, having closely looked at the reviews, why the assessment also includes an evaluation of “incomplete”. We gave considerable attention to this latter aspect of the assessment in our revision. In addition to providing additional data and analyses that we believe strengthen our case, we also include a much more substantial review and critique of existing methods in the IB literature to make clear exactly the gap our work fills and the advance it makes. (Indeed, if it is appropriate to say this here, we believe one key aspect of our work that is missing from the assessment is our inclusion of ‘absent’ trials, which is what allows us to make the crucial claims about conservative reporting of awareness in IB for the first time.) Moreover, we refocus our discussion on only our most central claims, and weaken several of our secondary claims so that the data we’ve collected are better aligned with the conclusions we draw, to ensure that the case we now make is in fact complete. Specifically, our two core claims are (1) that there is residual sensitivity to visual features for subjects who would ordinarily be classified as inattentionally blind (whether this sensitivity is conscious or not), and (2) that there is a tendency to respond conservatively on yes/no questions in the context of IB. We believe we have very compelling support for these two core claims, as we explain in detail below and also through revisions to our manuscript.

      Given the combination of strengthened and clarified case, as well as the weakening of any conclusions that may not have been fully supported, we believe and hope that these efforts make our contribution “solid”, “convincing”, or even “compelling” (especially because the “compelling” assessment characterizes contributions that are “more rigorous than the current state-of-the-art”, which we believe to be the case given the issues that have plagued this literature and that we make progress on).

      Reviewer #1 (Public review):

      Summary:

      In the abstract and throughout the paper, the authors boldly claim that their evidence, from the largest set of data ever collected on inattentional blindness, supports the views that "inattentionally blind participants can successfully report the location, color, and shape of stimuli they deny noticing", "subjects retain awareness of stimuli they fail to report", and "these data...cast doubt on claims that awareness requires attention." If their results were to support these claims, this study would overturn 25+ years of research on inattentional blindness, resolve the rich vs. sparse debate in consciousness research, and critically challenge the current majority view in cognitive science that attention is necessary for awareness.

      Unfortunately, these extraordinary claims are not supported by extraordinary (or even moderately convincing) evidence. At best, the results support the more modest conclusion: If sub-optimal methods are used to collect retrospective reports, inattentional blindness rates will be overestimated by up to ~8% (details provided below in comment #1). This evidence-based conclusion means that the phenomenon of inattentional blindness is alive and well as it is even robust to experiments that were specifically aimed at falsifying it. Thankfully, improved methods already exist for correcting the ~8% overestimation of IB rates that this study successfully identified.

      We appreciate here the reviewer’s recognition of the importance of work on inattentional blindness, and the centrality of inattentional blindness to a range of major questions. We also recognize their concerns with what they see as a gap between our data and the claims made on their basis. We address this in detail below (as well as, of course, in our revised manuscript). However, from the outset we are keen to clarify that our central claim is only the first one the reviewer mentions — and the one which appears in our title — namely that, as a group, participants can successfully report the location, color, and shape of stimuli they deny noticing, and thus that there is “Sensitivity to visual features in inattentional blindness”. This is the claim that we believe is strongly supported by our data, and all the more so after revising the manuscript in light of the helpful comments we’ve received.

      By contrast, the other claims the reviewer mentions, concerning awareness (as opposed to residual sensitivity–which might be conscious or unconscious) were intended as both secondary and tentative. We agree with the referee that these are not as strongly supported by our data (and indeed we say so in our manuscript), whereas we do think our data strongly support the more modest — and, to us central — claim that, as a group, inattentionally blind participants can successfully report the location, color, and shape of stimuli they deny noticing. 

      We also feel compelled to resist somewhat the reviewer’s summary of our claims. For example, the reviewer attributes to us the claim that “subjects retain awareness of stimuli they fail to report”; but while that phrase does appear in our abstract, what we in fact say is that our data are “consistent with an alternative hypothesis about IB, namely that subjects retain awareness of stimuli they fail to report”. We do in fact believe that our data are consistent with that hypothesis, whereas earlier investigations seemed not to be. We mention this only because we had used that careful phrasing precisely for this sort of reason, so that we wouldn’t be read as saying that our results unequivocally support that alternative.

      Still, looking back, we see how we may have given more emphasis than we intended to some of these more secondary claims. So, we’ve now gone through and revised our manuscript throughout to emphasize that our main claim is about residual sensitivity, and to make clear that our claims about awareness are secondary and tentative. Indeed, we now say precisely this, that although we favor an interpretation of “our results in terms of residual conscious vision in IB … this claim is tentative and secondary to our primary finding”. We also weaken the statements in the abstract that the reviewer mentions, to better reflect our key claims.

      Finally, we note one further point: Dialectically, inattentional blindness has been used to argue (e.g.) that attention is required for awareness. We think that our data concerning residual sensitivity at least push back on the use of IB to make this claim, even if (as we agree) they do not provide decisive evidence that awareness survives inattention. In other words, we think our data call that claim into question, such that it’s now genuinely unclear whether awareness does or does not survive inattention. We have adjusted our claims on this point accordingly as well.

      Comments:

      (1) In experiment 1, data from 374 subjects were included in the analysis. As shown in figure 2b, 267 subjects reported noticing the critical stimulus and 107 subjects reported not noticing it. This translates to a 29% IB rate, if we were to only consider the "did you notice anything unusual Y/N" question. As reported in the results text (and figure 2c), when asked to report the location of the critical stimulus (left/right), 63.6% of the "non-noticer" group answered correctly. In other words, 68 subjects were correct about the location while 39 subjects were incorrect. Importantly, because the location judgment was a 2-alternative-forced-choice, the assumption was that if 50% (or at least not statistically different than 50%) of the subjects answered the location question correctly, everyone was purely guessing. Therefore, we can estimate that ~39 of the subjects who answered correctly were simply guessing (because 39 guessed incorrectly), leaving 29 subjects from the nonnoticer group who may have indeed actually seen the location of the stimulus. If these 29 subjects are moved to the noticer group, the corrected rate of IB for experiment 1 is 21% instead of 29%. In other words, relying only on the "Y/N did you notice anything" question leads to an overestimate of IB rates by 8%. This modest level of inaccuracy in estimating IB rates is insufficient for concluding that "subjects retain awareness of stimuli they fail to report", i.e. that inattentional blindness does not exist.

      In addition, this 8% inaccuracy in IB rates only considers one side of the story. Given the data reported for experiment 1, one can also calculate the number of subjects who answered "yes, I did notice something unusual" but then reported the incorrect location of the critical stimulus. This turned out to be 8 subjects (or 3% of the "noticer" group). Some would argue that it's reasonable to consider these subjects as inattentionally blind, since they couldn't even report where the critical stimulus they apparently noticed was located. If we move these 8 subjects to the non-noticer group, the 8% overestimation of IB rates is reduced to 6%.

      The same exercise can and should be carried out on the other 4 experiments, however, the authors do not report the subject numbers for any of the other experiments, i.e., how many subjects answered Y/N to the noticing question and how many in each group correctly answered the stimulus feature question. From the limited data reported (only total subject numbers and d' values), the effect sizes in experiments 2-5 were all smaller than in experiment 1 (d' for the non-noticer group was lower in all of these follow-up experiments), so it can be safely assumed that the ~6-8% overestimation of IB rates was smaller in these other four experiments. In a revision, the authors should consider reporting these subject numbers for all 5 experiments.

      We now report, as requested, all these subject numbers in our supplementary data (see Supplementary Tables 1 and 2 in our Supplementary Materials).

      However, we wish to address the larger question the reviewer has raised: Do our data only support a relatively modest reduction in IB rates? Even if they did, we still believe that this would be a consequential result, suggesting a significant overestimation of IB rates in classic paradigms. However, part of our purpose in writing this paper is to push back against a certain binary way of thinking about seeing/awareness. Our sense is that the field has conceived of awareness as “all or nothing”: You either see a perfectly clear gorilla right in front of you, or you see nothing at all. Our perspective is different: We think there can be degraded forms of awareness that fall into neither of those categories. For that reason, we are disinclined to see our results in the way that the reviewer suggests, namely as simply indicating that fewer subjects fail to see the stimulus than previously assumed. To think that way is, in our view, to assume the orthodox binary position about awareness. If, instead, one conceives of awareness as we do (and as we believe the framework of signal detection theory should compel us to), then it isn’t quite right to think of the proportion of subjects who were aware, but rather (e.g.) the sensitivity of subjects to the relevant stimulus. This is why we prefer measures like d′ over % noticing and % correct. We understand that the reviewer may not think the same way about this issue as we do, but part of our goal is to promote that way of thinking in general, and so some of our comments below reflect that perspective and approach.

      For example, consider how we’d think about the single subject case where the task is 2afc detection of a low contrast stimulus in noise. Suppose that this subject achieves 70% correct. One way of thinking about that is that the subject sees the stimulus on 40% of trials (achieving 100% correct on those) and guesses blindly on the other 60% (achieving 50% correct on those) for a total of 40% + 30% = 70% overall. However, this is essentially a “high threshold” approach to the problem, in contrast to an SDT approach. On an SDT approach (an approach with tremendous evidential support), on every trial the subject receives samples from probabilistic distributions corresponding to each interval (one noise and one signal + noise) and determines which is higher according to the 2afc decision rule. Thus, across trials they have access to differentially graded information about the stimulus. Moreover, on some trials they may have significant information from the stimulus (perhaps, well above their single interval detection criterion) but still decide incorrectly because of high noise from the other spatial interval. From this perspective, there is no non-arbitrary way of saying whether the subject saw/did not see on a given trial. Instead, we must characterize the subject’s overall sensitivity to the stimulus/its visibility to them in terms of a parameter such as d′ (here, ~ 0.7).

      We take the same attitude to our super subject. Instead of saying that some subjects saw/failed to see the stimuli, instead we suggest that the best way to characterize our results is that across subjects (and so trials also) there was differential graded access to information from the stimulus best represented in terms of the group-level sensitivity parameter d′.

      We acknowledge that (despite ourselves) we occasionally fell into an all-too-natural binary/high threshold way of thinking, as when we suggested that our data show that “inattentionally blind subjects consciously perceive these stimuli after all” and “the inattentionally blind can see after all." (p.17) We have removed such problematic phrasing as well as other problematic phrasing as noted below.

      (2) Because classic IB paradigms involve only one critical trial per subject, the authors used a "super subject" approach to estimate sensitivity (d') and response criterion (c) according to signal detection theory (SDT). Some readers may have issues with this super subject approach, but my main concern is with the lack of precision used by the authors when interpreting the results from this super subject analysis.

      Only the super subject had above-chance sensitivity (and it was quite modest, with d' values between 0.07 and 0.51), but the authors over-interpret these results as applying to every subject. The methods and analyses cannot determine if any individual subject could report the features above-chance. Therefore, the following list of quotes should be revised for accuracy or removed from the paper as they are misleading and are not supported by the super subject analysis: "Altogether this approach reveals that subjects can report above-chance the features of stimuli (color, shape, and location) that they had claimed not to notice under traditional yes/no questioning" (p.6)

      "In other words, nearly two-thirds of subjects who had just claimed not to have noticed any additional stimulus were then able to correctly report its location." (p.6)

      "Even subjects who answer "no" under traditional questioning can still correctly report various features of the stimulus they just reported not having noticed, suggesting that they were at least partially aware of it after all." (p.8)

      "Why, if subjects could succeed at our forced-response questions, did they claim not to have noticed anything?" (p.8)

      "we found that observers could successfully report a variety of features of unattended stimuli, even when they claimed not to have noticed these stimuli." (p.14)

      "our results point to an alternative (and perhaps more straightforward) explanation: that inattentionally blind subjects consciously perceive these stimuli after all... they show sensitivity to IB stimuli because they can see them." (p.16)

      "In other words, the inattentionally blind can see after all." (p.17)

      We thank the reviewer for pointing out how these quotations may be misleading as regards our central claim. We intended them all to be read generically as concerning the group, and not universally as claiming that all subjects could report above-chance/see the stimuli etc. We agree entirely that the latter universal claim would not be supported by our data. In contrast, we do contend that our super-subject analysis shows that, as a group, subjects traditionally considered intentionally blind exhibit residual sensitivity to features of stimuli (color, shape, and location) that they had all claimed not to notice, and likewise that as a group they could succeed at our forced-choice questions. 

      To ensure this claim is clear throughout the paper, and that we are not interpreted as making an unsupported universal claim we have revised the language in all of the quotations above, as follows, as well as in numerous other places in the paper.

      “Altogether this approach reveals that subjects can report above-chance the features of stimuli (color, shape, and location) that they had claimed not to notice under traditional yes/no questioning” (p.6) => “Altogether this approach reveals that as a group subjects can report above-chance the features of stimuli (color, shape, and location) that they had all claimed not to notice under traditional yes/no questioning” (p.6)

      “Even subjects who answer “no” under traditional questioning can still correctly report various features of the stimulus they just reported not having noticed, suggesting that they were at least partially aware of it after all.” (p.8) => “... even subjects who answer “no” under traditional questioning can, as a group, still correctly report various features of the stimuli they just reported not having noticed, indicating significant group-level sensitivity to visual features. Moreover, these results are even consistent with an alternative hypothesis about IB, that as a group, subjects who would traditionally be classified as inattentionally blind are in fact at least partially aware of the stimuli they deny noticing.” (p.8)

      “Why, if subjects could succeed at our forced-response questions, did they claim not to have noticed anything?” (p.8) => “Why, if subjects could succeed at our forcedresponse questions as a group, did they all individually claim not to have noticed anything?” (p.8)

      “we found that observers could successfully report a variety of features of unattended stimuli, even when they claimed not to have noticed these stimuli.” (p.14) => “we found that groups of observers could successfully report a variety of features of unattended stimuli, even when they all individually claimed not to have noticed those stimuli.” (p.14)

      “our results point to an alternative (and perhaps more straightforward) explanation: that inattentionally blind subjects consciously perceive these stimuli after all... they show sensitivity to IB stimuli because they can see them.” (p.16) => “our results just as easily raise an alternative (and perhaps more straightforward) explanation: that inattentionally blind subjects may retain a degree of awareness of these stimuli after all.” (p.16) Here deleting: “they show sensitivity to IB stimuli because they can see them.”

      “In other words, the inattentionally blind can see after all.” (p.17) => “In other words, as a group, the inattentionally blind enjoy at least some degraded or partial sensitivity to the location, color and shape of stimuli which they report not noticing.” (p.17)

      In one case, we felt the sentence was correct as it stood, since it simply reported a fact about our data:

      “In other words, nearly two-thirds of subjects who had just claimed not to have noticed any additional stimulus were then able to correctly report its location.” (p.6)

      After all, if subjects were entirely blind and simply guessed, it would be true to say that 50% of subjects would be able to correctly report the stimulus location (by guessing).

      In addition to these and numerous other changes, we also added the following explicit statement early in the paper to head-off any confusion on this point: “Note that all analyses reported here relate to this super subject as opposed to individual subjects”. 

      (3) In addition to the d' values for the super subject being slightly above zero, the authors attempted an analysis of response bias to further question the existence of IB. By including in some of their experiments critical trials in which no critical stimulus was presented, but asking subjects the standard Y/N IB question anyway, the authors obtained false alarm and correct rejection rates. When these FA/CR rates are taken into account along with hit/miss rates when critical stimuli were presented, the authors could calculate c (response criterion) for the super subject. Here, the authors report that response criteria are biased towards saying "no, I didn't notice anything". However, the validity of applying SDT to classic Y/N IB questioning is questionable.

      For example, with the subject numbers provided in Box 1 (the 2x2 table of hits/misses/FA/CR), one can ask, 'how many subjects would have needed to answer "yes, I noticed something unusual" when nothing was presented on the screen in order to obtain a non-biased criterion estimate, i.e., c = 0?' The answer turns out to be 800 subjects (out of the 2761 total subjects in the stimulus-absent condition), or 29% of subjects in this condition.

      In the context of these IB paradigms, it is difficult to imagine 29% of subjects claiming to have seen something unusual when nothing was presented. Here, it seems that we may have reached the limits of extending SDT to IB paradigms, which are very different than what SDT was designed for. For example, in classic psychophysical paradigms, the subject is asked to report Y/N as to whether they think a threshold-level stimulus was presented on the screen, i.e., to detect a faint signal in the noise. Subjects complete many trials and know in advance that there will often be stimuli presented and the stimuli will be very difficult to see. In those cases, it seems more reasonable to incorrectly answer "yes" 29% of the time, as you are trying to detect something very subtle that is out there in the world of noise. In IB paradigms, the stimuli are intentionally designed to be highly salient (and unusual), such that with a tiny bit of attention they can be easily seen. When no stimulus is presented and subjects are asked about their own noticing (especially of something unusual), it seems highly unlikely that 29% of them would answer "yes", which is the rate of FAs that would be needed to support the null hypothesis here, i.e., of a non-biased criterion. For these reasons, the analysis of response bias in the current context is questionable and the results claiming to demonstrate a biased criterion do not provide convincing evidence against IB.

      We are grateful to the reviewer for highlighting this aspect of our data. We agree with several of these points. For example, it is indeed striking that — given the corresponding hit rate — a false alarm rate of 29% would be needed to obtain an unbiased criterion. At the same time, we would respectfully push back on other points above. In our first experiment that uses the super-subject analysis, for example, d′ is 0.51 and highly significant; to describe that figure, as the reviewer does, as “slightly above zero” seemed not quite right to us (and all the more so given that these experiments involve very large samples and preregistered analysis plans). 

      We also respectfully disagree that our data call into question the validity of applying SDT to classic yes/no IB questioning. The mathematical foundations of SDT are rock solid, and have been applied far more broadly than we have applied them here. In fact, in a way we would suggest that exactly the opposite attitude is appropriate: rather than thinking that IB challenges an immensely well-supported, rigorously tested and broadly applicable mathematical model of perception, we think that the conflict between our SDT-based model of IB and the standard interpretation constitutes strong reason to disfavor the standard interpretation. Several points are worth making here.

      First, it is already surprising that 11.03% of our subjects in E2 (46/417) and 7.24% of our subjects in E5 (200/2761) E5 reported noticing a stimulus when no stimulus was present. But while this may have seemed unlikely in advance of inquiry, this is in fact what the data show and forms the basis of our criterion calculations. Thus, our criterion calculations already factor in a surprising but empirically verified high false alarm rate of subjects answering “yes” when no stimulus was presented and were asked about their noticing. (We also note that the only paper we know of to report a false alarm rate in an IB paradigm, though not one used to calculate a response criterion, found a very consistent false alarm rate of 10.4%. See Devue et al. 2009.)

      Second, while the reviewer is of course correct that a common psychophysical paradigm involves detection of a “threshold-level”/faint stimulus in noise, it is widely recognized that SDT has an extremely broad application, being applicable to any situation in which two kinds of event are to be discriminated (Pastore & Scheirer 1975) and being “almost universally accepted as a theoretical account of decision making in research on perceptual detection and recognition and in numerous extensions to applied domains” quite generally (Estes 2002, see also: Wixted 2020). Indeed, cases abound in which SDT has been successfully applied to situations which do not involve near threshold stimuli in noise. To pick two examples at random, SDT has been used in studying acceptability judgments in linguistics (Huang and Ferreira 2020) and the assessment of physical aggression in childstudent interactions (Lerman et al. 2010; for more general discussion of practical applications, see Swets et al. 2000). Given that the framework of SDT is so widely applied and well supported, and that we see no special reason to make an exception, we believe it can be relied on in the present context.

      Finally, we note that inattentional blindness can in many ways be considered analogous to “near threshold” detection since inattention is precisely thought to degrade or even abolish awareness of stimuli, meaning that our stimuli can be construed as near threshold in the relevant sense. Indeed, our relatively modest d′ values suggest that under inattention stimuli are indeed hard to detect. Thus, even were SDT more limited in its application, we think it still would be appropriate to apply to the case of IB.

      (4) One of the strongest pieces of evidence presented in the entire paper is the single data point in Figure 3e showing that in Experiment 3, even the super subject group that rated their non-noticing as "highly confident" had a d' score significantly above zero. Asking for confidence ratings is certainly an improvement over simple Y/N questions about noticing, and if this result were to hold, it could provide a key challenge to IB. However, this result hinges on a single data point, it was not replicated in any of the other 4 experiments, and it can be explained by methodological limitations. I strongly encourage the authors (and other readers) to follow up on this result, in an in-person experiment, with improved questioning procedures.

      We agree that our finding that even the super-subject group that rated their non-noticing as “highly confident” had a d' score significantly above zero is an especially strong piece of evidence, and we thank the reviewer for highlighting that here. At the same time, we note that while the finding is represented by a single marker in Figure 3e, it seemed not quite right to call this a “single data point”, as the reviewer does, given that it derives from a large pre-registered experiment involving some 7,000 subjects total, with over 200 subjects in the relevant bin — both figures being far larger than a typical IB experiment. It would of course be tremendous to follow up on this result – and we certainly hope our work inspires various follow-up studies. That said, we note that recruiting the necessary numbers of in person subjects would be an absolutely enormous, career-level undertaking – it would involve bringing more than the entire undergraduate population at our own institution, Johns Hopkins, into our laboratory! While those results would obviously be extremely valuable, we wouldn’t want to read the reviewer’s comments as implying that only an experiment of that magnitude — requiring thousands upon thousands of in-person subjects — could make progress on these issues. Indeed, because every subject can only contribute one critical trial in IB, it has long been recognized as an extremely challenging paradigm to study in a sufficiently well-powered and psychophysically rigorous way. We believe that our large preregistered online approach represents a major leap forward here, even if it involves certain trade-offs.

      In the current Experiment 3, the authors asked the standard Y/N IB question, and then asked how confident subjects were in their answer. Asking back-to-back questions, the second one with a scale that pertains to the first one (including a tricky inversion, e.g., "yes, I am confident in my answer of no"), may be asking too much of some subjects, especially subjects paying half-attention in online experiments. This procedure is likely to introduce a sizeable degree of measurement error.

      An easy fix in a follow-up study would be to ask subjects to rate their confidence in having noticed something with a single question using an unambiguous scale:

      On the last trial, did you notice anything besides the cross?

      (1): I am highly confident I didn't notice anything else

      (2): I am confident I didn't notice anything else

      (3): I am somewhat confident I didn't notice anything else

      (4): I am unsure whether I noticed anything else

      (5): I am somewhat confident I noticed something else

      (6): I am confident I noticed something else

      (7): I am highly confident I noticed something else

      If we were to re-run this same experiment, in the lab where we can better control the stimuli and the questioning procedure, we would most likely find a d' of zero for subjects who were confident or highly confident (1-2 on the improved scale above) that they didn't notice anything. From there on, the d' values would gradually increase, tracking along with the confidence scale (from 3-7 on the scale). In other words, we would likely find a data pattern similar to that plotted in Figure 3e, but with the first data point on the left moving down to zero d'. In the current online study with the successive (and potentially confusing) retrospective questioning, a handful of subjects could have easily misinterpreted the confidence scale (e.g., inverting the scale) which would lead to a mixture of genuine high-confidence ratings and mistaken ratings, which would result in a super subject d' that falls between zero and the other extreme of the scale (which is exactly what the data in Fig 3e shows).

      One way to check on this potential measurement error using the existing dataset would be to conduct additional analyses that incorporate the confidence ratings from the 2AFC location judgment task. For example, were there any subjects who reported being confident or highly confident that they didn't see anything, but then reported being confident or highly confident in judging the location of the thing they didn't see? If so, how many? In other words, how internally (in)consistent were subjects' confidence ratings across the IB and location questions? Such an analysis could help screen-out subjects who made a mistake on the first question and corrected themselves on the second, as well as subjects who weren't reading the questions carefully enough.

      As far as I could tell, the confidence rating data from the 2AFC location task were not reported anywhere in the main paper or supplement.

      We are grateful to the reviewer for raising this issue and for requesting that we report the confidence rating data from our 2afc location task in Experiment 3. We now report all this data in our Supplementary Materials (see Supplementary Table 3).

      We of course agree with the reviewer’s concern about measurement error, which is a concern in all experiments. What, then, of the particular concern that some subjects might have misunderstood our confidence question? It is surely impossible in principle to rule out this possibility; however, several factors bear on the plausibility of this interpretation. First, we explicitly labeled our confidence scale (with 0 labeled as ‘Not at all confident’ and 3 as ‘Highly confident’) so that subjects would be very unlikely simply to invert the scale. This is especially so as it is very counterintuitive to treat “0” as reflecting high confidence. However, we accept that it is a possibility that certain subjects might nonetheless have been confused in some other way.

      So, we also took a second approach. We examined the confidence ratings on the 2afc question of subjects who reported being highly confident that they didn't notice anything.

      Reassuringly, the large majority of these high confidence “no” subjects (~80%) reported low confidence of 0 or 1 on the 2afc question, and the majority (51%) reported the lowest confidence of 0. Only 18/204 (9%) subjects reported high confidence on both questions. 

      Still, the numbers of subjects here are small and so may not be reliable. This led us to take a third approach. We reasoned that any measurement error due to inverting or misconstruing the confidence scale should be symmetric. That is: If subjects are liable to invert the confidence scale, they should do so just as often when they answer “yes” as when they answer “no” – after all the very same scale is being used in both cases. This allows us to explore evidence of measurement error in relation to the much larger number of highconfidence “yes” subjects (N = 2677), thus providing a much more robust indicator as to whether subjects are generally liable to misconstrue the confidence scale. Looking at the number of such high confidence noticers who subsequently respond to the 2afc question with low-confidence, we found that the number was tiny. Only 28/2677 (1.05%) of highconfidence noticers subsequently gave the lowest level of confidence on the 2afc question, and only 63/2677 (2.35%) subjects gave either of the two lower levels of confidence. In this light, we consider any measurement error due to misunderstanding the confidence scale to be extremely minimal.

      What should we make of the 18 subjects who were highly confident non-noticers but then only low-confidence on the 2afc question? Importantly, we do not think that these 18 subjects necessarily made a mistake on the first question and so should be excluded. There is no a priori reason why one’s confidence criterion in a yes/no question should carry over to a 2afc question. After all, it is perfectly rationally coherent to be very confident that one didn’t see anything but also very confident that if there was anything to be seen, it was on the left. Moreover, these 18 subjects were not all correct on the 2afc question despite their high confidence (4/18 or 22% getting the wrong answer). 

      Nonetheless, and again reassuringly, we found that the above-chance patterns in our data remained the same even excluding these 18 subjects. We did observe a slight reduction in percent correct and d′ but this is absolutely what one should expect since excluding the most confident performers in any task will almost inevitably reduce performance.

      In this light, we consider it unlikely that measurement error fully explains the residual sensitivity found even amongst highly confident non-noticers. That said, we appreciate this concern. We now raise the issue and the analysis of high confidence noticers which addresses it in our revised manuscript. We also thank the reviewer for pressing us to think harder about this issue, which led directly to these new analyses that we believed have strengthened the paper.

      (5) In most (if not all) IB experiments in the literature, a partial attention and/or full attention trial (or set of trials) is administered after the critical trial. These control trials are very important for validating IB on the critical trial, as they must show that, when attended, the critical stimuli are very easy to see. If a subject cannot detect the critical stimulus on the control trial, one cannot conclude that they were inattentionally blind on the critical trial, e.g., perhaps the stimulus was just too difficult to see (e.g., too weak, too brief, too far in the periphery, too crowded by distractor stimuli, etc.), or perhaps they weren't paying enough attention overall or failed to follow instructions. In the aggregate data, rates of noticing the stimuli should increase substantially from the critical trial to the control trials. If noticing rates are equivalent on the critical and control trials one cannot conclude that attention was manipulated.

      It is puzzling why the authors decided not to include any control trials with partial or full attention in their five experiments, especially given their online data collection procedures where stimulus size, intensity, eccentricity, etc. were uncontrolled and variable across subjects. Including such trials could have actually helped them achieve their goal of challenging the IB hypothesis, e.g., excluding subjects who failed to see the stimulus on the control trials might have reduced the inattentional blindness rates further. This design decision should at least be acknowledged and justified (or noted as a limitation) in a revision of this paper.

      We acknowledge that other studies in the literature include divided and full attention trials, and that they could have been included in our work as well. However, we deliberately decided not to include such control trials for an important reason. As the referee comments, the main role of such trials in previous work has been to exclude from analysis subjects who failed to report the unexpected stimulus on the divided and/or full attention control trials.

      (For example, as Most et al. 2001 write: “Because observers should have seen the object in the full-attention trial (Mack & Rock, 1998), we used this trial as a control … Accordingly, 3 observers who failed to see the cross on this trial were replaced, and their data were excluded from the analyses.") As the reviewer points out, excluding such subjects would very likely have ‘helped' us. However, the practice is controversial. Indeed, in a review of 128 experiments, White et al. 2018 argue that the practice has “problematic consequences” and “may lead researchers to understate the pervasiveness of inattentional blindness". Since we wanted to offer as simple and demanding a test of residual sensitivity in IB as possible, we thus decided not to use any such exclusions, and for that reason decided not to include divided/full attention trials. 

      As recommended, we discuss this decision not to include divided/full attention trials and our logic for not doing so in the manuscript. As we explain, not having those conditions makes it more impressive, not less impressive, that we observed the results we in fact did — it makes our results more interpretable, not less interpretable, and so absence of such conditions from our manuscript should not (in our view) be considered any kind of weakness.

      (6) In the discussion section, the authors devote a short paragraph to considering an alternative explanation of their non-zero d' results in their super subject analyses: perhaps the critical stimuli were processed unconsciously and left a trace such that when later forced to guess a feature of the stimuli, subjects were able to draw upon this unconscious trace to guide their 2AFC decision. In the subsequent paragraph, the authors relate these results to above-chance forced-choice guessing in blindsight subjects, but reject the analogy based on claims of parsimony.

      First, the authors dismiss the comparison of IB and blindsight too quickly. In particular, the results from experiment 3, in which some subjects adamantly (confidently) deny seeing the critical stimulus but guess a feature at above-chance levels (at least at the super subject level and assuming the online subjects interpreted and used the confidence scale correctly), seem highly analogous to blindsight. Importantly, the analogy is strengthened if the subjects who were confident in not seeing anything also reported not being confident in their forced-choice judgments, but as mentioned above this data was not reported.

      Second, the authors fail to mention an even more straightforward explanation of these results, which is that ~8% of subjects misinterpreted the "unusual" part of the standard IB question used in experiments 1-3. After all, colored lines and shapes are pretty "usual" for psychology experiments and were present in the distractor stimuli everyone attended to. It seems quite reasonable that some subjects answered this first question, "no, I didn't see anything unusual", but then when told that there was a critical stimulus and asked to judge one of its features, adjusted their response by reconsidering, "oh, ok, if that's the unusual thing you were asking about, of course I saw that extra line flash on the left of the screen". This seems like a more parsimonious alternative compared to either of the two interpretations considered by the authors: (1) IB does not exist, (2) super-subject d' is driven by unconscious processing. Why not also consider: (3) a small percentage of subjects misinterpreted the Y/N question about noticing something unusual. In experiments 4-5, they dropped the term "unusual" but do not analyze whether this made a difference nor do they report enough of the data (subject numbers for the Y/N question and 2AFC) for readers to determine if this helped reduce the ~8% overestimate of IB rates.

      Our primary ambition in the paper was to establish, as our title suggests, residual sensitivity in IB. The ambition is quite neutral as to whether the sensitivity reflects conscious or unconscious processing (i.e. is akin to blindsight as traditionally conceived). We were evidently not clear about this, however, leading to two referees coming away with an impression of our claims that is different than we intended. We have revised our manuscript throughout to address this. But we also want to emphasize here that we take our data primarily to support the more modest claim that there is residual sensitivity (conscious or unconscious) in the group of subjects who are traditionally classified as inattentionally blind. We believe that this claim has solid support in our data.

      We do in the discussion section offer one reason for believing that there is residual awareness in the group of subjects who are traditionally classified as inattentionally blind. However, we acknowledge that this is controversial and now emphasize in the manuscript that this claim “is tentative and secondary to our primary finding”. We also emphasize that part of our point is dialectical: Inattentional blindness has been used to argue (e.g.) that attention is required for awareness. We think that our data concerning residual sensitivity at least push back on the use of IB to make this claim, even if they do not provide decisive evidence (as we agree) that awareness survives inattention. (Cf. here, Hirshhorn et al. 2024 who take up a common suggestion in the field that awareness is best assessed by using both subjective and objective measures, with claims about lack of awareness ideally being supported by both; our data suggest at a minimum that in IB objective measures do not neatly line up with subjective measures.)

      We hope this addresses the referee’s concern that we dismiss the “the comparison of IB and blindsight too quickly”. We do not intend to dismiss that comparison at all, indeed we raise it because we consider it a serious hypothesis. Our aim is simply to raise one possible consideration against it. But, again, our main claim is quite consistent with sensitivity in IB being akin to “blindsight”.

      We also agree with the referee that a possible explanation of why some subjects say they do not notice something unusual in IB paradigms, is not because they didn’t notice anything but because they didn’t consider the unexpected stimulus sufficiently unusual. However, the reviewer is incorrect that we did not mention this interpretation; to the contrary, it was precisely the kind of concern which led us to be dissatisfied with standard IB methods and so motivated our approach. As we wrote in our main text: “However, yes/no questions of this sort are inherently and notoriously subject to bias…   For example, observers might be under-confident whether they saw anything (or whether what they saw counted as unusual); this might lead them to respond “no” out of an excess of caution.” On our view, this is exactly the kind of reason (among other reasons) that one cannot rely on yes/no reports of noticing unusual stimuli, even though the field has relied on just these sorts of questions in just this way.

      We do not, however, think that this explanation accounts for why all subjects fail to report noticing, nor do we think that it accounts for our finding of above-chance sensitivity amongst non-noticers. This is for two critical reasons. First, whereas the word “unusual” did appear in the yes/no question in our Experiments 1-3, it did not appear in our Experiments 4 and 5 on dynamic IB. (In both cases, we used the exact wording of such questions in the experiments we were basing our work on.) And, of course, we still found significant residual sensitivity amongst non-noticers in Experiments 4 and 5. Second, in relation to our confidence experiment, we think it unlikely that subjects who were highly confident that they did not notice anything unusual only said that because they thought what they had seen was insufficiently unusual. Yet even in this group of subjects who were maximally confident that they did not notice anything unusual, we still found residual sensitivity.

      (7) The authors use sub-optimal questioning procedures to challenge the existence of the phenomenon this questioning is intended to demonstrate. A more neutral interpretation of this study is that it is a critique on methods in IB research, not a critique on IB as a manipulation or phenomenon. The authors neglect to mention the dozens of modern IB experiments that have improved upon the simple Y/N IB questioning methods. For example, in Michael Cohen's IB experiments (e.g., Cohen et al., 2011; Cohen et al., 2020; Cohen et al., 2021), he uses a carefully crafted set of probing questions to conservatively ensure that subjects who happened to notice the critical stimuli have every possible opportunity to report seeing them. In other experiments (e.g., Hirschhorn et al., 2024; Pitts et al., 2012), researchers not only ask the Y/N question but then follow this up by presenting examples of the critical stimuli so subjects can see exactly what they are being asked about (recognition-style instead of free recall, which is more sensitive). These follow-up questions include foil stimuli that were never presented (similar to the stimulus-absent trials here), and ask for confidence ratings of all stimuli. Conservative, pre-defined exclusion criteria are employed to improve the accuracy of their IB-rate estimates. In these and other studies, researchers are very cautious about trusting what subjects report seeing, and in all cases, still find substantial IB rates, even to highly salient stimuli. The authors should consider at least mentioning these improved methods, and perhaps consider using some of them in their future experiments.

      The concern that we do not sufficiently discuss the range of “improved” methods in IB studies is well-taken. A similar concern is raised by Reviewer #2 (Dr. Cohen). To address the concern, we have added to our manuscript a substantial new discussion of such improved methods. However, although we do agree that these methods can be helpful and may well address some of the methodological concerns which our paper raises, we do not think that they are a panacea. Thus, our discussion of these methods also includes a substantial discussion of the problems and pitfalls with such methods which led us to favor our own simple forced-response and 2afc questions, combined with SDT analysis. We think this approach is superior both to the classic approach in IB studies and to the approach raised by the reviewers.

      In particular, we have four main concerns about the follow up questions now commonly used in the field:

      First, many follow up questions are used not to exclude people from the IB group but to include people in the IB group. Thus, Most et al. 2001 asked follow up questions but used these to increase their IB group, only excluding subjects from the IB group if they both reported seeing and answered their follow ups incorrectly: “Observers were regarded as having seen the unexpected object if they answered 'yes' when asked if they had seen anything on the critical trial that had not been present before and if they were able to describe its color, motion, or shape." This means that subjects who saw the object but failed to see its color, say, would be treated as inattentionally blind. This has the purpose of inflating IB rates, in exactly the way our paper is intended to critique. So, in our view this isn’t an improvement but rather part of the approach we take issue with.

      Second, many follow up questions remain yes/no questions or nearby variants, all of which are subject to response bias. For example, in Cohen’s studies which the reviewer mentions, it is certainly true that “he uses a carefully crafted set of probing questions to conservatively ensure that subjects who happened to notice the critical stimuli have every possible opportunity to report seeing them.” We agree that this improves over a simple yes/no question in some ways. However, such follow up probes nonetheless remain yes/no questions, subject to response bias, e.g.:

      (1) “Did you notice anything strange or different about that last trial?”

      (2) “If I were to tell you that we did something odd on the last trial, would you have a guess as to what we did?”

      (3) “If I were to tell you we did something different in the second half of the last trial, would you have a guess as to what we did?”

      (4) “Did you notice anything different about the colors in the last scene?”

      Indeed, follow up questions of this kind can be especially susceptible to bias, since subjects may be reluctant to “take back” their earlier answers and so be conservative in responding positively to avoid inconsistency or acknowledgement of earlier error. This may explain why such follow up questions produce remarkable consistency despite their rather different wording. Thus, Simons and Chabris (1999) report: “Although we asked a series of questions escalating in specificity to determine whether observers had noticed the unexpected event, only one observer who failed to report the event in response to the first question (“did you notice anything unusual?'') reported the event in response to any of the next three questions (which culminated in “did you see a ... walk across the screen?''). Thus, since the responses were nearly always consistent across all four questions, we will present the results in terms of overall rates of noticing.” Thus, while there are undoubtedly merits to these follow ups, they do not resolve problems of bias.

      This same basic issue affects the follow up question used in Pitts et al. 2012 which the reviewer mentions. Pitts et al. write: “If a participant reported not seeing any patterns and rated their confidence in seeing the square pattern (once shown the sample) as a 3 or less (1 = least confident, 5 = most confident), she or he was placed in Group 1 and was considered to be inattentionally blind to the square patterns.” The confidence rating follow-up question here remains subject to bias. Moreover, and strikingly, the inclusion criterion used means that subjects who were moderately confident that they saw the square pattern when shown (i.e. answered 3) were counted as inattentionally blind (!). We do not think this is an appropriate inclusion criterion.

      The third problem is that follow up questions are often free/open-response. For instance, Most et al. (2005) ask the follow up question: "If you did see something on the last trial that had not been present during the first two trials, what color was it? If you did not see something, please guess." This is a much more difficult and to that extent less sensitive question than our binary forced-response/2afc questions. For this reason, we believe our follow up questions are more suitable for ascertaining low levels of sensitivity.

      The fourth and final issue is that whereas 2afc questions are criterion free (in that they naturally have an unbiased decision rule), this is in fact not true of n_afc questions in general, nor is it true in general of _delayed n-alternative match to sample designs. Thus, even when limited response options are given, they are not immune to response biases and so require SDT analysis. Moreover, some such tasks can involve decision spaces which are often poorly understood or difficult to analyze without making substantial assumptions about observer strategy. 

      This last point (as well as the first) is relevant to Hirshhorn et al. 2024. Hirshhorn et al. write that they “used two awareness measures. Firstly, participants were asked to rate stimulus visibility on the Perceptual Awareness Scale (PAS, a subjective measure of awareness: Ramsøy & Overgaard, 2004), and then they were asked to select the stimulus image from an array of four images (an objective measure: Jakel & Wichmann, 2006).”

      While certainly an improvement on simple yes/no questioning, the PAS remains subject to response bias. On the other hand, we applaud Hirshhorn et al.’s use of objective measures in the context of IB which of course our design implements. However, while Hirshhorn et al. 2024 suggest that their task is a spatial 4afc following the recommendation of this design by Jakel & Wichmann (2006), it is strictly a 4-alternative delayed match to sample task, so it is doubtful if it can be considered a preferred psychophysical task for the reasons Jakel & Wichmann offer. Regardless, the more crucial point is that observers in such a task might be biased towards one alternative as opposed to another. Thus, use of d′ (as opposed to percent correct as in Hirshhorn et al. 2024) is crucial in assessing performance in such tasks.

      For all these reasons, then, while we agree that the field has taken significant steps to move beyond the simple yes/no question traditionally used in IB studies (and we have revised our manuscript to make this clear); we do not think it has resolved the methodological issues which our paper seeks to highlight and address, and we believe that our approach contributes something additional that is not yet present in the literature. We have now revised our manuscript to make these points much more clearly, and we thank the reviewer for prompting these improvements.

      Reviewer #2 (Public review):

      In this study, Nartker et al. examine how much observers are conscious of using variations of classic inattentional blindness studies. The key idea is that rather than simply asking observers if they noticed a critical object with one yes/no question, the authors also ask follow-up questions to determine if observers are aware of more than the yes/no questions suggest. Specifically, by having observers make forced choice guesses about the critical object, the authors find that many observers who initially said "no" they did not see the object can still "guess" above chance about the critical object's location, color, etc. Thus, the authors claim, that prior claims of inattentional blindness are mistaken and that using such simple methods has led numerous researchers to overestimate how little observers see in the world. To quote the authors themselves, these results imply that "inattentionally blind subjects consciously perceive these stimuli after all... they show sensitivity to IB stimuli because they can see them."

      Before getting to a few issues I have with the paper, I do want to make sure to explicitly compliment the researchers for many aspects of their work. Getting massive amounts of data, using signal detection measures, and the novel use of a "super subject" are all important contributions to the literature that I hope are employed more in the future.

      We really appreciate this comment and that the reviewer found our work to make these important contributions to the literature. We wrote this paper expecting not everyone to accept our conclusions, but hoping that readers would see the work as making a valuable contribution to the literature promoting an underexplored alternative in a compelling way. Given that this reviewer goes on to express some skepticism about our claims, it is especially encouraging to see this positive feedback up top!

      Main point 1: My primary issue with this work is that I believe the authors are misrepresenting the way people often perform inattentional blindness studies. In effect, the authors are saying, "People do the studies 'incorrectly' and report that people see very little. We perform the studies 'correctly' and report that people see much more than previously thought." But the way previous studies are conducted is not accurately described in this paper. The authors describe previous studies as follows on page 3:

      "Crucially, however, this interpretation of IB and the many implications that follow from it rest on a measure that psychophysics has long recognized to be problematic: simply asking participants whether they noticed anything unusual. In IB studies, awareness of the unexpected stimulus (the novel shape, the parading gorilla, etc.) is retroactively probed with a yes/no question, standardly, "Did you notice anything unusual on the last trial which wasn't there on previous trials?". Any subject who answers "no" is assumed not to have any awareness of the unexpected stimulus.

      If this quote were true, the authors would have a point. Unfortunately, I do not believe it is true. This is simply not how many inattentional blindness studies are run. Some of the most famous studies in the inattentional blindness literature do not simply as observes a yes/no question (e.g., the invisible gorilla (Simons et al. 1999), the classic door study where the person changes (Simons and Levin, 1998), the study where observers do not notice a fight happening a few feet from them (Chabris et al., 2011). Instead, these papers consistently ask a series of follow-up questions and even tell the observers what just occurred to confirm that observers did not notice that critical event (e.g., "If I were to tell you we just did XYZ, did you notice that?"). In fact, after a brief search on Google Scholar, I was able to relatively quickly find over a dozen papers that do not just use a yes/no procedure, and instead as a series of multiple questions to determine if someone is inattentionally blind. In no particular order some papers (full disclosure: including my own):

      (1) Most et al. (2005) Psych Review

      (2) Drew et al. (2013) Psych Science

      (3) Drew et al. (2016) Journal of Vision

      (4) Simons et al. (1999) Perception

      (5) Simons and Levin (1998) Perception

      (6) Chabris et al. (2011) iPerception

      (7) Ward & Scholl (2015) Psych Bulletin and Review

      (8) Most et al. (2001) Psych Science

      (9) Todd & Marois (2005) Psych Science

      (10) Fougnie & Marois (2007) Psych Bulletin and Review

      (11) New and German (2015) Evolution and Human Behaviour

      (12) Jackson-Nielsen (2017) Consciousness and cognition

      (13) Mack et al. (2016) Consciousness and cognition

      (14) Devue et al. (2009) Perception

      (15) Memmert (2014) Cognitive Development

      (16) Moore & Egeth (1997) JEP:HPP

      (17) Cohen et al. (2020) Proc Natl Acad Sci

      (18) Cohen et al. (2011) Psych Science

      This is a critical point. The authors' key idea is that when you ask more than just a simple yes/no question, you find that other studies have overestimated the effects of inattentional blindness. But none of the studies listed above only asked simple yes/no questions. Thus, I believe the authors are mis-representing the field. Moreover, many of the studies that do much more than ask a simple yes/no question are cited by the authors themselves! Furthermore, as far as I can tell, the authors believe that if researchers do these extra steps and ask more follow-ups, then the results are valid. But since so many of these prior studies do those extra steps, I am not exactly sure what is being criticized.

      To make sure this point is clear, I'd like to use a paper of mine as an example. In this study (Cohen et al., 2020, Proc Natl Acad Sci USA) we used gaze-contingent virtual reality to examine how much color people see in the world. On the critical trial, the part of the scene they fixated on was in color, but the periphery was entirely in black and white. As soon as the trial ended, we asked participants a series of questions to determine what they noticed. The list of questions included:

      (1) "Did you notice anything strange or different about that last trial?"

      (2) "If I were to tell you that we did something odd on the last trial, would you have a guess as to what we did?"

      (3) "If I were to tell you we did something different in the second half of the last trial, would you have a guess as to what we did?"

      (4) "Did you notice anything different about the colors in the last scene?"

      (5) We then showed observers the previous trial again and drew their attention to the effect and confirmed that they did not notice that previously.

      In a situation like this, when the observers are asked so many questions, do the authors believe that "the inattentionally blind can see after all?" I believe they would not say that and the reason they would not say that is because of the follow-up questions after the initial yes/no question. But since so many previous studies use similar follow-up questions, I do not think you can state that the field is broadly overestimating inattentional blindness. This is why it seems to me to be a bit of a strawman: most people do not just use the yes/no method.

      We appreciate this reviewer raising this issue. As he (Dr. Cohen) states, his “primary issue” concerns our discussion of the broader literature (which he worries understates recent improvements made to the IB methodology), rather than, e.g., the experiments we’ve run. We take this concern very seriously and address it comprehensively here.

      A very similar issue is identified by Reviewer #1, comment (7). To review some of what we say in reply to them: To address the concern we have added to our manuscript a substantial new discussion of such improved methods. However, although we do agree that these methods can be helpful and may well address some of the methodological concerns which our paper raises, we do not think that they are a panacea. Thus, our discussion of these methods also includes a substantial discussion of the problems and pitfalls with such methods which led us to favor our own simple forced-response and 2afc questions, combined with SDT analysis. We think this approach is superior both to the classic approach in IB studies and to the approach raised by the reviewers.

      In particular, we have three main concerns about the follow up questions now commonly used in the field:

      First, many follow up questions are used not to exclude subjects from the IB group but to include subjects in the IB group. Thus, Most et al. (2001) asked follow up questions but used these to increase their IB group, only excluding subjects from the IB group if they both reported seeing and failed to answer their follow ups correctly: “Observers were regarded as having seen the unexpected object if they answered 'yes' when asked if they had seen anything on the critical trial that had not been present before and if they were able to describe its color, motion, or shape." This means that subjects who saw the object but failed to describe it in these respects would be treated as inattentionally blind. This is problematic since failure to describe a feature (e.g., color, shape) does not imply a complete lack of information concerning that feature; and even if a subject did lack all information concerning these features of an object, this would not imply a complete failure to see the object. Similarly, Pitts et al. (2012) asked subjects to rate their confidence in their initial yes/no response from 1 = least confident to 5 = most confident, and used these ratings to include in the IB group those who rated their confidence in seeing at 3 or less. This is evidently problematic, since there is a large gap between being under confident that one saw something and being completely blind to it. More generally, using follows up to inflate IB rates in such ways raises precisely the kinds of issues our paper is intended to critique. So in our view this isn’t an improvement but rather part of the approach we take issue with.

      Second, many follow up questions remain yes/no questions or nearby variants, all of which are subject to response bias. For example, in the reviewer’s own studies (Cohen et al. 2020, 2011; see also: Simons et al., 1999; Most et al., 2001, 2005; Drew et al., 2013; Memmert, 2014) a series of follow up questions are used to try and ensure that subjects who noticed the critical stimuli are given the maximum opportunity to report doing so, e.g.:

      (1) “Did you notice anything strange or different about that last trial?”

      (2) “If I were to tell you that we did something odd on the last trial, would you have a guess as to what we did?”

      (3) “If I were to tell you we did something different in the second half of the last trial, would you have a guess as to what we did?”

      (4) “Did you notice anything different about the colors in the last scene?”

      We certainly agree that such follow up questions improve over a simple yes/no question in some ways. However, such follow up probes nonetheless remain yes/no questions, intrinsically subject to response bias. Indeed, follow up questions of this kind can be especially susceptible to bias, since subjects may be reluctant to “take back” their earlier answers and so be conservative in responding positively to avoid inconsistency or acknowledgement of earlier error. This may explain why such follow up questions produce remarkable consistency despite their rather different wording. Thus, Simons and Chabris (1999) report: “Although we asked a series of questions escalating in specificity to determine whether observers had noticed the unexpected event, only one observer who failed to report the event in response to the first question (“did you notice anything unusual?'') reported the event in response to any of the next three questions (which culminated in “did you see a ... walk across the screen?''). Thus, since the responses were nearly always consistent across all four questions, we will present the results in terms of overall rates of noticing.” Thus, while there are undoubtedly merits to these follow ups, they do not resolve problems of bias.

      It is also important to recognize that whereas 2afc questions are criterion free (in that they naturally have an unbiased decision rule), this is not true of n_afc nor delayed _n-alternative match to sample designs in general. Performance in such tasks thus requires SDT analysis – which itself may be problematic if the decision space is not properly understood or requires making substantial assumptions about observer strategy.

      Third, and finally, many follow up questions are insufficiently sensitive (especially with small sample sizes). For instance, Todd, Fougnie & Marois (2005) used a 12-alternative match-tosample task (see similarly: Fougnie & Marois, 2007; Devue et al., 2009). And Most et al. (2005) asked an open-response follow-up: “If you did see something on the last trial that had not been present during the first two trials, what color was it? If you did not see something, please guess.” These questions are more difficult and to that extent less sensitive than binary forced-response/2afc questions of the sort we use in our own studies – a difference which may be critical in uncovering degraded perceptual sensitivity.

      For all these reasons, then, while we agree that the field has taken significant steps to move beyond the simple yes/no question traditionally used in IB studies (and we have revised our manuscript to make this clear); we do not think it has resolved the methodological issues which our paper seeks to highlight and address, and we believe that our approach of using 2afc or forced-response questions combined with signal detection analysis is an important improvement on prior methods and contributes something additional that is not yet present in the literature. We have now revised our manuscript to make these points much clearer.

      Other studies that improve on the standard methodology

      This reviewer adds something else, however: A very helpful list of 18 papers which include follow ups and that he believes overcome many of the issues we raise in our paper. To just state our reaction bluntly: We are familiar with every one of these papers (indeed, one of them is a paper by one of us!), and while we think these are all very valuable contributions to the literature, it is our view that none of these 18 papers resolves the worries that led us to conduct our work.  

      Here we briefly comment on the relevant pitfalls in each case. We hope this serves to underscore the importance of our methodological approach.

      (1) Most et al. (2005) Psych Review

      Either a 2-item or 5-item questionnaire was used. The 2-item questionnaire ran as follows:

      (1) On the last trial, did you see anything other than the 4 circles and the 4 squares (anything that had not been present on the original two trials)? Yes No 

      (2) If you did see something on the last trial that had not been present during the original two trials, please describe it in as much detail as possible.

      This clearly does not substantially improve on the traditional simple yes/no question. Moreover, the second question (as well as being open-ended) was used to include additional subjects in the IB group, in that participants were counted as having seen the object only if they responded “yes” to Q1 and in addition “were able to report at least one accurate detail” in response to Q2. In other words, either a subject says “no” (and is treated as unaware), or says “yes” and then is asked to prove their awareness, as it were. If anything, this intensifies the concerns we raise, by inflating IB rates. 

      The 5-item questionnaire looked like this: 

      (1) On the last trial, did you see anything other than the black and white L’s and T’s (anything that had not been present on the first two trials)?

      (2) If you did see something on the last trial that had not been present during the first two trials, please describe it.

      (3) If you did see something on the last trial that had not been present during the first two trials, what color was it? If you did not see something, please guess. (Please indicate whether you did see something or are guessing)

      (4) If you did see something during the last trial that had not been present in the first two trials, please draw an arrow on the “screen” below showing the direction in which it was moving. If you did not see something, please guess. (Please indicate whether you did see something or are guessing)

      (5) If you did see something during the last trial that had not been present during the first two trials, please circle the shape of the object below [4 shapes are presented to choose from]. If you did not see anything, please guess. (Please indicate whether you did see something or are guessing)

      Q5 was not used for analysis purposes. (It suffers from the second issue raised above.) Q1 is the traditional y/n question. Qs 2&3 are open ended. It is unclear how responses to Q4 were analyzed (at the limit it could be considered a helpful, forced-choice question – though it again would suffer from the second issue raised above). However, as noted with respect to the 2-item questionnaire, these responses were not used to exclude people from the IB group but to include people in it. So again, this approach does not in any way address the issues we are concerned about, and if anything, only makes them worse. 

      (2)  Drew et al. (2013) Psych Science

      All follow ups were yes/no: “we asked a series of questions to determine whether they noticed the gorilla: ‘Did the final trial seem any different than any of the other trials?’, ‘Did you notice anything unusual on the final trial?’, and, finally, ‘Did you see a gorilla on the final trial?’”. So, this paper essentially implements the standard methodology we mention (and criticize). 

      (3)  Drew et al. (2016) Journal of Vision

      Follow up questions were used, but the reported procedure does not provide sufficient details to evaluate them (we are only told: “After the final trial, they were asked: ‘On that last trial of the task, did you notice anything that was not there on previous trials?’ They then answered questions about the features of the unexpected stimulus on a separate screen (color, shape, movement, and direction of movement).”). It is not clear that these follow ups were used to exclude any subjects from the analysis. Finally, given that the unexpected object could be the same color as the targets/distractors, it is clear that biases would have been introduced which would need to be considered (but which were not).

      (4)  Simons & Chabris (1999) Perception

      All follow ups were yes/no: “observers were … asked to provide answers to a surprise series of additional questions. (i) While you were doing the counting, did you notice anything unusual on the video? (ii) Did you notice any- thing other than the six players? (iii) Did you see anyone else (besides the six players) appear on the video? (iv) Did you see a gorilla [woman carrying an umbrella] walk across the screen? After any “yes'' response, observers were asked to provide details of what they noticed. If at any point an observer mentioned the unexpected event, the remaining questions were skipped.” As noted previously, the analyses in fact did not use these questions to exclude subjects since answers were so consistent.

      (5)  Simons and Levin (1998) Perception

      This is a change detection paradigm, not a study of inattentional blindness. And in any case, one yes/no follow up was used: “Did you notice that I'm not the same person who approached you to ask for directions?”

      (6)  Chabris et al. (2011) iPerception

      Two yes/no questions were asked: “we asked whether the subjects had seen anything unusual along the route, and then whether they had seen anyone fighting.” It seems that follow up questions (a request to describe the fight) were asked only of those who said yes.

      This is in fact a common procedure – follow up questions only being asked of the “yes” group. As discussed, it is sometimes used to increase rates of IB, compounding the problem we identify in our paper. So this is another example of a follow-up question that makes the problem we identify worse, not better.

      (7) Ward & Scholl (2015) Psych Bulletin and Review

      Two yes/no questions were used: “...observers were asked whether they noticed ‘anything … that was different from the first three trials’ — and if so, to describe what was different. They were then shown the gray cross and asked if they had noticed it—and if so, to describe where it was and how it moved. Only observers who explicitly reported not noticing the cross were counted as ‘nonnoticers’ to be included in the final sample (N = 100).” In each case, combining the traditional noticing question with a request to describe and identify may have induced conservative response biases in the noticing question, since a subject might consider being able to describe or identify the unexpected stimulus a precondition of giving a positive answer to the noticing question.

      (8) Most et al. (2001) Psych Science

      The same 5-item questionnaire discussed above in relation to Most et al. (2005) was used: 

      (1) On the last trial, did you see anything other than the black and white L’s and T’s (anything that had not been present on the first two trials)?

      (2)   If you did see something on the last trial that had not been present during the first two trials, please describe it.

      (3) If you did see something on the last trial that had not been present during the first two trials, what color was it? If you did not see something, please guess. (Please indicate whether you did see something or are guessing)

      (4) If you did see something during the last trial that had not been present in the first two trials, please draw an arrow on the “screen” below showing the direction in which it was moving. If you did not see something, please guess. (Please indicate whether you did see something or are guessing)

      (5) If you did see something during the last trial that had not been present during the first two trials, please circle the shape of the object below [4 shapes are presented to choose from]. If you did not see anything, please guess. (Please indicate whether you did see something or are guessing)

      Q5 was not used for analysis purposes. (It suffers from the second issue raised above.) Q1 is the traditional yes/no question. Qs 2&3 are open ended. It is unclear how responses to Q4 were analyzed (at the limit it could be considered a helpful, forced-choice question – though it again would suffer from the second issue raised above). However, as noted with respect to the two item questionnaire in Most et al. 2005, these responses were not used to exclude people from the IB group but to include people in it. So again this approach does not in any way address the issues we are concerned about, and if anything only makes them worse.

      (9) Todd, Fougnie & Marois (2005) Psych Science

      “participants were probed with three questions to determine whether they had detected the critical stimulus ... .The first question assessed whether subjects had seen anything unusual during the trial; they responded ‘‘yes’’ or ‘‘no’’ by pressing the appropriate key on the keyboard. The second question asked participants to select which stimulus they might have seen among 12 possible objects and symbols selected from MacIntosh font databases. The third question asked participants to select the quadrant in which the critical stimulus may have appeared by pressing one of four keys, each of which corresponded to one of the quadrants.”

      These follow ups were used to include people in the IB group: “In keeping with previous studies (Most et al., 2001), participants were considered to have detected the critical stimulus successfully if they (a) reported seeing an unexpected stimulus and (b) correctly selected its quadrant location.” In line with our third point about sensitivity, the object identity test transpired to be “too difficult even under full-attention conditions … Thus, performance with this question was not analyzed further.”

      (10) Fougnie & Marois (2007) Psych Bulletin and Review

      Same exact methods and problems as with Todd & Marois (2005) Psych Science, just discussed.

      (11) New and German (2015) Evolution and Human Behaviour

      “After the fourth trial containing the additional experimental stimulus, the participant was asked, “Did you see anything in addition to the cross on that trial?” and which quadrant the additional stimulus appeared in. They were then asked to identify the stimulus in an array which in Experiment 1 included two variants chosen randomly from the spider stimuli and the two needle stimuli. Participants in Experiment 2 picked from all eight stimuli used in that experiment.”

      Our second concern about response biases and the need for appropriate SDT analysis of the 4/8 alternative tasks applies to all these questions. We also note that analyses were only performed on groups separately (those who detected/failed to detect, those who located/failed to locate, and those who identified/failed to identify) and on the group which did all three/failed to do any one of the three. Especially in light of the fact that some subjects could clearly detect the stimulus without being able to identity it (e.g.), the most stringent test given our concerns (which were not obviously New and German’s comparative concerns), would be to consider the group which could not detect, identify or localize.

      (12) Jackson-Nielsen (2017) Consciousness and cognition

      This is a very interesting example of a follow-up which used a 3-AFC recognition test:

      “participants were immediately asked, ‘‘which display looks most like what you just saw?’ from 3 alternatives”. However, though such an objective test is definitely to be preferred in our view to an open-ended series of probes, the 3-AFC test administered clearly had issues with response biases, as discussed, and actually yielded significantly below chance performance in one of the experiments.

      (13) Mack et al. (2016) Consciousness and cognition

      The follow ups here were essentially yes/no combined with an assessment of surprise. Participants were asked to enter letters into a box, and if they did so “were immediately asked by the experimenter whether they had noticed anything different about the array on this last trial and if they did not, they were told that there had been no letters and their responses to that news were recorded. Clearly, if they expressed surprise, this would be compelling evidence that they were unaware of the absence of the letters. Those observers who did not enter letters and realized there were no letters present were considered aware of the absence.” So, this again has all of the same problems we identify, considering subjects unaware because they expressed surprise.

      (14) Devue et al. (2009) Perception

      An 8-alternative task was used. The authors were primarily interested in a comparative analysis and so did not use this task to exclude subjects. We note that an 8 alternative task is very demanding – compare the 12-alternative task used in Todd, Fougnie & Marois (2005). There was an attempt to investigate biases in a separate bias trial, however SDT measures were not used.

      (15) Memmert (2014) Cognitive Development

      “After watching the video and stating the number of passes, participants answered four questions (following Simons & Chabris, 1999): (1) While you were counting, did you perceive anything unusual on the video? (2) Did you perceive anything other than the six players? (3) Did you see anyone else (besides the six players) appear on the video? (4) Did you notice a gorilla walk across the screen? After any “yes” reply, children were asked to provide details of what they noticed. If at any point a child mentioned the unexpected event, the remaining questions were omitted.” All of these follow-up questions are yes/no judgments, used to determine awareness in exactly the way we critique as problematic.

      (16) Moore & Egeth (1997) JEP:HPP

      This study (which includes one of us, Egeth, as author) did use forced choice questions. In one case, the question was 2-alternative, in the other it was 4-alternative. In the latter case, SDT would have been appropriate but was not used. In the former case, it may have been that a larger sample would have revealed evidence of sensitivity to the background pattern (as it stood 55% answered the 2-alternative question correctly). Although these results have been replicated, unfortunately the replication in Wood and Simons 2019 used a 6-alternative recognition task and this was not analyzed using SDT. We also note that the task is rather difficult in this study. Wood and Simons report: “Exclusion rates were much higher than anticipated, primarily due to exclusions when subjects failed to correctly report the pattern on the full-attention trial; we excluded 361 subjects, or 58% of our sample.”

      (17) Cohen et al. (2020) Proc Natl Acad Sci

      While this paper improves over a simple yes/no question in some ways, especially in that it used the follow up questions to exclude subjects from the unaware (IB) group, the follow up probes nonetheless remain yes/no questions, subject to response bias, e.g.:

      (1) “Did you notice anything strange or different about that last trial?”

      (2) “If I were to tell you that we did something odd on the last trial, would you have a guess as to what we did?”

      (3) “If I were to tell you we did something different in the second half of the last trial, would you have a guess as to what we did?”

      (4) “Did you notice anything different about the colors in the last scene?”

      Follow up questions of this kind can be especially susceptible to bias, since subjects may be reluctant to “take back” their earlier answers and so be conservative in responding positively to avoid inconsistency or acknowledgement of earlier error. This may explain why such follow up questions can produce remarkable consistency despite their rather different wording. 

      (18) Cohen et al. (2011) Psych Science

      Here are the probes used in this study:

      (1) Did you notice anything different on that trial?

      (2) Did you notice something different about the background stream of images?

      (3) Did you notice that a different type of image was presented in the background that was unique in some particular way?

      (4) Did you see an actual photograph of a natural scene in that stream?

      (5) If I were to tell you that there was a photograph in that stream, can you tell me what it was a photograph of?

      Qs 1-4 are yes/no. Q5 is yes/no with an open-ended response. After this, a 5 or 6-alternative recognition test was administered. So again, this faces the same issues, since y/n questions are subject to bias in the way we have described, and many-alternative tests are more problematic than 2afc tests.

      In summary

      We really appreciate the care that went into compiling this list, and we agree that these papers and the improved methods they contain are relevant. But as hopefully made clear above, the approaches in each of these papers simply don’t solve the foundational issues our critique is aimed at (though they may address other issues). This is why we felt our new approach was necessary. And we continue to feel this way even after reading and incorporating these comments from Dr. Cohen.

      Nevertheless, there is clearly lots for us to do in light of these comments. And so as noted earlier we have now added a very substantial new section to our discussion section to more fairly and completely portray the state of the art in this literature. This is really to our benefit in the end, since we now not only better acknowledge the diverse approaches present, but also set up ourselves to make our novel contribution exceedingly clear.

      Main point 2: Let's imagine for a second that every study did just ask a yes/no question and then would stop. So, the criticism the authors are bringing up is valid (even though I believe it is not). I am not entirely sure that above chance performance on a forced choice task proves that the inattentionally blind can see after all. Could it just be a form of subliminal priming? Could there be a significant number of participants who basically would say something like, "No I did not see anything, and I feel like I am just guessing, but if you want me to say whether the thing was to the left or right, I will just 100% guess"? I know the literature on priming from things like change and inattentional blindness is a bit unclear, but this seems like maybe what is going on. In fact, maybe the authors are getting some of the best priming from inattentional blindness because of their large sample size, which previous studies do not use.

      I'm curious how the authors would relate their studies to masked priming. In masked priming studies, observers say the did not see the target (like in this study) but still are above chance when forced to guess (like in this study). Do the researchers here think that that is evidence of "masked stimuli are truly seen" even if a participant openly says they are guessing?

      We’re grateful to the reviewer for raising this question. As we say in response to Reviewer #1, our primary ambition in the paper is to establish, as our title suggests, residual sensitivity in IB. The ambition is quite neutral as to whether the sensitivity reflects conscious or unconscious processing (i.e. is akin to blindsight as traditionally conceived, or what the reviewer here suggests may be happening in masked priming). Since we were evidently insufficiently clear about this we have revised our manuscript in several places to clarify that we take our data primarily to support the more modest claim that there is residual sensitivity (conscious or unconscious) in the group of subjects who are traditionally classified as inattentionally blind. We believe that this claim has much more solid support in our data than our secondary and tentative suggestion about awareness.

      This said, we do consider masked priming studies to be susceptible to the critique that performance may reflect degraded conscious awareness which is unreported because of conservative response criteria. There is good evidence that response criteria tend to be conservative near threshold (Björkman et al. 1993; see also: Railo et al. 2020), including specifically in masked priming studies (Sand 2016, cited in Phillips 2021). So, we consider it a perfectly reasonable hypothesis that subjects who say they feel they are guessing in fact have conscious access to a degraded signal which is insufficient to reach a conservative response criterion but nonetheless sufficient to perform above chance in 2afc detection. Of course, we appreciate that this hypothesis is controversial, so it is not one we argue for in our paper (though we are happy to share our feelings about it here).

      Main point 3: My last question is about how the authors interpret a variety of inattentional blindness findings. Previous work has found that observers fail to notice a gorilla in a CT scan (Drew et al., 2013), a fight occurring right in front of them (Chabris et al., 2011), a plane on a runway that pilots crash into (Haines, 1991), and so forth. In a situation like this, do the authors believe that many participants are truly aware of these items but simply failed to answer a yes/no question correctly? For example, imagine the researchers made participants choose if the gorilla was in the left or right lung and some participants who initially said they did not notice the gorilla were still able to correctly say if it was in the left or right lung. Would the authors claim "that participant actually did see the gorilla in the lung"? I ask because it is difficult to understand what it means to be aware of something as salient as a gorilla in a CT scan, but say "no" you didn't notice it when asked a yes/no question. What does it mean to be aware of such important, ecologically relevant stimuli, but not act in response to them and openly say "no" you did not notice them?

      Our view is that in such cases, observers may well have a “degraded” percept of the relevant feature (gorilla, plane, fight etc.). But crucially we do not suggest that this percept is sufficient for observers to recognize the object/event as a gorilla, plane, fight etc. Our claim is only that, in our studies at least, observers (as a group) do have enough information about the unexpected stimuli to locate them, and discriminate certain low level features better than chance. Crudely, it may be that subjects see the gorilla simply as a smudge or the plane as a shadowy patch etc. (One of us who is familiar with the gorilla CT scan stimuli notes that the gorilla is in fact rather hard to see even when you know which slide it is on, suggesting that they are not as “salient” as the reviewer suggests!) 

      More precisely, in the paper we write that in our view perhaps “...unattended stimuli are encoded in a partial or degraded way. Here we see a variety of promising options for future work to investigate. One is that unattended stimuli are only encoded as part of ensemble representations or summary scene statistics (Rosenholtz, 2011; Cohen et al., 2016). Another is that only certain basic “low-level” or “preattentive” features (see Wolfe & Utochkin, 2019 for discussion) can enter awareness without attention. A final possibility consistent with the present data is that observers can in principle be aware of individual objects and higher-level features under inattention but that the precision of the corresponding representations is severely reduced. Our central aim here is to provide evidence that awareness in inattentional blindness is not abolished. Further work is needed to characterize the exact nature of that awareness.” We hope this sheds light on our perspective while still being appropriately cautious not to go too far beyond our data.

      Overall: I believe there are many aspects of this set of studies that are innovative and I hope the methods will be used more broadly in the literature. However, I believe the authors misrepresent the field and overstate what can be interpreted from their results. While I am sure there are cases where more nuanced questions might reveal inattentional blindness is somewhat overestimated, claims like "the inattentionally blind can see after all" or "Inattentionally blind subjects consciously perceive thest stimuli after all" seem to be incorrect (or at least not at all proven by this data).

      Once again, we would like to thank this reviewer for his feedback, which obviously comes from a place of tremendous expertise on these issues. We appreciate his assessment that our studies are innovative and that our methodological advances will be of use more broadly. We also hear the reviewer loud and clear about the passages in question, which on reflection we agree are not as central to our case as the other claims we make (regarding residual sensitivity and conservative responding), and so we have now edited them accordingly to refocus our discussion on only those claims that are central and supported. Thank you for making our paper stronger!

      Reviewer #3 (Public review):

      Summary:

      Authors try to challenge the mainstream scientific as well as popularly held view that Inattentional

      Blindness (IB) signifies subjects having no conscious awareness of what they report not seeing (after being exposed to unexpected stimuli). They show that even when subjects indicate NOT having seen the unexpected stimulus, they are at above chance level for reporting features such as location, color or movement of these stimuli. Also, they show that 'not seen' responses are in part due to a conservative bias of subjects, i.e. they tend to say no more than yes, regardless of actual visibility. Their conclusion is that IB may not (always) be blindness, but possibly amnesia, uncertainty etc.

      We just thought to say that we felt this was a very accurate summary of our claims, and in ways underscore the modesty we had hoped to convey. This is especially true of the reviewer’s final sentence: “Their conclusion is that IB may not (always) be blindness, but possibly amnesia, uncertainty etc.”; as we noted in response to other reviewers, our claim is not that IB doesn’t exist, that subjects are always conscious of the stimulus, etc.; it is only that the cohort of IB subjects show sensitivity to the unattended stimulus in ways that suggest they are not as blind as traditionally conceived. Thank you for reading us as intended!

      Strengths:

      A huge pool of (25.000) subjects is used. They perform several versions of the IB experiments, both with briefly presented stimuli (as the classic Mack and Rock paradigm), as well as with prolonged stimuli moving over the screen for 5 seconds (a bit like the famous gorilla version), and all these versions show similar results, pointing in the same direction: above chance detection of unseen features, as well as conservative bias towards saying not seen.

      We’re delighted that the reviewer appreciated these strengths in our manuscript!

      Weaknesses:

      Results are all significant but effects are not very strong, typically a bit above chance. Also, it is unclear what to compare these effects to, as there are no control experiments showing what performance would have been in a dual task version where subjects have to also report features etc for stimuli that they know will appear in some trials

      The backdrop to the experiments reported here is the “consensus view” (Noah & Mangun, 2020) according to which inattention completely abolishes perception, such that subjects undergoing IB “have no awareness at all of the stimulus object” (Rock et al., 1992) and that “one can have one’s eyes focused on an object or event … without seeing it at all” (Carruthers, 2015). In this context, we think our findings of significant above-chance sensitivity (e.g., d′ = 0.51 for location in Experiment 1; chance, of course, would be d′ = 0 here) are striking and constitute strong evidence against the consensus view. We of course agree that the residual sensitivity is far lower than amongst subjects who noticed the stimulus. For this reason, we certainly believe that inattention has a dramatic impact on perception. To that extent, our data speak in favor of a “middle ground” view on which inattention substantially degrades but crucially does not abolish perception/explicit encoding. We see this as an importantly neglected option in a literature which has overly focused on seen/not seen binaries (see our section ‘Visual awareness as graded’).

      Regarding the absence of a control condition, we think those conditions wouldn’t have played the same role in our experiments as they typically play in other experiments. As Reviewer #1 comments, the main role of such trials in previous work has been to exclude from analysis subjects who failed to report the unexpected stimulus on the divided and/or full attention control trials. As Reviewer #1 points out, excluding such subjects would very likely have ‘helped’ us. However, the practice is controversial. Indeed, in a review of 128 experiments, White et al. 2018 argue that the practice has “problematic consequences” and “may lead researchers to understate the pervasiveness of inattentional blindness". Since we wanted to offer as simple and demanding a test of residual sensitivity in IB as possible, we thus decided not to use any exclusions, and for that reason decided not to include divided/full attention trials.

      As recommended, we discuss this decision not to include divided/full attention trials and our logic for not doing so in the manuscript. As we explain, not having those conditions makes it more impressive, not less impressive, that we observed the results we in fact did — it makes our results more interpretable, not less interpretable, and so absence of such conditions from our manuscript should not (in our view) be considered any kind of weakness.

      There are quite some studies showing that during IB, neural processing of visual stimuli continues up to high visual levels, for example, Vandenbroucke et al 2014 doi:10.1162/jocn_a_00530 showed preserved processing of perceptual inference (i.e. seeing a kanizsa illusion) during IB. Scholte et al 2006 doi: 10.1016/j.brainres.2005.10.051 showed preserved scene segmentation signals during IB. Compared to the strength of these neural signatures, the reported effects may be considered not all that surprising, or even weak.

      We agree that such evidence of neural processing in IB is relevant to — and perhaps indeed consistent with — our picture, and we’re grateful to the reviewer for pointing out further studies along those lines. Previously, we mentioned a study from Pitts et al., 2012 in which, as we wrote, “unexpected line patterns have been found to elicit the same Nd1 ERP component in both noticers and inattentionally blind subjects (Pitts et al., 2012).” We have added references to both the studies which the reviewer mentions – as well as an additional relevant study – to our manuscript in this context. Thank you for the helpful addition.

      We do however think that our studies are importantly different to this previous work. Our question is whether processing under IB yields representations which are available for explicit report and so would constitute clear evidence of seeing, and perhaps even conscious experience. As we discuss, evidence for this kind of processing remains wanting: “A handful of prior studies have explored the possibility that inattentionally blind subjects may retain some visual sensitivity to features of IB stimuli (e.g., Schnuerch et al., 2016; see also Kreitz et al., 2020, Nobre et al., 2020). However, a recent meta-analysis of this literature (Nobre et al., 2022) argues that such work is problematic along a number of dimensions, including underpowered samples and evidence of publication bias that, when corrected for, eliminates effects revealed by earlier approaches, concluding “that more evidence, particularly from well-powered pre-registered experiments, is needed before solid conclusions can be drawn regarding implicit processing during inattentional blindness” (Nobre et al., 2022).” Our paper is aimed at addressing this question which evidence of neural processing can only speak to indirectly.

      Recommendations for the authors:  

      Reviewer #1 (Recommendations for the authors):

      (1) Please report all of the data, especially the number of subjects in each experiment that answered Y/N and the numbers of subjects in each of the Y and N groups that guessed a feature correctly/incorrectly on the 2AFC tasks. And also the confidence ratings for the 2AFC task (for comparison with the confidence ratings on the Y/N questions).

      We now report all this data in our (revised) Supplementary Materials. We agree that this information will be helpful to readers.

      (2) Consider adding a control condition with partial attention (dual task) or full attention (single task) to estimate the rates of seeing the critical stimulus when it's expected.

      This is the only recommendation we have chosen not to implement. The reason, as we explain in detail above (especially in response to Reviewer #1 comment 5), is that this would not in fact be a “control condition” in our studies, and indeed would only inflate the biases we are concerned with in our work. As the referee comments, the main role of such trials in previous work has been to exclude from analysis subjects who failed to report the unexpected stimulus on the divided and/or full attention control trials. And the practice is controversial: Indeed, in a review of 128 experiments, White et al. 2018 argue that the practice has “problematic consequences” and “may lead researchers to understate the pervasiveness of inattentional blindness" (emphasis added). So, our choice not to have such conditions ensures an especially stringent test of our central claim. Not having those conditions (and their accompanying exclusions) makes our results more interpretable, not less interpretable, and so the absence of such conditions from our manuscript should not (in our view) be considered any kind of weakness.

      We have added a paragraph to our “Design and analytical approach” section explaining the logic behind our deliberate decision not to include divided or full attention trials in our experiments. (For even fuller discussion, see our response to Reviewer #1’s comment 5 above.)

      (3) Consider revising the interpretations to be more precise about the distinction between the super subject being above chance versus each individual subject who cannot be at chance or above chance because there was only a single trial per subject.

      We have now done this throughout the manuscript, as discussed above. We have also added a substantive additional discussion to our “Design and analytical approach” section discussing what should be said about individual subjects in light of our group level data.

      This was a very helpful point, and greatly clarifies the claims we wish to make in the paper. Thank you for this comment, which has certainly made our paper stronger.

      Reviewer #2 (Recommendations for the authors):

      I would be curious to hear the authors' response to two points:

      (1) What do they have to say about prior studies that do more than just ask yes/no questions (and ask several follow-ups)? Are those studies "valid"?

      A very substantial new discussion of this important point has been added. As you will see above, we comment on every one of the 18 papers this reviewer raised (as well as the general argument made); we contend that while many of these papers improve on past methodology in various ways, most in fact do “just ask yes/no questions”, and none of them makes the methodological advance we offer in our manuscript. However, this discussion has helped us clarify that very advance, and so working through this issue has really helped us improve our paper and make its relation to existing literature that much clearer. Thank you for raising this crucial point.

      (2) Do the authors think it is possible that in many cases, people are just guessing about a critical item's location or color and this is at least in part a form of priming?

      We have clarified our discussion in numerous places to further emphasize that our main point concerns above-chance sensitivity, not awareness. Given this, we take very seriously the hypothesis that something like priming of a kind sometimes proposed to occur in cases of blindsight or other putative cases of unconscious perception could be what is driving the responses in non-noticers.

      Reviewer #3 (Recommendations for the authors):

      (1) Control dual task version with expected stimuli would be nice

      We have added a paragraph to our “Design and analytical approach” section explaining the logic behind our deliberate decision not to include divided or full attention trials, which would not in fact be a “control” task in our experiments. For full discussion, see our response to Reviewer 3 above, as well as our summary here in the Recommendations for Authors section in responding to Reviewer 1, recommendation (2).

      (2) Please do a better job in discussing and introducing experiments about neural signatures during IB.

      A discussion of Vandenbroucke et al. 2014 and Scholte et al. 2006 has been added to our discussion of neural signatures in IB, as well as an additional reference to an important early study of semantic processing in IB (Rees et al., 1999). Thank you for these very helpful suggestions!

    1. Reviewer #1 (Public review):

      Summary:

      This manuscript seeks to estimate the causal effect of genes on disease. To do so, they introduce a novel algorithm, termed the Root Causal Strength using Perturbations (RCSP) algorithm. RCSP uses perturb-seq to first estimate the gene regulatory network structure among genes, and then uses bulk RNA-seq with phenotype data on the samples to estimate causal effects of genes on the phenotype conditional on the learned network structure. The authors assess the performance of RCSP in comparison to other methods via simulation. Next, they apply RCSP to two real human datasets: 513 individuals age-related macular degeneration and 137 individuals with multiple sclerosis.

      Strengths:

      The authors tackle an important and ambitious problem - the identification of causal contributors to disease in the context of a causal inference framework. As the authors point out, observational RNA-seq data is insufficient for this kind of causal discovery, since it is very challenging to recover the true underlying graph from observational data; interventional data are needed. However, little perturb-seq data has been generated with annotated phenotype data, and much bulk RNA-seq data has already been generated, so it is useful to propose an algorithm to integrate the two as the authors have done.

      The authors also offer substantial theoretical exposition for their work, bringing to bear both the literature on causal discovery as well as literature on the genetic architecture of complex traits. They also benchmark RCSP under multiple challenging simulation settings, including an analysis of RCSP when the underlying graph is not a DAG.

      Weaknesses:

      The notion of a "root" causal gene - which the authors define based on a graph theoretic notion of topologically sorting graphs - requires a graph that is directed and acyclic. It is the latter that constitutes an important weakness here - it simply is a large simplification of human biology to draw out a DAG including hundreds of genes and a phenotype Y and to claim that the true graph contains no cycles. For example - consider the authors' analysis of T cell infiltration in multiple sclerosis (MS). CD4+ effector T cells have the interesting property that they are stimulated by IL2 as a growth factor; yet IL2 also stimulates the activation of (suppressive) regulatory T cells. What does it mean to analyze CD4+ regulation in disease with a graph that does not consider IL2 (or other cytokine) mediated feedback loops/cycles? To the authors' credit, in the supplementary materials they do consider a simulated example with a cyclic underling causal graph, finding that RCSP performed well comparison to an implementation of the additive noise model (ANM), LiNGAM, CausalCell, and two simpler approaches based on linear regression.

      I also encourage the authors to consider more carefully when graph structure learned from perturb-seq can be ported over to bulk RNA-seq. Consider again the MS CD4+ example - the authors first start with a large perturb-seq experiment (Replogle et al., 2022) performed in K562 cells. To what extent are K562 cells, which are derived from a leukemia cell line, suitable for learning the regulatory structure of CD4+ cells from individuals with an MS diagnosis? Presumably this structure is not exactly correct - to what extent is the RCSP algorithm sensitive to false edges in this graph? The authors perform an analysis of this scenario in Supplementary Figure 4, which shows that RCSP is robust to some degree of departure from the underlying true structure. And although challenging - it would be ideal for the RCSP to model or reflect the challenges in correctly identifying the regulatory structure.

      It should also be noted that in most perturb-seq experiments, the entire genome is not perturbed, and frequently important TFs (that presumably are very far "upstream" and thus candidate "root" causal genes) are not expressed highly enough to be detected with scRNA-seq. In that context - perhaps slightly modifying the language regarding RCSP's capabilities might be helpful for the manuscript - perhaps it would be better to describe it has an algorithm for causal discovery among a set of genes that were perturbed and measured, rather than a truly complete search for causal factors. Perhaps more broadly - it would also benefit the manuscript to devote slightly more text to describing the kinds of scenarios where RCSP (and similar ideas) would be most appropriately applied - perhaps a well-powered, phenotype annotated perturb-seq dataset performed in a disease relevant primary cell.

    1. Author response:

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

      Public Reviews:

      Reviewer #1 (Public review):

      Summary:

      In this manuscript, Dong et al. study the directed cell migration of tracheal stem cells in Drosophila pupae. The migration of these cells which are found in two nearby groups of cells normally happens unidirectionally along the dorsal trunk towards the posterior. Here, the authors study how this directionality is regulated. They show that inter-organ communication between the tracheal stem cells and the nearby fat body plays a role. They provide compelling evidence that Upd2 production in the fat body and JAK/STAT activation in the tracheal stem cells play a role. Moreover, they show that JAK/STAT signalling might induce the expression of apicobasal and planar cell polarity genes in the tracheal stem cells which appear to be needed to ensure unidirectional migration. Finally, the authors suggest that trafficking and vesicular transport of Upd2 from the fat body towards the tracheal cells might be important.

      Strengths:

      The manuscript is well written. This novel work demonstrates a likely link between Upd2JAK/STAT signalling in the fat body and tracheal stem cells and the control of unidirectional cell migration of tracheal stem cells. The authors show that hid+rpr or Upd2RNAi expression in a fat body or Dome RNAi, Hop RNAi, or STAT92E RNAi expression in tracheal stem cells results in aberrant migration of some of the tracheal stem cells towards the anterior. Using ChIP-seq as well as analysis of GFP-protein trap lines of planar cell polarity genes in combination with RNAi experiments, the authors show that STAT92E likely regulates the transcription of planar cell polarity genes and some apicobasal cell polarity genes in tracheal stem cells which appear to be needed for unidirectional migration. Moreover, the authors hypothesise that extracellular vesicle transport of Upd2 might be involved in this Upd2-JAK/STAT signalling in the fat body and tracheal stem cells, which, if true, would be quite interesting and novel.

      Overall, the work presented here provides some novel insights into the mechanism that ensures unidirectional migration of tracheal stem cells that prevents bidirectional migration. This might have important implications for other types of directed cell migration in invertebrates or vertebrates including cancer cell migration.

      Weaknesses:

      It remains unclear to what extent Upd2-JAK/STAT signalling regulates unidirectional migration. While there seems to be a consistent phenotype upon genetic manipulation of Upd2-JAK/STAT signalling and planar cell polarity genes, as in the aberrant anterior migration of a fraction of the cells, the phenotype seems to be rather mild, with the majority of cells migrating towards the posterior.

      We agree that the phenotype is mild, as perturbing JAK/STAT signaling in the progenitors specifically affects the coordinated migration of the cells rather than alters their direction or completely blocks migration. Our data indicate that inter-organ communication ensures coordinated behavior of the progenitor cells, although the differential responses exhibited by individual cells represent an interesting unresolved issue that awaits future in-depth investigation.

      While I am not an expert on extracellular vesicle transport, the data presented here regarding Upd2 being transported in extracellular vesicles do not appear to be very convincing.

      We performed additional PLA experiments which support the interaction between Upd2 and the core components of extracellular vesicles (revised Figure 8). Furthermore, we performed electron microscopy to visualize the Lbm-containing vesicles in fat body (Figure 8-figure supplement 1D).

      These data are now provided in the revised manuscript.

      Major comments:

      (1) The graphs showing the quantification of anterior (and in some cases also posterior migration) are quite confusing. E.g. Figure 1F (and 5E and all others): These graphs are difficult to read because the quantification for the different conditions is not shown separately. E.g. what is the migration distance for Fj RNAi anterior at 3h in Fig5E? Around -205micron (green plus all the other colors) or around -70micron (just green, even though the green bar goes to -205micron). If it's -205micron, then the images in C' or D' do not seem to show this strong phenotype. If it's around -70, then the way the graph shows it is misleading, because some readers will interpret the result as -205. Moreover, it's also not clear what exactly was quantified and how it was quantified. The details are also not described in the methods. It would be useful, to mark with two arrowheads in the image (e.g. 5 A' -D') where the migration distance is measured (anterior margin and point zero).

      Overall, it would be better, if the graph showed the different conditions separately. Also, n numbers should be shown in the figure legend for all graphs.

      We apologize for those inappropriate presentation and insufficient description and thank you for kindly pointing them out. We used different colors to represent different genotypes, and the columns were superimposed. we chose to show the quantification in different conditions separately in the revised Figures. The anterior migration distance for Fj RNAi is around 70 µm.

      We now provided detailed description in the revised methods. For migration distance measurement, we took snapshots at 0hr\ 1hr\ 2hr and 3hr, and measured the distance from the starting point (the junction of TC and DT) to the leading edge of progenitor clusters. The velocity formula: v=d (micrometer)/t (min). As you kindly suggested, we indicated the anterior margin and point zero in the corresponding panels. We have added n number in the legends.

      (2) Figure 2-figure supplement 1: C-L and M: From these images and graph it appears that Upd2 RNAi results in no aberrant anterior migration. Why is this result different from Figures 2D-F where it does?

      The fat body-expressing lsp2-Gal4 was used in Figure 2-figure supplement 1C-L and Figure 2D-F, while trachea specific btl-Gal4 was used in Figure 2-figure supplement 1K-L. The lsp2-Gal4-driven but not btl-Gal4-driven upd2RNAi causes aberrant anterior migration, suggesting that fat bodyderived Upd2 plays a role. We have further clarified this in the text.

      (3) Figure 5F: The data on the localisation of planar cell polarity proteins in the tracheal stem cell group is rather weak. Figure 5G and J should at least be quantified for several animals of the same age for each genotype. Is there overall more Ft-GFP in the cells on the posterior end of the cell group than on the opposite side? Or is there a more classic planar cell polarity in each cell with FtGFP facing to the posterior side of the cell in each cell? Maybe it would be more convincing if the authors assessed what the subcellular localisation of Ft is through the expression of Ft-GFP in clones to figure out whether it localises posteriorly or anteriorly in individual cells.

      We staged the animals, measured several animals for each genotype and provided the quantifications in the revised manuscript. The level of Ft-GFP is higher in the cells at the frontal edge. We tried to examine the expression of Ft-GFP at single-cell level. However, this turned out to be technically difficult because the tracheal stem cells are not regularly arranged as epithelial cells and the proximal-distant axis of the tracheal stem cells remains unclear. We thus decided to measure the fluorescence signal of groups of stem cells along the DT regardless of their individual polarity within cells.

      (4) Regarding the trafficking of Upd2 in the fat body, is it known, whether Grasp65, Lbm, Rab5, and 7 are specifically needed for extracellular vesicle trafficking rather than general intracellular trafficking? What is the evidence for this?

      In our experiments, knocking down rab5, rab7, grasp65 or lbm in trachea using btl-Gal4 did not cause abnormality in the disciplined migration, which excludes their intracellular contribution in the trachea (Figure 7-figure supplement 1). Perturbation of Grasp65 or Lbm in fat body increased intracellular upd2-containing vesicles, indicating that intracellular production is functional (Figure 6J). The Grasp65 is specifically required for Upd2 production. Lbm, Rab5 and Rab7 are important of vesicle trafficking. Our conclusion does not pertain to extracellular or intracellular compartment.

      (5) Figure 8A-B: The data on the proximity of Rab5 and 7 to the Upd2 blobs are not very convincing.

      The confocal images indicate the proximity of Rab5 and Rab7 to the Upd2 vesicles. We interpret the proximity together with the results from Co-IP and PLA data (Figure 8E-K).

      (6) The authors should clarify whether or not their work has shown that "vesicle-mediated transport of ligands is essential for JAK/STAT signaling". In its current form, this manuscript does not appear to provide enough evidence for extracellular vesicle transport of Upd2.

      Lbm belongs to the tetraspanin protein family that contains four transmembrane domains, which are the principal components of extracellular vesicles. We show that Lbm interacts with Upd2. The JAK/STAT signaling depends on the Upd2 in the fat body as well as vesicle trafficking machinery. Furthermore, we performed electron microscopy and show the presence of Lbm-containing vesicles in fat body (Figure 8-figure supplement 1D).

      (7) What is the long-term effect of the various genetic manipulations on migration? The authors don't show what the phenotype at later time points would be, regarding the longer-term migration behaviour (e.g. at 10h APF when the cells should normally reach the posterior end of the pupa). And what is the overall effect of the aberrant bidirectional migration phenotype on tracheal remodelling?

      We observed that the integrity of tracheal network especially the dorsal trunk was impaired, which may be due to incomplete regeneration (Figure 3-figure supplement1E-I).

      (8) The RNAi experiments in this manuscript are generally done using a single RNAi line. To rule out off-target effects, it would be important to use two non-overlapping RNAi lines for each gene.

      We validated the phenotype using several independent RNAi alleles.

      Reviewer #2 (Public review):

      Summary:

      This work by Dong and colleagues investigates the directed migration of tracheal stem cells in Drosophila pupae, essential for tissue homeostasis. These cells, found in two nearby groups, migrate unidirectionally along the dorsal trunk towards the posterior to replenish degenerating branches that disperse the FGF mitogen. The authors show that inter-organ communication between tracheal stem cells and the neighboring fat body controls this directionality. They propose that the fat body-derived cytokine Upd2 induces JAK/STAT signaling in tracheal progenitors, maintaining their directional migration. Disruption of Upd2 production or JAK/STAT signaling results in erratic, bidirectional migration. Additionally, JAK/STAT signaling promotes the expression of planar cell polarity genes, leading to asymmetric localization of Fat in progenitor cells. The study also indicates that Upd2 transport depends on Rab5- and Rab7-mediated endocytic sorting and Lbm-dependent vesicle trafficking. This research addresses inter-organ communication and vesicular transport in the disciplined migration of tracheal progenitors.

      Strengths:

      This manuscript presents extensive and varied experimental data to show a link between Upd2JAK/STAT signaling and tracheal progenitor cell migration. The authors provide convincing evidence that the fat body, located near the trachea, secretes vesicles containing the Upd2 cytokine. These vesicles reach tracheal progenitors and activate the JAK-STAT pathway, which is necessary for their polarized migration. Using ChIP-seq, GFP-protein trap lines of planar cell polarity genes, and RNAi experiments, the authors demonstrate that STAT92E likely regulates the transcription of planar cell polarity genes and some apicobasal cell polarity genes in tracheal stem cells, which seem to be necessary for unidirectional migration.

      Weaknesses:

      Directional migration of tracheal progenitors is only partially compromised, with some cells migrating anteriorly and others maintaining their posterior migration.

      Our results suggest that Upd2-JAK/STAT signaling is required for the consistency of disciplined migration. Although only a few tracheal progenitors display anterior migration, these cells lose the commitment of directional movement. We acknowledge that the phenotype is moderate.

      Additionally, the authors do not examine the potential phenotypic consequences of this defective migration.

      We examined the long-term effects of the aberrant migration and observed an impairment of tracheal integrity and melanized tracheal branches (Figure 3-figure supplement1E-I).

      It is not clear whether the number of tracheal progenitors remains unchanged in the different genetic conditions. If there are more cells, this could affect their localization rather than migration and may change the proposed interpretation of the data.

      We examined the progenitor cell number in bidirectional movement samples and control group. The results show that cell number does not exhibit a significant difference between control and bidirectional movement groups (Figure 3-figure supplement 1).

      Upd2 transport by vesicles is not convincingly shown.

      We performed additional PLA experiments to further support the interaction between Upd2 and the core components of extracellular vesicles. Furthermore, we performed electron microscopy and show the presence of Lbm-containing vesicles in fat body (Figure 8-supplement 1D). Additional experiments such as colocalization and Co-IP assay and better quantification are provided in the revised manuscript (see revised Figure 8).

      Data presentation is confusing and incomplete.

      We used different colors to represent different genotypes, and the columns were superimposed. we changed the graphs to show the quantification in different conditions separately. We revised data presentation to avoid confusing.

      Reviewer #3 (Public review):

      Summary:

      Dong et al tackle the mechanism leading to polarized migration of tracheal progenitors during Drosophila metamorphosis. This work fits in the stem cell research field and its crucial role in growth and regeneration. While it has been previously reported by others that tracheal progenitors migrate in response to FGF and Insulin signals emanating from the fat body in order to regenerate tracheal branches, the authors identified an additional mechanism involved in the communication of the fat body and tracheal progenitors.

      Strengths:

      The data presented were obtained using a wide range of complementary techniques combining genetics, molecular biology, quantitative, and live imaging techniques. The authors provide convincing evidence that the fat body, found in close proximity to the trachea, secrete vesicles containing the Upd2 cytokine that reach tracheal progenitors leading to JAK-STAT pathway activation, which is required for their polarized migration. In addition, the authors show that genes regulating planar cell polarity are also involved in this inter-organ communication.

      Weaknesses:

      (1) Affecting this inter-organ communication leads to a quite discrete phenotype where polarized migration of tracheal progenitors is partially compromised. The study lacks data showing the consequences of this phenotype on the final trachea morphology, function, and/or regeneration capacities at later pupal and adult stages. This could potentially increase the significance of the findings.

      Regarding your kind suggestion, we examined the long-term effects of the aberrant migration and observed the impairment of tracheal integrity and melanized tracheal branches (Figure 3-figure supplement1E-I).

      (2) The conclusions of this paper are mostly well supported by data, but some aspects of data acquisition and analysis need to be clarified and corrected, such as recurrent errors in plotting of tracheal progenitor migration distance that mislead the reader regarding the severity of the phenotype.

      We used different colors to represent different genotypes, and the columns were superimposed. we changed the graphs to show the quantification in different conditions separately. We thank you for kindly pointing it out.

      (3) The number of tracheal progenitors should be assessed since they seem to be found in excess in some genetic conditions that affect their behavior. A change in progenitor number could lead to crowding, thus affecting their localization rather than migration capacities, thereby changing the proposed interpretation. In addition, the authors show data suggesting a reduced progenitor migration speed when the fat body is affected, which would also be consistent with a crowding of progenitors.

      We examined the cell number in bidirectional movement samples and control group. We examined cell number and cell proliferation and observed that there was no significance between control and bidirectional movement groups (Figure 3-figure supplement 2).

      (4) The authors claim that tracheal progenitors display a polarized distribution of PCP proteins that is controlled by JAK-STAT signaling. However, this conclusion is made from a single experiment that is not quantified and for which there is no explanation of how the plot profile measurements were performed. It also seems that this experiment was done only once. Altogether, this is insufficient to support the claim. Finally, a quantification of the number of posterior edges presenting filopodia rather than the number of filopodia at the anterior and posterior leading edges would be more appropriate.

      We staged the animals, measured several animals for each genotype and provided the quantifications in the revised manuscript. The level of Ft-GFP is higher in the cells at the frontal edge. We tried to examine the expression of Ft-GFP at single-cell level. However, this turned out to be difficult due to the fact that the tracheal stem cells are not regularly patterned as epithelial cells and the proximaldistant axis of tracheal stem cells is not well defined. We thus decided to measure the fluorescence signal of groups of stem cells along the DT regardless of their individual polarity.

      (5) The authors demonstrate that Upd2 is transported through vesicles from the fat body to the tracheal progenitors where they propose they are internalized. Since the Upd2 receptor Dome ligand binding sites are exposed to the extracellular environment, it is difficult to envision in the proposed model how Upd2 would be released from vesicles to bind Dome extracellularly and activate the JAK-STAT pathway. Moreover, data regarding the mechanism of the vesicular transport of Upd2 are not fully convincing since the PLA experiments between Upd2 and Rab5, Rab7, and Lbm are not supported by proper positive and negative controls and co-immunoprecipitation data in the main figure do not always correlate to the raw data.

      We use molecular modeling to show that Upd2 and Lbm intermingle, and Upd2 is not entirely encapsulated in vesicles (Figure 8-supplement 1E). We performed PLA experiments using the animals not expressing upd2-Cherry as negative control (Figure 8 E-J). We corrected the Co-IP panel and apologize for this error.

      Recommendations for the authors:

      Reviewer #1 (Recommendations for the authors):

      Minor comments:

      (1) Figure 1-figure supplement 1: E: How was the migration velocity assessed? By live imaging individual cells or following the cell front of the group? Over what time period? Do the data points in the graph correspond to individual cells or the cell group? It would be important to show confocal images that go along with this quantification.

      We took snapshots of pupae at 0hr\ 1hr\ 2hr and 3hr, and measured the distance covered by the migrating progenitor cells from the start place (the junction of TC and DT) to the leading edge of progenitor groups. We then calculated the migration rate by v=d (micrometer)/t (min). As the progenitor cells revolve around and migrate along the DT, tracking single tracheoblast through intact cuticle is technically challenging. We have therefore measured the leading edge as a proxy to the whole cell group. We agree with you that time-lapse imaging is favorable for analysis of migration.

      (2) Figure 1-figure supplement 1: F: Why is there Gal80ts in the genotype? (and in Figure 1H). Also, what pupal age was used for this quantification?

      Expression of hid and rpr in L3 stage impaired fat body integrity and adipocyte abundance, and caused lethality. Gal80ts was used for controlling the expression of rpr.hid. The pupal at 0hr APF were used in EdU experiment.

      (3) Figure 2C: what is shown in the 6 columns (why 3 each for control and rpr/hid)?

      We conducted 3 replicates of each group for control and rpr.hid.

      (4) In the methods, several Drosophila stocks are listed as 'source:" from a particular person (e.g. Dr Ma). Please list the real source of this stock, e.g. Bloomington stock number, or the lab and publication in which the stock was originally made.

      We provide the information on these stocks in the revised methods.

      (5) The SKOV3 carcinoma cell and S2 cell work is not described in the methods.

      We added detailed description of this experiment in the revised method-Cell culture and transfection. 

      (6) Figure 6 (F) 'Bar graph plots the abundance of Upd2-mCherry-containing vesicles in progenitors.' What does abundance mean? What was quantified, the number of vesicles, or the mean intensity? This is also not mentioned in the methods.

      We counted the number of Upd2-mCherry-containing vesicles in fat body cells and trachea progenitors and added the description of measurement in the method.

      (7) There are a few language mistakes throughout the manuscript. E.g.

      (a) Line 117 and other places: Language: 'fat body' should be 'the fat body'.

      We thank you for pointing out these errors and corrected it accordingly.

      (b) Line 1276 Language mistakes: 'Video 1 3D-view of confocal image stacks of tracheal progenitors and fat body. Scale bar: 100 μm. Genotypes: UAS-mCD8-GFP/+;lsp2-Gal4,P[B123]-RFP-moe/+.' :stacks and genotypes should be singular.

      We fixed these errors and thank you for kindly pointing them out. We also proofread the entire manuscript to assure accuracy.

      (8) In general, it is hard to figure out the exact genotypes used in experiments. This is mostly not written very clearly in the figure legends. E.g. Figure 2: genotype for A-C missing in figure legend (is B from control animals?)

      We added genotypes in the figure legends. For Figure 2, A and C lsp2-Gal4,P[B123]-RFP-moe/+ for control, UAS-rpr-hid/+;Gal80ts/+;lsp2-Gal4,P[B123]-RFP-moe/+ for rpr.hid; B from control animals.

      Reviewer #2 (Recommendations for the authors):

      Major comments:

      (1) The phenotype resulting from Upd2 downregulation by RNAi is subtle and shown by unconvincing images. In addition, these phenotypes are analyzed using only one RNAi line.

      We used two independent alleles of upd2RNAi from THFC (THU1288 and THU1331), and observed similar phenotype. For RNAi experiments, we always use multiple independent alleles.

      (2) The authors should analyze the phenotypic consequences of directional migration changes. Is there an effect on tracheal remodeling?

      We observed that the integrity of tracheal network especially the dorsal trunk was impaired and that melanized tracheal branches were present, which may be due to incomplete regeneration (Figure 3figure supplement1E-I).

      (3) The number of tracheal progenitors should be quantified, as some genetic conditions may affect cell numbers, as is apparent in some panels.

      We examined cell number and cell proliferation and observed that there was no significance between control and bidirectional movement groups (Figure 3-figure supplement 1).

      (4) The data on PCP protein distribution are unconvincing, unquantified, and insufficient to support one of the main conclusions of the study, which is stated in the abstract: "JAK/STAT signaling promotes the expression of genes involved in planar cell polarity, leading to asymmetric localization of Fat in progenitor cells."

      We staged the animals, measured several animals for each genotype and provided the quantifications in the revised manuscript. The level of Ft-GFP is higher in the cells at the frontal edge. We tried to examine the expression of Ft-GFP at single-cell level. However, this turned out to be difficult due to the fact that the tracheal stem cells are not regularly patterned as epithelial cells and the proximaldistant axis of tracheal stem cells is not well defined. We thus decided to measure the fluorescence signal of groups of stem cells along the DT regardless of their individual polarity.

      Minor comments:

      (1) Language should be revised. In many places in the manuscript, starting in line 113, "fat body" should be "the fat body".

      Thank you for pointing out this error. We corrected it accordingly.

      (2) Genotypes used in experiments should be described.

      We added all the genotypes. We proofread the entire manuscript to complete the figure legends for genotypes.

      (3) Line 67, the reference to "The progenitor cells reside in Tr4 and Tr5 metameres and start to move along the tracheal branch" should include (Chen and Krasnow, Science 2014).

      We added the reference in the manuscript.

      (4) Line 1081, Figure 7 Legend. "Bar graph plots the abundance of Upd2-mCherry-containing vesicles" Abundance is the number of vesicles? The graph displays the average number of vesicles? Please explain and describe the quantification.

      The bar graph represents the number of Upd2-mCherry-containing vesicles in different conditions. We quantified the number of vesicles per area.

      (5) Figure 1 (I-J) What is shown on the panels? Progenitors marked with? This information is not present in the figure or figure legend. Same for Figure 2 (D-E).

      Figure 1I-J show the vector of migrating progenitors. We added the information in the legends. The tracheal cells were labeled by nls-mCherry in Figure 1I-J. In Figure 2D-E, the progenitors were marked with P[B123]-RFP-moe.

      (6) Figure 3 Q, Stat92E-GFP values in the graph are not well-explained. What do the numbers in the y-axis refer to?

      y-axis represents the intensity of Stat92E-GFP normalized to control. We have changed the y-axis label to ‘normalized Stat92E-GFP intensity’ in the legends.

      (7) In general, figures and figure legends must be revised. Sometimes stainings are not well-defined, some scale bars are missing and plots do not say what the values are.

      We apologized for inadequate information and have revised the figures and legends accordingly.

      Reviewer #3 (Recommendations for the authors):

      Several points should be addressed by the authors in order to improve their manuscript.

      Major points:

      (1) The phenotype obtained from decreasing the inter-organ signaling is quite discrete. It is further weakened by the fact that the images chosen to illustrate the measures are not really convincing. No image at 1h APF shows any clear anterior migration. Based on the scale, most of the images at 3h APF do not show a striking difference compared to the control, and in any case, stronger phenotypes would be missed anteriorly since they would thus be out of frame. In addition, at 3h APF, progenitors migrating anteriorly from Tr5 position get mixed with those migrating posteriorly from Tr4 so it is not clear how measurements were made. Given that most phenotypes are observed upon the use of RNAis, it is possible that phenotypes are weak due to persistent gene expression. Using null clones for dome, hop, or stat in progenitors could therefore aggravate the phenotypes and support further the significance of the study. Finally, assessing the consequences of compromised fat body-tracheal communication on trachea morphology, function, and regeneration later in pupal development and on adult flies would also help strengthen the importance of the findings.

      We agree with you that anteriorly migrated Tr5 progenitors adjoining Tr4 progenitor hinders measurements and that mutants may give stronger phenotype than RNAi lines. We only measured Tr4 progenitors (instead of Tr5) when assessing anterior migration. Thus, we performed experiments using mutant alleles, which gave aberrant migration of tracheal progenitors (Figure 3-figure supplement1A-D). We can now show that the integrity of tracheal network especially dorsal trunk was impaired, which may be due to incomplete regeneration (Figure 3-figure supplement1E-I).

      (2) Although the authors did not observe defects in tracheal progenitor proliferation, progenitors seem to be present in excess in some key genetic background (e.g, upon expression of rpr.hid, statRNAi, Rab-RNAi or in the presence of BFA). This excess could be the result of another mechanism than proliferation (recruitment of extra progenitors since it is not clear how they originate, defect in apoptosis...) and could impact the localization of progenitors, those being pushed anteriorly as a consequence of crowding. A proper characterization of tracheal progenitor number would thus help to discriminate between defects in migration or crowding. This point could also be addressed by performing individual tracking of tracheal progenitors, to find out whether each progenitor is indeed migrating in the wrong direction or if the movement assessed by the global tracking method that is used is just a consequence of progenitor excess.

      We examined the cell number in bidirectional movement samples and control group. The results show that there was no significance between control and bidirectional movement groups (Figure 3figure supplement 1). We also tried to follow every progenitor, but were unable to obtain convincing results with P[B123]-RFP-moe, as tracking single tracheoblast through intact cuticle is technically challenging.

      (3) Regarding the ChIP-seq experiment, an explanation of why choosing the "establishment of planar polarity" family should be provided since data indicate a quite low GeneRatio. Indeed, the "cell adhesion" family seems a more obvious candidate, which would be further supported by the fact that the JAK-STAT pathway has been shown to affect cell adhesion components such as ECadherin and FAK (Silver and Montell 2001, Mallart et al 2024). Also, have these known targets of JAK-STAT signaling been found in the ChIP-seq data? Since filopodia polarization is affected in tracheal progenitors when JAK-STAT signaling is decreased, the same question also applies to enabled, which is involved in filopodia formation and has been recently identified as a target of JAK-STAT signaling.

      As you kindly suggested, we tested a number of cell adhesion-related genes such as E-Cadherin (shg), fak, robo2 and enabled (ena). We did not observe an apparent aberrancy in the migration of tracheal progenitors (Figure 5-supplement 1J).

      (4) Data investigating PCP protein distribution is not convincing, not quantified, and not sufficient to draw one of the main conclusions of the study, which is even written in the abstract "JAK/STAT signaling promotes the expression of genes involved in planar cell polarity leading to asymmetric localization of Fat in progenitor cells."

      We better quantified the abundance of Ft in in the progenitors in the frontal edge and those lagging behind. The traces plot multiple replicates in the figures. The level of Ft-GFP is higher in the cells at the frontal edge.

      (5) Overall, the figures together with their caption and/or the material and methods section lack some important information for the reader to fully understand the data. In addition, some errors are found in multiple plots throughout the article and must be corrected. Here are some examples:

      According to your suggestion, we revised legends and methods section to include sufficient information.

      (a) Migration distance plots from Figure 3E do not match the data presented in the source data file. It seems that, when creating the plot, instead of superimposing the bars, bars were stacked. This should be corrected for all migration distance plots from Figure 3E onward, including in supplementary figures.

      We apologized for misleading representation. We revised it accordingly and show the quantification in different conditions separately.

      (b) The number of analyzed flies and/or clusters of tracheal progenitors from different flies should be stated for all quantification or observations made on images. This information is lacking for all migration distance plots, for progenitor migration tracking (Figure 1 I, J), for DIPF reporter in Figure 2J, for plot profiles (Figure 5G, J), for Upd2-Rab5/Rab7/Lbm co-detections, PLA, CoIP, and lbm-pHluorin experiments. This also applies to RNA seq, ChIP seq, and surface proteomics, for which the number of pupae and number of replicates is not indicated.

      We changed the graphs to show the quantification and n number in different conditions separately.

      We also added the n number of replicates in methods.

      (c) How quantifications were performed is not sufficiently explained. For example, the reference point for migration distance measurement is not defined, and neither is whether the measures were made on fixed or live imaging samples. In fluorescence intensity measurements and Upd2 vesicle counting, information on whether measures were made on a single z slice or on a projection of several z slices should be stated together with what ROI and which FIJI tool for quantification were used. For plot profiles, the same information regarding z slices misses together with how the orientation, the thickness, and the length of the line were chosen, and again the number of times the experiment was conducted should be mentioned and error bars should appear on graphs.

      We thank this reviewer for the suggestions which help clarify the methodology of our experiments and improve presentation of our data. We have made the changes according to the suggestions and modified our methods section and the related figures to incorporate these changes.

      For measuring the migration distance of tracheal progenitors, we took snapshots of living pupae at 0hr\ 1hr\ 2hr and 3hr APF, and measured the migration distance of tracheal progenitors from the start place (the junction of TC and DT) to the leading edge of progenitor groups.

      For the measurements of fluorescent intensity of stat92E-GFP and DIPF, we took z-stack confocal images of samples and quantified the fluorescent intensity using FIJI. Specifically, intensity was quantified for regions of interest, using the Analysis and Measurement tools. To quantify Upd2mCherry vesicles, z-stack confocal images of fat body were taken and the cell counting function of FIJI was used to measure the vesicle number.

      To quantify the fluorescent intensity of in vivo tagged Ds, Ft and Fj proteins, a single z slice was used. The expression level of the protein was assessed as the integrated fluorescent intensity normalized to area.

      For the measurement of Ft-GFP distribution, a single z slice of the progenitors immediately proximal to the DT was imaged. An arbitrary line was drawn along the migration direction from the starting TC-DT junction to the leading front (the length of the line corresponds to the distribution range of tracheal stem cell clusters). Then, fluorescent intensity along the line was automatically calculated with the imbedded measurement function of Zeiss confocal software.

      Minor points:

      (1) In several instances, the authors generalize that stem cells migrate to leave their niche, but this is not the case for all stem cells.

      The phenomenon that stem cells leave their niche when they are activated is commonly observed. We interpreted the general mechanism from our system of tracheal stem cells. We fully agree with you that it may not be the case for all stem cells. We modified the text accordingly.

      (2) Line 122 -a reference paper or an image showing the expression pattern of the lsp2-Gal4 driver is missing.

      We added the reference in the manuscript.

      (3) Line 136 - The term "traces of individual progenitors" is overstated and should be reformulated as the method used does not seem to be individual cell tracking.

      We rephrased accordingly in the revised manuscript.

      (4) Line 146 - Fat body and tracheal progenitors are qualified as interdependent organs, in which aspect do tracheal progenitors affect the fat body?

      Current knowledge suggests a close inter-organ crosstalk between trachea and fat body: The fly trachea provides oxygen to the body and influences the oxidation and metabolism of the whole body. When the trachea is perturbed, the body is in hypoxia, which causes inflammatory response in adipose tissue as an important immune organ (Shin et al., 2024).

      (5) Line 163 - Not all the genes tested are cytokines, so the sentence should be reformulated. In addition, in supplementary Fig2-1 C-J, the KD of hh seems to abolish completely tracheal progenitor migration, which is not commented on.

      According to your suggestion, we revised the description on information of the genes tested. We added comments in the revised manuscript regarding phenotypes of hh knockdown. 

      (6) Line 180 - Conclusion is made on Dome expression while using a dome-Gal4 construct, which does not necessarily recapitulate the endogenous pattern of dome expression, so it should be reformulated. Ideally, dome expression should be assessed in another way. Also, it is not clear whether GFP is present only in progenitors since images are zoomed.

      We revised statement and provided larger view of dome>GFP that shows an enriched expression in the tracheal progenitors (Figure 2-figure supplement 2E), an expression pattern that is consistent with FlyBase.

      (7) Line 199 - Is it upd-Gal4 or upd2-Gal4 that is used? Since the conclusion of the experiment is made on upd2, the use of upd-gal4 would not be relevant. If upd2-gal4 is used, it should be corrected. In general, the provenance of the Gal4 lines should be provided. In addition, a strong GFP signal in the trachea is visible on the image in Supplementary Figure 2-2F but not commented on and seems contradictory with the conclusion mentioning that fat body and gut are the main source of Upd2 production.

      We removed data obtained from the use of this irrelevant upd-Gal4 line.

      (8) Figures:

      -  Figure 1 G, H - Scale bar is missing.

      We added it accordingly.

      -  Figure 1 I, J - The information on the staining is missing.

      We added it in the revised manuscript.

      -  Figure 2A - Providing explanations of the terms "Count" and "Gene ratio" in the caption would be helpful for readers who are not used to this kind of data. In addition, the color code is confusing since the same color is used for the selected gene family and for high p-values (the same applies to other similar graphs).

      Gene ratio refers to the proportion of genes in a dataset that are associated with a particular biological process, function, or pathway. Count indicates the number of genes from input gene list that are associated with a specific GO term. We used redness to indicate a smaller p-value and a higher significance.

      -  Figure 2 B, C - What does the color scale represent? What do the columns in C correspond to, different time points, different replicates?

      The color scale represents the normalized expression. The columns in C correspond to different replicates of control and rpr.hid.

      -  Figure 2 F - The error bars on the 3h APF posterior bars are missing.

      We added error bars accordingly.

      -  Figure 2 G - The legend "Down-Stable-Up" is in comparison to what?

      The control group was generated from the reaction without H2O2. The comparison was relative to the control group.

      -  Figure 2 J - The specificity of the DIPF tool that has been created should be validated in other tissues displaying known JAK-STAT activity and/or in conditions of decreased JAK-STAT signaling. In addition, the added value of the tool as compared to the JAK-STAT activity reporter used later, which has been well characterized, is not obvious.

      We added the signal of DIPF in fat body and salivary gland, both of which harbor active JAK/STAT signaling (Figure 2-figure supplement 2F-H). As opposed to the well characterized Stat92E-GFP reporter that assays the downstream transcription activity, the DIPF reporter measures the upstream event of receptor dimerization.

      -  Figure 3 I-P - Reporter tool validation in Images I-L could be moved to supplementary data. In images M-P, staining of nuclei and/or membranes would be useful to assess cell integrity.

      We revised the figures accordingly.

      -  Figure 3Q and similar plots in the following figures do not explain the normalization performed and how it can be higher than 1 in control conditions.

      In these figures, we normalized the signal relative to control groups, e.g., The value of Stat92E-GFP in btl-GFP control group was set to 1 in the previous Figure 3Q (revised Figure 3-supplementary

      Figure B-J).

      -  Figure 4C - These representations lack explanations to be fully understood by a broad audience.

      The figure showing that Stat92E binding was detected in the promoters and intronic regions (the orange peaks) of genes functioning in distal-to-proximal signaling, such as ds, fj, fz, stan, Vang and fat2. We added the information in figure legend according to your suggestion.

      -  Figure 5 K,L - What is the x-axis missing, together with the method of tracking used?

      The x-axis refers to time of recording from a t stack series with a time interval of 5 min. We revised method section and provide detailed procedure of this experiment.

      -  Figures 6 and 8- The overall figures lack a wider view of the cells/tissues/organs and/or additional staining to understand what is presented.

      We showed preparation of fat body. In order to obtain the high resolution of vesicles, we used high magnification. We now added wider views of the tissues under investigation (e.g. Figure 6-figure supplement 1).

      -  Figure 6 D,E - The scale bar is missing.

      We added it accordingly.

      -  Figure 8 O-S - What is the blue staining?

      The blue staining shows DAPI-stained nuclei. We have added the information in the legend.

      -  PLA experiments can give a lot of non-specific background. What kind of controls have been used in Figure 8 F-J? Negative controls should be done on cells that do not express upd2-mCherry using both antibodies to detect non-specific background, which does not usually appear completely black.

      If possible, a positive control using a known protein interacting with Rab5-GFP should be included.

      We used the control samples without one of the primary antibodies in previous Figure 8. In the revised Figure 8, we conducted experiment as you suggested with controls that do not express upd2mCherry (Figure 8 E-J).

      -  Co-IP experiments - The raw data file for blots is quite hard to read through. Some legends are not facing the right lane and some blots presented in the main figure are difficult to track since several blots are presented in the raw data file. e.g.

      (a)  Raw blot for Figure 8 K: the band for mCherry in the IP anti-GFP blot (lane one in K) is not convincing, it is not distinguishable from other aspecific bands. On the reverse IP presented only in raw data, on the input from blot IB anti-mCherry, both lanes present exactly the same bands at 72kb when one of the lanes corresponds to extract from flies not expressing upd2-mCherry.

      We thank you for pointing out the incorrect labels. We apologized for the errors and corrected it accordingly.

      (b)  Raw blot for Figure 8 L: on the input blot IB anti-GFP, there is a band corresponding to Rab7-GFP in the lane of the extract from flies not expressing Rab7-GFP.

      We corrected it.

      (c)  Raw data for Figure 8 M: on the last blot, legends are missing above the input Ib anti-GFP blot.

      We added the missing legends in the figure.

      Shin, M., Chang, E., Lee, D., Kim, N., Cho, B., Cha, N., Koranteng, F., Song, J.J., and Shim, J. (2024). Drosophila immune cells transport oxygen through PPO2 protein phase transition. Nature 631, 350-359.

    1. Kwasy tłuszczowe omega-3 wydają się spowalniać procesy starzenia
      • Daily intake of 1g of omega-3 fatty acids can slow down aging, especially when combined with vitamin D and exercise - as demonstrated by a 3-year study on 777 Swiss seniors[1][2].

      • Epigenetic clocks were used to measure the pace of aging, showing up to 4 months of biological age reduction[1][3].

      • Additional benefits included:

        • 61% reduction in invasive cancer risk[4]
        • 10% reduction in falls[4]
        • 13% decrease in infection rates[4]
      • Sources of omega-3: fish, seafood, rapeseed and flaxseed oil, walnuts, supplements[7][9]

      Citations: [1] https://www.sci.news/medicine/omega-3-supplementation-biological-aging-13635.html [2] https://www.news-medical.net/news/20250204/Omega-3-fatty-acids-and-vitamin-D-slow-biological-aging-in-older-adults.aspx [3] https://www.nature.com/articles/s43587-024-00793-y [4] https://www.medonet.pl/leki-od-a-do-z/witaminy-i-mineraly,wystarczy-gram-dziennie--spowolnisz-starzenie-nawet-o-cztery-miesiace,artykul,86261609.html [5] https://www.eurekalert.org/news-releases/1072552 [6] https://www.national-geographic.pl/nauka/kwasy-omega-3-to-pigulka-mlodosci-zwlaszcza-w-polaczeniu-z-witamina-d/ [7] https://dzienniknaukowy.pl/kwasy-tluszczowe-omega-3-wydaja-sie-spowalniac-procesy-starzenia [8] https://translate.google.com/?prev=_t&hl=pl&ie=UTF-8&eotf=1&sl=en&tl=zh-CN [9] https://www.businessinsider.com/taking-omega-3-supplements-may-slow-down-aging-study-2025-2 [10] https://pubmed.ncbi.nlm.nih.gov/36629088/ [11] https://www.sciencedaily.com/releases/2025/02/250204132423.htm

    1. Author response:

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

      Reviewer #1 (Public review):

      Weaknesses:

      In my estimation, the following would improve this manuscript:

      (1) The physiological relevance of these data could be better highlighted. For instance, future work could revolve around incubating oocytes with oviduct fluid (or OVGP1) to reduce polyspermy in porcine IVF, and naturally improve sperm selection in human IVF.

      Thank you for the suggestions. We have added these physiological relevance points at the end of the discussion.

      (2) Biological and technical replicate values for each experiment are unclear - for semen, oocytes, and oviduct fluid pools. I suggest providing in the Materials and Methods and/or Figure legends.

      Biological and technical replicates are now indicated in M&M. Number of oocytes or ZPs used were already indicated in every Supplementary Table.

      (3) Although differences presented in the bar charts seem obvious, providing statistical analyses would strengthen the manuscript.

      Statistical analyses are now indicated in each bar chart.

      (4) Results are presented as {plus minus} SEM (line 677); however, I believe standard deviation is more appropriate.

      This was a mistake; all the results are indicated as standard deviation.

      (5) Given the many independent experimental variables and combinations, a schematic depiction of the experimental design may benefit readers.

      A schematic depiction of the experimental design is now included as Figure 1. This new Figure modifies the number assigned to the rest of Figures.

      (6) Attention to detail can be improved in parts, as delineated in the "author recommendation" review section.

      Done

      Reviewer #2 (Public review):

      Weaknesses:

      The authors postulate a role for oviductal fluid in species-specific fertilization, but in my opinion, they cannot rule out hormonal effects or differences in the method of oocyte maturation employed.

      As we indicate below, the effect of hormones has been analyzed, and we have demonstrated that it is not the cause of zona pellucida specificity.

      They also cannot unequivocally prove that OVGP1 is the oviductal protein involved in the effect. Additional experiments are necessary to rule out these alternative explanations.

      Our work does not demonstrate that other proteins could be involved, but it does show that OVGP1 is involved in the process.

      When performing the EZPT assay on mouse oocytes obtained either from the ovary or from the oviduct, the oocytes obtained from the ovary came from mice primed with eCG, whereas the ones collected from the oviduct were obtained from superovulated mice (eCG plus hCG). This difference in the hormonal environment may make a difference in the properties of the ZP. Additionally, the ones obtained from the ovary were in vitro matured, which is also different from the freshly ovulated eggs and, again, may change the properties of the ZP. I suggest doing this experiment superovulating both groups of mice but collecting the fully matured MII eggs from the ovary before they get ovulated. In that way the hormonal environment will be the same in both groups and in both groups, oocytes will be matured in vivo. Hence, the only difference will be the exposure to oviductal fluids.

      In Figure 2, we compare ZPs from murine oocytes obtained from the ovary using only PMSG with ZPs from oviductal oocytes treated with both HCG and PMSG. But in Figure 7, however, we compared ZPs from murine oocytes exposed only to PMSG, with the only difference being whether or not they had been in contact with OVGP1. This shows that it is not the effect of the hormone but rather the contact with OVGP1 that determines their specificity.

      Mice with OVGP1 deletion are viable and fertile. It would be quite interesting to investigate the species-specificity of sperm-ZP binding in this model. That would indicate whether OVGP1 is the only glycoprotein involved in determining species-specificity. Alternatively, the authors could immunodeplete OVGP1 from oviductal fluid and then ascertain whether this depleted fluid retains the ability to impede cross-species fertilization.

      We agree with the reviewer that it would be interesting to investigate sperm-ZP binding in this model. Unfortunately, we do not have the OVGP1 knockout mouse strain. We also believe that immunodepletion of OVGP1 would not completely remove the protein, so its effect would likely not be entirely eliminated.

      What is the concentration of OVGP1 in the oviduct? How did the authors decide what concentration of protein to use in the experiments where they exposed ZPs to purified OVGP1? Why did they use this experimental design to check the structure of the ZP by SEM? Why not do it on oocytes exposed to oviductal fluid, which would be more physiological?

      We have included in the manuscript that the concentration of OVGP1 in the oviductal fluid was quantified using ImageJ software by comparing the mean gray value of the band in the oviductal fluid to the band in the recombinant protein lane. By establishing this relationship, along with the known concentration of protein amount in the recombinant one and in the total protein amount of oviductal fluid, the concentration of OVGP1 in the oviductal fluid was determined as the average of three western blots. The concentration of OVGP1 in oviductal fluids was in the range of 100-150 ng/µl in mice and 150-200 ng/µL in cow. We have included also in the manuscript the concentration that we have use for the EZPTs, 30 ng/µL of recombinants OVGP1 (bovine, murine and human) for 30 minutes in 20µL drops. With this concentration, we observed a clear effect on zona specificity with no negative impact on the gametes.

      As you can see in supplementary Fig S8B, we already realized SEM of oocytes exposed to oviductal fluid.

      None of the figures show any statistical analysis. Please perform analysis for all the data presented, include p values, and indicate which statistical tests were performed. The Statistical analysis section in the Methods indicating that repeated measures ANOVA was used must refer to the tables. Was normality tested? I doubt all the data are normally distributed, in which case using ANOVA is not appropriate.

      Statistical results are now included in each Figure and Table. All the statistical analysis are included, all the data pass normality, homogeneity of variance and independence; for this reason the data analysis was conducted by using a one-way ANOVA, followed by Tukey´s post hoc test. Significance level was set at p <0.05.

      Why was OVGP1 selected as the probable culprit of the species specificity? In the Results section entitled "Homology of bovine, human and murine OVGP1 proteins..." the authors delve into the possible role of this protein without any rationale for investigating it. What about other oviductal proteins?

      A sentence indicating this rationale for investigating OVGP1 has been introduced in this paragraph.

      Reviewer #3 (Public review):

      Weaknesses:

      The manuscript began with a well-written introduction, but problems started to surface in the Results section, in the Discussion, as well as in the Materials and Methods. Major concerns include inconsistencies, misinterpretation of results, lacking up-to-date literature search, numerous errors found in the figure legends, misleading and incorrect information given in the Materials and Methods, missing information regarding statistical analysis, and inadequate discussion. These concerns raise questions regarding the authenticity of the study, reliability of the findings, and interpretation of the results. The manuscript does not provide solid and convincing findings to support the conclusion.

      We have modified and clarified all the issues, some of which are misunderstandings, we have also performed the suggested experiment of putting sperm in contact with OVGP1.

      Recommendations for the authors:

      Reviewer #1 (Recommendations for the authors):

      (1) Ensure consistency in (past) tense, for example, "decondensed" (line 102), "induced" (line 103), and elsewhere.

      Done

      (2) Replace "table" with "Table" throughout.

      Done

      (3) The authors often refer to "co-incubation". I believe this should read "incubation". My understanding is that oocytes were incubated with oviduct fluid or sperm but never both simultaneously as "co-incubation" implies.

      Done

      (4) Synonymous terms "OVGP1" and "oviductin" are used interchangeably. Consider using one or the other for consistency.

      We believe that by using both terms, reading is more fluid.

      (5) Delete "around" on line 256 and "approximately" on line 263 and provide actual percentages.

      Done

      (6) The point of the sentence on lines 311-313 is unclear to me.

      Rewritten

      (7) Suggest specifying "wildtype" on line 419.

      All the mice used in this work are wildtype

      (8) Do the authors have details regarding cattle oocyte donor breeds?

      Done

      (9) What do the authors mean by "strengthen" on line 500?

      The word strengthen has been changed to carefully isolated

      (10) Ponceau and vinculin (Figure 3) details are not provided in the manuscript.

      Ponceau and vinculin details are now included in the manuscript

      (11) Address formatting issues (e.g. citation 26 among others).

      Done

      (12) Primary and secondary antibody controls for immunofluorescent imaging (to fully exclude autofluorescence) are lacking.

      Controls for immunofluorescent imaging are indicated in Supplementary Figure S7.

      (13) The corresponding author on the manuscript and in the eLife submission system are different

      It was a problem during submission, now it is corrected.

      Reviewer #2 (Recommendations for the authors):

      (1) For the experiment depicted in Figures 3C and D, the authors need to perform a negative control to demonstrate that this fluorescent signal is specific. What happens if they express a different FLAG-tagged protein instead of bOVGP1 and mOVGP1? FLAG antibodies give quite strong non-specific binding. Or if they expressed untagged bovine and mouse OVGP1?

      The negative controls are in the supplementary Figure S7. A rabbit polyclonal antibody to the human OVGP1 was used for murine and bovine IVM ZPs from ovaries and murine superovulated ZPs recovered from mouse oviducts. There is a remarkable difference in the ones that are not incubated with any OVGP1 and the endogenous one, given the specificity of the antibody.

      Also, IVM mouse and bovine oocytes incubated or not with OF were immunoblotted with anti-Flag-tag antibody. Since any of them present OVGP1 tagged to Flag, there is not signal in the immunofluorescence.

      (2) For the Western blots of recombinant proteins, why are the authors not showing the blots using His and FLAG tag antibodies? Is the 50-kDa band observed for the mouse OVGP1 detected with His-Tag antibody?

      We have included a supplementary figure S6 with the western blot with anti-His and anti-Flag. The protein around 50 kDa is not a specific band (there is not signal with anti-Flag). This new figure modifies the number assigned to the rest of supplementary figures (S6-S8).

      (3) How was the estrous cycle stage determined in mice? It is not described in the Methods.

      Estrous cycle stage was determined in mice by visual examination of the vaginal opening and cytological examination of the vagina smear. This is now included in the M&M

      (4) For sperm binding, what does the percentage mean?

      It was a mistake, percentages were related to pronuclear formation and cleavage not to sperm binding, this is now corrected.

      (5) In Figure 3A, the labels for regions C, D, and E are mixed up. It is regions A and C that are conserved (or orange and blue, if the letters are incorrect). The purple region is only present in the mouse (E?), and the red region (D?) is only in the human form. Also, the legend for this panel is repeated verbatim in the Results section. Please remove one of them.

      Errors in Figure 3a have been corrected. Legend repetition is removed.

      (6) In the title of Figure 1B and in different places in the text, it should be mouse (not mice) oocytes.

      Done

      (7) In line 140, I would change the part indicating "We extracted the cytoplasmic contents from the oocytes". It is not only the cytoplasm, but all the oocyte, including the nucleus and membranes, that are being removed.

      Done

      (8) Please rephrase the sentence in lines 245-247, as it is quite confusing.

      Done

      (9) In line 236, the authors indicate that "During in vitro maturation (IVM), oocytes displayed a porous ZP structure...". Do they mean after IVM? When were those oocytes collected for SEM?

      The sentence has been modified by “after IVF”. Bovine oocytes were collected from slaughterhouse ovaries and were similar to those used in the rest of the experiments in the manuscript.

      (10) In the legend of Figure 1, please indicate what the parthenogenic group is.

      Done

      (11) In the legend to Figure 1G, the text indicates "Note sperm only appear outside the zona". However, I cannot see any sperm in that image.

      The phrase has been removed, as when enlarging the image to better see the sperm that are inside the area, the vision of those that are outside has been lost.

      (12) In the legend to Figure 2 describing the different zona pictures, the letters of the panels are not correct.

      Done

      (13) In line 999, please provide the right concentration for NMase (it indicates 10 μ/mL).

      Done

      (14) Where does the model depicted at the end of the manuscript go? Is it a Figure? A graphical abstract? In that model, please correct some typos: it should be "ZP obtained from ovarian oocytes"; and change specie for species in all three panels.

      Done. It is a model (Fig. 10)

      (15) The FITC-PNA staining to visualize acrosomes is not described in the Methods section.

      Done

      Reviewer #3 (Recommendations for the authors):

      The present study reports findings from a series of experiments suggesting that bovine oviductal fluid and species-specific oviductal glycoprotein (OVGP1 or oviductin) from bovine, murine, or human sources modulate the species specificity of bovine and murine oocytes. The manuscript began with a well-written introduction, but problems started to surface in the Results section, Discussion as well as in the Materials and Methods. Major concerns include inconsistencies, misinterpretation of results, lacking up-to-date literature search, numerous errors found in the figure legends, misleading and incorrect information given in the Materials and Methods, missing information regarding statistical analysis, and inadequate discussion.

      We have modified and clarified all the issues, some of which are misunderstandings, we have also performed the suggested experiment of putting sperm in contact with OVGP1.

      Specific comments:

      (1) Lines 142 to 143 on page 5: It is stated that "Because this experiment was done on empty ZPs, we called this test "empty zona penetration test" (EZPT)". In fact, the experiment was not actually done on empty ZPs, but on oocytes with the ooplasm extracted. Therefore, the zona pellucidae used in the experiment were not empty but contained an intact zona matrix of glycoproteins. The term "EZPT" used by the authors in the manuscript is a misnomer. A better term should be used to reflect the ZPs which were intact and not empty.

      We extracted the cytoplasmic containing all the organelles, nucleus and membranes, and the polar body. This has been clarified in the text.

      (2) The authors need to distinguish between sperm penetration and sperm binding in the manuscript. In lines 169 to 177 on page 6, the authors mixed up the terms "penetration" and "binding" in the text. In writing about events leading to fertilization in reproductive biology, the term "sperm binding" refers to the interaction between the sperm plasma membrane and the oocyte zona pellucida (ZP), whereas the term "sperm penetration" refers to the passage of the sperm through the ZP. Therefore, the statements in lines 169 to 177 describing the binding of bovine, murine, and human sperm to bovine oocytes with and without prior treatment with oviductal fluid are misleading and not correct. In fact, Figure 2 and Table 6 show sperm penetration and not sperm binding.

      Figure 2A and B (now 3A and 3B), and Tables S6 show both sperm penetration (% penetration rate and average sperm in penetrated ZPs) and sperm binding (average sperm bound to ZPs). Throughout the manuscript, a clear distinction is made between sperm attached to the ZP and sperm that have penetrated it.

      (3) Lines 182 to 187 on page 6: What is being described in the text here does not match what is being shown in Figure 3A. As a result, the information provided in lines 182 to 187 is not correct and misleading. For example, it is stated in lines 182 to 183 that "As depicted in Fig. 3A, the sequences of these three OVGP1 have five distinct regions (A, B, C, D and E)." However, Figure 3A shows that hOVGP1 and mOVGP1 both have only 4 regions and bOVGP1 has only 3 regions. None of the three has 5 regions. In lines 183 to 184, the authors continued to state that "Regions A and D are conserved in the different mammals." This statement is also not true because Figure 3A shows that only region A is conserved in all three species but not region D which is found only in the human. What is stated in lines 186 to 187 is also not correct based on the information provided in Figure 3A. It is stated here that "Region C is an insertion present only in the mouse (Mus) and region E is typical of human oviductin." However, based on the color codes provided in Figure 3A, region C is present in all three species while region E is present only in the mouse.

      Errors with naming regions in Figure 3A (now 4A) have been corrected.

      (4) In lines 195 to 197 on page 6, the authors stated that "Western blots of the three OVGP1 recombinants indicated expected sizes based on those of the proteins: 75 kDa for human and murine OVGP1 and around 60 kDa for bovine OVGP1 (Fig. 3B)." However, the expected size of the recombinant human OVGP1 is not in agreement with what has been published in literature regarding the molecular weight of recombinant human OVGP1. It has been previously reported that a single protein band of approximately 110-150 kDa was detected for recombinant human OVGP1 using an antibody against human OVGP1. The authors provided Western blots of murine oviductal fluid and bovine oviductal fluid in Figure 3B but not a Western blot of native human oviductal fluid. The latter should have been included for a comparison with the recombinant human OVGP1.

      We do not have human oviductal fluid, but we have included now a supplementary figure 6S of a western blot with antibody again His and Flag (present in the recombinant OVGP1) which shows that the size of the recombinant protein is as indicated in the Figure 3B (now 4B).

      (5) Lines 220 to 229 on page 7: In this experiment, the authors conducted the EZPT using ZPs from bovine oocytes that were either treated with or without bOVGP1 followed by incubation, respectively, with homologous sperm (bovine) and heterologous sperm (human and murine). This is a logical experiment to determine if OVGP1 plays a species-specific role in setting the specificity of the zona pellucida. However, in the in vivo situation, sperm that reach the lumen of the ampulla region of the oviduct where fertilization takes place are also exposed to oviductal fluid of which OVGP1 is a major constituent. Therefore, an additional experiment in which sperm are treated with OVGP1 prior to incubation with ZP should be carried out for a comparison.

      The additional experiment in which sperm are treated with OVGP1 prior to incubation with ZP has been done (Table S9). No effects were observed. This is now included in the manuscript.

      (6) Regarding the results obtained with the use of neuraminidase (lines 278 to 293 on pages 8 to 9), if neuraminidase treatment of bovine ZP prevented bovine sperm penetration regardless of whether ZPs had been or had not been in contact with OVGP1, that means OVGP1 is not responsible for penetration despite the description of earlier findings in the manuscript. Sialic acid is likely associated with the sugar side chains of ZP glycoproteins and not sugar side chains of OVGP1. To attribute the species-specific property of sialic acid to OVGP1 for sperm binding, an experiment in which OVGP1 will be treated with neuraminidase prior to performing the EZPT is needed.

      We conducted the experiment by treating only OVGP1 with neuraminidase and then isolating OVGP1 from the enzyme previously to incubate treated OVGP1 with ZPs. The results agree with our previous findings, indicating the importance of sialic acid on OVGP1 for sperm binding and penetration, and confirming that OVGP1 is responsible for species-specific penetration. Results are shown in Fig. 9 and Table S14.

      (7) The Discussion appears superficial and a more in-depth discussion regarding the results obtained in the present study in relation to other reports about OVGP1 published in literature is needed (e.g. a recent paper published by Kenji Yamatoya et al. (2023) Biology of Reproduction https://doi.org/10.1093/biolre/ioad159). Lines 317 to 342 of the Discussion on pages 10 to 11 should belong to the Introduction.

      Results of Yamatoya are now included in discussion. Part of the discussion from 317 to 342 are now in the introduction

      (8) In is not clear what the authors exactly want to say in lines 343 to 344 of the Discussion on page 11. It is stated here that "The empty zona penetration test (EZPT) enables heterologous sperm to overcome the oocyte's second barrier, the plasma membrane or oolemma." Do the authors mean that the sperm can now enter the empty space encircled by the ZP without having to go through the plasma membrane or oolemma? In Figure S4 which depicts the method used to empty the ooplasm in the bovine oocyte, does the method extract only the ooplasm (or cytoplasmic contents) leaving behind the intact plasma membrane or oolemma? This needs to be clearly shown and clearly explained. High magnifications of the zona pellucida are also needed to show whether the plasma membrane (or oolemma) is still present and intact after extraction of the ooplasm.

      This is clearly explained in the text. To obtain empty ZP, everything except ZP (nucleus, organelles, membranes and cytoplasmic contents of the oocytes) was removed using a micromanipulator, following the procedure outlined in Figure S4.

      (9) The authors stated in the Discussion in lines 383 to 383 on page 12 that "After ovulation, the changes reported in the carbohydrate composition of the ZP (3, 25) are likely induced by the addition of glycoproteins of oviductal origin, as we have seen here with OVGP1." There is no evidence in the present study to suggest that OVGP1 or glycoproteins of oviductal origin have changed or can change the carbohydrate composition of the ZP. At present, it is not known if OVGP1 or glycoproteins of oviductal origin directly interact with ZP glycoproteins (including ZP1, ZP2, ZP3 and/or ZP4) that make up the zona matrix.

      There is scientific evidence suggesting that oviductal glycoproteins, including OVGP1, interact with the zona pellucida (ZP) glycoproteins of the oocyte. Studies have shown that OVGP1 binds to the ZP of the oocyte. Specifically, OVGP1 is thought to interact with ZP glycoproteins, such as ZP2 and ZP3, in a way that may help stabilize the oocyte or modify the ZP structure during its passage through the oviduct. This interaction is believed to influence processes like sperm binding, oocyte maturation, and potentially the prevention of polyspermy during fertilization. For example, in several studies, the absence of OVGP1 in knockout animals (such as in Ovgp1-KO hamsters) has been associated with impaired fertilization and embryonic development, which indicates the importance of this interaction. However, the detailed molecular mechanisms and functional significance of these interactions require further exploration. We have use the work “likely” to soften this statement.

      Velásquez, J. G., Canovas, S., Barajas, P., Marcos, J., Jiménez‐Movilla, M., Gallego, R. G., ... & Coy, P. (2007). Role of sialic acid in bovine sperm–zona pellucida binding. Molecular reproduction and development, 74(5), 617-628.

      Kunz, P., et al. (2013). "The role of oviductal glycoprotein 1 in sperm–egg interaction and early embryonic development." Reproduction, 145(3), 225-233. DOI: 10.1530/REP-12-0300

      Yamatoya, K., Kurosawa, M., Hirose, M., Miura, Y., Taka, H., Nakano, T., ... & Araki, Y. (2024). The fluid factor OVGP1 provides a significant oviductal microenvironment for the reproductive process in golden hamster. Biology of reproduction, 110(3), 465-475.

      (10) Lines 390 to 391 page 12: The statement "This determines that OVGP1 modifications are critical to define the barrier among the different species of mammals." needs to be rephrased because there is no evidence in the present study showing that OVGP1 has been modified. There are many concerns with errors, important information that is missing, and inconsistencies as well as wrong and misleading information in the Materials and Methods which are troublesome. These concerns raise questions regarding the authenticity and reliability of the study. Some of the major concerns are listed below:

      All concerns have been fixed

      (11) It says in line 399 on page 13 that "Human semen samples were obtained from a normozoospermic donor...". Do the authors really mean that the semen samples were obtained from only one donor?

      Samples were obtained from 3 normozoospermic donor, this is now indicated in M&M

      (12) In lines 409 to 411 on page 13, what do the authors mean by "...the samples were frozen into pellets..."? Was centrifugation of the samples carried out prior to freezing the samples? Secondly, what do the authors mean by "....and stored in liquid nitrogen at -196{degree sign}C or lower.", particularly what do the authors mean by "or lower"? The temperature of liquid nitrogen is -196{degree sign}C. What is the "lower" temperature?

      Centrifugation of the samples were no carried out at this time. A more detailed protocol is now included The word lower has been removed.

      (13) Line 424 on page 13: Provide the full name of "M2" when it is first used in the text then followed by the abbreviation.

      Done

      (14) Is there a reason why different counting chambers were used to determine sperm concentrations? In line 432 on page 13, a Thomas cell counting chamber was used to determine the sperm count of epididymal mouse sperm whereas it is mentioned in line 441 on page 14 that a Neubauer cell counting chamber was used to determine epididymal cat sperm. Furthermore, where did the cat's sperm come from?

      The cat sperm was obtained and processed at the Faculty of Veterinary Medicine and the rest of the samples were processed in the INIA-CSIC lab, and different chambers were used in both places.

      (15) The mention of the use of cat spermatozoa in line 439 on page 14 is a worrisome problem of the manuscript. The present study used bovine, mouse, and human sperm and not cat. Therefore, the sudden mentioning of the use of cat spermatozoa in the Materials and Methods is troublesome and worrisome. It appears that the paragraph from lines 439 to 450 was directly copied and pasted from previously published work. Furthermore, lines 441 to 445 do not flow and do not make sense. In fact, what is described in this paragraph (lines 439 to 450) does not appear to correspond to the method(s) used to obtain the results presented in the Results section of the manuscript.

      I don't understand why the reviewer says we don't use cat sperm. This study uses cat sperm. Results of cat sperm are indicated in the Figure 1A (now 2A). We have modified the M&M to clarify frozen description.

      (16) Similarly, several problems are also found in the paragraphs (lines 453-478 on page 14) describing the methods and procedures to obtain homologous and heterologous IVF of bovine oocytes. Firstly, it is mentioned here (in line 460) that COCs were co-incubated with selected sperm without removing the cumulus cells. However, the results of the sperm penetration experiment indicated otherwise. Figures 2 and 3 show that the oocytes were denuded of cumulus cells. Secondly, it is very worrisome and troublesome to read what is written in line 468 on page 14 that "...from other species (cat, human, mouse, and rabbit)." One wonders where the cat and rabbit came from. Again, it appears that this paragraph was directly copied and pasted from previously published work.

      Cat sperm was used in this manuscript and it is correctly indicated in every section and figures. About IVF and EZPT protocols, in the protocol of IVF for bovine oocytes, COCs were used without removing the cumulus cells. For the EZPT cumulus cells were removed, this is described in the following sections of the material and methods. The word rabbit was a mistake and it has been removed.

      (17) In lines 468 to 469 on page 14, it is mentioned that "Sperm-egg interactions were assessed through a sperm-ZP binding assay...". The authors only examined sperm penetration in their study. Therefore, this needs to be specified in the Materials and Methods. Secondly, the authors did not use the conventional sperm-ZP binding assay in their study. Instead, they used the EZPT in their study. There appear to be many inconsistencies throughout the manuscript.

      When the IVF experiments using bovine COCs were done (Fig 2A and C, Fig 1S to 3S, and Tables 1S to 4S) conventional sperm-egg interaction was assessed at 2.5 hours after IVF. EZPT was used in the rest of experiments. IVF with COCs and EZPT with ZPs are different experiments.

      (18) Lines 480 to 489 on page 15 under the sub-heading of "In vitro culture of presumptive zygotes to first cleavage embryos on Day 2" do not provide the correct methodology used for obtaining the results presented in the manuscript. In line 482, it is not clear where the "synthetic oviductal fluid" came from. In fact, in the Results section, none of the results came from the use of synthetic oviductal fluid. In line 487, humans and rabbits are mentioned here. However, human and rabbit oocytes were not used in the present study. It is very strange indeed to read human and rabbit in the sentence.

      SOF reference is now included. Human results are in Fig 1A; the sentence is referred about the cultures of bovine oocytes inseminated with sperm of bull, human, mouse or cat). Rabbit word is a mistake and is now eliminated of the manuscript.

      (19) In line 500 on page 15, what do the authors mean by "Each oviduct was strengthen by removing the adjacent tissue..."?

      The sentence has been modified.

      (20) On page 15 in the Materials and Methods, the authors described the collection of bovine and mouse oviductal fluid. However, there is no mention of human oviductal fluid and how it was collected. This important information is missing.

      We have not use human oviductal fluid in this manuscript.

      (21) Line 510 on page 15: The sub-heading of "Preparation of empty zonae pellucidae from bovine ovarian oocytes" should be rephrased. As pointed out earlier in my review, the ZPs prepared by the authors were intact and not "empty". It was the oocyte which was empty after extraction of the ooplasm.

      Everything except the ZP were removed from the oocyte, this is now clarified in the manuscript.

      (22) Line 518 on page 16 and line 553 on page 17: "Figure S5" should be "Figure 4S".

      Done

      (23) Line 538 and line 547 on page 16: "mice oocytes" should be "mouse oocytes".

      Done

      (24) On page 17, the procedures for in vitro fertilization, sperm penetration, and binding assessment in mice were described here in lines 560 to 574. Several problems are noted in this paragraph as listed below:<br /> a. As mentioned earlier the authors in the present manuscript mixed up sperm penetration and sperm binding which are two separate events. Based on the results presented in the manuscript, they represent sperm penetration and not sperm binding. Therefore, the authors need to precisely explain in the manuscript whether the results presented refer to sperm penetration or sperm binding.

      Both sperm penetration and binding have been analyzed in this work.

      b. In line 570 on page 17, the term "insemination" is wrongly used here. Insemination is the introduction of semen into the female reproductive tract either through sexual intercourse or through an instrument. The procedure used in the present study was carried out in vitro in a co-incubation manner and not by transferring sperm into the female reproductive tract.

      The word insemination has been changed to incubation

      c. Information regarding procedures for treatment with various oviductal fluid and OVGP1s are all missing in the Materials and Methods.

      This information is now in M&M

      d. The concentrations of various oviductal fluids and OVGP1s used and the number of ZPs used in each incubation are also missing.

      Concentrations are now indicated in the manuscript. All the numbers and ZPs used are indicated in supplementary figures.

      (25) Lines 577 to 603 on pages 17 to 18: Were recombinant bovine and murine glycoproteins prepared using the same methodology? In line 595 on page 18, it is stated that "Supernatant was saved in subsequent experiments." It is not clear exactly what experiments the supernatant was subsequently used in.

      Details about how the bovine and murine glycoproteins were prepared are now included. Sentence about subsequent experiment is delete; supernatant was used for the next steps of protein purification.

      (26) What is being described in lines 604 to 609 on page 18 is problematic. The paragraph starts by saying that "Human recombinant oviductin was obtained from Origene Technologies....". Strangely, the paragraph continues by saying that the recombinant proteins were produced by transfection in HEK293T...". If recombinant human OVGP1 had already been obtained from Origene Technologies, why did the authors want to produce it again? It does not make sense.

      We briefly described the method that Origene used for the production of the human recombinant OVGP1

      (27) In lines 626 to 627 on page 18, it is stated that "Zonae pellucidae previously incubated with OVGP1 proteins from several species and murine oviductal fluid...". Were the zonae pellucidae previously incubated with only murine oviductal fluid or also with others?

      It was only incubated with OVGP1 or with oviductal fluid, this is now clarified in the text.

      (28) In lines 638 and 639 on page 19, can the authors please explain the difference between "endogenous OVGP1 and bOVGP1" and "exogenous recombinant hOVGP1 and mOVGP1"?

      This is now clarified

      (29) As stated in lines 676 to 679 on page 20, statistical analysis was performed in the study. Strangely, no "n" numbers and p values were provided in any of the figures that require statistical analysis. This is problematic.

      Statistical analysis and significant differences are now included in the figures, all the numbers used are included in the supplementary tables that are related with the figures.

      There are also many errors noted in the Figure Legends. These concerns raise questions regarding the reliability of the findings and interpretation of the results. Some major ones that require attention are listed below:

      (30) Figure legend 1 on page 27: In line 912, where did the "cat sperm" come from? In line 913, where did the "feline sperm" come from? In line 918, as pointed out earlier, the term "empty zona penetration test (EZPT)" is a misnomer and should be replaced with a better term. In line 924, it is stated that "Note sperm only appear outside the zona." However, no sperm can be seen outside the zona pellucida shown in Figure 1.

      Cat sperm is used in this manuscript. Term EZPT is now clarified The sentence about sperm outside of ZP is removed

      (31) Figure legend 2 on page 27 (lines 928 to 940) needs to be rewritten. Some of the sentences are not clearly written. Authors, please check all the capital labeling letters some of which appear to be wrong.

      Done

      (32) As is written, Figure legend 3 on pages 28 and 29 (lines 943 to 959) presents many problems:

      a. Contrary to what is stated in the figure legend, not all five regions are present in the hOVGP1, mOVGP1, and bOVGP1.

      Done

      b. Contrary to what is stated in line 946, region D is not conserved in the mouse and bull as shown in Figure 3A, and region C is not present only in the mouse.

      Done

      c. Based on what is shown in Figure 3A, region E is present only in the mouse and not in the human.

      Done

      d. What is stated in line 951 that "Proteins were expressed in mammalian cells..." is not correct. Based on the information provided in the manuscript, recombinant human OVGP1 was obtained from Origene Technologies and was not expressed in mammalian cells as claimed.

      All the recombinant proteins were produced in mammalian cells.

      (33) Figure legend 6 on page 28: In lines 985 to 986, what do the authors mean by "...and combinations of the three oviductins with sperm of the three species."? As is written, it appears that the bovine ZPs were pretreated with a combination of all three oviductins and then co-incubated with sperm from the bull, mouse and human together.

      We have clarified this sentence

      (34) What is described in the figure legend for the supplemental figure (Figure S7) does not make sense.

      Legend of Fig S7 (now S8) is related to pictures A to E, the legend is now clarified.

      (35) In addition to the figures and supplemental figures provided in the manuscript, there is also an additional figure labeled with "Model" showing three diagrams. Strangely, there is no mention of this additional figure in the manuscript. There is no figure legend for or description of this figure. It is not clear what is being shown in this figure, and it is not clear about the purpose of the use of this figure.

      We have included a legend to the model that is now Figure 10.

    1. “Desafortunadamente, nuestros sistemas de evaluación siguen estando estrechamente ligados a la publicación de nuestros trabajos y a métricas controvertidas como el factor de impacto

      Esto depende las disciplinas, en Ciencias Sociales o Humanidades es posible hacer una carrera académica exitosa sin nunca haber publicado en revistas de "alto factor de impacto".

    1. Une autre stratégie visant à renforcer l’adoption de l’IA consiste à présenter les fonctionnalités qui mobilisent de l’IA générative comme des extensions de la productivité, de la créativité et de l’intelligence des personnes, qui leur donne de nouvelles compétences (ex : générer du code) ou plus de temps (ex : générer des notes de réunion). Les fonctionnalités ne présentent pas l’IA comme un assistant personnalisé qui ferait à notre place, mais affichent plutôt un panel d’outils qui étendent les compétences (ici professionnelles) des utilisateurs: les actions possibles réalisables grâce à l’IA sont représentées grâce à des icônes traditionnels d’outils logiciels, plutôt qu’un champ textuel libre. Cette évocation de l’outil participe à présenter l’utilisation de l’IA comme un facteur de compétitivité professionnelle, où la rapidité de réalisation des tâches et le spectre de savoirs-faires sont valorisés. C’est une manière d’imposer l’IA plus indirecte et pernicieuse, en jouant sur la concurrence entre travailleurs.

      Il y a aussi la tension que j'évoquais sur Element de confusion avec de la commande macro simple.

      -> ça fait basculer toute forme d'automatisation du côté de l'IA. Sans permettre de distinguer notamment ce qui est déterministe de ce qui est probabiliste.

    2. avant par la rupture dans l’expérience

      Utiliser un mot plus fort et qui signifie mieux : ? l'interruption ?

      J'ai l'impression qu'il y a un mot plus familier que je trouve pas pour la personne chiante qui s'insère dans une discussion qui pourrait marcher

    3. Poussées par des investissements massifs qu’il faut rentabiliser, par la promesse de nouvelles sources de profits ou par la peur de se voir dépassées, de nombreuses entreprises du numérique ont commencé à mettre en œuvre des fonctionnalités alimentées par l’IA.

      Il y a aussi clairement des enjeux géopolitiques, on a pas trop parlé car on est centré sur les usages grand public, mais c'est quand même principalement pour faire la guerre :/

    1. Los fármacos constituyen la piedra angular de la terapéutica moderna; aunque, médicos y legos reconocen que los resultados de la farmacoterapia varían sobremanera con cada persona. Aunque esta variabilidad se ha percibido como un aspecto impredecible y, por lo tanto, inevitable de la farmacoterapia, en realidad no es así.

      Farmacologia Clinica

    1. y uniting diverse parties who are each other’s “environment,” we enable people toexperience themselves connected to a larger whole rather than talk about it as something “outthere.

      [C] The underlying concept of focusing on the positive reminds me of appreciative inquiry. I think that even though this process is very different, (so far) I have taken away that by awknowledging our shared experiences and not what is different is a similar moral of AI.

    1. Author response:

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

      eLife Assessment

      The study presents important findings on inositol-requiring enzyme (IRE1α) inhibition on diet-induced obesity (overnutrition) and insulin resistance where IRE1α inhibition enhances thermogenesis and reduces the metabolically active and M1-like macrophages in adipose tissue. The evidence supporting the conclusions is convincing but can be enhanced with information/data on the validity, specificity, selectivity, and toxicity of the IRE1α inhibitor and supported with more detail on the mechanisms by which adipose tissue macrophages influence adipocyte metabolism. The work will be of interest to cell biologists and biochemists working in metabolism, insulin resistance, and inflammation.

      We thank the editors for the assessment and appreciation of our findings in this study. In the revision, we have added the information on the validity, selectivity and toxicity of IRE1α inhibitor. In addition, we also discussed the likelihood that suppression of metabolically activated proinflammatory macrophage population in adipose tissue on the reversal of adipose remodeling and thermogenesis. In the revision, we have improved the manuscript significantly throughout the text and figures following the recommends by the reviewers.

      Public Reviews:

      Reviewer #1 (Public review):

      First, the authors confirm the up-regulation of the main genes involved in the three branches of the Unfolded Protein Response (UPR) system in diet-induced obese mice in AT, observations that have been extensively reported before. Not surprisingly, IRE1a inhibition with STF led to an amelioration of the obesity and insulin resistance of the animals. Moreover, non-alcoholic fatty liver disease was also improved by the treatment. More novel are their results in terms of thermogenesis and energy expenditure, where IRE1a seems to act via activation of brown AT. Finally, mice treated with STF exhibited significantly fewer metabolically active and M1-like macrophages in the AT compared to those under vehicle conditions. Overall, the authors conclude that targeting IRE1a has therapeutical potential for treating obesity and insulin resistance.

      The study has some strengths, such as the detailed characterization of the effect of STF in different fat depots and a thorough analysis of macrophage populations. However, the lack of novelty in the findings somewhat limits the study´s impact on the field.

      We thank the reviewer for the appreciation of our findings and the comments about the novelty. Regarding the novelty, we would emphasize several novelties presented in this manuscript. First, as the reviewer correctly pointed out, we discovered that IRE1 inhibition by STF activates brown AT and promotes thermogenesis and that IRE1 inhibition not only significantly attenuated the newly discovered CD9+ ATMs and the “M1-like” CD11c+ ATMs but also diminished the M2 ATMs for the first time. These discoveries are very important and novel. In obesity, it was originally proposed that ATM undergoes M1/M2 polarization from an anti-inflammatory M2 to a classical pro-inflammatory M1 state. It was further reported that IRE1 deletion improves thermogenesis by boosting M2 population which then synthesize and secrete catecholamines to promote thermogenesis. It is now known that M2 macrophages do not synthesize catecholamines or promote thermogenesis. In this study, we discovered that IRE1 inhibition doesn’t increase (but instead decrease) the M2 population and that IRE1 inhibition promotes thermogenesis likely by suppressing pro-inflammatory macrophage populations including the M1-like ATMs and most importantly the newly identified metabolically active macrophages, given that ATM inflammation has been reported to suppress thermogenesis. Second, this study presented the first characterization of relationship between the more classical M1-like ATMs and the newly discovered metabolically active ATMs, showing that the CD11c+ M1-like ATMs are largely overlapping with but yet non-identical to CD9+ ATMs in the eWAT under HFD. Third, although upregulation of ER stress response genes in the adipose tissues of diet-induced obese mice have been extensively reported, it doesn’t necessarily mean that targeting IRE1a or ER stress can reverse existing insulin resistance and obesity. It is not uncommon that a therapy doesn’t yield the desired effect as expected. For instance, amyloid plaques are a hallmark of Alzheimer's disease (AD), interventions that prevent or reverse beta amyloid deposition have been expected to prevent progression or even reverse cognitive impairment in AD patients. However, clinical trials on such therapies have been disappointing. In essence, experimental demonstration of effectiveness or feasibility for any potential therapeutic targets is a first step for any future clinical implementation.

      Reviewer #2 (Public review):

      The manuscript by Wu et al demonstrated that IRE1a inhibition mitigated insulin resistance and other comorbidities through increased energy expenditure in DIO mice. In this reviewer's opinion, this timely study has high significance in the field of metabolism research for the following reasons.

      (1) The authors' findings are significant and may offer a new therapeutic target to treat metabolic diseases, including diabetes, obesity, NAFLD, etc.

      (2) The authors carefully profiled the ATMs and examined the changes in gene expression after STF treatment.

      (3) The authors presented evidence collected from both systemic indirect calorimetry and individual tissue gene expression to support the notion of increased energy expenditure.

      Overall, the authors have presented sufficient background in a clear and logically organized structure, clearly stated the key question to be addressed, used the appropriate methodology, produced significant and innovative main findings, and made a justified conclusion.

      We thank the reviewer for the appreciation of our work.

      Reviewer #3 (Public review):

      Summary:

      The manuscript by Wu D. et al. explores an innovative approach to immunometabolism and obesity by investigating the potential of targeting macrophage Inositol-requiring enzyme 1α (IRE1α) in cases of overnutrition. Their findings suggest that pharmacological inhibition of IRE1α could influence key aspects such as adipose tissue inflammation, insulin resistance, and thermogenesis. Notable discoveries include the identification of High-Fat Diet (HFD)-induced CD9+ Trem2+ macrophages and the reversal of metabolically active macrophages' activity with IRE1α inhibition using STF. These insights could significantly impact future obesity treatments.

      Strengths:

      The study's key strengths lie in its identification of specific macrophage subsets and the demonstration that inhibiting IRE1α can reverse the activity of these macrophages. This provides a potential new avenue for developing obesity treatments and contributes valuable knowledge to the field.

      Weaknesses:

      The research lacks an in-depth exploration of the broader metabolic mechanisms involved in controlling diet-induced obesity (DIO). Addressing this gap would strengthen the understanding of how targeting IRE1α might fit into the larger metabolic landscape.

      Impact and Utility:

      The findings have the potential to advance the field of obesity treatment by offering a novel target for intervention. However, further research is needed to fully elucidate the metabolic pathways involved and to confirm the long-term efficacy and safety of this approach. The methods and data presented are useful, but additional context and exploration are required for broader application and understanding.

      We thank the reviewer for the appreciation of strengths in our manuscript. In particular, we appreciate the reviewer’s recommendation on the exploration of broader metabolic landscape, such as the effect of IRE1 inhibition on non-adipose tissue macrophages and metabolism. We agree that achieving these will certainly broaden the therapeutic potential of IRE1 inhibition to larger metabolic disorders and we will pursue these explorations in future studies.

      Recommendations for the authors:

      Reviewer #1 (Recommendations for the authors):

      A list of recommendations for the authors is presented below:

      (1) Please, update the literature review to include more recent studies relevant to the topic.

      We thank the reviewer’s suggestions. We have added more references from recent studies.

      (2) Please, provide a detailed explanation of how STF functions, including potential off-target effects or issues related to specificity.

      We thank the reviewer’s suggestions. STF is a small-molecule inhibitor designed to selectively inhibit the RNase activity of IRE1a. Once IRE1a is activated (e.g., in obesity), its RNase domain initiates the unconventional splicing of the transcription factor X-box binding protein 1 (XBP1) mRNA and the Regulated IRE1-Dependent Decay (RIDD) of microRNAs, which is detrimental if prolonged. IRE1a RNase inhibitors including STF engage the RNase-active site of IRE1a with high affinity and specificity by exploiting a shallow complementary pocket through pi-stacking interactions with His910 and Phe889 and an essential Schiff base interaction between the aldehyde moiety of the inhibitor and the side chain amino group of Lys907 (Sanches et al., NComm 2014, PMID: 25164867). This specific and high affinity binding blocks the IRE1a RNase activity, preventing the splicing of XBP1 mRNA and RIDD. As IRE1a has been shown to be activated in multiple tissues under various pathological conditions and to be responsible for the progression of the pathological conditions, inhibition of IRE1a by pharmacological agents including STF has the great potential for the treatment of various pathological disorders. Several studies have reported that STF shows no overt toxicity when administered systemically (Madhavan, Aparajita, et al.2022, PMID 35105890; Herlea-Pana et al., 2021, PMID 34675883; Papandreou et al., 2011, PMID 21081713; Tufanli et al., 2017, PMID 28137856).

      (3) Lines 263-266 require a reference.

      We thank the reviewer’s suggestion. A reference has been added.

      (4) Stromal vascular fraction (SVF) also contains a significant amount of preadipocytes and stem cells, not only macrophages, which might affect the conclusions reached by the authors.

      We thank the reviewer’s comments. It is true that SVF consists of multiple cell types, including endothelial cells, macrophages, preadipocytes, and various stem cell populations. In HFD-induced obesity, adipose tissue undergoes significant remodeling, and the percentage of macrophages in the SVF of obese adipose tissue increases significantly relative to other cell types. In our studies, SVFs from adipose tissues of obese mice were isolated, cultured, and treated with STF for overnight.  We observed that IRE1 RNase activity in SVFs was inhibited by STF treatment, and that ATM population and the expression of pro-inflammatory genes were downregulated by STF. Given the short-term treatment, the parsimonious interpretation of the data would be that STF directly acts on ATMs.  However, we note that the possibility that the effect of STF on other cell types might influence the ATM and inflammatory gene expression can’t be totally ruled out. As such, we have modified our conclusion from “these results indicate that STF acts directly on ATMs to regulate inflammation” to “these results indicate that STF likely acts directly on ATMs to regulate inflammation”.

      (5) Figures 1A and G: It is common practice to present the XBP1s/XBP1u ratio; consider using this standard measure.

      We thank the reviewer’s comments. Regarding the XBP1 mRNA splicing, we see both ways of presentation in publications. There are quite a number of papers, for instance, PMID25018104, 2014, Cell; PMID23086298, 2012, NCB, that used the XBP1s/ (XBP1s+XBP1u) ratio. We preferred this way of presentation as it shows the ratio of spliced XBP1 (XBP1s) relative to the total XBP1 mRNA (XBP1s+XBP1u).

      (6) Figure 1F: please indicate the type of AKT phosphorylation assessed.

      We thank the reviewer’s comments. We have added Ser473 as the phosphorylation site at in both figure legend and figure.

      (7) Figures 2E-H: please clearly indicate the specific fat depots analyzed in each figure.

      We thank the reviewer’s comments. We have added the information in the figure legends and figures.

      (8) Figures 1I and 3A, and Supplementary Figures 6D-E: please include a quantification analysis of the images presented.

      We thank the reviewer’s suggestion. We have added the quantifications of the images.

      (9) In Figure 3D the image corresponding to the merge for the STF condition is a duplication of the control, please correct this.

      We thank the reviewer for pointing this out. We have replaced it with the correct image.

      (10) Figures 4B-F: please provide individual data points in the graphs to show variability and sample distribution.

      We thank the reviewer’s suggestion. We have re-plotted the graphs in Fig. 4B-F with the individual data points.

      (11) Figure 4I: it is rather unusual to have such a strong signal of UCP1 in ND conditions, please explain.

      We thank the reviewer for the comment. We wish to point out that the images were taken from BAT slides. UCP1 is expected to show strong staining in BAT under DN condition, which as expected is weakened under HFD condition. STF treatment was able to correct the HFD-induced weakening of UCP1 staining in BAT.

      (12) Supplementary Figures 2C-D: please provide representative images for better clarity and interpretation.

      We thank the reviewer for the comment. The representative images for Supplementary Figures 2C-D were actually shown in Figures 2C and F. Supplementary Figures 2C-D were the mere quantification for adipocyte areas for Figures 2C and F.

      (13) Supplementary Table 3 is repeated, please remove.

      We thank the reviewer for the comment. We have deleted this repetition.

      Reviewer #2 (Recommendations for the authors):

      The manuscript can be further strengthened with more clarification on the following points.

      (1) The use of IRE1a pharmacological inhibitor STF-083010 (STF) needs to be validated. How was the dose determined? Were there any dose-dependent studies? Under the current dosing regimen, what are the specificity, selectivity, and toxicity of STF? Also, were the serine/threonine kinase and RNase activities measured in the adipocytes and ATMs of the animals dosed with the compound? What's the PK data?

      We thank the reviewer for the comments. In the animal study, we used STF 10 mg/kg for intraperitoneal injection. This dose was adopted from several recent studies (Madhavan, Aparajita, et al.2022, PMID 35105890; Herlea-Pana et al., 2021, PMID 34675883; Papandreou et al., 2011, PMID 21081713; Tufanli et al., 2017, PMID 28137856), in which STF treatment showed beneficial effect in their respective disease models. STF didn’t compromise cell viability or induce any other toxicity at the dose or concentration used in these studies (Papandreou I, et al., 2011; Upton JP, et al., 2012; Lerner AG, et al., 2012; Kemp KL, et al., 2013; Cross BC, et al., 2012). In our study, we didn’t observe any apparent toxicity on mice at this dose. Importantly, we did observe that STF inhibited IRE1 RNase activity in adipose tissues (F1G, S1D) and ATMs (F6Q, S8C, G, I) of the animals at this dose. As the IRE1 inhibitors including STF has been extensively examined and shown to have no effect on the kinase function of IRE1 (Cross et al., 2012, PMID: 22315414; Tufanli et al., 2017, PMID 28137856), we didn’t perform the assay on Ire1 kinase activity. Additionally, as the chemical has been administered into several animal models, with significant beneficial effects, one would assume decent pharmacokinetic parameters being achieved with the current dose. It would be important and necessary to have systematic PK studies in the future if clinical trials are to be considered.

      (2) The statistical method for individual panels in each figure needs to be specified.

      We thank the reviewer for the suggestion. We have specified the statistical method in the figure legends.

      (3) In Figure 1E, there's no difference in fasting insulin levels, though a difference was detected after the glucose load. This suggests an effect on insulin secretion but not insulin sensitivity.

      We thank the reviewer for the comments. The insulin levels are still different between Veh and STF groups at fasting, just not reaching statistically significant. Under glucose stimulation, the insulin levels all showed the same trend, which is, the STF group is lower than the Veh group. Even if the fasting insulin levels showed no difference between the two groups regardless of glucose stimulation, the fact that the blood glucose levels at all the time points are lower in STF group than Veh group (Fig. 1C) indicates that insulin sensitivity is improved. In our study, the insulin levels were lower in STF group, but the blood glucose levels were still lowered by STF, further strengthening the notion that STF treatment improves insulin sensitivity. This is indeed further corroborated by the ITT results (Fig. 1D).

      (4) Figure 2 and S2A did not show a decrease in BW but rather BW gain. The statement (line 308) needs to be edited. As a result of this, the relative fat mass measurement (% of BW) needs to be presented in addition to Figure 2B.

      We thank the reviewer for the comments/suggestions. As shown in Figs. 2A and S2A, we observed a slight decrease in body weight (~2g reduction) in STF-treated mice while Veh group increased body weight by ~3.5g, at the end of 4 weeks of treatment. As shown in Fig. 2B, this difference in body weight between Veh and STF groups was primarily due to a reduction in fat tissue. In the revision, we also added the percentages of fat and lean masses over total body weight in Supplemental Fig. 2B, which show the similar trend.

      (5) The measurement of blood lipid levels in Figure 3F-H is informative. More importantly, hepatic lipid content needs to be measured.

      We thank the reviewer for and agree on the comments. As this study is more focused on the insulin resistance and adipose tissue remodeling, we didn’t go deep into the comorbidities beyond the reported observations. It will be interesting to explore the effects of IRE1 inhibition on the obesity/insulin resistance comorbidities including hepatic lipid content measurement in future study.

      Minor corrections:

      (1) Line 261: "(spliced".

      Done. We have corrected it.

      (2) Line 334: spell out "PEPCK".

      We have added the full name “Phosphoenolpyruvate carboxykinase”. Thanks!

      (3) Line 478: please rephrase.

      We thank the reviewer for the comment. We have rephrased the sentence as following: “These results reveal that STF treatment suppresses the adipose tissue inflammation and the accumulation of pro-inflammatory ATM with augmenting (suppressing instead) M2-like ATMs.”

      (4) Figure 4L: "pGC1-a".

      We thank the reviewer for pointing this out. We have corrected the name.

      (5) Figure 4O: missing Y-axis label.

      We have added the label. Thanks!

      Reviewer #3 (Recommendations for the authors):

      The observations presented by Wu D. et al. in the manuscript are potentially interesting and relevant. The current study seeks to build upon previous findings, specifically from the work titled, "Silencing IRE1α using myeloid-specific cre suppresses alternative activation of macrophages and impairs energy expenditure in obesity." By using a pharmacological inhibitor to modulate IRE1α activity in adipose tissue macrophages (ATMs), the authors aim to develop therapeutics that could significantly impact the treatment of obesity and metabolic disease.

      The authors have performed some satisfactory experiments related to liver steatosis. However, the manuscript would benefit from a more comprehensive exploration of the mechanisms by which ATMs influence adipocyte metabolism, particularly in epididymal white adipose tissue (eWAT). In particular, the study should investigate how adiposity and lipid droplet size change in response to alterations in lipolysis and adipogenesis, as this could provide insights into how these processes contribute to the amelioration of the obesity phenotype.

      Several issues should be addressed to strengthen the manuscript and make the study more convincing. Below are specific comments and recommendations:

      Major:

      (1) The indirect calorimetric data should be normalized for dependent variables such as body weight, lean mass, and fat mass+ lean mass to accurately interpret the results. The results for 24-hour energy expenditure should be included in Figure 4B-F to provide a more comprehensive analysis. It is recommended to plot bar graphs with all individual data points for the energy expenditure (EE) results shown in Figure 4B-F, to offer a clearer and more detailed presentation of the data (Figure 4B-F).

      We thank the reviewer for the comments. Data analysis on the indirect calorimetric studies has evolved over the years. One common practice was/is to normalize the data by body weight. However, this approach was deemed improper some years ago (Tschop et al Nature Methods 2012, PMID: 22205519). Tschop paper also pointed out the shortcomings associated with normalization by lean mass. Instead, it concludes that “generalized linear model is the most appropriate statistical approach to accommodate discrete (genotype) and continuous (body mass) traits, rather than using a simple division by BW or lean BW”. In our study, we used CalR, an improved generalized linear model (which includes ANOVA and ANCOVA) (Mina et al Cell Metabolism 2018, PMID: 30017358) for all our energy expenditure data analysis (shown in Fig. 4A-E). In the revision, we also included data analysis normalized by BW (Fig. S2F-H’), which actually shows even wider difference between Veh and STF groups than the data shown in Fig. 4A-F. As STF decreased the fat mass and had little effect on lean mass, the difference would be more drastic for normalization with fat mass and with fat mass+ lean mass than the data shown in Fig. 4A-E and would be similar to the data shown in Fig. 4A-E for normalization with lean mass. In addition, we replotted the graphs in Fig. 4B, D, F-H with the individual data points.

      (2) At the thermoneutral point (30{degree sign}C), the study could benefit from testing the indirect calorimetric models of human energy physiology. Future studies could also explore this to evaluate the implications for drug development.

      We agree with the reviewer on the comments. In the future study, it will be very informative to investigate the effects of STF under thermoneutral conditions, which could provide more consistent data on how drugs affect metabolic processes in humans, improving translational research.

      (3) The current study missed the opportunity to investigate the effects of STF on non-adipose tissue (non-AT) resident macrophage populations, such as those in bone marrow or lymph-node macrophages. Understanding how STF modulates macrophage metabolism in these contexts would be valuable.

      We thank the reviewer for and agree on the comments. As this study is more focused on the insulin resistance and adipose tissue remodeling, we were mostly restricted to adipose tissue macrophage populations. In the future, it would be interesting to investigate the effect of STF on macrophages in other non-adipose tissues, which will provide a more comprehensive understanding of STF's effects on immune cell metabolism, which could inform its application in various therapeutic areas.

      (4) The study should explore how STF influences the expression of CD9, Trem2, (positive subpopulations), and the secretion of pro-inflammatory cytokines by macrophages, particularly in response to LPS and IFNγ activation in stromal vascular fraction (SVF) cells and bone marrow-derived macrophages (BM-Macrophages).

      We appreciate the reviewer for the comments. Under obesity, the ATM does not undergo the classical M1/M2 polarization; instead, both M1-like/pro-inflammatory macrophages and M2 macrophages increase drastically in obesity. It will be interesting to investigate the effects of STF on the newly identified CD9- and Trem2-positive macrophage subpopulations in SVF and bone marrow macrophages in response to LPS and IFNγ stimulation in the future, although these studies might not faithfully reflect the changes in adipose tissue under obesity as these stressors typically induce classical M1/M2 polarization.

      (5) Additional macrophage gating is necessary better to understand adipose tissue macrophage (ATM) inflammation. Specifically, CD11c−MHC2 low macrophages represent a newly identified inflammatory and dynamic subset in murine adipose tissue. These ATMs accumulate rapidly after ten days of a high-fat diet (HFD) and should increase further with prolonged HFD. For this study, CD11c−MHC2 low ATMs could be subdivided for flow cytometry analysis based on their MHC2 expression, distinguishing them from CD11c−MHC2 high ATMs. All macrophage subtypes categorized here can be studied for metabolic health using seahorse analysis as well.

      We appreciate the reviewer for the comments. It will be interesting to investigate the effects of STF on the newly identified CD11c−MHC2 low macrophage subpopulation in the future. Future studies certainly can include metabolic analysis with Seahorse which can corroborate the energy metabolism at the cellular level with organismal thermogenesis. 

      (6) All flow cytometry histograms - are they showing mean fluorescence intensity or cell# per population? Please specify. All flow cytometry dot plots - It would be helpful for readers to see populations plotted as bar graphs next to respective flow plots, as opposed to being shown as supplemental tables. Additionally, labeling dot plots with the parent population from which cells were gated on would also help readers understand faster what we're looking at.

      We appreciate the reviewer for the comments. In flow cytometry histograms, we used “normalized to mode”. The mode is often used to compare the distribution of fluorescence intensity between different samples. It focuses on the shape of the distribution (with a max of 100%) rather than the absolute cell counts, which helps remove variations caused by different cell numbers or sample sizes, making it easier to compare populations based on fluorescence intensity. When normalizing to the mode, the highest peak in the histogram is scaled to 100%, and all other values are scaled relative to that peak. This allows for easy comparison of multiple histograms, even if the total number of cells (or events) differs between samples.

      (7) The results appear to confuse the actual sample size and p-value. Please carefully review the statistical analyses to ensure that biological replicates are accurately represented. Additionally, include p-values alongside fold change data in the text for clarity represented.

      We appreciate the reviewer for the comments. We have rechecked the statistical analyses confirming that the biological replicates are now properly represented. The exact number of biological replicates for each experiment is now clearly specified in both the methods section and figure legends.

      (8) To further validate the findings, consider using Seahorse analysis at the cellular level in future experiments. This could confirm indirect calorimetric data and thermogenesis responses to cold stimulation.

      We appreciate the reviewer for the comments. Yes, Seahorse analysis at the cellular level will be conducted in future experiments.

      (9) Please ensure the use of person-first language, avoiding labels or adjectives that define individuals based on a condition or characteristic.

      We appreciate the reviewer for the comments. We have changed the descriptions by using person-first language.

      (10) The manuscript does not demonstrate how STF inhibition of IRE1α in ATM, specifically through CD9 and Trem2, controls diet-induced obesity. This aspect should be further elucidated.

      We appreciate the reviewer for the comment. In this study, we observed that STF inhibits IRE1α RNase activity in SVF and in sorted ATMs as well as in adipose tissue. The improvement in diet-induced obesity can be attributable to IRE1α inhibition in both adipocytes and macrophages as shown previously by myeloid and adipocyte-specific knockouts of IRE1α. To conclude whether the IRE1α in CD9- and/or Trem2-positive ATMs controls diet-induced obesity, genetic means would be needed to generate CD9- and/or Trem2-positive ATMs-specific deletion of IRE1α, which will be technically challenging at this moment as there is no CD9 or Trem2-specific Cre lines available.

      Minor:

      (1) Line 43-44: Update terminology to "MASLD" instead of "NAFLD."

      We thank the reviewer for pointing these out. We have changed the terminology in the revision.

      (2) Line 58-59: Add a reference for the mentioned text.

      We thank the reviewer for the comment. Added a reference in the text in the revision.

      (3) Was the antibody used to detect CD9 and Trem2 validated for FACS and other analyses?

      We thank the reviewer for the comment. In our studies, we determined CD9 and Trem2 expression through flow cytometry and immunostaining staining. In flow experiment, CD9 and Trem2 were acquired from Biolegend: PE/Dazzle™ 594 anti-mouse CD9 (BioLegend Cat# 124821, RRID:AB_2800601); APC-conjugated Trem2 (R&D Systems Cat# FAB17291N, RRID:AB_3646995), which were validated for FACS. For immunostaining: CD9  (Abcam Cat# ab223052, RRID:AB_2922392). and Trem2 (R&D Systems Cat# MAB17291, RRID:AB_2208679).

      (4) Studies were limited to male mice; this should be noted in the title and discussed as a limitation.

      We thank the reviewer for the comment. We have modified the wording in the revision.

      (5) Ensure all reagents are fully described with preparation details and identifiable numbers for reproducibility and/or submit the FACS protocol to any protocol archives.

      We thank the reviewer for the suggestions. Yes, we have modified the wording in the revision.

      (6) Provide the correct version numbers for all software used (FlowJo, Prism, etc.).

      We thank the reviewer for the suggestions. We have provided the correct version numbers for softwares for FlowJo and Prism.

      (7) Specify section size (µm) and blocking agent used for eWAT immunofluorescence (Line 207).

      We thank the reviewer for the suggestions. We have added this information.

      (8) Add gene accession numbers to Supplementary Table 3.

      We thank the reviewer for the suggestions. We have added this information.

      (9) Figure 2: Clarify HFD and treatment timelines with a schematic diagram.

      We thank the reviewer for the suggestions. We have added a schematic diagram in Supplemental Figure 1C.

      (10) For histology analysis, the minimum combined data from triplicate images is shown in Figure 2C-2H. For Figures 2E and H, provide complete methods for histology analysis.

      We thank the reviewer for the comments. For the histology analysis shown in Figures 2C–2H, we used a minimum of three mice per treatment group. For each mouse, 3–5 images were taken for analysis. All histology analyses were conducted using ImageJ for image quantification, and the data were processed and organized using Excel and Graphpad.

      (11) Figure 3D Macrophage markers F4/80 stained differently in Figure 5B; to avoid false positive staining, show isotype control to confirm actual staining. For eWAT immunofluorescence (Figures 3D, 5B, 6E)., counterstaining is needed in addition to macrophages, such as for adipocytes-perilipin, and phalloidin for total cells.

      We thank the reviewer for the comments. Yes, Figures 3D macrophage marker F4/80 stained is differently from that of Figure 5B, as they are in different tissues, with Figure 3D in liver samples while Figure 5B in adipose tissues. In the liver, subsets of macrophages are known as Kupffer cells. Kupffer cells have distinct morphology and behavior compared to other tissue-resident macrophages. When stained with F4/80 in the liver, the pattern may reflect the specialized role of Kupffer cells, typically showing a more diffuse or localized staining around blood vessels and sinusoids. In adipose tissue, macrophages tend to accumulate around dead or dying adipocytes, forming what is known as "crown-like structures" (CLS). The F4/80 staining in adipose tissue shows a more clustered pattern, particularly around areas of fat tissue undergoing remodeling or inflammation. In adipose tissue, you can still see clear, defined cells even without counterstaining like perilipin, and importantly, adipocytes are generally way larger than macrophages in size. Yes, we agree that if with counterstaining it would enhance the accuracy. In the future study, we will use perilipin staining to make it easier to differentiate adipocytes from other structures and provide stronger data.

      (12) Insert scale bars in the original images for Figures 3D, 4I, 4M, 5B, 6E, S3B, S6D-E, and S7A-B. All images added a scale bar not inserted while acquiring the image or using imaging software.

      We thank the reviewer for the suggestions. The resolution for the scale bars in the images obtained during acquisition, somehow, isn’t sufficient enough to be clearly visible and requires the enlargement of the images to be seen clearly. In the revision, we have manually added the scale bars for clarity.

      (13) Figure 5E: Please label X-axis as F4/80.

      We thank the reviewer for pointing this out. The label has been added in the revision.

      (14) Figure 5F: It is specified in the legend that cells were gated on F4/80+CD11b+CD11c+, but there is a CD11c- population shown in the histogram...How is this population appearing if all cells should be CD11c+?

      We thank the reviewer for pointing this out. We gated against CD11c in F4/80+CD11b+ population. As such, we have corrected the description in the legend.

      (15) Figure 5G: What is the F4/80+CD11b+CD11c-CD206- population gated in quadrants?

      We thank the reviewer for the comment. The F4/80+CD11b+CD11c-CD206- population was shown in Figure 5G on the lower left side, with the percentages being 15.7% for ND, 5.54% for Veh-HFD, and 26% for STF-HFD.

      (16) Figure 6J: Flow cytometry gates seem slightly misplaced and the sample appears to be overcompensated - were FMOs included in this experiment to establish proper gates? If so, please include.

      We thank the reviewer for the comment. In the study, we did include Fluorescence Minus One (FMO) control in the experiment to establish proper gating. We have included this information in the methods section.

      (17) Table 1-3: Indicate the number of replicates (n=) used in all tables.

      We thank the reviewer for the suggestion. We have provided the specific number of mice used in the study within the figure legends.

    1. Voici un document de synthèse pour un briefing, basé sur les sources fournies :

      Thème central : La fragilité psychique des jeunes, mythe ou réalité ?

      • Un webinaire, organisé par LISA et le Learning Planet Institute, a exploré la question de la fragilité psychique des jeunes, en se demandant si elle est un mythe ou une réalité.

      L'objectif était de nuancer cette vision et de mieux comprendre les dimensions sociales, éducatives et psychologiques qui influencent le bien-être des jeunes.

      Le dispositif LISA

      • LISA est un dispositif conçu pour repérer et comprendre les besoins et les forces des élèves afin de mieux accompagner leur bien-être et leur santé mentale.

      • Il est composé d'une plateforme numérique, d'un parcours de formation pour les enseignants et d'un réseau de collaboration multi-acteurs.

      • Le projet LISA France 2030 vise à améliorer le bien-être d'environ 70 000 élèves dans 190 établissements.

      • Le projet s'articule autour de la question de comment favoriser le bien-être de tous les élèves afin de leur permettre d'apprendre et de progresser ensemble en s'épanouissant.

      • LISA offre des formations, des outils et des ressources élaborées à partir d'une collaboration interdisciplinaire d'enseignants, de cliniciens, de chercheurs et d'ingénieurs.

      • Une plateforme numérique comprend un outil de repérage et une base de ressources avec des interventions concrètes pour la classe.

      • Un questionnaire "facettes" de 12 questions permet aux enseignants de faire une observation structurée de chaque élève.

      Un tableau de bord individuel est généré avec des suggestions pour les enseignants sous forme de fiches pratiques.

      • Lisapédia est une base de ressources avec des fiches pratiques et des liens vers des ressources externes.

      • Des formations pratiques en ligne et en présentiel sont proposées aux enseignants.

      • La démarche s'appuie sur un programme de recherche mené par des spécialistes de la santé mentale et du neurodéveloppement, avec un comité éthique scientifique international.

      Intervention de Christophe Ferveur

      • Christophe Ferveur, psychologue clinicien, psychanalyste et psychodramatiste, a été invité à s'exprimer sur la question.

      Il est spécialisé dans la prévention des risques psychosociaux.

      • Il travaille à la Fondation Santé des étudiants de France et dans un dispositif appelé "relais étudiant lycéen" avec une consultation pluridisciplinaire.

      • L'adolescence et la jeunesse sont des périodes d'incertitude, un "entre-deux" avec un travail intérieur important.

      • Ce travail comprend des aspects corporels, la recherche d'idéaux ou leur désidéalisation, l'affiliation à des groupes, et un apprentissage par tentatives et erreurs.

      • Aujourd'hui, il y a une prolongation de l'immaturité combinée avec une précocité, créant des déséquilibres.

      • Les jeunes sont confrontés à une injonction paradoxale : "fais ce que tu veux, mais sois performant".

      • Le contexte sociétal est anxiogène, avec un climat de "tout s'effondre".

      • Il y a un idéal contemporain du potentiel caché, où il faut s'épanouir et s'accomplir sans défaillance.

      • On observe un désendettement du collectif au profit de l'individu.

      • La société actuelle est marquée par l'accélération, avec l'idée de "ne rien manquer", créant des vulnérabilités.

      • Cette accélération conduit à une fragmentation cognitive, rendant difficiles l'attention, la mémorisation et l'exécution des tâches.

      • Le vécu de l'ennui est devenu un vide, et non plus un temps de maturation.

      Il y a une dispersion et une simplification de la pensée, avec un risque de ne pas vérifier les informations.

      • Il y a une idéologie de la connexion permanente, mais les relations sont labiles et fragiles.

      • Les jeunes ont des difficultés à renoncer et sont plus dans le besoin et l'envie que dans le désir.

      • Il y a une montée des "addictions" et des passages à l'acte, avec une intolérance à la frustration.

      • Les jeunes sont dans une revendication de soutien car leur socle narcissique est fragile.

      • Les jeunes ont des difficultés à se projeter dans l'avenir, pouvant mener à un enfermement dans une "rêvasserie".

      • Le syndrome "hikikomori" (repli sur soi) est un exemple de refus de rentrer dans le monde adulte.

      • Il faut se demander si cette génération est réellement si "fragile" ou si elle est "fragilisée" par son environnement. Il ne s'agit pas d'une fragilité de fond, mais d'une réaction à un environnement anxiogène.

      • On observe une montée des troubles anxio-dépressifs.

      • Une personne sur cinq rencontrera un épisode de trouble psychique nécessitant une prise en charge au cours de sa vie.

      • 40% des jeunes déclarent être en mal-être ou en détresse psychologique.

      • La santé mentale est une construction intérieure complexe dépendant de l'histoire personnelle, de l'environnement et des aspects biologiques.

      • Le trouble psychique est une zone grise où il faut intervenir sans tomber dans le catastrophisme.

      • Dans les troubles psychiques, plus on va mal, moins on demande de l'aide.

      Ils sont souvent invisibles, cachés, et fluctuants.

      • Les troubles sont multifactoriels, liés à l'environnement familial, social et à la projection dans l'avenir.

      • Le retrait social et le désinvestissement des études sont des signaux d'alerte majeurs.

      • Les jeunes tentent souvent des auto-résolutions, notamment par des substances, avant de demander de l'aide.

      Pistes d'action

      • Repérer le plus tôt possible les signaux d'alerte, sans dramatiser.

      • Proposer un accès rapide à une consultation, mais dans un cadre déstigmatisé.

      • Ne pas se focaliser uniquement sur l'individu, mais considérer le contexte familial et social.

      • En cas de retrait social, ne pas retirer brutalement les outils numériques, car ils peuvent être la seule connexion avec le monde pour les jeunes en difficulté.

      • Il n'y a pas de recette miracle pour prévenir le mal-être, car les jeunes ne peuvent pas être super protégés de tout.

      • Il faut permettre aux jeunes de rencontrer les difficultés et d'apprendre par eux-mêmes.

      • Ne pas forcer un jeune à consulter, mais ne pas le laisser seul avec sa détresse.

      • Proposer des cadres de consultation adaptés, comme le relais étudiant lycéen.

      • La France est un pays où la prescription de médicaments est élevée.

      La psychiatrie évolue vers la pharmacologie et la résolution rapide des symptômes.

      La société se désengage et l'individu doit trouver des solutions rapidement.

      Conclusion

      • La question de la fragilité psychique des jeunes est complexe.

      Il faut nuancer cette vision et comprendre l'impact de l'environnement sur leur bien-être.

      • Il est important de repérer les signaux d'alerte, de proposer des cadres de consultation adaptés, et de ne pas stigmatiser les jeunes.

      • Il faut aussi tenir compte du contexte sociétal et de ses injonctions paradoxales.

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

      Learn more at Review Commons


      Reply to the reviewers

      1. General Statements [optional]

      Our manuscript initially entitled “Ribosomal RNA synthesis by RNA polymerase I is regulated by premature termination of transcription” investigates the regulation of the initial steps of ribosome biogenesis – the synthesis of large ribosomal RNA precursor by RNA polymerase I.

      In our manuscript, we demonstrate for the first time that RNA Polymerase I (Pol I) can prematurely release nascent transcripts at the 5' end of ribosomal DNA transcription units in vivo. This achievement was made possible by comparing wild-type Pol I with a mutant form of Pol I, hereafter called SuperPol previously isolated in our lab (Darrière at al., 2019). By combining in vivo analysis of rRNA synthesis (using pulse-labelling of nascent transcript and cross-linking of nascent transcript - CRAC) with in vitro analysis, we could show that Superpol reduced premature transcript release due to altered elongation dynamics and reduced RNA cleavage activity. Such premature release could reflect regulatory mechanisms controlling rRNA synthesis. Importantly, This increased processivity of SuperPol is correlated with resistance with BMH-21, a novel anti-cancer drugs inhibiting Pol I, showing the relevance of targeting Pol I during transcriptional pauses to kill cancer cells. This work offers critical insights into Pol I dynamics, rRNA transcription regulation, and implications for cancer therapeutics.

      We sincerely thank the three reviewers for their insightful comments and recognition of the strengths and weaknesses of our study. Their acknowledgment of our rigorous methodology, the relevance of our findings on rRNA transcription regulation, and the significant enzymatic properties of the SuperPol mutant is highly appreciated. We are particularly grateful for their appreciation of the potential scientific impact of this work. Additionally, we value the reviewer’s suggestion that this article could address a broad scientific community, including in transcription biology and cancer therapy research. These encouraging remarks motivate us to refine and expand upon our findings further.

      All three reviewers acknowledged the increased processivity of SuperPol compared to its wild-type counterpart. However, two out of three questions our claims that premature termination of transcription can regulate ribosomal RNA transcription. This conclusion is based on SuperPol mutant increasing rRNA production. Proving that modulation of early transcription termination is used to regulate rRNA production under physiological conditions is beyond the scope of this study. Therefore, we propose to change the title of this manuscript to focus on what we have unambiguously demonstrated:

      “Ribosomal RNA synthesis by RNA polymerase I is subjected to premature termination of transcription”.

      Reviewer 1 main criticisms centers on the use of the CRAC technique in our study. While we address this point in detail below, we would like to emphasize that, although we agree with the reviewer’s comments regarding its application to Pol II studies, by limiting contamination with mature rRNA, CRAC remains the only suitable method for studying Pol I elongation over the entire transcription units. All other methods are massively contaminated with fragments of mature RNA which prevents any quantitative analysis of read distribution within rDNA. This perspective is widely accepted within the Pol I research community, as CRAC provides a robust approach to capturing transcriptional dynamics specific to Pol I activity.

      We hope that these findings will resonate with the readership of your journal and contribute significantly to advancing discussions in transcription biology and related fields.

      2. Description of the planned revisions

      Despite numerous text modification (see below), we agree that one major point of discussion is the consequence of increased processivity in SuperPol mutant on the “quality” of produced rRNA. Reviewer 3 suggested comparisons with other processive alleles, such as the rpb1-E1103G mutant of the RNAPII subunit (Malagon et al., 2006). This comparison has already been addressed by the Schneider lab (Viktorovskaya OV, Cell Rep., 2013 - PMID: 23994471), which explored Pol II (rpb1-E1103G) and Pol I (rpa190-E1224G). The rpa190-E1224G mutant revealed enhanced pausing in vitro, highlighting key differences between Pol I and Pol II catalytic rate-limiting steps (see David Schneider's review on this topic for further details).

              Reviewer 2 and 3 suggested that a decreased efficiency of cleavage upon backtracking might imply an increased error rate in SuperPol compared to the wild-type enzyme. Pol I mutant with decreased rRNA cleavage have been characterized previously, and resulted in increased error-rate. We already started to address this point. Preliminary results from *in vitro* experiments suggest that SuperPol mutants exhibit an elevated error rate during transcription. However, these findings remain preliminary and require further experimental validation to confirm their reproducibility and robustness. We propose to consolidate these data and incorporate into the manuscript to address this question comprehensively. This could provide valuable insights into the mechanistic differences between SuperPol and the wild-type enzyme. SuperPol is the first pol I mutant described with an increased processivity *in vitro* and *in vivo*, and we agree that this might be at the cost of a decreased fidelity.
      

      Regulatory aspect of the process:

      To address the reviewer’s remarks, we propose to test our model by performing experiments that would evaluate PTT levels in Pol I mutant’s or under different growth conditions. These experiments would provide crucial data to support our model, which suggests that PTT is a regulatory element of Pol I transcription. By demonstrating how PTT varies with environmental factors, we aim to strengthen the hypothesis that premature termination plays an important role in regulating Pol I activity.

      We propose revising the title and conclusions of the manuscript. The updated version will better reflect the study's focus and temper claims regarding the regulatory aspects of termination events, while maintaining the value of our proposed model.

      __ __

      3. Description of the revisions that have already been incorporated in the transferred manuscript

      Some very important modifications have now been incorporated:



      Statistical Analyses and CRAC Replicates:

      Unlike reviewers 2 and 3, reviewer 1 suggests that we did not analyze the results statistically. In fact, the CRAC analyses were conducted in biological triplicate, ensuring robustness and reproducibility. The statistical analyses are presented in Figure 2C, which highlights significant findings supporting the fact WT Pol I and SuperPol distribution profiles are different. We CRAC replicates exhibit a high correlation and we confirmed significant effect in each region of interest (5’ETS, 18S.2, 25S.1 and 3’ ETS, Figure 1) to confirm consistency across experiments. We finally took care not to overinterpret the results, maintaining a rigorous and cautious approach in our analysis to ensure accurate conclusions.

      CRAC vs. Net-seq:

      Reviewer 1 ask to comment differences between CRAC and Net-seq. Both methods complement each other but serve different purposes depending on the biological question on the context of transcription analysis. Net-seq has originally been designed for Pol II analysis. It captures nascent RNAs but does not eliminate mature ribosomal RNAs (rRNAs), leading to high levels of contamination. While this is manageable for Pol II analysis (in silico elimination of reads corresponding to rRNAs), it poses a significant problem for Pol I due to the dominance of rRNAs (60% of total RNAs in yeast), which share sequences with nascent Pol I transcripts. As a result, large Net-seq peaks are observed at mature rRNA extremities (Clarke 2018, Jacobs 2022). This limits the interpretation of the results to the short lived pre-rRNA species. In contrast, CRAC has been specifically adapted by the laboratory of David Tollervey to map Pol I distribution while minimizing contamination from mature rRNAs (The CRAC protocol used exclusively recovers RNAs with 3′ hydroxyl groups that represent endogenous 3′ ends of nascent transcripts, thus removing RNAs with 3’-Phosphate, found in mature rRNAs). This makes CRAC more suitable for studying Pol I transcription, including polymerase pausing and distribution along rDNA, providing quantitative dataset for the entire rDNA gene.

      CRAC vs. Other Methods:

      Reviewer 1 suggests using GRO-seq or TT-seq, but the experiments in Figure 2 aim to assess the distribution profile of Pol I along the rDNA, which requires a method optimized for this specific purpose. While GRO-seq and TT-seq are excellent for measuring RNA synthesis and co-transcriptional processing, they rely on Sarkosyl treatment to permeabilize cellular and nuclear membranes. Sarkosyl is known to artificially induces polymerase pausing and inhibits RNase activities which are involved in the process. To avoid these artifacts, CRAC analysis is a direct and fully in vivo approach. In CRAC experiment, cells are grown exponentially in rich media and arrested via rapid cross-linking, providing precise and artifact-free data on Pol I activity and pausing.

      Pol I ChIP Signal Comparison:

      The ChIP experiments previously published in Darrière et al. lack the statistical depth and resolution offered by our CRAC analyses. The detailed results obtained through CRAC would have been impossible to detect using classical ChIP. The current study provides a more refined and precise understanding of Pol I distribution and dynamics, highlighting the advantages of CRAC over traditional methods in addressing these complex transcriptional processes.

      BMH-21 Effects:

      As highlighted by Reviewer 1, the effects of BMH-21 observed in our study differ slightly from those reported in earlier work (Ref Schneider 2022), likely due to variations in experimental conditions, such as methodologies (CRAC vs. Net-seq), as discussed earlier. We also identified variations in the response to BMH-21 treatment associated with differences in cell growth phases and/or cell density. These factors likely contribute to the observed discrepancies, offering a potential explanation for the variations between our findings and those reported in previous studies. In our approach, we prioritized reproducibility by carefully controlling BMH-21 experimental conditions to mitigate these factors. These variables can significantly influence results, potentially leading to subtle discrepancies. Nevertheless, the overall conclusions regarding BMH-21's effects on WT Pol I are largely consistent across studies, with differences primarily observed at the nucleotide resolution. This is a strength of our CRAC-based analysis, which provides precise insights into Pol I activity.

      We will address these nuances in the revised manuscript to clarify how such differences may impact results and provide context for interpreting our findings in light of previous studies.

      Minor points:

      Reviewer #1:

      • In general, the writing style is not clear, and there are some word mistakes or poor descriptions of the results, for example: On page 14: "SuperPol accumulation is decreased (compared to Pol I)". • *On page 16: "Compared to WT Pol I, the cumulative distribution of SuperPol is indeed shifted on the right of the graph." *

      We clarified and increased the global writing style according to reviewer comment.

      • *There are also issues with the literature, for example: Turowski et al, 2020a and Turowski et al, 2020b are the same article (preprint and peer-reviewed). Is there any reason to include both references? Please, double-check the references. *

      This was corrected in this version of the manuscript.

      • *In the manuscript, 5S rRNA is mentioned as an internal control for TMA normalisation. Why are Figure 1C data normalised to 18S rRNA instead of 5S rRNA? *

      Data are effectively normalized relative to the 5S rRNA, but the value for the 18S rRNA is arbitrarily set to 100%.

      • Figure 4 should be a supplementary figure, and Figure 7D doesn't have a y-axis labelling.

      The presence of all Pol I specific subunits (Rpa12, Rpa34 and Rpa49) is crucial for the enzymatic activity we performed. In the absence of these subunits (which can vary depending on the purification batch), Pol I pausing, cleavage and elongation are known to be affected. To strengthen our conclusion, we really wanted to show the subunit composition of the purified enzyme. This important control should be shown, but can indeed be shown in a supplementary figure if desired.

      Y-axis is figure 7D is now correctly labelled

      • *In Figure 7C, BMH-21 treatment causes the accumulation of ~140bp rRNA transcripts only in SuperPol-expressing cells that are Rrp6-sensitive (line 6 vs line 8), suggesting that BHM-21 treatment does affect SuperPol. Could the author comment on the interpretation of this result? *

      The 140 nt product is a degradation fragment resulting from trimming, which explains its lower accumulation in the absence of Rrp6. BMH21 significantly affects WT Pol I transcription but has also a mild effect on SuperPol transcription. As a result, the 140 nt product accumulates under these conditions.

      Reviewer #2:

      • *pp. 14-15: The authors note local differences in peak detection in the 5'-ETS among replicates, preventing a nucleotide-resolution analysis of pausing sites. Still, they report consistent global differences between wild-type and SuperPol CRAC signals in the 5'ETS (and other regions of the rDNA). These global differences are clear in the quantification shown in Figures 2B-C. A simpler statement might be less confusing, avoiding references to a "first and second set of replicates" *

      According to reviewer, statement has been simplified in this version of the manuscript.


      • *Figures 2A and 2C: Based on these data and quantification, it appears that SuperPol signals in the body and 3' end of the rDNA unit are higher than those in the wild type. This finding supports the conclusion that reduced pausing (and termination) in the 5'ETS leads to an increased Pol I signal downstream. Since the average increase in the SuperPol signal is distributed over a larger region, this might also explain why even a relatively modest decrease in 5'ETS pausing results in higher rRNA production. This point merits discussion by the authors. *

      We agree that this is a very important discussion of our results. Transcription is a very dynamic process in which paused polymerase is easily detected using the CRAC assay. Elongated polymerases are distributed over a much larger gene body, and even a small amount of polymerase detected in the gene body can represent a very large rRNA synthesis. This point is of paramount importance and, as suggested by the reviewer, is now discussed in detail.


      • *A decreased efficiency of cleavage upon backtracking might imply an increased error rate in SuperPol compared to the wild-type enzyme. Have the authors observed any evidence supporting this possibility? *

      Reviewer suggested that a decreased efficiency of cleavage upon backtracking might imply an increased error rate in SuperPol compared to the wild-type enzyme. We already started to address this point. Preliminary results from in vitro experiments suggest that SuperPol mutants exhibit an elevated error rate during transcription. However, these findings remain preliminary and require further experimental validation to confirm their reproducibility and robustness. We propose to consolidate these data and incorporate into the manuscript to address this question comprehensively.


      • *pp. 15 and 22: Premature transcription termination as a regulator of gene expression is well-documented in yeast, with significant contributions from the Corden, Brow, Libri, and Tollervey labs. These studies should be referenced along with relevant bacterial and mammalian research. *

      According to reviewer suggestion, we referenced these studies.


      • *p. 23: "SuperPol and Rpa190-KR have a synergistic effect on BMH-21 resistance." A citation should be added for this statement. *

      This represents some unpublished data from our lab. KR and SuperPol are the only two known mutants resistant to BMH-21. We observed that resistance between both alleles is synergistic, with a much higher resistance to BMH-21 in the double mutant than in each single mutant (data not shown). Comparing their resistance mechanisms is a very important point that we could provide upon request. This was added to the statement.


      • *p. 23: "The released of the premature transcript" - this phrase contains a typo *

      This is now corrected.


      Reviewer #3:

      • *Figure 1B: it would be opportune to separate the technique's schematic representation from the actual data. Concerning the data, would the authors consider adding an experiment with rrp6D cells? Some RNAs could be degraded even in such short period of time, as even stated by the authors, so maybe an exosome depleted background could provide a more complete picture. Could also the authors explain why the increase is only observed at the level of 18S and 25S? To further prove the robustness of the Pol I TMA method could be good to add already characterized mutations or other drugs to show that the technique can readily detect also well-known and expected changes. *

      The precise objective of this experiment is to avoid the use of the Rrp6 mutant. Under these conditions, we prevent the accumulation of transcripts that would result from a maturation defect. While it is possible to conduct the experiment with the Rrp6 mutant, it would be impossible to draw reliable conclusions due to this artificial accumulation of transcripts.


      • *Figure 1C: the NTS1 probe signal is missing (it is referenced in Figure 1A but not listed in the Methods section or the oligo table). If this probe was unused, please correct Figure 1A accordingly. *

      __We corrected Figure 1A. __


      • *Figure 2A: the RNAPI occupancy map by CRAC is hard to interpret. The red color (SuperPol) is stacked on top of the blue line, and we are not able to observe the signal of the WT for most of the position along the rDNA unit. It would be preferable to use some kind of opacity that allows to visualize both curves. Moreover, the analysis of the behavior of the polymerase is always restricted to the 5'ETS region in the rest of the manuscript. We are thus not able to observe whether termination events also occur in other regions of the rDNA unit. A Northern blot analysis displaying higher sizes would provide a more complete picture. *

      We addressed this point to make the figure more visually informative. In Northern Blot analysis, we use a TSS (Transcription Start Site) probe, which detects only transcripts containing the 5' extremity. Due to co-transcriptional processing, most of the rRNA undergoing transcription lacks its 5' extremity and is not detectable using this technique. We have the data, but it does not show any difference between Pol I and SuperPol. This information could be included in the supplementary data if asked.


      • *"Importantly, despite some local variations, we could reproducibly observe an increased occupancy of WT Pol I in 5'-ETS compared to SuperPol (Figure 1C)." should be Figure 2C. *

      Thanks for pointing out this mistake. it has been corrected.


      • *Figure 3D: most of the difference in the cumulative proportion of CRAC reads is observed in the region ~750 to 3000. In line with my previous point, I think it would be worth exploring also termination events beyond the 5'-ETS region. *

      We agree that such an analysis would have been interesting. However, with the exception of the pre-rRNA starting at the transcription start site (TSS) studied here, any cleaved rRNA at its 5' end could result from premature termination and/or abnormal processing events. Exploring the production of other abnormal rRNAs produced by premature termination is a project in itself, beyond this initial work aimed at demonstrating the existence of premature termination events in ribosomal RNA production.


      • *Figure 4: should probably be provided as supplementary material. *

      As lmentioned earlier (see comments), ____the presence of all Pol I specific subunits (Rpa12, Rpa34 and Rpa49) is crucial for the enzymatic activity we performed. This important control should be shown, but can indeed be shown in a supplementary figure if desired.


      • *"While the growth of cells expressing SuperPol appeared unaffected, the fitness of WT cells was severely reduced under the same conditions." I think the growth of cells expressing SuperPol is slightly affected. *

      We agree with this comment and we modified the text accordingly.


      • *Figure 7D: the legend of the y-axis is missing as well as the title of the plot. *

      Legend of the y-axis and title of the plot are now present.


      • The statements concerning BMH-21, SuperPol and Rpa190-KR in the Discussion section should be removed, or data should be provided.

      This was discussed previously. See comment above.


      • *Some references are missing from the Bibliography, for example Merkl et al., 2020; Pilsl et al., 2016a, 2016b. *

      Bibliography is now fixed

      __ __

      4. Description of analyses that authors prefer not to carry out

      Does SuperPol mutant produces more functional rRNAs ?

      As Reviewer 1 requested, we agree that this point requires clarification. In cells expressing SuperPol, a higher steady state of (pre)-rRNAs is only observed in absence of degradation machinery suggesting that overproduced rRNAs are rapidly eliminated. We know that (pre)-rRNas are unable to accumulate in absence of ribosomal proteins and/or Assembly Factors (AF). In consequence, overproducing rRNAs would not be sufficient to increase ribosome content. This specific point is further address in our lab but is beyond the scope of this article.

      __Is premature termination coupled with rRNA processing __

      We appreciate the reviewer’s insightful comments. The suggested experiments regarding the UTP-A complex's regulatory potential are valuable and ongoing in our lab, but they extend beyond the scope of this study and are not suitable for inclusion in the current manuscript.

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      Referee #3

      Evidence, reproducibility and clarity

      In the manuscript "Ribosomal RNA synthesis by RNA polymerase I is regulated by premature termination of transcription", Azouzi and co-authors investigate the regulatory mechanisms of ribosomal RNA (rRNA) transcription by RNA Polymerase I (RNAPI) in the budding yeast S. cerevisiae. They follow up on exploring the molecular basis of a mutant allele of the second largest subunit of RNAPI, RPA135-F301S, also dubbed SuperPol, that they had previously reported (Darrière et al, 2019), and which was shown to rescue Rpa49-linked growth defects, possibly by increasing rRNA production.

      Through a combination of genomic and in vitro approaches, the authors test the hypothesis that RNAPI activity could be subjected to a Premature Transcription Termination (PPT) mechanism, akin to what is observed for RNA Polymerase II (RNAPII), and which is suggested to be an important step for the quality control of rRNA transcripts. SuperPol is proposed to lack such a regulatory mechanism, due to an increased processivity. In agreement, SuperPol is shown to be resistant to BMH-21, a drug previously shown to impair RNAPI elongation.

      Overall, the experiments are performed with rigor and include the appropriate controls and statistical analysis. Both the figures and the text present the data clearly. The Material and Methods section is detailed enough. The reported results are interesting; however, I am not fully convinced of the existence of PPT of RNAPI, and even less of its utmost importance. The existence of PPT of RNAPI would entail an intended regulatory mechanism. The authors propose that PPT could serve as quality control step for the UTP-A complex loading on the rRNA 5'-end. While this hypothesis is enticing and cautiously phrased by the authors, the lack of evidence showing a specific regulatory function (such as UTP-A loading checkpoint or else) limits these termination events to possibly abortive actions of unclear significance. The auhors may want to consider comparisons to other processive alleles, such as the rpb1-E1103G mutant of the RNAPII subunit (Malagon et al, 2006) or the G1136S allele of E. coli RNAP (Bar-Nahum et al., 2005). While clearly mechanistically distinct, these mutations result in similarly processive enzymes that achieve more robust transcription, possibly at the cost of decreased fidelity. Indeed, an alternative possibility explaining these transcripts could be that they originate from unsuccessful resumption of transcription after misincorporation (see below).

      I suggest reconsidering the study's main conclusions by limiting claims about the regulatory function of these termination events (the title of the manuscript should be changed accordingly). Alternatively, the authors should provide additional investigation on their regulatory potential, for example by assessing if indeed this quality control is linked to the correct assembly of the UTP-A complex. The expectation would be that SuperPol should rescue at least to some extent the defects observed in the absence of UTP-A components. Moreover, the results using the clv3 substrate suggest the possibility that SuperPol might simply be more able to tolerate mismatches, thus be more processive in transcribing, because not subjected to proof-reading mechanisms, similarly to what observed in Schwank et al., 2022. This could explain many of the observations, and I think it is worth exploring by assessing the fidelity of the enzyme, especially in the frame of suggesting a regulatory function for these termination events.

      Minor comments

      1. Figure 1B: it would be opportune to separate the technique's schematic representation from the actual data. Concerning the data, would the authors consider adding an experiment with rrp6D cells? Some RNAs could be degraded even in such short period of time, as even stated by the authors, so maybe an exosome depleted background could provide a more complete picture. Could also the authors explain why the increase is only observed at the level of 18S and 25S? To further prove the robustness of the Pol I TMA method could be good to add already characterized mutations or other drugs to show that the technique can readily detect also well-known and expected changes.
      2. Figure 1C: the NTS1 probe signal is missing (it is referenced in Figure 1A but not listed in the Methods section or the oligo table). If this probe was unused, please correct Figure 1A accordingly.
      3. Figure 2A: the RNAPI occupancy map by CRAC is hard to interpret. The red color (SuperPol) is stacked on top of the blue line, and we are not able to observe the signal of the WT for most of the position along the rDNA unit. It would be preferable to use some kind of opacity that allows to visualize both curves. Moreover, the analysis of the behavior of the polymerase is always restricted to the 5'ETS region in the rest of the manuscript. We are thus not able to observe whether termination events also occur in other regions of the rDNA unit. A Northern blot analysis displaying higher sizes would provide a more complete picture.
      4. "Importantly, despite some local variations, we could reproducibly observe an increased occupancy of WT Pol I in 5'-ETS compared to SuperPol (Figure 1C)." should be Figure 2C.
      5. Figure 3D: most of the difference in the cumulative proportion of CRAC reads is observed in the region ~750 to 3000. In line with my previous point, I think it would be worth exploring also termination events beyond the 5'-ETS region.
      6. Figure 4: should probably be provided as supplementary material.
      7. "While the growth of cells expressing SuperPol appeared unaffected, the fitness of WT cells was severely reduced under the same conditions." I think the growth of cells expressing SuperPol is slightly affected.
      8. Figure 6B: can the authors explain why most of bands detected in their Pol I TMA assay in Figure 6B are unchanged? It is unclear to me why only the 18S and 25S bands are decreased following BMH-21 treatment. Moreover, this experiment lacks the corresponding quantification and statistical tests.
      9. Figure 7D: the legend of the y-axis is missing as well as the title of the plot.
      10. The statements concerning BMH-21, SuperPol and Rpa190-KR in the Discussion section should be removed, or data should be provided.
      11. Some references are missing from the Bibliography, for example Merkl et al., 2020; Pilsl et al., 2016a, 2016b.

      Significance

      Azouzi and co-authors' work builds on their previous study (Darrière et al, 2019) of RPA135-F301S (SuperPol), a mutant allele of the second largest RNAPI subunit, which was shown to compensate for Rpa49 loss, potentially by increasing rRNA production. The work advances the mechanistic understanding of the the SuperPol allele, demonstrating the increased processivity of this enzyme compared to its wild-type counterpart. Such increased processivity "desensitizes" RNAPI from abortive transcription cycles, the existence of which is clearly shown, though the biological significance of this phenomenon remains unclear. The lack of evidence for a regulatory mechanism behind these early termination events is, in my opinion, a limitation of this study, as it does not allow for differentiation between an intended regulatory process and a byproduct of an imperfect system.

      This work is of interest for researchers studying transcription regulation, particularly those interested in understanding RNAPI's role and fidelity. Demonstrating PPT as a regulatory quality control for RNAPI could point to common strategies in between RNAPI and RNAPII regulation, where premature termination has been extensively documented. However, without evidence of a specific regulatory function, these findings may currently be limited to descriptive insights.

      My expertise lies is RNAPII transcription, transcription termination, and genomic approaches to studying transcription.

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      Referee #1

      Evidence, reproducibility and clarity

      The study characterises an RNA polymerase (Pol) I mutant (RPA135-F301S) named SuperPol. This mutant was previously shown to increase yeast ribosomal RNA (rRNA) production by Transcription Run-On (TRO). In this work, the authors confirm this mutation increases rRNA transcription using a slight variation of the TRO method, Transcriptional Monitoring Assay (TMA), which also allows the analysis of partially degraded RNA molecules. The authors show a reduction of abortive rRNA transcription in cells expressing the SuperPol mutant and a modest occupancy decrease at the 5' region of the rRNA genes compared to WT Pol I. These results suggest that the SuperPol mutant displays a lower frequency of premature termination. Using in vitro assays, the authors found that the mutation induces an enhanced elongation speed and a lower cleavage activity on mismatched nucleotides at the 3' end of the RNA. Finally, SuperPol mutant was found to be less sensitive to BMH-21, a DNA intercalating agent that blocks Pol I transcription and triggers the degradation of the Pol I subunit, Rpa190. Compared to WT Pol I, short BMH-21 treatment has little effect on SuperPol transcription activity, and consequently, SuperPol mutation decreases cell sensitivity to BMH-21.

      I'd suggest the following points to be taken into consideration:

      Major points:

      1. The differences in the transcriptionally engaged WT Pol I and SuperPol profiles (Figure 2) are very modest, without any statistical analyses. What is the correlation between CRAC replicates? Are they separated in PCA analyses? Please, include more quality control information. In my opinion, these results are not very convincing. Similarly, the effect of BMH-21 on WT Pol I activity (Figure 7) is also very subtle and doesn't match the effect observed in a previous study [1]. Could the author comment on the reasons for these differences? These discrepancies raise concerns about the methodology. In addition, according to the laboratory's previous work [2], Pol I ChIP signal at rDNA is not significantly different in cells expressing WT Pol I and SuperPol. How can these two observations be reconciled? I would suggest using an independent methodology to analyse Pol I transcription, for example, GRO-seq or TT-seq.
      2. While the experiments clearly show SuperPol mutant increases nascent transcription and decreases the production of abortive promoter-proximal transcripts compared to WT Pol I. RPA135-F301S mutation has a minor impact on total rRNA levels, at least those shown in Figure 3B. Are steady-state rRNA levels higher in cells expressing SuperPol mutant? It would be interesting to know if SuperPol mutant produces more functional rRNAs.

      Minor points

      1. In general, the writing style is not clear, and there are some word mistakes or poor descriptions of the results, for example:<br /> On page 14: "SuperPol accumulation is decreased (compared to Pol I)". On page 16: "Compared to WT Pol I, the cumulative distribution of SuperPol is indeed shifted on the right of the graph."
      2. There are also issues with the literature, for example: Turowski et al, 2020a and Turowski et al, 2020b are the same article (preprint and peer-reviewed). Is there any reason to include both references? Please, double-check the references.
      3. In the manuscript, 5S rRNA is mentioned as an internal control for TMA normalisation. Why are Figure 1C data normalised to 18S rRNA instead of 5S rRNA?
      4. Figure 4 should be a supplementary figure, and Figure 7D doesn't have a y-axis labelling.
      5. In Figure 7C, BMH-21 treatment causes the accumulation of ~140bp rRNA transcripts only in SuperPol-expressing cells that are Rrp6-sensitive (line 6 vs line 8), suggesting that BHM-21 treatment does affect SuperPol. Could the author comment on the interpretation of this result?

      References

      1. Jacobs RQ, Huffines AK, Laiho M & Schneider DA (2022) The small-molecule BMH-21 directly inhibits transcription elongation and DNA occupancy of RNA polymerase I in vivo and in vitro. J. Biol. Chem. 298: 101450
      2. Darrière T, Pilsl M, Sarthou M-K, Chauvier A, Genty T, Audibert S, Dez C, Léger-Silvestre I, Normand C, Henras AK, Kwapisz M, Calvo O, Fernández-Tornero C, Tschochner H & Gadal O (2019) Genetic analyses led to the discovery of a super-active mutant of the RNA polymerase I. PLoS Genet. 15: e1008157

      Significance

      The work further characterises a single amino acid mutation of one of the largest yeast Pol I subunits (RPA135-F301S). While this mutation was previously shown to increase rRNA synthesis, the current work expands the SuperPol mutant characterisation, providing details of how RPA135-F301S modifies the enzymatic properties of yeast Pol I. In addition, their findings suggest that yeast Pol I transcription can be subjected to premature termination in vivo. The molecular basis and potential regulatory functions of this phenomenon could be explored in additional studies.

      Our understanding of rRNA transcription is limited, and the findings of this work may be interesting to the transcription community. Moreover, targeting Pol I activity is an open strategy for cancer treatment. Thus, the resistance of SuperPol mutant to BMH-21 might also be of interest to a broader community, although these findings are yet to be confirmed in human Pol I and with more specific Pol I inhibitors in future.

      My expertise is human Pol II and Pol III transcription regulation.

    1. Background In recent years, Large Language Models (LLMs) have shown promise in various domains, notably in biomedical sciences. However, their real-world application is often limited by issues like erroneous outputs and hallucinatory responses.Results We developed the Knowledge Graph-based Thought (KGT) framework, an innovative solution that integrates LLMs with Knowledge Graphs (KGs) to improve their initial responses by utilizing verifiable information from KGs, thus significantly reducing factual errors in reasoning. The KGT framework demonstrates strong adaptability and performs well across various open-source LLMs. Notably, KGT can facilitate the discovery of new uses for existing drugs through potential drug-cancer associations, and can assist in predicting resistance by analyzing relevant biomarkers and genetic mechanisms. To evaluate the Knowledge Graph Question Answering (KGQA) task within biomedicine, we utilize a pan-cancer knowledge graph to develop a pan-cancer question answering benchmark, named the Pan-cancer Question Answering (PcQA).Conclusions The KGT framework substantially improves the accuracy and utility of LLMs in the biomedical field. This study serves as a proof-of-concept, demonstrating its exceptional performance in biomedical question answering.

      This work has been peer reviewed in GigaScience (see , https://doi.org/10.1093/gigascience/giae082), which carries out open, named peer-review. These reviews are published under a CC-BY 4.0 license and were as follows:

      Reviewer: Linhao Luo

      Summary: This paper proposes a novel framework called KGT that integrates Large Language Models (LLMs) with Knowledge Graphs (KGs) for pan-cancer question answering. The KGT framework can effectively retrieve knowledge from KGs and improve the accuracy of LLMs for question answering. Moreover, it can provide interpretable and faithful explanations with the help of structured KGs. Comments: 1. This paper construct a new dataset denoted as PcQA form a customized KG called SOKG for the evaluation of pan-cancer question answering. This is a great contribution to the community. However, it is unclear how to constuct such a dataset. More details about the construnction process and statistics of the final datasets should be disscussed in the paper. For example, how to generate the natural language questions and answers? How to link the question with relatived KG information (i.e., entity and relation)? How many questions can be answered by the KGs (i.e., answer converage rate). How many questions have been generated? What is the ratio of each quetion types defined in Table 2? 2. In Table2, the author define 4 reasoning types. How about other reasoning types such union and negation? Can we incorpate these tpes into the datasets? 3. The propsed method is novel and interesting. However some details are unclear. In the candidate path search, do we want to search reasoning paths or relational chains? The definition of these two paths are also unclear. Please give clear definition of them in prelimary. If is the reasoning paths, do we only keep the type information during the BFS? 4. I do not understand why we need to generatea cypher query to retrieve subgraph then construct relation paths from KG. We can directly retrieved relational paths from KGs by BFS. What are the benefits and motivations of using this two-stage pipeline? 5. What are the meanings of the X and √ in the figure. How to get them? 6. In experiments, other advanced KGQA methods can be compared, e.g., RoG [1] and ToG [2]. 7. The analysis of used token, time, and cost should be disscussed in the paper. 8. Can we apply the proposed metod to other KGs (i.e., SynLethKG, and SDKG) or KGQA tasks (MetaQA, and FACTKG) to show the generability. [1] LUO, L., Li, Y. F., Haf, R., & Pan, S. Reasoning on Graphs: Faithful and Interpretable Large Language Model Reasoning. In The Twelfth International Conference on Learning Representations. [2] Sun, J., Xu, C., Tang, L., Wang, S., Lin, C., Gong, Y., ... & Guo, J. (2023). Think-on-graph: Deep and responsible reasoning of large language model with knowledge graph. arXiv preprint arXiv:2307.07697

    1. Author response:

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

      Public Reviews:

      Reviewer #1 (Public Review):

      Summary:

      In this work, Huang et al used SMRT sequencing to identify methylated nucleotides (6mA, 4mC, and 5mC) in Pseudomonas syringae genome. They show that the most abundant modification is 6mA and they identify the enzymes required for this modification as when they mutate HsdMSR they observe a decrease of 6mA. Interestingly, the mutant also displays phenotypes of change in pathogenicity, biofilm formation, and translation activity due to a change in gene expression likely linked to the loss of 6mA. Overall, the paper represents an interesting set of new data that can bring forward the field of DNA modification in bacteria.

      Thank you for your valuable feedback on our paper exploring the impact of 6mA modification in P. syringae.

      Major Concerns:

      Most of the authors' data concern Psph pathovar. I am not sure that the authors' conclusions are supported by the two other pathovars they used in the initial 2 figures. If the authors want to broaden their conclusions to Pseudomonas syringe and not restrict it to Psph, the authors should have stronger methylation data using replicates. Additionally, they should discuss why Pss is so different than Pst and Psph. Could they do a blot to confirm it is really the case and not a sequencing artifact? Is the change of methylation during bacterial growth conserved between the pathovar? The authors should obtain mutants in the other pathovar to see if they have the same phenotype. The authors have a nice set of data concerning Psph but the broadening of the results to other pathovar requires further investigation.

      We appreciate the reviewer’s insightful comments. While the majority of our data focuses on the Psph, we recognize the importance of validating these findings in Pss and Pst. To this end, we have performed additional experiments using dot blot and mutant construction to enhance our conclusions in other pathovars.

      We agree that we should discuss why Pss is different from Psph and Pst. We performed a dot blot assay using genome DNA in Pss and Pst, presented in Figure S5A. Meanwhile, we compared the 6mA modification level of Pss and Pst in different growth phases. As shown in Figure S5A, the change of methylation during bacterial growth is conserved in Pst. The change was not obvious in Pss, which might be due to the lack of a type I R-M system.

      “In accordance with previous studies showing that growth conditions affect the bacterial methylation status, we applied dot blot experiments using the same amount of DNA (1 μg) from these three P. syringae strains to detect the 6mA levels during both logarithmic and stationary phases. The results revealed that 6mA levels in the stationary phase were much higher compared to the logarithmic phase in Psph and Pst, but no significant change in Pss. Additionally, we found that during the stationary phase, 6mA methylation levels in Psph and Pst were higher than those in Pss. These findings were consistent with the MTases predication on these three strains, since Pss does not harbor any type I R-M systems, which are important for 6mA medication in bacteria.”

      Please see Figure S5A and Lines 220-228 in the revised manuscript.

      We also tried to construct an HsdM mutant in Pst to explore whether the influence of 6mA methylation was conserved in P. syringae, but it failed after multiple attempts. We did not construct a Pss mutant because no type I R-M system was predicted, and few methylation sites were identified via SMRT-seq in this strain. Therefore, we overexpressed HsdM in Pst instead. We have performed additional experiments in WT and the HsdM overexpression strains, including dot blot and growth curve assays.

      Please see Figures S5B-C and Lines228-232 in the revised manuscript.

      The authors should include proper statistical analysis of their data. A lot of terms are descriptive but not supported by a deeper analysis to sustain the conclusions. For example, in Figure 4E, we do not know if the overlap is significant or not. Are DEGs more overlapping to 6mA sites than non-DEGs? Here is a non-exhaustive list of terms that need to be supported by statistics: different level (L145), greater conservation (L162), significant conservation (L165), considerable similarity (L175), credible motifs (L189), Less strong (L277) and several "lower" and "higher" throughout the text.

      Thank you for the insightful feedback. We have made the following revisions in the manuscript to ensure that the terms are more precise and do not require statistical significance testing.

      (1) Statistical analysis: We have added statistical tests for the overlap between DEGs and 6mA sites in Figure 4E. We performed the Fisher test, and we found the overlap was not significant (p> 0.05). DEGs and non-DEGs were both non-significant overlapped 6mA sites. Please see Figure 4E and Lines 261-262.

      “Less strong” was used to indicate the influence of HsdM on biofilm in Figure 5D. All Figures with “*” labels were analyzed using students' two-tailed t-tests with a significant change (p < 0.05).

      (2) Revised wording: For terms used to describe comparisons, we have revised the wording to be clearer and ensure that the terminology used did not imply the need for statistical significance testing when not required. For example:

      “Different level” has been removed. Please see Lines 143-144.

      “Greater conservation” has been revised to “more conserved functional terms”. Please see Lines 161-162.

      “Significant conservation” has been revised to “notable conservation”. Please see Line 165.

      “Credible motifs” has been revised to “identified motifs”. Please see Line 186.

      The authors performed SMRT sequencing of the delta hsdMSR showing a reduction of 6mA. Could they include a description of their results similar to Figures 1-2. How reduced is the 6mA level? Is it everywhere in the genome? Does it affect other methylation marks? This analysis would strengthen their conclusions.

      Yes, we agree. We have provided additional analysis and descriptions to strengthen the conclusions regarding these valuable comments. We determined three methylation sites in the HsdMSR mutant strain and compared the overlapped genes within these modification patterns. Specifically, we focused on the 6mA sites in Psph WT, HsdMSR mutant, and HsdM motif CAGCN<sub>(6)</sub>CTC. As expected, we found almost all of the reduction 6mA sites in the ΔhsdMSR were from motif CAGCN<sub>(6)</sub>CTC. We also noticed that 5mC and 4mC sites in the mutant were relatively similar to that in WT, and the slight difference might be caused by sequencing errors. Overall, we propose that HsdMSR only catalyze the 6mA located on the motif CAGCN<sub>(6)</sub>CTC, but does not affect other 6mA sites and other modification types.

      Please see Figures S4D-E and Lines 212-218 in the revised manuscript.

      In Figure 6E to conclude that methylation is required on both strands, the authors are missing the control CAGCN6CGC construct otherwise the effect could be linked to the A on the complementary strand.

      Thank you for your valuable suggestions. We have provided the control result on the complementary strand. Please see Figure 6C. The new result evidences the conclusion that 6mA methylation regulates gene transcription based on methylation on both strands.

      Please see Figure 6C and Lines 329-330 in the revised manuscript.

      Reviewer #2 (Public Review):

      In the present manuscript, Huang et.al. revealed the significant roles of the DNA methylome in regulating virulence and metabolism within Pseudomonas syringae, with a particular focus on the HsdMSR system in this model strain. The authors used SMRT-seq to profile the DNA methylation patterns (6mA, 5mC, and 4mC) in three P. syringae strains (Psph, Pss, and Psa) and displayed the conservation among them. They further identified the type I restriction-modification system (HsdMSR) in P. syringae, including its specific motif sequence. The HsdMAR participated in the process of metabolism and virulence (T3SS & Biofilm formation), as demonstrated through RNA-seq analyses. Additionally, the authors revealed the mechanisms of the transcriptional regulation by 6mA. Strictly from the point of view of the interest of the question and the work carried out, this is a worthy and timely study that uses third-generation sequencing technology to characterize the DNA methylation in P. syringae. The experimental approaches were solid, and the results obtained were interesting and provided new information on how epigenetics influences the transcription in P. syringae. The conclusions of this paper are mostly well supported by data, but some aspects of data analysis and discussion need to be clarified and extended.

      Thank you for your positive feedback and recognition of the importance of our study. We appreciate the suggestions for further clarification and extension of some aspects of data analysis and discussion. We added further analysis of the SMRT-seq result of the ΔhsdMSR and overexpressed HsdM in Pst to provide more information on conservation. We added these contents to the discussion in the revised manuscript. Please see Figure 6C and  Figure S5.

      Reviewer #3 (Public Review):

      Summary:

      The article by Huang et.al. presents an in-depth study on the role of DNA methylation in regulating virulence and metabolism in Pseudomonas syringae, a model phytopathogenic bacterium. This comprehensive research utilized single-molecule real-time (SMRT) sequencing to profile the DNA methylation landscape across three model pathovars of P. syringae, identifying significant epigenetic mechanisms through the Type-I restriction-modification system (HsdMSR), which includes a conserved sequence motif associated with N6-methyladenine (6mA). The study provides novel insights into the epigenetic mechanisms of P. syringae, expanding the understanding of bacterial pathogenicity and adaptation. The use of SMRT sequencing for methylome profiling, coupled with transcriptomic analysis and in vivo validation, establishes a robust evidence base for the findings

      Strengths:

      The results are presented clearly, with well-organized figures and tables that effectively illustrate the study's findings.

      Weaknesses:

      It would be helpful to add more details, especially in the methods, which make it easy to evaluate and enhance the manuscript's reproducibility.

      Thank you for the positive evaluation of our study, as well as the constructive feedback provided. We have added more details in methods for RNA-seq analysis and Ribo-seq analysis. Please see Lines 484-515.

      “Briefly, bacteria were cultured to an OD<sub>600</sub> of 0.4, at which point chloramphenicol was added to a final concentration of 100 µg/mL for 2 minutes. Cells were then pelleted and washed with pre-chilled lysis buffer [25 mM Tris-HCl, pH 8.0; 25 mM NH4Cl; 10 mM MgOAc; 0.8% Triton X-100; 100 U/mL RNase-free DNase I; 0.3 U/mL Superase-In; 1.55 mM chloramphenicol; and 17 mM GMPPNP]. The pellet was resuspended in lysis buffer, followed by three freeze-thaw cycles using liquid nitrogen. Sodium deoxycholate was then added to a final concentration of 0.3% before centrifugation. The resulting supernatant was adjusted to 25 A260 units and mixed with 2 mL of 500 mM CaCl<sub>2</sub> and 12 µL MNase, making up a total volume of 200 µL. After the digestion, the reaction was quenched with 2.5 mL of 500 mM EGTA. Monosomes were isolated using Sephacryl S400 MicroSpin columns, and RNA was purified using the miRNeasy Mini Kit (Qiagen). rRNA was removed using the NEBNext rRNA Depletion Kit, and the final library was constructed with the NEBNext Small RNA Library Prep Kit. For each sample, ribosome footprint reads were mapped to the Psph 1448A reference genome, and the translational efficiency was calculated by dividing the normalized Ribo-seq counts by the normalized RNA counts. Two biological replicates were performed for all experiments.”

      Recommendations For The Authors:

      Reviewer #1 (Recommendations For The Authors):

      I would recommend the authors limit their manuscript to Psph pathovar and include statistical analysis supporting their conclusions.

      Thank you for your suggestion.

      Minor

      • L104: "significantly" please add a p-value and explain the analysis.

      Sorry for the confusion. We have added the p-value and explained the analysis in the method section. The p-value used for SMRT-seq was the modification quality value (QV) score, which were used to call the modified bases A (QV=50) and C (QV=100). Please see Lines 452-454.

      • Figures 1B, D, F, and Figure 2A: make the Venn diagram to scale

      Yes, we have revised.

      • L110-111: missing p-value to say that the authors observe a bigger overlap in Pst than Psph as they observed more modified sites in Pst

      Sorry for the confusion. We said it had a bigger overlap in Pst because the number 17.7 was bigger than the number of 15 in Psph. To avoid misunderstanding, we revised the wording to “more genes equipped with all three modification types were detected in Pst than Psph”. Please see Lines 110-111.

      • L112: missing description of their Pss analysis (IDP, sites...)

      We have added the information for Pss in the revised manuscript.

      “Additionally, the methylome atlas of Pss revealed a lower incidence of methylation than those of Psph and Pst, particularly in terms of 6mA modifications, which were only seen in 457 significant 6mA occurrences under the same threshold (IPD > 1.5) and a total of 2,853 and 1,438 methylation sites were detected as 5mC and 4mC, respectively”. Please see Lines 114-116.

      • L118: "modification" to "modified "

      We have revised. Please see Line 119.

      • L120: "modification sites" to "modified nucleotides"

      We have revised. Please see Line 121.

      • L142: correct the title "Methylated genes revealed highly functional conservation among three P. syringae strains" maybe to "Methylated genes are functionally conserved among ..."

      We have revised. Please see Line 142.

      • Figure 2C: not easy to read and interpret

      Sorry for the confusion. Figure 2C revealed the significantly enriched functional pathways in GO and KEGG databases among three P. syringae strains. The specific names of each pathway were listed on the left, and each column with dots indicated the number of genes within one kind of methylation in one of three P. syringae strains. The larger the size, the bigger the number.

      We have revised the legend of Figure 2C. Please see Lines 575-579.

      “The dot plot revealed the significantly enriched functional pathways in GO and KEGG databases among three P. syringae strains. The specific names of each pathway were listed on the left, and each column with dots indicated the number of genes within one kind of methylation in one of three P. syringae strains. The size of the dots indicates the number of related genes.”

      • Figure 6B-C: what is the difference between B 24h and C?

      Figure 6B revealed the expression difference between WT and mutant during 24 hours. Figure 6C only showed a time point in 24 hours. To avoid repetition, we have removed Figure 6C.

      • Figure 6C-D: if the same maybe remove Figure 2C

      We have removed Figure 6D.

      Reviewer #2 (Recommendations For The Authors):

      The manuscript could be improved by addressing the following concerns:

      (1) In line 146: How to understand the percentage conserved in "more than two of the strains"?

      Sorry for the confusion, we planned to indicate the pattern that conserved in two strains and three strains. We have revised it to: “Notable, about 25% to 45% of methylated genes were conserved in two and three strains”. Please see Line 145.

      (2) In line 178: Five conserved sequence motifs should be replaced by "Six conserved sequence motifs".

      We have revised. Please see Line 176.

      (3) In Figure 2B, specify the C1, C2 and C3. "m6A" should be replaced by "6mA".

      Yes, we have revised.

      (4) In Figure S2, "m6A" should be replaced by "6mA".

      Yes, we have revised.

      (5) In line 212, please add references for the previous studies showing that growth conditions affect bacterial methylation status.

      Thank you for your suggestion. We have added the relevant references (Gonzalez and Collier, 2013), (Krebes et al., 2014), (Sanchez-Romero and Casadesus, 2020).

      (6) In line 217, "illustrate" should be "illustrated".

      Yes, we have revised. Please see Line 210.

      (7) There are some genes colored in grey, revealing bigger differences between the two strains than those related to ribosomal protein, T3SS, and alginate synthesis in Fig. 4A. Do they have important functional roles as well?

      Thank you for your suggestion. A total of 116 genes with bigger differences (|Log<sub>2</sub>FC| > 2) except for genes related to ribosomal protein, T3SS, and alginate synthesis. Among these genes, 31 were annotated as hypothetical proteins and 4 as transcription factors with unknown functions, and the remaining genes mostly encoded metabolism-related enzymes. These enzymes might have effects on growth defects in ΔhsdMSR. We added this information in the revised manuscript. Please see Line 249-254.

      (8) The authors should discuss what will be the potential signals or factors that can regulate the activity of HsdMSR. In other words, what can decide the extent of methylation through activating or suppressing the expression of HsdMSR?

      Thank you for your valuable suggestion. We have added this part in the discussion part. Please see Lines 404-415.

      “Apart from the established roles of 6mA and HsdMSR in P. syringae, certain signals or factors may influence HsdMSR expression. For instance, we confirmed that the growth phase affects methylation levels in P. syringae. Previous studies have shown that increased temperatures can reduce methylation levels, as observed in PAO1(Doberenz et al., 2017). These findings suggest that HsdMSR expression may be responsive to both intrinsic cellular states and extrinsic environmental conditions. To further explore potential upstream TFs regulating the expression of HsdMSR, we searched for TF-binding sites in the HsdMSR promoter using our own databases (Fan et al., 2020; Shao et al., 2021; Sun et al., 2024). As a result, we found three candidate TFs (PSPPH_0061, PSPPH_3268, and PSPPH_3504) that might directly bind and regulate HsdMSR expression. Future studies on these TFs and their interactions with the HsdMSR promoter would help clarify the regulatory network governing HsdMSR activity.”

      Reviewer #3 (Recommendations For The Authors):

      (1) Some figures contain dense information, which may be overwhelming for readers. Streamlining the legend for Figure 1 and resizing the Venn diagrams within it could enhance clarity and visual appeal.

      Thank you for your suggestion. We have scaled all the Venn plots in the revised version.

      (2) Incorporating a discussion about the role of the restriction-modification (RM) system in bacterial defense against phage infection into the discussion section could enrich the manuscript's context and relevance.

      Thank you for your valuable suggestion. We have added this part in the Discussion part. Please see Lines 416-427.

      “RM systems are known for their intrinsic role as innate immune systems in anti-phage infection, and present in around 90% of bacterial genomes(Oliveira et al., 2014). RM systems protect bacteria self by recognizing and degrading foreign phage DNA via methylation-specific site and restriction endonucleases (REases) (Loenen et al., 2014). In addition, other phage-resistance systems are similar to RM systems but carry extra genes. One is called the phage growth limitation (Pgl) system, which modifies and cleaves phage DNA. However, the Pgl only modifies the phage DNA in the first infection cycle, and cleaves phage DNA in the subsequent infections, which gives a warn to the neighboring cells(Hampton et al., 2020; Hoskisson et al., 2015). To counteract RM and RM-like systems, phages have evolved strategies, including unusual modifications such as hydroxymethylation, glycosylation, and glucosylation. They can also encode their own MTases to protect their DNA or employ strategies to evade restriction systems and other anti-RM defenses.(Iida et al., 1987; Murphy et al., 2013; Vasu and Nagaraja, 2013).

      (3) In line 152: What is the importance of the mentioned example of Cro/CI family TF?

      Thank you for your comments. The Cro/CI are important TFs present in phages. The interaction between Cro and CI affects bacteria immunity status in Enterohemorrhagic Escherichia coli (EHEC) strains(Jin et al., 2022). RM systems are known as a kind of phage-defense system, and hence we mentioned it here. We have added this description in the revised manuscript. Please see Lines 152-154.

      Reference:

      (1) Doberenz, S., Eckweiler, D., Reichert, O., Jensen, V., Bunk, B., Sproer, C., Kordes, A., Frangipani, E., Luong, K., Korlach, J., et al. (2017). Identification of a Pseudomonas aeruginosa PAO1 DNA Methyltransferase, Its Targets, and Physiological Roles. mBio 8. 10.1128/mBio.02312-16.

      (2) Fan, L., Wang, T., Hua, C., Sun, W., Li, X., Grunwald, L., Liu, J., Wu, N., Shao, X., Yin, Y., et al. (2020). A compendium of DNA-binding specificities of transcription factors in Pseudomonas syringae. Nat Commun 11, 4947. 10.1038/s41467-020-18744-7.

      (3) Gonzalez, D., and Collier, J. (2013). DNA methylation by CcrM activates the transcription of two genes required for the division of Caulobacter crescentus. Mol Microbiol 88, 203-218. 10.1111/mmi.12180.

      (4) Hampton, H.G., Watson, B.N., and Fineran, P.C. (2020). The arms race between bacteria and their phage foes. Nature 577, 327-336.

      (5) Hoskisson, P.A., Sumby, P., and Smith, M.C. (2015). The phage growth limitation system in Streptomyces coelicolor A (3) 2 is a toxin/antitoxin system, comprising enzymes with DNA methyltransferase, protein kinase and ATPase activity. Virology 477, 100-109.

      (6) Iida, S., Streiff, M.B., Bickle, T.A., and Arber, W. (1987). Two DNA antirestriction systems of bacteriophage P1, darA, and darB: characterization of darA− phages. Virology 157, 156-166.

      (7) Jin, M., Chen, J., Zhao, X., Hu, G., Wang, H., Liu, Z., and Chen, W.-H. (2022). An engineered λ phage enables enhanced and strain-specific killing of enterohemorrhagic Escherichia coli. Microbiology Spectrum 10, e01271-01222.

      (8) Krebes, J., Morgan, R.D., Bunk, B., Sproer, C., Luong, K., Parusel, R., Anton, B.P., Konig, C., Josenhans, C., Overmann, J., et al. (2014). The complex methylome of the human gastric pathogen Helicobacter pylori. Nucleic Acids Res 42, 2415-2432. 10.1093/nar/gkt1201.

      (9) Loenen, W.A., Dryden, D.T., Raleigh, E.A., Wilson, G.G., and Murray, N.E. (2014). Highlights of the DNA cutters: a short history of the restriction enzymes. Nucleic Acids Res 42, 3-19.

      (10) Murphy, J., Mahony, J., Ainsworth, S., Nauta, A., and van Sinderen, D. (2013). Bacteriophage orphan DNA methyltransferases: insights from their bacterial origin, function, and occurrence. Appl Environ Microb 79, 7547-7555.

      (11) Oliveira, P.H., Touchon, M., and Rocha, E.P. (2014). The interplay of restriction-modification systems with mobile genetic elements and their prokaryotic hosts. Nucleic Acids Res 42, 10618-10631.

      (12) Sanchez-Romero, M.A., and Casadesus, J. (2020). The bacterial epigenome. Nature reviews. Microbiology 18, 7-20. 10.1038/s41579-019-0286-2.

      (13) Shao, X., Tan, M., Xie, Y., Yao, C., Wang, T., Huang, H., Zhang, Y., Ding, Y., Liu, J., Han, L., et al. (2021). Integrated regulatory network in Pseudomonas syringae reveals dynamics of virulence. Cell Rep 34, 108920. 10.1016/j.celrep.2021.108920.

      (14) Sun, Y., Li, J., Huang, J., Li, S., Li, Y., Lu, B., and Deng, X. (2024). Architecture of genome-wide transcriptional regulatory network reveals dynamic functions and evolutionary trajectories in Pseudomonas syringae. bioRxiv, 2024.2001. 2018.576191.

      (15) Vasu, K., and Nagaraja, V. (2013). Diverse functions of restriction-modification systems in addition to cellular defense. Microbiol Mol Biol Rev 77, 53-72. 10.1128/MMBR.00044-12.

    1. Reviewer #1 (Public review):

      This paper presents a model of the whole somatosensory non-barrel cortex of the rat, with 4.2 million morphologically and electrically detailed neurons, with many aspects of the model constrained by a variety of data. The paper focuses on simulation experiments, testing a range of observations. These experiments are aimed at understanding how multiscale organization of the cortical network shapes neural activity.

      Strengths

      • The model is very large and detailed. With 4.2 million neurons and 13.2 billion synapses, as well as the level of biophysical realism employed, it is a highly comprehensive computational representation of the cortical network.

      • Large scope of work - the authors cover a variety of properties of the network structure and activity in this paper, from dendritic and synaptic physiology to multi-area neural activity.

      • Direct comparisons with experiments, shown throughout the paper, are laudable.

      • The authors make a number of observations, like describing how high-dimensional connectivity motifs shape patterns of neural activity, which can be useful for thinking about the relations between the structure and the function of the cortical network.

      • Sharing the simulation tools and a "large subvolume of the model" is appreciated.

      Weaknesses

      • A substantial part of this paper - the first few figures - focuses on single-cell and single-synapse properties, with high similarity to what was shown in Markram et al., 2015. Details may differ, but overall it is quite similar.

      • Although the paper is about the model of the whole non-barrel somatosensory cortex, out of all figures, only one deals with simulations of the whole non-barrel somatosensory cortex. Most figures focus on simulations that involve one or a few "microcolumns". Again, it is rather similar to what was done in Markram et al., 2015 and constitutes relatively incremental progress.

      • With a model like this, one has an opportunity to investigate computations and interactions across an extensive cortical network in an in vivo-like context. However, the simulations presented are not addressing realistic specific situations corresponding to animals performing a task or perceiving a relevant somatosensory stimulus. This makes the insights into roles of cell types or connectivity architecture less interesting, as they are presented for relatively abstract situations. It is hard to see their relationship to important questions that the community would be excited about - theoretical concepts like predictive coding, biophysical mechanisms like dendritic nonlinearities, or circuit properties like feedforward, lateral, and feedback processing across interacting cortical areas. In other words, what do we learn from this work conceptually, especially, about the whole non-barrel somatosensory cortex?

      • Most of comparisons with in vivo-like activity are done using experimental data for whisker deflection (plus some from the visual stimulation in V1). But this model is for the non-barrel somatosensory cortex, so exactly the part of the cortex that has less to do with whiskers (or vision). Is it not possible to find any in vivo neural activity data from non-barrel cortex?

      • The authors almost do not show raw spike rasters or firing rates. I am sure most readers would want to decide for themselves whether the model makes sense, and for that the first thing to do is to look at raster plots and distributions of firing rates. Instead, the authors show comparisons with in vivo data using highly processed, normalized metrics.

      • While the authors claim that their model with one set of parameters reproduces many experimentally established metrics, that is not entirely what one finds. Instead, they provide different levels of overall stimulation to their model (adjusting the target "P_FR" parameter, with values from 0 to 1, and other parameters), and that influences results. If I get this right (the figures could really be improved with better organization and labeling), simulations with P_FR closer to 1 provide more realistic firing rate levels for a few different cases, however, P_FR of 0.3 and possibly above tends to cause highly synchronized activity - what the authors call bursting, but which also could be called epileptic-like activity in the network.

      • The authors mention that the model is available online, but the "Resource availability" section does not describe that in substantial detail. As they mention in the Abstract, it is only a subvolume that is available. That might be fine, but more detail in appropriate parts of the paper would be useful.

      Comments on revisions:

      The authors addressed all my comments by revising and adding text as well as revising and adding some figures and videos. The limitations described in my previous review (above) mostly remain, but they are much better acknowledged and described now. These limitations can be addressed in the future work, whereas the current paper represents a step forward relative to the state of the art and provides a useful resource for the community.

      Two minor points about the new additions to the paper:

      (1) Something does not seem right in the sentence, "Unlike the Markram et al. (2015) model, the new model can also be exploited by the community and has already been used in a number of follow up papers studying (Ecker et al., 2024a,b; ...)". Should the authors remove "studying"?

      (2) It is great that the authors added more plots and videos of the firing rates, but most of them show maximum-normalized rates, which sort of defeats the purpose. No scale on the y-axis is shown (it can be useful even for normalized data). And it is impossible to see anything for inhibitory populations.

      These are minor points that may not need to be addressed. Overall, it is a nice study that is certainly useful for the field.

      A great improvement is that the model is made fully available to the public.

    1. La conclusion, un monde "sans racisme", montre bien le problème. Le racisme c'est d'abord une société fantasmée comme attribuant des inégalités spirituelles et sociales à l'appartenance raciale. Cet arrière plan, le "racisme structurel", commande la signification du mot, que ce soit pour le "blanc" (qui ne veut pas se mélanger) et le "noir" (qui se victimise).

      Cette réalité de la barrière ne se manifeste que dans les sociétés "mixtes", quand le territoire doit être partagé et que l'on ressent une pression dans un sens ou dans l'autre, que ce soit la submersion par le peuple prolifique ou l'écrasement par des lois majoritaires insupportables, les deux sentiments symétriques et consécutifs étant attribuables aux deux parties.

      La submersion est d'abord vécue par le blanc, qui craint sa conséquence, la soumission aux lois noires et la submersion réélle vécue par le noir minoritaire dans le monde banc refuse la réalité des lois blanches jugées étrangères et injustes.

      Le fond de l'affaire est que la notion de race, quelle qu'elle soit, induit une impossibilité du partage national et familial. Elle est ce qu'il n'est pas possible d'oublier, la transmission de soi. Il y a donc bien dans un monde mixte nécessairement asymétrique, 1) une volonté du maintien de cette asymétrie là 2) une discrimination nécessaire qui lui est liée.

      Chacun chez soi, et le "racisme" discriminateur et cruel ne peut qu'être atténué par les droits humains minimaux et le respect des personnes, et certainement pas par l'abolition d'une réalité.

    1. explore la question de la valeur de l’éducation privée.

      L’intervenant remet en question l’idée répandue que les écoles privées produisent automatiquement des élèves sûrs d’eux, soulignant que la confiance en soi est souvent liée au contexte socio-économique familial, plutôt qu’à l’école elle-même.

      Il critique le biais de confirmation qui nous pousse à remarquer les réussites des élèves issus de milieux privilégiés tout en ignorant les échecs.

      Finalement, la discussion aborde le rôle complexe des écoles privées dans la société, leur coût croissant et l’incertitude quant à leur valeur réelle pour les familles.

      Voici une synthèse des points clés concernant l'enseignement privé, basée sur les sources fournies :

      • Confiance et origine sociale :

      L'une des raisons souvent évoquées pour le choix de l'enseignement privé est le développement de la confiance chez l'enfant.

      Cependant, les études montrent que cette confiance est souvent liée au milieu socio-économique d'origine.

      Les enfants issus de milieux privilégiés, ayant grandi avec moins d'inquiétudes financières, ont tendance à être plus confiants, ce qui n'est pas forcément dû à leur scolarisation dans le privé.

      • Biais de confirmation :

      On a tendance à remarquer les exemples de personnes ayant réussi, comme Boris Johnson, qui a fréquenté Eton, mais on oublie les enfants issus de l'enseignement privé qui rencontrent des difficultés.

      On entend moins parler des élèves en échec, ceux qui ont du mal à interagir avec des personnes d'autres milieux ou qui ont des problèmes liés à la pression scolaire.

      • Expériences personnelles contrastées :

      Un témoignage personnel relate une expérience dans une école publique diversifiée et une école privée, toutes deux avec leurs bons et mauvais côtés.

      Le témoin souligne avoir vu plus de consommation d'alcool et de drogues dans l'école privée.

      Des études confirment que les élèves du privé ont tendance à commencer à boire plus tôt et à prendre des risques.

      • Complexité du choix de l'enseignement privé :

      Le choix de l'enseignement privé est complexe et ne se résume pas à la qualité de l'éducation.

      Les raisons sont variées et dépassent souvent le cadre scolaire.

      Les parents cherchent parfois un réseau social ou des opportunités spécifiques pour leurs enfants.

      • Impact du réseau social :

      Les personnes issues d'écoles privées comme Eton peuvent avoir tendance à privilégier d'autres personnes issues du même milieu.

      Il y a une prise de conscience croissante de ce phénomène.

      • Avenir de l'enseignement privé :

      L'avenir de l'enseignement privé est incertain.

      Avec des propositions de taxes sur les écoles privées, les parents devront se poser des questions importantes sur l'intérêt de ces établissements.

      Les parents doivent considérer ce qu'ils recherchent vraiment pour leurs enfants, car la perception de l'enseignement privé varie.

      Il est difficile de savoir si l'enseignement privé est vraiment rentable, car chaque parent a une perception différente de ce qu'il recherche.

      • Problèmes sociétaux plus vastes:

      Le débat sur l'enseignement privé reflète des problèmes plus vastes dans la société, comme des problèmes structurels.

      Il faut que les entreprises, les universités et les différents acteurs de la société aillent au-delà du simple parcours scolaire pour identifier les personnes compétentes.

    1. Voici un sommaire minuté basé sur la transcription de la conférence sur le conflit sévère de séparation :

      • 0:04-0:17: Introduction et remerciements par l'animateur de l'événement. Il remercie Michel Rabitail et Vanessa Lete pour l'organisation et l'animation.
      • 0:17-0:55: L'animateur rappelle qu'il s'agit de la deuxième conférence sur les conflits sévères de séparation en peu de temps et que ce sujet est devenu une priorité au Québec, notamment parce que c'était le seul motif de signalement à la hausse pendant la pandémie. Il souligne que la plupart des professionnels n'ont pas reçu de formation spécifique sur les approches familiales et systémiques.
      • 1:55-2:47 L'animateur explique l'objectif de cette journée scientifique et clinique. Il mentionne les distinctions entre le conflit sévère de séparation, l'aliénation parentale, et la violence conjugale. Il annonce une présentation de projets appliqués et des approches basées sur des données probantes comme la médiation, les approches systémiques et les approches sensibles aux traumatismes.
      • 2:54-4:54: Introduction des trois conférencières : Elisabeth Godbout, professeure spécialiste des transitions familiales, des séparations et des conflits sévères de séparation ; Amylie Paquin Boudreau, psychologue clinicienne spécialiste des enjeux de conflits sévères de séparation et de violence familiale ; et Catherine Turbide, professeure spécialiste des conflits sévères de séparation, de violence conjugale post-séparation et des trajectoires de service en protection de la jeunesse.
      • 4:54-5:29: L'animateur souligne l'expertise des conférencières et l'importance de cette thématique.
      • 5:29-6:03: Elisabeth Godbout prend la parole et remercie l'Institut Universitaire jeunes en difficulté pour l'invitation. Elle souligne que le conflit de séparation est un sujet de prédilection dans leurs travaux.
      • 6:53-7:25 : Présentation du plan de la conférence pour les 3 heures à venir..
      • 7:25-8:38: Introduction au sujet des conflits sévères de séparation, problématique apparue il y a une trentaine d'années, d'abord identifiée par les professionnels de la justice et des services psychosociaux. Le premier article sur le sujet est celui de Janet Johnston en 1994. La problématique a pris de l'ampleur dans les années 2000, avec une augmentation des écrits scientifiques et des dossiers judiciaires. Il est noté que cette problématique représente un défi pour l'intervention.
      • 9:45-10:09: Il est souligné que la distinction entre conflit sévère de séparation, violence conjugale et contrôle coercitif est primordiale.
      • 10:09-11:17: Présentation des objectifs de la journée : faire le point sur les connaissances, présenter les services existants au Québec, et aborder les bases d'une évaluation appropriée.
      • 11:17-12:09 : Présentation d'un programme et d'une intervention en soulignant l'importance des approches pour comprendre la complexité du problème.
      • 12:09-12:42: Définition du conflit sévère de séparation : un terme "parapluie" qui englobe plusieurs problématiques de nature et de sévérité différentes. Il se manifeste par une hostilité importante entre les parents, rendant la communication difficile, voire inexistante.
      • 13:54-14:06: Le conflit sévère de séparation inclut des signalements répétés à la protection de la jeunesse, des recours fréquents aux tribunaux, parfois malintentionnés.
      • 14:06-15:34: Les conflits sévères de séparation sont des dossiers qui encombrent le système et consomment beaucoup de ressources. Les enfants sont affectés par l'exposition à des conflits répétés et intenses.
      • 15:34-17:31: La distinction entre les conflits de séparation et la violence conjugale est complexe : il est nécessaire de distinguer les manifestations de violence liées au conflit de celles liées à un déséquilibre de pouvoir. Il est important de souligner que la violence dans le contexte du conflit peut être sporadique ou situationnelle, alors que dans la violence conjugale il y a un déséquilibre de pouvoir.
      • 17:31-18:49: Le conflit sévère de séparation est souvent chronique et enlisée dans le temps, les parents restent en colère comme au premier jour de leur séparation. Une distinction est faite entre les conflits normaux lors d'une séparation et les situations qui perdurent. La durée des conflits dans le temps reste mal connue.
      • 18:49-20:27: Les différentes dimensions du conflit sont présentées, notamment la judicialisation importante des litiges. Les conflits judiciaires sont à distinguer des difficultés parentales. Présentation d'une étude avec un échantillon de parents québécois récemment séparés.
      • 20:27-21:24: Les analyses ont permis de dégager un sous-groupe de parents vivant des conflits élevés au plan judiciaire et des difficultés parentales. Il s'agit d'une zone "très préoccupante" de 13% des parents.
      • 22:00-24:03: Un tiers des parents de ce sous-groupe ont eu une présentation en cour, 30% ont eu recours à une expertise en matière de garde. Les dimensions du conflit sont enchevêtrées. Il y a une persistance dans le temps pour un sous-groupe de ce 13%.
      • 24:03-25:08: Les données des dossiers judiciaires montrent qu'une minorité de parents va à procès, la majorité règle les questions entourant leur séparation durant les procédures.
      • 25:08-27:45 : Les données de la protection de la jeunesse montrent qu'une minorité de dossiers comportent un litige actif en matière de garde d'enfants. Ces dossiers sont plus souvent réouverts avec une détérioration du fonctionnement des enfants. Au Québec, 15,7 % des évaluations faites par les services de protection de la jeunesse ont été faites en raison de mauvais traitements psychologiques, dont 14,8% comportaient des conflits de séparation. L'exposition à la violence conjugale demeure le motif le plus souvent signalé, suivi des conflits de séparation.
      • 27:45-28:46: Présentation d'un panorama des services au Québec pour traiter ou prévenir le conflit sévère de séparation.
      • 28:46-30:06: Les services sont divisés en différents types et niveaux, allant de la prévention à l'intervention spécialisée. Les services de première ligne sont peu documentés et il manque d'outils.
      • 30:06-31:20: Présentation de la séance d'information de groupe sur la parentalité après la rupture, un service universel mais surtout utilisé par les parents en litige. Ce service n'a pas été évalué..
      • 31:20-32:54 : La médiation familiale est un service très utilisé et apprécié. Par contre, son efficacité à prévenir la détérioration des conflits reste à être étudiée.
      • 32:54-33:51 : Il existe des services juridiques de prévention et de règlement des différends, offerts en pratique privée. Leur efficacité à prévenir les conflits n'a pas été examinée.
      • 33:51-34:53 : Des programmes de groupes plus ciblés sont offerts par des organismes communautaires et par le CUS centre sud de l'île de Montréal. Un silence est fait sur l'action du milieu communautaire qui est jugée très importante, mais non nommée.
      • 34:53-36:03: Les services de première ligne sont peu documentés. Il y a un manque d'outils pour les professionnels pour intervenir dans ce type de problématique. Plus on augmente en sévérité, plus les interventions sont contraignantes. On utilise le judiciaire comme levier d'intervention.
      • 36:03-37:04 : Les services indiqués pour les conflits chroniques incluent l'expertise, des programmes spécialisés, la supervision des échanges de garde et la coordination parentale. Les services les plus spécialisés sont concentrés dans les grands centres.
      • 37:04-38:32: Les services offerts par les intervenants psychosociaux en pratique privée se développent. Un programme du ministère de la Justice à Montréal est en cours d'évaluation. Le coaching parental est aussi une intervention privée.
      • 38:32-41:08 : Des programmes sociojudiciaires importants tels que le programme parentalité conflit résolution et le programme d'intervention sociojudiciaire en conflit sévère de séparation sont présentés. Ces programmes ont fait l'objet d'évaluations et ont montré un potentiel intéressant.
      • 41:08-42:02: L'offre de services est morcelée, manque d'intégration et ne traite pas le conflit sévère de séparation comme un problème de santé publique. Une réelle prévention des conflits de séparation devrait être le défi des prochaines années.
      • 42:02-43:31 : Il faut rehausser les pratiques et les évaluations et s'appuyer sur les programmes dont l'efficacité a été démontrée. Il manque de données sur l'efficacité des services les plus accessibles, tandis qu'il y a plus de données sur les services spécialisés. Les services les plus spécialisés sont souvent offerts par des praticiens en pratique privée.
      • 43:31-44:02: Les conférencières souhaitent savoir ce qui amène les participants à suivre cette formation.
      • 44:02-44:44 : Les conférencières demandent aux participants de se situer dans l'écosystème des services.
      • 44:44-45:05 : Transition vers une séquence sur l'évaluation qui sera présentée par Catherine Turbide.
      • 45:05-47:53: Avant l'évaluation, on doit réfléchir aux questions que l'on doit se poser en tant qu'intervenant. Il existe des biais documentés sur ces questions, notamment, la question de savoir si un conflit est toujours un "tango" qui se danse à deux.
      • 47:53-49:03: On constate souvent que l'on croit que les parents utilisent les services pour se venger l'un de l'autre. Or, les écrits montrent que c'est beaucoup plus complexe. Une étude a montré que seulement 13 % des allégations sont malicieuses.
      • 49:03-51:07: Ce n'est pas notre prémisse de base, mais il faut y être attentif. Il y a plusieurs raisons qui expliquent l'utilisation des services, comme une mauvaise gestion des émotions ou un manque de moyens.
      • 51:07-51:19: Amélie Paquin prend la parole et souligne l'importance de prendre le temps de faire une bonne évaluation avant d'intervenir.
      • 51:19-52:16 : L'évaluation permet d'élaborer un plan d'intervention ajusté aux besoins des familles, à leurs ressources et aux limites de la pratique. Les situations de conflits sévères de séparation sont variées..
      • 52:16-53:00: La plupart des services actuels sont fondés sur le principe de coparentalité, un idéal qui n'est pas toujours réaliste. Par principe de coparentalité on entend : la prise de décisions négociées, la communication fonctionnelle et la coopération.
      • 53:00-57:37: La littérature scientifique soulève un problème avec cet idéal de coparentalité. Pour certaines familles, l'intervention familiale n'est pas appropriée, notamment en présence de violence conjugale. Il faut être attentif quand on travaille une rupture de lien parent enfant.
      • 57:37-59:10 : Il faut prendre conscience qu'il y a des prérequis pour avoir une coparentalité coopérative. Il y a des parents qui n'ont pas les ressources pour y arriver. Les parents qui sont à l'extrémité du continuum sur le conflit sévère de séparation n'ont pas nécessairement ces ressources.
      • 59:10-1:00:47 : Il faut être nuancé dans la présentation de ces prérequis, ce n'est pas un objectif qui est réaliste tout le temps. Il y a un travail de deuil de la relation, un travail de régulation des émotions qui doit être fait avant de vouloir travailler la coparentalité.
      • 1:00:47-1:01:24: Présentation d'une figure exclusive, démontrant la réflexion sur l'intégration de l'évaluation et de la séquence d'intervention.
      • 1:01:24-1:02:11: Il faut être neutre et objectif dans l'évaluation, et distinguer si l'objectif est de traiter la famille ou de la structurer..
      • 1:02:11-1:03:26: L'objectif de structuration vise à mettre des limites, des règles claires et s'assurer de leur respect. L'objectif de traitement vise à apporter des changements profonds dans les interactions familiales. Certains parents vivent un deuil compliqué de la relation, d'autres ont des problèmes psychosociaux complexes ou des antécédents traumatiques.
      • 1:03:26-1:05:30: Quand on est dans un objectif de traitement, on a confiance dans les ressources des parents pour faire des changements profonds. Quand on est dans une logique de structuration, on fait face à des parents qui n'ont pas la collaboration ou la motivation pour changer. Il faut être réaliste face à ces familles et partir de leurs ressources.
      • 1:05:30-1:05:57: Transition vers la présentation de deux programmes en protection de la jeunesse, par Catherine Turbide.
      • 1:05:57-1:06:26 : Les programmes sont déployés en région..
      • 1:06:26-1:07:37 : Présentation du projet PCS, initiative du 6 de la Mauricie qui a constaté un besoin de mieux intervenir auprès de ces familles.
      • 1:07:37-1:08:16 : Les objectifs du programme sont de connaître, d'évaluer et d'intervenir auprès des familles en conflit de séparation. Il s'agit d'un projet qui vise à donner des outils pour faire l'évaluation du conflit.
      • 1:08:16-1:09:39: Le programme comprend une trousse de soutien, des groupes de soutien pour les intervenants et une communauté de pratique.
      • 1:09:39-1:10:12: Présentation d'une évaluation du point de vue des intervenants, qui montre que l'utilisation du programme est variable.
      • 1:10:12-1:11:59: Les intervenants trouvent que le programme a amélioré leur pratique, mais n'a pas diminué le temps consacré aux dossiers..
      • 1:11:59-1:12:31: Il y a eu une amélioration individuelle et collective.
      • 1:12:31-1:14:06 : Les capsules de la trousse sont utiles, elles couvrent une vaste gamme de thématiques associées aux conflits de séparation. Elles permettent de mieux comprendre certaines dynamiques..
      • 1:14:06-1:15:22 : Le groupe de soutien est la modalité qui fait le plus consensus. Il permet aux intervenants de se sentir soutenus, de concrétiser l'utilisation de la trousse, de démythifier les problématiques, de baisser le stress. Par contre, il n'est pas toujours disponible selon les besoins.
      • 1:15:22-1:16:46: La communauté de pratique augmente le sentiment de compétence et permet de bonifier les connaissances, mais peut ajouter une surcharge de travail.
      • 1:16:46-1:18:15 : Facteurs qui facilitent l'utilisation des outils : sentiment de spécialisation, cointervention. Les facteurs qui font obstacle : la surcharge de travail, l'instabilité des équipes, le contexte organisationnel.
      • 1:18:15-1:19:23: Recommandations : désigner des leaders, favoriser la participation des nouveaux employés, réduire le nombre de dossiers, favoriser la cointervention, prévoir des modalités plus souples..
      • 1:19:23-1:21:34: Présentation du programme d'intervention sociojudiciaire en conflit sévère de séparation, qui est un programme de partenariat entre les milieux psychosocial et judiciaire. Il a été mis en place dans 36 établissements et est maintenant déployé dans 10 établissements. Le programme vise à concerter l'intervention de la protection de la jeunesse et le processus judiciaire. Son objectif est d'augmenter le nombre de situations qui se règlent par des mesures consensuelles.
      • 1:21:34-1:22:09 : Les familles ciblées sont celles où la sécurité et le développement de l'enfant sont compromis en raison d'un conflit sévère de séparation. Les parents doivent reconnaître la présence du conflit et son impact sur l'enfant.
      • 1:22:09-1:22:58: Le programme implique une trajectoire judiciaire adaptée et une intervention psychosociale avec des rencontres hebdomadaires pendant 6 à 9 mois.
      • 1:22:58-1:23:54: Présentation d'une évaluation du volet qualitatif du programme, auprès de 30 professionnels.
      • 1:23:54-1:24:43: Le programme permet de remettre aux parents le pouvoir de changer leur situation familiale. Il améliore la communication, recentre les parents sur les besoins des enfants et favorise la négociation..
      • 1:24:43-1:25:30 : Un enjeu demeure les critères d'admissibilité des familles. Le programme est difficile à mettre en œuvre pour les familles ayant peu de motivation. Un autre défi est le soutien des familles lorsque le programme est terminé.
      • 1:25:30-1:26:22: Il y a des difficultés liées à la concertation des interventions cliniques et judiciaires. Une évaluation est en cours pour étudier les effets du programme auprès des familles. Proposition d'une pause de 15 minutes.
      • 1:26:22-1:26:41: Amélie Paquin prend le relais pour la présentation des méthodes d'interventions auprès des familles vivant un conflit sévère de séparation..
      • 1:26:41-1:27:06 : Survol des groupes éducatifs pour les parents séparés, des interventions individuelles pour les parents et celles pour les enfants. Les interventions sont présentées en fonction de ce que la littérature dit sur qui est orienté vers ses services et qui ne l'est pas.
      • 1:27:06-1:28:01: Les groupes éducatifs pour parents séparés sont des interventions de groupes où les parents ont accès à plusieurs rencontres. Il y a beaucoup de travail à faire pour que ces groupes soient réellement efficaces. La forme des groupes est hétérogène. Ils ont surtout une portée préventive ou universelle.
      • 1:28:01-1:29:26: Les groupes éducatifs sont souvent offerts à tous les parents séparés, sans tenir compte des problématiques associées au conflit sévère de séparation. Ils ne sont pas toujours adaptés aux enjeux des parents dans cette situation, notamment les enjeux liés à la violence conjugale. Ils peuvent même être problématiques dans certains contextes.
      • 1:29:26-1:30:02 : La littérature scientifique propose d'adapter les groupes pour les familles en conflit sévère de séparation. Il y a de nombreuses recherches sur l'efficacité des groupes. La taille des effets est faible, notamment pour les familles avec un haut niveau de conflit. Les effets ne se maintiennent pas dans le temps.
      • 1:30:02-1:31:20 : Une réforme des groupes éducatifs est suggérée : se concentrer sur la parentalité plutôt que la coparentalité, accompagner les parents à développer leurs ressources individuelles, leur régulation émotionnelle. D'autres auteurs proposent d'ajouter de l'information sur la violence conjugale et la violence familiale.
      • 1:31:20-1:32:32: Il serait intéressant d'intégrer de l'information sur l'influence des traumas relationnels et des contextes d'adversité sur la régulation émotionnelle. Il est important de sortir du focus de la séparation pour comprendre la trajectoire relationnelle des familles.
      • 1:32:32-1:33:01: Présentation des interventions individuelles auprès des parents. C'est souvent un préalable nécessaire avant les interventions familiales.
      • 1:33:01-1:34:33 : Les séparations peuvent faire émerger des émotions difficiles, des traumas non-intégrés. Il est important de clarifier le mandat lors de ces interventions. Il y a des limites au travail individuel.
      • 1:34:33-1:35:51: Il est important de mettre les limites de l'intervention dès le début et de rester neutre face aux informations données sur l'autre parent. Il faut faire attention de ne pas prendre partie dans le conflit tout en amenant les parents à se protéger et à nuancer leurs convictions.
      • 1:35:51-1:37:16: Importance de l'approche systémique, de tenir compte des enfants dans la relation. On doit aider le parent à mieux comprendre la position de son enfant. C'est important de garder en tête l'approche sensible au trauma.
      • 1:37:16-1:39:07 : Il est primordial de considérer l'impact du contexte d'adversité et des traumas relationnels sur les parents, en plus de la séparation. Il faut être conscient du fonctionnement psychologique des parents, de leurs limites et pronostics d'évolution.
      • 1:39:07-1:40:26: Il faut reconnaître que tous les parents n'évolueront pas. Il existe des critères d'inclusion et d'exclusion à l'intervention individuelle. Présentation des interventions individuelles auprès des enfants.
      • 1:40:26-1:42:30 : Il faut se questionner sur les bénéfices d'une intervention individuelle pour l'enfant. L'intervention individuelle est appropriée si l'enfant est demandeur ou si le conflit a des impacts importants sur son bien-être. L'intervention individuelle peut aider l'enfant à développer des ressources, à mieux comprendre la situation et à nommer ses besoins et émotions.
      • 1:42:30-1:43:00 : L'enfant a besoin d'adultes qui lui permettent de nommer ses émotions et qui puissent valider son expérience..
      • 1:43:00-1:43:30 : Il est important de ne pas donner de faux espoirs et d'aider l'enfant à comprendre ses limites, ce qu'il peut en faire..
      • 1:43:30-1:44:45: Transition vers la présentation des approches qui peuvent être utiles par Catherine Turbide, notamment l'approche de négociation sur intérêt, ou approche de médiation.
      • 1:44:45-1:45:32: Cette approche a été utilisée en protection de la jeunesse autour de 2005, pour amener les intervenants à soutenir les familles dans leur propre démarche de résolution de problèmes.
      • 1:45:32-1:47:22: Cette approche a ensuite été déployée dans des contextes variés, notamment en protection de la jeunesse, elle est utile pour comprendre les enjeux du conflit de séparation. Le but est que les participants comprennent les grands principes et sachent dans quoi s'embarquent les parents qui travaillent avec cette approche.
      • 1:47:22-1:48:22: L'approche est une structure de résolution de problèmes et de communication coopérative qui implique la présence simultanée des parties. Il faut donc éviter de l'utiliser dans les cas de violence conjugale.
      • 1:48:22-1:49:58 : Une distinction doit être faite entre la médiation, un processus volontaire mené par un médiateur impartial, et l'approche de médiation, qui peut être utilisée dans des contextes non-volontaires. L'intervenant peut avoir un mandat et une partialité, mais doit quand même favoriser la résolution de problème de manière collaborative.
      • 1:49:58-1:50:37 : L'approche implique une évaluation, l'identification des besoins, le tour de toutes les options, l'évaluation de leur pertinence et l'élaboration d'une entente. La posture de l'intervenant est de soutien et d'accompagnement, et non de prescription de solution.
      • 1:50:37-1:51:47: Les principes de l'approche : il faut utiliser un vocabulaire positif, c'est mieux de parler de besoins que de problèmes, chaque personne doit avoir une place pour exprimer ses besoins et être responsabilisée, la confiance et le respect sont au premier plan.
      • 1:51:47-1:52:35: Le processus est centré sur les intérêts et non sur les positions. L'objectif est d'amener les parents à se centrer sur leurs besoins. Le processus doit favoriser la communication et la collaboration.
      • 1:52:35-1:53:38: Il est préférable que la solution soit choisie par les parties plutôt que d'être imposée. Le processus a pour objectif que les parents soient capables de reproduire les techniques et de les utiliser dans le futur.
      • 1:53:38-1:54:37: Les étapes de la négociation : l'accueil, la négociation des intérêts et les résultats. L'accueil est très structuré. Les règles doivent être très explicites.
      • 1:54:37-1:56:56 : La négociation des intérêts : décoder les besoins, repérer les besoins communs, prioriser les besoins, identifier les options et en faire un choix. Il faut identifier les besoins de manière précise et respectueuse.
      • 1:56:56-1:58:12 : Décoder les besoins est un travail de coconstruction avec les parents. Il faut valider le besoin, et sortir des positions pour aller dans l'intérêt. Il faut donner la possibilité aux personnes d'exprimer leur besoin..
      • 1:58:12-2:01:04 : L'outil du tableau est souvent utilisé pour repérer les besoins communs et les prioriser. Il faut faire une liste de solution exhaustive sans se soucier de sa pertinence.
      • 2:01:04-2:02:14: Choisir une option qui satisfait toutes les parties, l'entente peut être verbale ou écrite..
      • 2:02:14-2:02:31: Transition vers les approches transversales..
      • 2:02:31-2:04:29 : L'approche systémique et l'approche sensible aux traumas sont essentielles pour un travail pertinent auprès des familles en conflit sévère de séparation. L'approche systémique : considérer que l'intervention sur un individu peut avoir un impact sur le système. L'intervenant fait partie du système, ses caractéristiques et décisions vont influencer le système.
      • 2:04:29-2:06:06: Les professionnels influencent le fonctionnement familial et doivent être conscients de leurs propres caractéristiques, connaissances et biais. Ils doivent être humbles et avoir des réactions conscientes et inconscientes qui peuvent cristalliser le système..
      • 2:06:06-2:07:30: Il faut faire attention à l'optimisme excessif et avoir une posture de scepticisme saine. Être attentif aux situations de clivage ou au tout ou rien. Il faut se remettre en question et s'entourer pour réfléchir.
      • 2:07:30-2:09:50 : Il faut éviter de tomber dans le piège de l'impuissance, éviter la critique, et ne pas venir reproduire les dynamiques parentales. Les professionnels doivent se considérer comme faisant partie du système plutôt que comme un expert à l'extérieur.
      • 2:09:50-2:10:26 : L'approche sensible au trauma est appliquée récemment aux familles en conflit sévère de séparation. Il y a un historique de vie marqué par l'adversité et des traumas émotionnels et relationnels qui sont revivifiés lors de la séparation. La séparation vient déclencher des traumas plus précoces.
      • 2:10:26-2:11:54 : Le simple fait de se retrouver dans des procédures judiciaires ou dans une intervention de la DPJ peut être traumatisant. Il faut faire preuve de sensibilité et comprendre le vécu des familles.
      • 2:11:54-2:13:25 : Il faut éviter de voir les parents comme étant de mauvaise foi. Il faut voir le conflit comme un signe de souffrance, mettre de l'humanité, et sortir du "qui a raison" pour amener les parents à faire sens de leur vécu..
    2. Voici un document de synthèse pour un briefing sur le conflit sévère de séparation, basé sur les sources fournies :

      Définition et enjeux du conflit sévère de séparation

      • Le conflit sévère de séparation est une problématique relativement nouvelle, apparue il y a une trentaine d'années dans la littérature clinique et scientifique.
      • Il s'agit d'un terme « parapluie » regroupant un ensemble de problématiques variées, tant par leur nature que par leur sévérité. Il inclut des enjeux tels que la judiciarisation importante des litiges, des signalements répétés à la protection de la jeunesse, des recours fréquents aux tribunaux et des impacts négatifs pour les enfants.
      • Ces situations sont souvent décrites comme étant chroniques et enlisées dans le temps, avec des parents pouvant exprimer la même colère des années après la séparation.
      • Les professionnels peuvent se sentir submergés et impuissants face à la complexité de ces situations.
      • Il est crucial de distinguer le conflit sévère de séparation des situations de violence conjugale et de contrôle coercitif.

      Ampleur du phénomène

      • Le conflit sévère de séparation représente un défi important pour l'intervention et a été documenté dans plusieurs études.
      • Les intervenants décrivent souvent cette problématique comme difficile à cerner, mettant en échec certaines interventions.
      • Au Québec, les conflits sévères de séparation ont été en hausse lors de la pandémie.
      • Bien que l'exposition à la violence conjugale demeure le motif de signalement le plus fréquent, les conflits sévères de séparation représentent une part importante des évaluations en protection de la jeunesse.

      Services existants au Québec

      • L'offre de services est morcelée et il y a un manque d'intégration entre les services.
      • Il existe des solutions à la fois psychosociales et juridiques, avec des services parfois mixtes favorisant les partenariats sociojudiciaires.
      • Les services de base incluent la séance d'information sur la parentalité après la rupture et la médiation familiale, qui sont très utilisés.
      • Des programmes de groupe plus ciblés sont offerts par des organismes communautaires.
      • Les services plus pointus incluent l'expertise, des programmes de groupe spécialisés, la supervision des échanges de garde et la coordination parentale.
      • Les services les plus spécialisés sont concentrés dans les grands centres et souvent offerts en pratique privée.
      • Il y a un manque d'évaluation des services les plus accessibles, notamment en prévention.

      Bases d'une évaluation appropriée

      • L'évaluation est cruciale dans les situations de conflit sévère de séparation en raison de la diversité des enjeux et des besoins des familles.
      • Il est important de ne pas appliquer systématiquement l'idéal de la coparentalité et de prendre en compte la dynamique relationnelle entre les parents, leurs ressources et leur motivation.
      • Il faut distinguer les situations où la coparentalité est possible de celles où elle ne l'est pas, en considérant notamment les antécédents de violence, les troubles de santé mentale sévères et l'échec d'un parent à collaborer.
      • L'évaluation doit être neutre et objective, en prenant le temps de comprendre les points de vue de chacun.
      • Il est important de distinguer les parents qui ont des conflits liés à un deuil compliqué de ceux qui ont un historique de problèmes psychosociaux complexes.

      Interventions et approches

      • Les groupes éducatifs pour parents séparés ont souvent une portée préventive ou universelle et peuvent ne pas être adaptés aux enjeux du conflit sévère de séparation. Il est donc important de les adapter.
      • Il est crucial de clarifier son mandat lors d'interventions individuelles avec les parents et de rester neutre par rapport aux informations données sur l'autre parent.
      • Il est important de tenir compte du fonctionnement psychologique des parents et des pronostics d'évolution.
      • Les interventions individuelles auprès des enfants sont appropriées si l'enfant est demandeur ou si le conflit a des impacts importants sur son bien-être.
      • L'approche de médiation ou de négociation sur intérêts peut être utilisée en protection de la jeunesse mais doit être évitée dans les situations de violence conjugale.
      • Il est nécessaire de favoriser la collaboration et le respect, tout en centrant le processus sur les besoins plutôt que les positions.
      • L'approche systémique et l'approche sensible aux traumas sont essentielles pour un travail pertinent auprès des familles vivant un conflit sévère de séparation.
      • Il faut tenir compte du fait que les professionnels font partie du système familial et peuvent influencer son fonctionnement.

      Points saillants

      • Le conflit sévère de séparation est un problème complexe et croissant, avec des défis importants pour l'intervention.
      • L'évaluation et l'adaptation des interventions aux besoins spécifiques des familles sont primordiales.
      • Les professionnels doivent adopter une posture réflexive, être conscients de leur influence et tenir compte du vécu des familles.

      Ce document de synthèse met en évidence la complexité du conflit sévère de séparation et la nécessité d'une approche multidimensionnelle pour accompagner les familles touchées par ce problème. Il est important que les professionnels comprennent les enjeux, les services disponibles et les différentes approches pour intervenir de manière efficace.

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

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      Reply to the reviewers

      Response to Reviewers

      We thank the three reviewers for their insightful and constructive comments, which have helped improve the manuscript. Our replies to each comment are provided below.

      Reviewer #1

      Evidence, reproducibility and clarity

      The abscission checkpoint, also known as NoCut, is a genome protection mechanism that remains poorly understood. This pathway is conserved from yeast to humans and protects the genome against chromosome bridges, a dangerous missegregation event that can have catastrophic consequences on genome stability. Dam et al now report the role of Srs2, a DNA helicase, as a key factor in the abscission checkpoint. The authors establish Srs2 as bona fide factor in this pathway by showing its involvement in abscission delays when chromatin bridges are induced. Importantly, yeast defective for Srs2 show increased levels of DNA damage when the frequency of chromatin bridges is increased. The authors also provide genetic evidence supporting a model whereby the interaction of SrS2 with PCNA s required for abscission regulation. In the second part of the manuscript, the authors study the human homologue of SRS2, PARI, in abscission regulation. The manuscript provides convincing evidence that PARI is also required for abscission delays in the presence of chromatin bridges. Critically, this role is specific for chromosome missegregation as abscission delays in response to nucleoporin depletion remain intact in PARI-depleted cells. Thus there is a conserved requirement for these DNA helicases in the abscission checkpoint.

      * Overall, these are important advances in our understanding of the abscission checkpoint. The data is high quality and convincing in general. However, the impact of PARI depletion on genome stability needs to be further demonstrated to support key claims in the manuscript. Specifically:*

      • Disruptions of the abscission checkpoint in human cells result in bi-nucleation or increased levels of DNA damage. In this context, the authors need to show that PARI-depleted cells with increased frequency of chromatin bridges exhibit increased levels of bi-nucleation, DNA damage or both.

      We thank the reviewer for its positive assessment of our work. While our data establish that Srs2 inhibits abscission to prevent DNA damage in yeast, we agree with the reviewer that we have not tested the consequences of PARI loss on DNA damage or cytokinesis failure in HeLa cells. We will address this in the revised version of our study.

      Significance

      The abscission checkpoint, remains poorly understood. There is evidence in the literature that disruptions in this pathway increase susceptibility to cancer. The identification of the Srs2/PARI helicases as key components in this pathway is a considerable step forward in this field.


      Reviewer #2

      __Evidence, reproducibility and clarity __

      The Aurora B-mediated abscission checkpoint ("NoCut" in yeast) prevents tetraploidization or chromatin breakage in the presence of chromatin bridges in cytokinesis and the mechanisms of its activation are a matter of active investigation. In the present study, Dam et al propose that the conserved Srs2/PARI DNA helicase is required for the activation of the abscission checkpoint in response to chromatin bridges generated by DNA replication stress or topoisomerase inhibition. This is a timely and very interesting topic and the potential identification of a novel regulatory protein that activates the abscission checkpoint would be important. However, in my opinion, some Figures are of relatively low quality and need improving, there are apparent discrepancies between data and important control experiments are missing, which preclude the reader from fully evaluating the conclusions of this study. Some direct evidence of the role of Srs2/PARI on DNA bridges is also required. Also, it would be nice to investigate mechanistic details of the potential Srs2/PARI functions in the abscission checkpoint, and how it fits with other recently published signaling pathways that activate the abscission checkpoint in cytokinesis.

      Specific comments: 1. The DNA channel (Ht2B-mCherry) in Figure 1A is of very low quality to be able to verify the authors interpretations of when the individual chromatin bridges are resolved (probably broken). For example, in the WT movie, they claim that the bridge is intact in frames 10 min and 14 min (yellow arrow) and that the bridge is resolved at 16 min (asterisk); however, I'm not convinced this is the case, because I can only see a very small portion of the bridge already at the 10 min and 14 min time-points. In my opinion, this bridge could have been broken much earlier, probably at 10 min. Also, WT +HU, is this bridge really intact at 10 min and at 14 min? In Srs2Δ + HU, the bridge appears broken to me much earlier, perhaps at 30 min. There is a distinct possibility that the authors could not calculate the resolution times accurately from these movies (please also see my next comment, #2). The authors could perhaps use a more sensitive bridge marker such as GFP-BAF.

      To clarify our approach, chromosome segregation was considered complete only when bridges were no longer detectable, while discontinuous or faint bridges were still classified as unresolved, as stretched DNA may result in weak nucleosome signals. This definition aligns with the bridge resolution times reported in Figure 1B-E. To improve clarity, we have revised the Results section to specify our classification criteria, and added all frames from the time-lapse movies in Figure 1A as a new figure (Supplementary Figure S1).

      In Figure 1B, they conclude that Srs2Δ cells treated with HU exhibit increased time from anaphase onset to bridge resolution compared with WT or Srs2Δ cells. This result appears at odds with data from Fig. 2C showing that Srs2Δ+HU finish abscission at similar times to WT or Srs2Δ cells as judged by plasma membrane morphology. (final cut). Given that the final cut of the plasma membrane should cause chromatin bridges to break, if Srs2 is required for an abscission delay in response to HU-induced chromatin bridges, I would expect Srs2Δ + HU cells to exhibit accelerated plasma membrane cut and also faster chromatin bridge resolution compared with controls. This discrepancy could at least in part be caused by the relatively low quality of movies used for the calculations in Fig. 1.

      This is a perceptive point. To clarify, we analyzed the timing of chromosome segregation, membrane ingression at the abscission site, and abscission relative to anaphase onset, as shown in the new Supplementary Figure S2. In HU-treated cells (both WT and srs2∆), bridge resolution and membrane ingression occur around the same time (~10 minutes after anaphase onset), with srs2∆ cells exhibiting slightly earlier membrane contraction. This suggests that bridges resolve during cytokinesis (see also our reply to the next comment) but does not distinguish whether they break prematurely or resolve normally. Our key finding is that membrane abscission is delayed in HU-treated cells in an Srs2-dependent manner, raising the question of whether this delay is important to prevent bridge breakage. This hypothesis is tested and supported by Figure 2D, where delaying cytokinesis (via cyk3∆) reveals the protective role of Srs2.

      Fig. 2 shows faster abscission times (membrane cut) in Srs2Δ+HU cells compared with WT+HU. The authors interpret this data as evidence for a role of Srs2 in abscission delay in response to HU-induced chromatin bridges (page 7 and elsewhere). However, there is no direct evidence that the cells analyzed in Fig.2 exhibited DNA bridges in cytokinesis. One could argue that HU-induced DNA replication stress caused DNA lesions at the nuclear chromatin, which affected completion of cytokinesis in the absence or presence of Srs2. What proportion of HU-treated cells in cytokinesis exhibit DNA bridges? Judging from Fig. 1D this could be as low as 0-20%. The authors should analyze HU-treated cells that clearly exhibit DNA bridges, either by live-cell imaging or in fixed cells experiments. As it stands and together with my previous comments #1 and 2, I'm not convinced this data fully supports a role for Srs2 in the abscission delay in response to HU-induced DNA bridges.

      We appreciate the reviewer's concern. The presence of chromatin bridges in HU-treated cells during cytokinesis (membrane ingression) is documented in the new Supplementary Figure S2, as noted in our response to the previous comment. Additionally, our previous study (Amaral 2016, PMID: 27111841, Figure 1D) demonstrated that under the same HU treatment conditions used here, >90% of wild-type cells exhibit chromatin bridges during cytokinesis. This strongly supports the conclusion that the effects observed in Figure 2 are linked to the presence of DNA bridges.

      In Fig. 2D, there is no evidence to support that Mre11 foci are caused by bridge breakage, and not by replication-stress induced DNA lesions at the main nucleus (no DNA bridge is evident, also see comment #3).

      The use of the cyk3 mutant in Figure 2D specifically addresses this concern. If Mre11 foci resulted from replication stress-induced lesions in the main nucleus, delaying cytokinesis should have no impact on damage levels. However, we observe that delaying cytokinesis via the cyk3 mutation significantly reduces Mre11 foci, strongly suggesting that these foci arise from chromatin bridge breakage rather than replication stress, and that delaying cytokinesis provides extra time to solve the chromosome segregation problem. This conclusion is further supported by previous studies showing that cyk3∆ delays cytokinesis (Amaral 2016, PMID: 27111841, Figure 2C; Onishi et al. 2013, PMID: 23878277). We have clarified this point in the revised text.

      Figure 3: the authors use a top2-4 mutant strain to generate DNA bridges from catenated DNA and investigate the potential role of Srs2 in the abscission delay. However, no DNA bridges are obvious in the cells shown in Fig. 3. What proportion of top2-4 mutant cells in cytokinesis exhibit DNA bridges? Does this explain the striking difference in the percentage of cells that haven't completed abscission after 30-60 min in WT+HU vs Top2-4 cells? Please also see my previous comments above.

      The top2-4 mutant is well-characterized, and under the conditions used here, 100% of cells exhibit DNA bridges during cytokinesis (see for example Amaral et al., 2016, Figure 3A). We have clarified this point in the revised text. Notably, previous work has shown that top2-4-induced bridges are thicker and more persistent than those caused by HU-induced replication stress. This difference might contribute to the more severe abscission defect observed in top2-4 cells, though we have not directly tested this.

      The authors propose that association of Srs2 with PCNA is required for complete inhibition of abscission in top2-4 mutant cells with chromatin bridges. Assuming a role for Srs2 in abscission timing in cytokinesis with chromatin bridges is fully proven, it is essential that the authors also investigate the localization of Srs2 and PCNA on chromatin bridges, using GFP-tagged proteins or appropriate antibodies in fixed and/or living cells. This would suggest a direct role of these proteins on chromatin bridges and considerably strengthen the authors hypothesis. Alternatively, Srs2 and PCNA may indirectly affect abscission timing through their well-established roles at nuclear chromatin.

      The perturbations used in Figure 4 have been previously shown to disrupt Srs2-PCNA and PCNA-chromatin interactions (Armstrong et al., 2012; Ayyagari et al., 1995; Johnson et al., 2016; Kubota et al., 2013), as referenced in our manuscript. Given this well-established evidence, we believe additional imaging experiments would be redundant. Moreover, we do not claim that Srs2 or PCNA must specifically localize to chromatin bridges for NoCut function. Instead, our data demonstrate their genetic requirement for abscission inhibition in the presence of bridges. Whether these proteins localize exclusively on bridges or more broadly on chromatin remains unresolved, a point we explicitly discuss in the manuscript.

      In Fig. 4D, the authors show an abscission delay in elg1Δ mutant cells in the presence of dicentric bridges compared with cytokinesis without bridges and interpret this as evidence that artificially retaining PCNA on dicentric chromatin bridges is sufficient to inhibit abscission. It is important that the authors demonstrate that PCNA localizes to dicentric bridges in elg1Δ mutant, but not in ELG1 control, cells, e.g., by immunofluorescence, to support their claim and their proposed model.

      As noted in our previous response, the association of PCNA with chromatin throughout the cell cycle and its regulation by Elg1 have been extensively characterized in prior studies. Given this established evidence, additional imaging experiments would be redundant.

      We also clarify that we do not claim that PCNA is specifically retained on chromatin bridges in elg1Δ mutants. Rather, our model is based on the overall retention of PCNA on chromatin in elg1Δ cells, as demonstrated in published studies.

      Notably, elg1Δ mutants without dicentric bridges retain PCNA on chromatin but do not exhibit delayed abscission. However, only elg1Δ mutants with chromatin bridges inhibit abscission, indicating that PCNA retention alone is not sufficient—it is the presence of a bridge with retained PCNA that is critical. This distinction has been clarified in the revised manuscript.

      In Fig. 5, the authors claim that HeLa cells treated with the Top2 inhibitor ICRF193 exhibit delayed midbody resolution compared with controls and that depletion of PARI by siRNA accelerates abscission in ICRF-treated cells. They interpret this as evidence for a role of PARI in the abscission delay in response to ICRF-induced chromatin bridges. However, no bridges are visible at any time-frame in cells in Fig. 5B raising the possibility that the observed time-differences are due to some effect of ICRF in cytokinesis without bridges. I'm also not convinced that in Fig. 5B the midbodies in NT/ICRF/230 min, siPARI/DMSO/110 min and siPARI/ICRF/150 min were resolved as indicated by the authors, as I can definitely see both midbody arms very clearly in these photos. The p-values are also just below the p

      We acknowledge that the chromatin bridges in Figure 5B are challenging to visualize and may appear discontinuous. This is not due to poor image quality but likely reflects the low chromatin density of these structures. To clarify this, we now include magnified and contrast-enhanced images to better highlight the bridges, and quantification in Fig. 5C. Additionally, in the revised manuscript, we will provide new images using GFP-BAF, which directly binds DNA, to more clearly demonstrate the presence of chromatin bridges in ICRF-treated cells. These data will confirm that most cytokinetic cells in ICRF-treated conditions exhibit bridges.

      Regarding the midbodies shown in Figure 5B, the presence of one or both arms intact does not indicate unresolved abscission but rather that the midbody has been severed, a distinction we explicitly describe in the manuscript.

      Concerning the statistical analysis, we note that the p-value threshold of 0.05 is a widely accepted convention for statistical significance, and we have applied it appropriately in our analysis.

      Finally, regarding the EM images in Figure 5C, these are single-section images, which do not allow us to determine definitively whether the bridges are physically broken when they appear discontinuous. It is possible that portions of the bridge extend outside the sectioned image. Regardless, we do not claim that these bridges are intact or broken. Rather, our key conclusion is that their presence at the abscission site in ICRF-treated cells is not affected by PARI knockdown, supporting our model.

      In Fig. 6, the authors examine actin patches in PARI-depleted and control cells as a marker of abscission. Although a role for PARI in actin patch formation would be very interesting, I'm not sure how it fits with the present story. The actin inside the intercellular canal described by Bai et al (removal of which correlates with abscission) appears very different to the accumulations of actin at the base of the intercellular canal described by Sreigemann et al and by Dandoulaki et al. I can definitely see actin patches (similar to the ones in Steigemann et al) in Fig. 6 NT/ICRF, but I can't see any at the other treatments (I disagree with the arrows). Incidentally, I can see a DNA bridge only in NT/ICRF, but not in the other treatments.

      We have revised our description of this figure for greater clarity. In control cells, actin accumulates at the cleavage furrow during anaphase and gradually disperses (clears) as cytokinesis progresses. We do not see patches in untreated cells, and we have updated the y-axis label in Figure 5B from “% of cells with actin patches” to “% of cells with actin clearance” to better reflect our observations.

      Actin patches were observed only in ICRF-193-treated cells and were often associated with chromatin bridges. Cells that successfully disassembled these actin patches were classified as having completed actin clearance. Our data indicate that PARI depletion increases the fraction of cells that clear chromatin from the division plane, facilitating actin patch disassembly.

      The actin patches observed in our study closely resemble those reported by Steigemann et al., and notably, we used the same cell line as in that study. Regarding Bai et al., they used both phalloidin and actin-GFP. For example, Figure 5C in Bai et al., shows examples of both actin patches near chromatin bridges, which resemble those in our study, and filamentous actin structures within the intercellular canal, which appear distinct.

      Finally, a bridge fragment lacking actin patches is visible in PARI knockdown cells treated with ICRF, and we have now highlighted this in the revised figure.

      1. Midbody resolutions are clearer in Fig. 7, perhaps with the exception of siPARI/DMSO. However, no DNA bridges are visible, raising again the possibility that the authors investigate effects in cytokinesis without DNA bridges.

      See our response to point 8: while bridges are difficult to visualize, our analysis confirms that ICRF treatment induces bridges that persist during cytokinesis.

      Can the authors investigate whether the helicase activity of PARI is required for the abscission checkpoint, by depletion-reconstitution experiments with a helicase-mutant protein?

      PARI lacks detectable Walker motifs and associated ATPase activity, suggesting PARI lacks helicase activity (Moldovan et al., 2012). Therefore, we have not pursued depletion-reconstitution experiments with a helicase-mutant protein.

      The authors should investigate localization of PARI to the midbody/ DNA bridge in cytokinesis with chromatin bridges. Recent reports have proposed that a Top2-MRN-ATM-Chk2 pathway activates the Aurora B-dependent abscission checkpoint in human cells (PMIDs: 37638884, 33355621). The authors should examine localization of Aurora B and some of the above proteins in control and PARI-deficient cells to establish if/how PARI fits in the above pathway.

      As noted in our manuscript, we attempted to visualize PARI at midbodies and DNA bridges but were unable to detect any signal. This could be due to either its absence in these regions or its low concentration, making detection challenging.

      We agree that investigating the Top2-MRN-ATM-Chk2 pathway in this context is important. We will examine the localization of key pathway components, including Aurora B, in control and PARI-deficient cells, and include the results in the revised manuscript.

      1. The authors use ICRF to generate chromatin bridges. If ICRF is continuously present in their assays, one would expect it to inhibit Top2 and impair the abscission checkpoint (PMIDs: 37638884, 33355621). How do the authors reconcile this with their proposed model?

      This is an important point. Studies from the Zachos lab have shown that Topoisomerase IIα-DNA covalent complexes (Top2ccs) accumulate near the midbody in cells with chromatin bridges and play a key role in initiating abscission checkpoint signaling by recruiting MRN, ATM, and Aurora B. Supporting this model, ICRF-193 treatment does not alter midbody disassembly timing in HeLa cells, as shown in Petsalaki et al., 2023 (Figure S4D).

      However, our results indicate that ICRF-193-treated HeLa cells exhibit delayed midbody severing, suggesting that at least some aspects of abscission checkpoint signaling remain active under these conditions. One possible explanation for this discrepancy is the difference in ICRF-193 concentration: our study uses a low dose (250 nM) versus 10 µM in the Zachos group study. We favor the hypothesis that this lower dose preserves sufficient Top2 activity to support some level of checkpoint signaling while still effectively generating chromatin bridges.

      Additional comments:

      Page 8: "Although SIM-defective Srs2 has a lower affinity to SUMOylated PCNA, it can still interact with PCNA". The authors should test this experimentally or provide appropriate references supporting this claim.

      We have clarified our statement and provided the reference: Although SIM-defective Srs2 has a lower affinity to SUMOylated PCNA, it can still interact with non-SUMOylated PCNA (Armstrong et al. 2012).

      1. Page 6: "Deletion of SRS2 further increased the fraction of anaphase cells with RPA foci, rising to approximately 30% in the absence of HU..."; however, this rise was not statistically significant as indicated in Fig. 1C.

      Thank you for noting this - we have removed this statement.

      Fig. 1C, D: SDs are missing. Fig. 1E: please show the p-values.

      These data in Figures 1C-D represent percentages from cells pooled from two independent experiments with similar results. P-values were calculated using Dunn’s multiple comparison test. Standard deviations are not applicable in this case. We have included the p-values for Figure 1E.

      Fig. 2D: please show SDs and individual values.

      These data represent percentages from cells pooled from independent experiments with similar results. P-values were calculated using Fisher’s exact test. Standard deviations and individual values are not applicable in this case.

      1. Why do the authors show the spindle pole body in their movies?

      We do this to infer the time of anaphase onset; see our response to points 1-3 and Fig. S2.

      Fig. 4A: WT and top2-4 cells have the same symbol in the graph.

      We have changed the symbols.

      Significance

      Strengths: potentially novel regulator of the abscission checkpoint. Timely and interesting topic of broad scientific interest.

      Limitations: problems with quality of some data and withy the interpretation. Also, more mechanistic evidence is required to significantly advance our knowledge in the field.

      Reviewer #3

      Evidence, reproducibility and clarity:

      Summary: Building on the specific connection between DNA bridges that bear marks of replication stress and the NoCut checkpoint (Amaral 2016, 2017), which prevents completion of cytokinesis, Dam et al. test the helicase Srs2/PARI for a role in this checkpoint pathway. The authors have produced a thorough study investigating the role of this helicase in both yeast and mammalian cells in the presence of DNA bridges. The manuscript includes clear evidence that Srs2 is important to resolve chromatin bridges, remove replication protein A (RPA) from chromatin, and delay cytokinesis under replication stress. Further, the authors show that loss of Srs2 under replication stress increases DNA damage, marked by elevated MRE11 foci in a manner dependent on cytokinesis (i.e., dependent on Cyk3). Srs2 deletion also partially abrogates the abscission delay seen upon topo-II inactivation. They further report that Srs2 must interact with PCNA to delay abscission in S. cerevisiae. While chromatin bridges formed when a dicentric chromosome is present escape detection by the NoCut checkpoint, inactivation of Elg1, which unloads PCNA and associated factors following DNA replication, results in delayed abscission. In HeLa cells, the Srs2 ortholog PARI is shown to similarly help promote abscission delay in the presence of DNA bridges following topoisomerase inhibition, as loss of PARI through siRNA knockdown prevents this abscission delay. Mechanistically, when PARI levels are reduced in HeLa cells, actin patches that function to stabilize the midbody and protect DNA bridges do not form/persist robustly as in cells with intact PARI. Consistent with a specific role in sensing the presence of a DNA bridge, depletion of PARI did not impact abscission checkpoint activity in response to depletion of the NPC component, Nup153. Finally, the authors show that PARI depletion reduced time to abscission to the same extent as treatment with an Aurora B inhibitor, and PARI depletion in conjunction with Aurora B inhibition did not reduce abscission timing further than singular treatments, suggesting that PARI works within the Aurora B-mediated NoCut signaling cascade.

      Major comments: The manuscript is well written and, in general, the conclusions are thoroughly supported, but there are a few recommendations for addition or revision.

      1. The first of these is for a more thorough introduction of helicases potentially involved in cytokinesis and more clear rationale for why the focus is on Srs2.

      We appreciate the reviewer’s suggestion and have expanded the introduction to better contextualize helicases in cytokinesis and clarify our focus on Srs2.

      Figure 1 E lacks statistical analysis. In addition, the text referring to 1E leads to confusion because the distinction between "RPA foci during anaphase" and "RPA coated chromatin bridges" is not made clear. The authors should clarify that the data presented in 1E shows quantification of cells with RPA foci during anaphase, not RPA coated chromatin bridges, and use consistent wording between the text and figure/figure legend. Further, how cells with RPA foci were identified, and what is classified as an RPA focus from images should be described in the methods.

      We appreciate the reviewer’s feedback. In the revised manuscript, we have included statistical analysis for Figure 1E and clarified the distinction between "RPA foci during anaphase" and "RPA-coated chromatin bridges" to ensure consistency. Additionally, we have updated the Methods section to specify how cells with RPA foci were identified and what criteria were used to classify RPA foci based on the imaging data.

      In some cases, it is unclear whether DNA bridge formation is prevented vs aberrantly broken. For example, under Top2 inactivation, does the absence of Srs2 prevent bridge formation or promote their breakage along with premature midbody abscission? Confirming the frequency of chromatin bridge formation would address this and, further, monitoring RPA persistence would validate whether RPA clearance from bridges is consistently correlated with Srs2 activity (an interesting observation from Figure 1 that is not followed up on). Similarly, other conditions that appear to interfere with abscission delay (e.g., disrupting Srs2-PCNA interaction) should be monitored for whether the formation of DNA bridges has been altered.

      We agree this is important and will address it in a full revision. We will quantify chromatin bridge formation under Top2 inactivation to determine whether Srs2 mutations affect bridge frequency or stability. Additionally, we will monitor RPA persistence in top2 cells to assess whether RPA clearance correlates with Srs2 activity. While we find it unlikely that bridge formation is prevented by srs2 mutations, as Top2 is essential for decatenation, our experiments will directly test this possibility.

      In Figure 4A, the data show that the PIP-box is required for timely abscission. Imaging data from yeast strains with the PIP-box deletion alone should be included, rather than only showing the deletion in combination with the SIM deletion.

      We agree with the reviewer’s suggestion, and will include imaging data from yeast strains with the PIP-box deletion alone in the revised manuscript.

      While the authors state that PARI and PCNA were not detectable at bridges in mammalian cells, it would be worth examining whether RPA is persistent on DNA bridges in mammalian cells depleted of PARI to understand how closely this pathway resembles the features found in yeast.

      Here too, we agree with the reviewer’s suggestion, and will include imaging data from HeLa cells visualizing RPA in the revised manuscript.

      In Figure 6, the authors should describe in the methods how cells with actin patches were identified and quantified and explain what criteria must be met to be identified as an actin patch. Actin patches were described as "disassembling more quickly" in PARI-depleted cells, but the images look as if actin patches are not forming properly in these cells. The images are crisp and clear, but a change in wording may be necessary to accurately describe the data.

      Thank you for pointing this out. We agree that the wording was confusing (see our reply to reviewer 2, comment 9) and have revised our description of this figure for greater clarity. In control cells, actin accumulates at the cleavage furrow during anaphase and gradually disperses (clears) as cytokinesis progresses. We do not see patches in untreated cells, and we have updated the y-axis label in Figure 5B from “% of cells with actin patches” to “% of cells with actin clearance” to better reflect our observations. Actin patches were observed only in ICRF-193-treated cells and were often associated with chromatin bridges. Cells that successfully disassembled these actin patches were classified as having completed actin clearance. Our data indicate that PARI depletion increases the fraction of cells that clear chromatin from the division plane, facilitating actin patch disassembly.

      Minor suggestions to improve the manuscript are:

      Include a diagram that shows hallmarks of cell division and what is being tracked in particular assays (e.g., DNA bridge duration vs time to abscission).

      Thank you for this suggestion, which we have implemented in Figure S2A.

      In the elegant CLEM experiments presented in Figure 5, organelle labels could be added to orient the readers.

      We added organelle labels to CLEM images.

      The data in supplemental Figure 2 should be moved to Figure 5. The fact that there are similar levels of chromatin bridges is vital information and stresses that the defect lies in detection and response to the bridge as opposed to formation of bridges when PARI is depleted.

      We agree, and have moved Figure S2 to Figure 5 (now Figure 5C).

      Significance

      The link between DNA bridges and NoCut/abscission checkpoint signaling is a fundamental aspect of cell cycle regulation. This manuscript makes a significant contribution to our understanding of this pathway by introducing a novel role for the helicase Srs2/PARI in execution of an abscission delay in the presence of DNA bridges. This is an important contribution as there is sparse information about cellular factors that mediate detection and response to DNA bridges, which is vital to protecting genome integrity. Although, as the authors themselves state, "the molecular mechanisms by which Srs2 and PARI function in NoCut remain unclear," this study, with some revisions, merits publication as it reveals a conserved role for a factor in this important response pathway and provides new insights into why certain DNA bridges (i.e., bridges formed by dicentric chromosomes) are not recognized by the NoCut pathway.

    1. Author response:

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

      Public Reviews:

      Reviewer #1 (Public review): 

      Hotinger et al. explore the population dynamics of Salmonella enterica serovar Typhimurium in mice using genetically tagged bacteria. In addition to physiological observations, pathology assessments, and CFU measurements, the study emphasizes quantifying host bottleneck sizes that limit Salmonella colonization and dissemination. The authors also investigate the genetic distances between bacterial populations at various infection sites within the host.

      Initially, the study confirms that pretreatment with the antibiotic streptomycin before inoculation via orogastric gavage increases the bacterial burden in the gastrointestinal (GI) tract, leading to more severe symptoms and heightened fecal shedding of bacteria. This pretreatment also significantly reduces between-animal variation in bacterial burden and fecal shedding. The authors then calculate founding population sizes across different organs, discovering a severe bottleneck in the intestine, with founding populations reduced by approximately 10^6-fold compared to the inoculum size. Streptomycin pretreatment increases the founding population size and bacterial replication in the GI tract. Moreover, by calculating genetic distances between populations, the authors demonstrate that, in untreated mice, Salmonella populations within the GI tract are genetically dissimilar, suggesting limited exchange between colonization sites. In contrast, streptomycin pretreatment reduces genetic distances, indicating increased exchange.

      In extraintestinal organs, the bacterial burden is generally not substantially increased by streptomycin pretreatment, with significant differences observed only in the mesenteric lymph nodes and bile. However, the founding population sizes in these organs are increased. By comparing genetic distances between organs, the authors provide evidence that subpopulations colonizing extraintestinal organs diverge early after infection from those in the GI tract. This hypothesis is further tested by measuring bacterial burden and founding population sizes in the liver and GI tract at 5 and 120 hours post-infection. Additionally, they compare orogastric gavage infection with the less injurious method of infection via drinking, finding similar results for CFUs, founding populations, and genetic distances. These results argue against injuries during gavage as a route of direct infection. 

      To bypass bottlenecks associated with the GI tract, the authors compare intravenous (IV) and intraperitoneal (IP) routes of infection. They find approximately a 10-fold increase in bacterial burden and founding population size in immune-rich organs with IV/IP routes compared to orogastric gavage in streptomycin-pretreated animals. This difference is interpreted as a result of "extra steps required to reach systemic organs."

      While IP and IV routes yield similar results in immune-rich organs, IP infections lead to higher bacterial burdens in nearby sites, such as the pancreas, adipose tissue, and intraperitoneal wash, as well as somewhat increased founding population sizes. The authors correlate these findings with the presence of white lesions in adipose tissue. Genetic distance comparisons reveal that, apart from the spleen and liver, IP infections lead to genetically distinct populations in infected organs, whereas IV infections generally result in higher genetic similarity. 

      Finally, the authors investigate GI tract reseeding, identifying two distinct routes. They observe that the GI tracts of IP/IV-infected mice are colonized either by a clonal or a diversely tagged bacterial population. In clonally reseeded animals, the genetic distance within the GI tract is very low (often zero) compared to the bile population, which is predominantly clonal or pauciclonal. These animals also display pathological signs, such as cloudy/hardened bile and increased bacterial burden, leading the authors to conclude that the GI tract was reseeded by bacteria from the gallbladder bile. In contrast, animals reseeded by more complex bacterial populations show that bile contributes only a minor fraction of the tags. Given the large founding population size in these animals' GI tracts, which is larger than in orogastrically infected animals, the authors suggest a highly permissive second reseeding route, largely independent of bile. They speculate that this route may involve a reversal of known mechanisms that the pathogen uses to escape from the intestine. 

      The manuscript presents a substantial body of work that offers a meticulously detailed understanding of the population dynamics of S. Typhimurium in mice. It quantifies the processes shaping the within-host dynamics of this pathogen and provides new insights into its spread, including previously unrecognized dissemination routes. The methodology is appropriate and carefully executed, and the manuscript is well-written, clearly presented, and concise. The authors' conclusions are well-supported by experimental results and thoroughly discussed. This work underscores the power of using highly diverse barcoded pathogens to uncover the within-host population dynamics of infections and will likely inspire further investigations into the molecular mechanisms underlying the bottlenecks and dissemination routes described here.

      Major point:

      Substantial conclusions in the manuscript rely on genetic distance measurements using the Cavalli-Sforza chord distance. However, it is unclear whether these genetic distance measurements are independent of the founding population size. I would anticipate that in populations with larger founding population sizes, where the relative tag frequencies are closer to those in the inoculum, the genetic distances would appear smaller compared to populations with smaller founding sizes independent of their actual relatedness. This potential dependency could have implications for the interpretation of findings, such as those in Figures 2B and 2D, where antibiotic-pretreated animals consistently exhibit higher founding population sizes and smaller genetic distances compared to untreated animals.

      Thank you for raising this important point regarding reliance on cord distances for gauging genetic distance in barcoded populations. The reviewer is correct that samples with more founders will be more similar to the inoculum and thus inherently more similar to other samples that also have more founders. However, creation of libraries containing very large numbers of unique barcodes can often circumvent this issue. In this case, the effect size of chance-based similarity is not large enough to change the interpretation of the data in Figures 2B and 2D. In our case, the library has ~6x10<sup>4</sup> barcodes, and the founding populations in Figure 2B are ~10<sup>3</sup>. Randomly resampling to create two populations of 10<sup>3</sup> cells from an initial population with 6x10<sup>4</sup> barcodes is expected to yield largely distinct populations with very little similarity. Thus, the similarity between streptomycin-treated populations in Figure 2D is likely the result of biology rather than chance.  

      Reviewer #2 (Public review):

      In this paper, Hotinger et. al. propose an improved barcoded library system, called STAMPR, to study Salmonella population dynamics during infection. Using this system, the authors demonstrate significant diversity in the colonization of different Salmonella clones (defined by the presence of different barcodes) not only across different organs (liver, spleen, adipose tissues, pancreas, and gall bladder) but also within different compartments of the same gastrointestinal tissue. Additionally, this system revealed that microbiota competition is the major bottleneck in Salmonella intestinal colonization, which can be mitigated by streptomycin treatment. However, this has been demonstrated previously in numerous publications. They also show that there was minimal sharing between populations found in the intestine and those in the other organs. Upon IV and IP infection to bypass the intestinal bottleneck, they were able to demonstrate, using this library, that Salmonella can renter the intestine through two possible routes. One route is essentially the reverse path used to escape the gut, leading to a diverse intestinal population; while the other, through the bile, typically results in a clonal population. Although the authors showed that the STAMPR pipeline improved the ability to identify founder populations and their diversity within the same animal during infections, some of the conclusions appear speculative and not fully supported.

      (1) It's particularly interesting how the authors, using this system, demonstrate the dominant role of the microbiota bottleneck in Salmonella colonization and how it is widened by antibiotic treatment (Figure 1). Additionally, the ability to track Salmonella reseeding of the gut from other organs starting with IV and IP injections of the pathogen provides a new tool to study population dynamics (Figure 5). However, I don't think it is possible to argue that the proximal and distal small intestine, Peyer's patches (PPs), cecum, colon, and feces have different founder populations for reasons other than stochastic variations. All the barcoded Salmonella clones have the same fitness and the fact that some are found or expanded in one region of the gastrointestinal tract rather than another likely results from random chance - such as being forced in a specific region of the gut for physical or spatial reasons-and subsequent expansion, rather than any inherent biological cause. For example, some bacteria may randomly adhere to the mucus, some may swim toward the epithelial layer, while others remain in the lumen; all will proliferate in those respective sites. In this way, different founder populations arise based on random localization during movement through the gastrointestinal tract, which is an observation, but it doesn't significantly contribute to understanding pathogen colonization dynamics or pathogenesis. Therefore, I would suggest placing less emphasis on describing these differences or better discussing this aspect, especially in the context of the gastrointestinal tract.

      Thank you for helping us identify this area for further clarification. We agree with the reviewer’s interpretation that seeding of proximal and distal small intestine, Peyer's patches (PPs), cecum, colon, and feces with different founder populations is likely caused by stochastic variations, consistent with separate stochastic bottlenecks to establishing these separate niches. To clarify this point we have modified the text in the results section, “Streptomycin treatment decreases compartmentalization of S. Typhimurium populations within the intestine”.

      Change to text:

      “Except for the cecum and colon, in untreated animals the S. Typhimurium populations in different regions of the intestine were dissimilar (Avg. GD ranged from 0.369 to 0.729, 2D left); i.e., there is little sharing between populations in the intestine. These data suggest that there are separate bottlenecks in different regions of the intestine that cause stochastic differences in the identity of the founders. Interestingly, when these founders replicate, they do not mix, remaining compartmentalized with little sharing between populations throughout the intestinal tract (i.e., barcodes found in one region are not in other regions, Figure S3). This was surprising as the luminal contents, an environment presumably conducive to bacterial movement, were not removed from these samples.”

      In this section we are interested in the underlying biology that occurs after the initial bottleneck to preserve this compartmentalization during outgrowth of the intestinal population. In other words, what prevents these separate populations from merging (e.g., what prevents the bacteria replicating in the proximal small intestine from traveling through the intestine and establishing a niche in the distal small intestine)? While we do not explore the mechanisms of compartmentalization, we observe that it is disrupted by streptomycin pretreatment, suggesting a microbiota-dependent biological cause. 

      (2) I do think that STAMPR is useful for studying the dynamics of pathogen spread to organs where Salmonella likely resides intracellularly (Figure 3). The observation that the liver is colonized by an early intestinal population, which continues to proliferate at a steady rate throughout the infection, is very interesting and may be due to the unique nature of the organ compared to the mucosal environment. What is the biological relevance during infection? Do the authors observe the same pattern (Figures 3C and G) when normalizing the population data for the spleen and mesenteric lymph nodes (mLN)? If not, what do the authors think is driving this different distribution?

      Thank you for raising this interesting point. These data indicate that the liver is seeded from the intestine early during infection. The timing and source of dissemination have relevance for understanding how host and pathogen variables control the spread of bacteria to systemic sites. For example, our conclusion (early dissemination) indicates that the immune state of a host at the time of exposure to a pathogen, and for a short period thereafter, are what primarily influence the process of dissemination, not the later response to an active infection. 

      We observe that the liver and mucosal environments within the intestine have similar colonization behaviors. Both niches are seeded early during infection, followed by steady pathogen proliferation and compartmentalization that apparently inhibits further seeding. This results in the identity of barcodes in the liver population remaining distinct from the intestinal populations, and the intestinal populations remaining distinct from each other.

      We observe a similar pattern to the liver in the spleen and MLN (the barcodes in the spleen and MLN are dissimilar to the population in the intestine). To clarify this point, we have modified the text (below) and added this analysis as a supplemental figure (S4).

      Change to text:

      Genetic distance comparison of liver samples to other sites revealed that, regardless of streptomycin treatment, there was very little sharing of barcodes between the intestine and extraintestinal sites (Avg. GD >0.75, Figure 3C). Furthermore, the MLN and spleen populations also lacked similarity with the intestine (Figure S4). These analyses strongly support the idea that S. Typhimurium disseminates to extraintestinal organs relatively early following inoculation, before it establishes a replicative niche in the intestine.

      (3) Figure 6: Could the bile pathology be due to increased general bacterial translocation rather than Salmonella colonization specifically? Did the authors check for the presence of other bacteria (potentially also proliferating) in the bile? Do the authors know whether Salmonella's metabolic activity in the bile could be responsible for gallbladder pathology?

      The reviewer raises interesting points for future work. We did not check whether other bacterial species are translocating during S. Typhimurium infection. The relevance of Salmonella’s metabolic activity is also very interesting, and we hope these questions will be answered by future studies.

      Recommendations for the authors:

      Reviewer #1 (Recommendations for the authors):

      Minor points:

      (1) P. 9/10 "... the marked delay in shedding after IP and IV relative to orogastric inoculation suggest that the S. Typhimurium population encounters substantial bottleneck(s) on the route(s) from extraintestinal sites back to the intestine.": Can you conclude that from the data? It could also be possible that there is a biological mechanism (other than chance events) that delays the re-entry to the intestine.

      We propose that the delay in shedding indicates additional obstacles that bacteria face when re-entering the intestine, and that there are likely biological mechanisms that cause this delay. However, these unknown mechanisms effectively act as additional bottlenecks by causing a stochastic loss of population diversity. 

      (2) P. 11 "...both organs would likely contain all 10 barcodes. In contrast, a library with 10,000 barcodes can be used to distinguish between a bottleneck resulting in Ns = 1,000 and Ns = 10,000, since these bottlenecks result in a different number of barcodes in output samples. Furthermore, high diversity libraries reduce the likelihood that two tissue samples share the same barcode(s) due to random chance, enabling more accurate quantification of bacterial dissemination.": I agree with the general analysis, but I find it misleading to talk about the presence of barcodes when the analyses in this manuscript are based on the much more powerful comparison of relative abundance of individual tags instead of their presence or absence.

      The reviewer raises an excellent point, and the distinction between relative abundance versus presence/absence is discussed extensively in the original STAMPR manuscript. Although relative abundance is powerful, the primary metric used in this study (Ns) is calculated principally from the number of barcodes, corrected (via simulations) for the probability of observing the same barcode across distinct founders. Although this correction procedure does rely on barcode abundance, the primary driver of founding population quantification is the number of barcodes.

      (3) P.14 "the library in LB supplemented with SM was not significantly different than the parent strain" and Figure 2C: How was significance tested? How many times were the growth curves recorded? On my print-out, the red color has different shades for different growth curves.

      Significance was tested with a Mann-Whitney and growth curves were performed 5 times. Growth curves are displayed with 50% opacity, and as a result multiple curves directly on top of each other appear darker. The legend to S2 has been modified accordingly.

      (4) P.16: close bracket in the equation for FRD calculation.

      Done

      (5) Figure 2C "Average CFU per founder": I found the wording confusing at first as I thought you divided the average bacterial burden per organ by Ns, instead of averaging the CFU/Ns calculated for each mouse.

      The wording has been clarified. 

      (6) Figure 3B: It would be helpful to include expected genetic distances in the schematic as it is difficult to infer the genetic distance when only two of three, respectively, different "barcode colors" are used. While I find the explanation in the main text intuitive, a graphical representation would have helped me.

      Thank you for the suggestion. Unfortunately, using colors to represent barcodes is imperfect and limits the diversity that can be depicted. We have modified Figure 3B to further clarify. 

      (7) Figure 3C: Why do you compare the genetic distance to the liver, when you discuss the genetic distance of the intestinal population? Is it not possible that the intestinal populations are similar to the extraintestinal organs except the liver?

      For clarity, we chose to highlight exclusively the liver. However, we observed a similar pattern to the liver in other extraintestinal organs. To clarify the generalizability of this point we have added a supplemental figure with comparisons to MLN and Spleen (Supplemental figure S4) as well as further text.

      (8) Figure 3C & S5A: I found "+SM" and "+SM, Drinking" confusing and would have preferred "+SM, Gavage" and "+SM, Drinking" for clarity.

      Done, thank you for the suggestion.

      (9) Figure 3G&H: I find it worthy of discussion that the bacterial burden increases over time, while the founding population decreases. Does that not indicate that replication only occurs at specific sites leading to the amplification of only a few barcodes and thereby a larger change of the relative barcode abundance compared to the inoculum?

      From 5h to 120h the size of the founding population decreases in multiple intestinal sites. This likely indicates that the impact of the initial bottleneck is still ongoing at 5h, although further temporal analysis would be required to define the exact timing of the bottleneck. Notably, the passage time through the mouse intestine is ~5h. Many of the founders observed at 5h could be a population that will never establish a replicative niche, and failing to colonize be shed in the feces, bottlenecking the population between 5h and 120h. To clarify this point we have added the following text:

      Section “S. Typhimurium disseminates out of the intestine before establishing an intestinal replicative niche”.

      “In contrast to the liver, there were more founders present in samples from the intestine (particularly in the colon) at 5 hours versus 120 hours (Figure 3H). These data likely indicate that many of the founders observed in the intestine at 5 hours are shed in the feces prior to establishing a replicative niche, and demonstrates that the forces restricting the S. Typhimurium population in the intestine act over a period of > 5 hours.”  

      (10) Figure S2A: I do not understand this figure. Why are there more than 70.000 tags listed? I was under the impression the barcode library in S. Typhimurium had 55.000 tags while only the plasmid pSM1 had more than 70.000 (but the plasmid should not be relevant here). Why are there distinct lines at approximately 10^-5 and a bit lower? I would have expected continuously distributed barcode frequencies.

      During barcode analysis, each library is mapped to the total barcode list in the barcode donor pSM1, which contains ~70,000 barcodes. This enables consistent analysis across different bacterial libraries. The designation “barcode number” refers to the barcode number in pSM1, meaning many of the barcodes in the Salmonella library are at zero reads. This graph type was chosen to show there was no bias toward a particular barcode, however there is significant overlap of the points, making individual barcode frequencies difficult to see. We have changed the x-axis to state “pSM1 Barcode Number” and clarified in the figure legend.

      Since the y-axes on these graphs is on a log10 scale, the lines represent barcodes with 1 read, 2 reads, 3 reads, etc. As the number of reads per barcode increases linearly, the space between them decreases on logarithmic axes.

      (11) There are a few typos in the figure legends of the supplementary material. For example Figure S2: S. Typhimurium not italicized, ~7x105 no superscript. Fig. S4&5 ", Open circles" is "O" is capitalized.

      Typos have been corrected.

    1. Voici un document de synthèse pour un briefing, basé sur les informations de la transcription de la vidéo "Research Seminar - A child is a person - an introduction to Sudbury Valley School, M. Vahid" et notre conversation précédente.

      Ce document met en lumière une approche alternative de l'éducation, contrastant avec les modèles traditionnels, et propose des pistes de réflexion pour une transformation éducative centrée sur l'enfant.

      Titre : L'Éducation Centrée sur l'Enfant : L'Approche de Sudbury Valley School

      Introduction

      • L'objectif principal est d'explorer une approche éducative qui place l'enfant au centre de son propre apprentissage, en le considérant comme une personne à part entière, digne de respect et de confiance.
      • L'approche de Sudbury Valley School remet en question les fondements de l'éducation traditionnelle, en prônant la liberté, l'autonomie et la responsabilité de l'apprenant.

      Principes Fondamentaux de Sudbury Valley School

      • L'enfant est une personne : L'idée centrale est que chaque enfant est un individu unique, avec ses propres besoins et aspirations, et qu'il doit être traité avec le même respect que tout autre être humain.
      • Respect absolu : Tous les membres de la communauté scolaire (enfants et adultes) sont égaux et ont une voix égale dans la prise de décision.
      • Absence de programme imposé : Il n'y a pas de programme scolaire standard, de notes, de classes d'âge, ou d'évaluations externes. Les enfants sont libres de choisir ce qu'ils veulent apprendre, quand et comment.
      • Apprentissage auto-dirigé : L'initiative de l'apprentissage vient de l'enfant lui-même. Les adultes sont là pour soutenir, guider et répondre aux demandes, mais ne forcent pas l'apprentissage.
      • Communauté démocratique : L'école fonctionne comme une démocratie où chaque membre a le droit de vote et participe aux décisions concernant tous les aspects de la vie scolaire.
      • Responsabilité personnelle : Les enfants sont responsables de leurs propres actions, de leurs décisions, et des conséquences qui en découlent.
      • L'importance du jeu et de l'exploration : L'école offre un environnement où les enfants peuvent explorer le monde à leur propre rythme et selon leurs propres intérêts, en prenant des risques calculés.

      Fonctionnement de l'École

      • Réunions scolaires : Toutes les décisions sont prises lors de réunions auxquelles tous les membres de la communauté scolaire participent et ont un vote.
      • Absence de structure traditionnelle : Pas de cours, de devoirs, de notes, d'examens, ni de séparation des élèves par âge ou niveau.
      • Rôle des adultes : Les adultes sont là pour fournir du soutien, des conseils et des ressources si on leur demande, mais ils ne sont pas là pour imposer un programme d'apprentissage.
      • Apprentissage par l'expérience : Les enfants apprennent en vivant dans un environnement stimulant et en interagissant avec les autres.
      • Gestion des conflits : Les conflits sont gérés par le biais du système judiciaire de l'école, où les enfants apprennent à résoudre leurs problèmes par eux-mêmes.
      • Pas d'évaluation standardisée : L'évaluation est inhérente au processus d'apprentissage lui-même. Les enfants évaluent leur propre progression en fonction de leurs objectifs personnels.

      Réactions et Défis

      • Questions fréquentes : Les gens se demandent souvent comment les enfants apprennent sans programme, comment ils sont évalués, et s'ils sont préparés à la vie adulte.
      • Idée fausse sur la paresse : L'idée que les enfants deviendront paresseux s'ils ne sont pas forcés d'apprendre est fausse. Les enfants sont naturellement curieux et actifs.
      • Préjugés sur la sécurité : Il est essentiel de différencier la sécurité de l'enfant et la responsabilité de son éducation. Les adultes interviennent pour assurer la sécurité, mais laissent les enfants gérer leur propre apprentissage.
      • Défis de l'évaluation : Évaluer les résultats de cette approche est complexe, car les compétences développées sont souvent qualitatives et individuelles. Il n'y a pas d'évaluation externe mais une auto-évaluation continue.

      Les Résultats et leur Interprétation

      • Développement de l'autonomie : Les élèves développent une forte capacité à s'auto-diriger, à prendre des initiatives, et à résoudre des problèmes.
      • Préparation à la vie adulte : Les diplômés de Sudbury Valley School sont généralement bien préparés à la vie adulte, car ils ont appris à se connaître, à identifier leurs forces et leurs faiblesses, et à s'adapter aux défis de la vie. Ils sont aptes à définir leurs objectifs, à identifier les étapes pour atteindre ces objectifs et à apprendre ce qui est nécessaire pour y parvenir.
      • Diversité des parcours : Les diplômés de Sudbury Valley School suivent des parcours professionnels très variés, certains allant à l'université, d'autres choisissant des voies alternatives.
      • Études et recherches : Bien qu'il existe peu d'études indépendantes sur Sudbury Valley School, les travaux de Peter Gray montrent que les enfants qui suivent des approches d'apprentissage auto-dirigé réussissent aussi bien que ceux qui sont scolarisés.

      Conclusion

      • Sudbury Valley School propose un modèle d'éducation radicalement différent, basé sur le respect de l'enfant et la confiance en sa capacité à apprendre.
      • Cette approche met l'accent sur l'apprentissage auto-dirigé, la responsabilité personnelle et la participation démocratique à la vie scolaire.
      • Bien que cette approche puisse être déroutante pour ceux qui sont habitués aux modèles d'éducation traditionnels, elle offre une perspective intéressante sur la manière d'éduquer les enfants pour un avenir incertain.
      • Il est essentiel de continuer à explorer ces alternatives pour construire un système éducatif plus adapté aux besoins des enfants et aux défis du monde contemporain.

      Ce document de synthèse met en évidence une approche éducative alternative, qui pourrait inspirer des réflexions sur les transformations à opérer dans le système éducatif pour le rendre plus centré sur l'enfant et sur ses besoins uniques.

      Il souligne l'importance d'une approche collaborative et inclusive pour construire un avenir éducatif plus durable et souhaitable pour tous.

    1. il y a un autre exemple à très vite qui est qui est assez remarquable aussi en France le lobby de Poissy qui a été couvert par une série de vidéos qui a été faite par Elsa Elsa Costa Granger là c'est l'histoire de petit enfant CM2 qui se qui s'intéressent à la terre et puis qui découvre ce qu'on les problèmes posés par les activités humaines donc qui font une BD sur la question cette BD remporte un prix alors ils vont rencontrer Hubert Reeves là-dessus ils vont franchir encore une étape ils vont rencontrer Nicolas Hulot et puis encore une étape et ils finissent par écrire une charte des droits de la planète et il présente et c'est la photo ici alors entre temps ils ont grandi ils sont deux ils sont ils sont montés en 6ème et puis après pour continuer leur travail ils ont créé une junior entreprise pour pouvoir se retrouver et continuer leur travail après les cours et cette photo montre la rencontre où représente leur charte de des droits de la planète à Ursula Vanden à au Parlement européen donc c'est quand même une histoire exceptionnelle surtout quand on sait que ils sont issus d'une d'une raie et que c'était pas du tout le destin qui leur était prévu donc c'est ça fait aussi ça fait aussi partie des éléments essentiels c'est que c'est possible pour tout le monde et en particulier dans les endroits les plus défavorisés comme je crois on vient de le voir sur l'ensemble des éléments il peut se passer des choses absolument partout et c'est ça qui est qui est important
    1. webinaire du Learning Planet Institute présente une discussion autour de l’« apprenance », concept central exploré à travers six citations de penseurs variés.

      L’objectif est de relier le désengagement à l’apprenance, en examinant les facteurs influençant l’engagement dans l’apprentissage, notamment chez les publics fragilisés.

      La discussion explore des thèmes tels que le rôle de la reconnaissance sociale, l’autodétermination (besoins d’autonomie, compétence et appartenance), la prise en compte des enjeux, ressources et contraintes de l’apprenant, et la posture du formateur comme accompagnateur plutôt que simple transmetteur de savoir.

      Finalement, un outil numérique, « Learning Coach », est présenté comme une solution pour soutenir l’autonomie et la performance de l’apprenant.

      Voici un document de synthèse pour un briefing, basé sur les informations de la transcription du webinaire "Chercheurs d'or #1" , en mettant l'accent sur les points clés :

      Titre : Convictions pédagogiques : du désengagement à l'apprenance

      Introduction

      • Le Learning Planet Institute est un institut de recherche pluridisciplinaire axé sur les pédagogies actives et la recherche participative.
      • Le webinaire "Chercheurs d'or" a pour but de créer un espace d'échange entre chercheurs et praticiens afin de partager des savoirs et de discuter des défis rencontrés dans l'accompagnement de l'apprentissage.
      • Le séminaire se concentre sur l'engagement dans les processus d'apprentissage, en particulier chez les personnes en situation de vulnérabilité.

      Problématique centrale

      • Comment passer du désengagement à l'apprenance, en particulier chez les personnes en situation non désirée, ayant des blocages liés à des expériences passées ou un manque de confiance en soi ?
      • Le désengagement est défini comme le fait de quitter une formation, tandis que le non-engagement est le fait de ne pas s'engager dans une formation. Les deux sont liés à la motivation et à la capacité de maintenir son intention.
      • L'apprenance est définie comme un ensemble de dispositions favorables à l'acte d'apprendre.

      Définition et composantes de l'apprenance

      • L'apprenance est composée de facteurs :
        • Affectifs (ce que j'aime ou pas apprendre)
        • Cognitifs (l'importance de l'apprentissage tout au long de la vie)
        • Conatifs (l'engagement et la capacité à saisir les opportunités d'apprentissage)
      • L'apprenance est un facteur interne qui favorise l'engagement et inhibe le désengagement, mais des facteurs externes (comme la distance, le contexte économique) influencent également l'apprentissage.

      Six phrases clés pour une vision pédagogique

      1. "On apprend toujours seul, mais jamais sans les autres." : Même en autodidaxie, on se base sur le travail des autres et la dimension sociale est importante. La reconnaissance par les autres est un facteur important pour l'engagement.
      2. "On apprend bien que ce qu'on a étudié par soi-même." : L'expérience personnelle et intime du savoir est essentielle pour l'intégrer. La connaissance est plus que le savoir, c'est l'expérience de l'objet étudié.
      3. "On apprend par intérêt, par valeur ou par besoin." : La motivation est un levier essentiel. Il y a trois niveaux principaux de motivation : l'intérêt (plaisir), les valeurs (convictions personnelles), et le besoin (nécessité). Il est important d'identifier les motivations profondes de l'apprenant.
      4. "On apprend selon ses enjeux, ses ressources et ses contraintes." : L'apprentissage est influencé par la perception de l'environnement. Il est important de prendre en compte les ressources, les contraintes et les enjeux de l'apprenant. Le sentiment d'efficacité personnelle (SEP) est important mais peut être un frein s'il est trop élevé.
      5. "On doit pouvoir apprendre aussi vite que le monde change." : La quantité de savoir augmente rapidement, il est donc nécessaire de développer les capacités d'apprentissage des individus.
      6. "Renoncer à fabriquer, accompagner la liberté d'apprendre." : Le rôle du formateur est d'accompagner l'apprenant vers sa propre liberté d'apprentissage, et non pas de le formater. Il est important de donner à l'apprenant les moyens de choisir et d'apprendre en toute autonomie.

      Outil "Learning Coach"

      • Un compagnon d'apprentissage qui vise à développer l'autonomie de l'apprenant et à supporter sa performance d'apprentissage.
      • Il offre des modules pour le profil de l'apprenant, un coach virtuel, des activités et une IA pédagogique.
      • Il permet de personnaliser l'accompagnement et s'adapte au profil de l'apprenant en utilisant l'IA.

      Enjeux et perspectives

      • Il est nécessaire d'adapter les formations aux besoins de chacun et de favoriser l'apprentissage collaboratif.
      • L'importance de l'autonomie de l'apprenant est centrale dans l'apprentissage tout au long de la vie.
      • La technologie peut être un outil pour aider les apprenants à apprendre à apprendre, mais il faut faire attention à ne pas exclure les personnes non numérisées.

      Ce document de synthèse devrait vous fournir une base solide pour votre briefing, en mettant en évidence les concepts clés et les enjeux liés à l'apprentissage et à l'apprenance.

    1. Voici un sommaire minuté de la transcription, mettant en évidence les idées importantes en gras :

      • 0:00 - 0:22 Vrai ou faux ?
        • Un élève peut se concentrer plus de 20 minutes sur certaines activités.
        • L'attention peut être entraînée par la volonté.
        • Les enseignants remarquent que les enfants ont plus de difficultés d'attention qu'avant.
        • Un cours magistral ne facilite pas nécessairement l'attention.
      • 0:30 - 1:22 Joker
        • Les écrans parasitent la capacité d'attention des jeunes.
        • On ne naît pas avec un niveau d'attention prédéterminé.
        • Le multitâche n'améliore pas l'attention.
        • L'âge ne limite pas la capacité attentionnelle.
        • La fatigue attentionnelle est un mythe.
        • On peut améliorer son attention avec de l'entraînement, ce n'est pas un muscle, mais ça s'améliore.
        • Tous les élèves n'apprennent pas de la même manière.
        • Le silence n'est pas toujours la meilleure condition pour apprendre.
      • 1:39 - 2:23 Introduction
        • Les enseignants sont confrontés à l'attention fluctuante des élèves.
        • La compétition pour l'attention est au cœur de notre société.
        • L'objectif est de déconstruire les mythes autour de l'attention.
      • 2:29 - 3:34 Qu'est-ce qu'un cerveau attentif?
        • Le cerveau est toujours attentif à quelque chose. L'attention est un processus de sélection de perceptions.
        • Même un enfant qui semble distrait est attentif à quelque chose.
        • Quand on dit à un élève qu'il n'est pas attentif, il faut lui préciser à quoi il n'est pas attentif.
        • Il y a un implicite : l'enfant n'est pas attentif à ce à quoi il devrait l'être.
      • 3:40 - 5:18 Identifier la cible
        • Il est important de savoir à quoi on veut que les élèves soient attentifs.
        • La cible peut être dynamique.
        • Il faut des critères pour savoir à quelle cible il faut se connecter. La cible est ce qu'il faut absolument percevoir.
      • 5:18 - 7:19 Exemples de cibles
        • Beaucoup d'exercices sont des recherches attentionnelles (ex: Où est Charlie ?).
        • Dans un devoir d'histoire-géographie, il faut analyser des documents pour trouver des informations.
        • L'attention doit être placée de manière à détecter l'information cherchée.
        • Il faut parfois faire attention à des choses qui ne sont pas immédiatement évidentes.
        • Dans un calcul de périmètre, la cible est très concrète (les petits côtés).
      • 7:19 - 8:33 Cibles plus complexes
        • Dans certaines matières, la cible est plus experte, plus complexe.
        • Le cerveau extrait des patterns (lettres, mots).
        • La cible de l'attention n'est pas la matière brute mais une matière déjà digérée.
        • Plus on monte dans les classes, plus les cibles sont abstraites.
      • 8:33 - 11:13 Inégalités à combler
        • Il y a des inégalités de capacités à viser certaines cibles.
        • Certains enfants peuvent activer un détecteur de fautes d'orthographe.
        • D'autres enfants doivent se concentrer sur des cibles plus concrètes (ex: terminaisons des verbes).
        • Il y a aussi la question de la stabilité de l'attention.
        • La stabilité de l'attention peut être entraînée si on est conscient de sa cible.
      • 11:13 - 13:02 Identifier la cible (suite)
        • En tant qu'enseignant, il faut être capable de donner à entendre la cible aux élèves.
        • Il faut cibler les situations problématiques.
        • Il faut commencer par des exercices avec des cibles concrètes.
        • Il faut développer une culture de la cible attentionnelle.
        • Il faut donner aux élèves une capacité métacognitive à réfléchir sur leur cible.
      • 13:19 - 17:01 Mythes et réalités
        • Se concentrer n'est pas forcément fatigant.
        • La distraction est souvent connotée positivement.
        • On se repose de la concentration en se concentrant sur autre chose.
        • La fatigue provient des processus cognitifs sollicités.
        • La concentration n'implique pas de crispation.
        • Le "flow" est un état de grande concentration, agréable.
      • 17:01 - 18:20 Le coût de renonciation
        • Dans le "flow", le calcul du coût de renonciation disparait.
        • Le coût de renonciation est ce qu'on perd à ne pas faire autre chose.
      • 18:30 - 21:29 L'école et le choix
        • L'état de "flow" est plus facile sur une tâche choisie.
        • À l'école, les élèves ne choisissent pas ce qu'ils font.
        • Il faut développer une attitude d'acceptation pour faire ce qu'on a à faire.
        • Le système scolaire teste l'adaptation à ce qu'on n'a pas choisi.
        • Il faut que l'élève trouve des moyens de rendre les choses plus amusantes.
      • 21:46 - 23:59 Crise de l'attention
        • La multiplication des sources d'information entraîne une dispersion de l'attention.
        • Le cerveau consacre moins de temps à chaque chose pour ne rien rater.
        • Il y a un refus de choisir et d'accepter de renoncer.
      • 24:04 - 26:16 Conséquences du zapping
        • Le temps passé sur une activité est lié à l'expertise.
        • Les contenus commerciaux génèrent une gratification immédiate.
        • Les apprentissages qui demandent du temps sont délaissés.
      • 26:16 - 29:38 Comment rectifier la donne
        • L'école simplifie le choix en limitant les distractions.
        • Les tâches sont obligatoires.
        • L'attention conjointe stabilise l'attention.
        • Le système de récompense et de punition colore les résultats.
      • 29:38 - 32:06 Rôle de l'école
        • L'école est un sanctuaire qui permet de développer des compétences nécessaires.
        • L'école ne peut pas être aussi attractive que les jeux vidéo.
        • Il faut une éducation de l'attention pour comprendre pourquoi l'école est comme elle est.
        • Il faut développer sa capacité à se concentrer pour la vie.
      • 32:13 - 35:38 Éducation à l'attention
        • L'éducation à l'attention est un enseignement métacognitif.
        • La métacognition est la conscience de ce qu'on fait dans sa tête.
        • Il faut s'intéresser à la technique cognitive.
        • Il faut un vocabulaire commun pour parler de la métacognition.
        • Il faut que l'enseignant s'interroge sur ses propres processus cognitifs.
        • Il faut prendre conscience de ce qui nous distrait.
        • Il faut expérimenter le plaisir de se concentrer.
      • 36:09 - 38:33 Écrans et attention
        • Les écrans sont source de compétition pour l'attention.
        • Les écrans sont un outil et un organe sensoriel.
        • Il faut utiliser les écrans de manière intentionnelle.
        • Il faut se poser les questions "Pourquoi faire ?" et "Pour combien de temps ?".
        • La perte de contrôle est problématique.
      • 38:53 - 41:40 Le programme Atole
        • Atole est un enseignement qui vise à développer la capacité à se concentrer.
        • Le programme est un changement de posture pour l'enseignant et l'élève.
        • On travaille sur la cible, la différence entre attention et concentration, la stabilité attentionnelle et l'intention claire.
        • On développe le sens de l'équilibre attentionnel.
        • Il faut avoir une intention claire avant d'aborder une tâche.
      • 41:40 - 43:03 Atole et inégalités scolaires
        • Atole réduit les inégalités scolaires.
        • Atole est déployé à l'école pour que tous les élèves en bénéficient.
        • Certaines familles valorisent l'attention plus que d'autres.
      • 43:03 - 44:05 Déploiement d'Atole
        • Atole a commencé en élémentaire, puis au collège.
        • Une version maternelle est en préparation.
      • 44:11 - 46:01 Mesure d'impact d'Atole
        • Une étude a comparé les classes ayant suivi Atole et les classes contrôles.
        • Atole améliore la réactivité aux appels à l'attention.
        • Atole améliore les capacités métacognitives des élèves.
        • Atole a un impact positif sur les gestes pédagogiques des enseignants.
        • Des études suisses ont confirmé les effets positifs d'Atole sur l'attention.
      • 46:07 - 47:01 Conclusion
        • Il faut tenir compte de la réalité de sa propre classe.
        • Les études d'impact favorisent le passage à l'action.

      Ce sommaire met en évidence les concepts clés et les arguments développés dans la transcription. Il permet de saisir rapidement les enjeux liés à l'attention et les solutions proposées, notamment à travers le programme Atole.

    1. Voici un sommaire minuté basé sur le webinaire LISA, avec les points clés et les temps approximatifs où ils sont abordés dans la source vidéo :

      Introduction (0:00 - 2:00) * Présentation du dispositif LISA, conçu pour améliorer le bien-être et la santé mentale des élèves. * L'objectif de LISA est de favoriser l'épanouissement des élèves au sein d'un collectif, tout en reconnaissant leur unicité. * Importance d'aborder la santé mentale à l'école, en complément des dispositifs existants comme les PAP. * Présentation des outils LISA : plateforme numérique, formations, ressources élaborées en collaboration interdisciplinaire.

      Outils et ressources LISA (2:00 - 4:00) * Description de la plateforme numérique, avec l'outil de repérage et la base de ressources. * Explication du questionnaire "Facettes" pour une observation structurée des élèves. * Génération d'un tableau de bord individuel avec les forces et les besoins de chaque élève. * Présentation de la base de ressources Lzapédia et des formations proposées. * Mention du programme de recherche mené par des spécialistes, avec un comité éthique scientifique.

      Le monde digital et ses enjeux (4:00 - 10:00) * Constat de la transformation des modes de communication par les outils digitaux. * Distinction entre les différents services digitaux : jeux vidéo, réseaux sociaux, sites de rencontre, etc.. * Les réseaux sociaux répondent au besoin essentiel de relations sociales, mais introduisent la notion de métrique. * Les services digitaux sont vivants, s'adaptent aux usages et peuvent maintenir l'utilisateur engagé. * Risque de sollicitation permanente, d'univers permanents et de réponses immédiates. * Le scrolling et l'excitation qu'il procure sont décrits comme des facteurs qui peuvent maintenir l'usage.

      Usage des écrans chez les jeunes (10:00 - 16:00) * Les adolescents sont majoritairement exposés à la pornographie, aux réseaux sociaux et aux jeux vidéo. * Certains passent plus de 20h par semaine sur ces services, ce qui a suscité des inquiétudes. * La question de l'addiction à Internet a émergé, en particulier pour les jeux vidéo. * Une étude longitudinale montre que les usages importants se maintiennent pour certains jeunes, alors que d'autres diminuent. * L'activation de l'aire striatale ventrale est liée à la sévérité de la conduite addictive à la pornographie. * Les usages de la pornographie restent relativement stables chez les adolescents sur 3 ans.

      Caractéristiques des services digitaux et notion d'addiction (16:00 - 23:00) * Les services digitaux ont des caractéristiques communes et spécifiques. * La question centrale est de savoir ce qui est considéré comme "trop de temps". * L'usage peut être utile et s'inscrire dans une passion harmonieuse. * Les critères d'addiction classiques sont appliqués au domaine des écrans : préoccupation, sevrage, tolérance, etc.. * Tous ces critères ne sont pas équivalents pour identifier une conduite addictive. * L'OMS a reconnu le trouble du jeu vidéo comme une addiction. * Les caractéristiques personnelles et les motivations (gratification, compensation) contribuent au maintien de l'usage. * Diverses motivations à jouer aux jeux vidéo sont identifiées : avancement, compétition, relations sociales, évasion. * L'évasion est un facteur qui revient souvent et qui est lié aux conduites addictives.

      Réseaux sociaux et bien-être digital (23:00 - 30:00) * Les jeunes sont très connectés en ligne et communiquent souvent via les réseaux sociaux. * Les motivations à utiliser les réseaux sociaux sont sociales, de conformité, de régulation émotionnelle, de connaissance et d'excitation. * Le bien-être digital est devenu central, car le bien-être général passe par le numérique. * Définition du bien-être : expérience et fonctionnement psychologique optimaux. * Importance de trouver un équilibre entre les avantages et inconvénients de la connectivité. * Les difficultés fonctionnelles incluent les conduites addictives, le harcèlement, les difficultés de gestion des conflits.

      Régulation et outils (30:00 - 36:00) * Les outils de contrôle du temps d'écran sont disponibles sur les smartphones. * Ces outils sont utilisés mais leur efficacité est jugée modérée. * Il faut optimiser l'usage, en étant conscient des avantages et inconvénients pour que les outils digitaux soient au service du bien-être. * Nécessité de prendre du recul sur l'usage et d'identifier les changements possibles. * La diversité des personnes, des besoins et des ressources est importante à considérer.

      Questions et réponses (36:00 - Fin) * Discussion sur la manière de lutter contre la surexposition aux écrans des élèves. * Importance d'échanger avec les jeunes sur leur usage, leurs perceptions et les priorités à gérer. * Les parents doivent être des modèles dans leur utilisation des écrans. * Recommandations sur la gestion du temps de jeu après l'école : il faut considérer les autres intérêts et priorités de l'enfant et mettre en balance le temps passé sur les écrans avec les autres aspects de sa vie. * Il faut s'interesser à ce que l'enfant apprécie dans ses jeux, et envisager d'autres activités. * Comment convaincre les adolescents malgré de bons résultats scolaires : il faut maintenir une discussion ouverte et explorer d'autres activités. * Comment intégrer les compétences numériques à l'école sans créer d'addiction : il faut inclure la question de l'usage harmonieux et la conscience de l'usage. * Les effets de la lumière des écrans : il y a des liens entre les troubles de l'attention et l'usage des écrans, mais il est difficile de dire quelle est la cause et la conséquence. * L'approche de la régulation du temps d'écran varie selon l'âge de l'enfant. Pour les plus jeunes, il faut diversifier les activités et les inviter à participer à d'autres types d'activités, tandis que pour les plus âgés, on peut les impliquer davantage dans la réflexion. * Les activités appréciées hier ne disparaissent pas, mais il faut diversifier et proposer des activités avec des composantes ludiques ou de partage social.

      Ce sommaire minuté offre une vue d'ensemble des points abordés lors du webinaire, permettant de retrouver facilement les informations importantes. Les éléments en gras mettent en évidence les points clés et les recommandations.

    2. Voici un document de synthèse pour un briefing, basé sur les informations du webinaire LISA avec Yasser Khazaal, concernant le rapport des jeunes aux écrans :

      Introduction au projet LISA * LISA est un dispositif conçu pour identifier et comprendre les besoins et les forces des élèves afin d'améliorer leur bien-être et leur santé mentale. * Il se compose d'une plateforme numérique, d'un parcours de formation pour les enseignants et d'un réseau de collaboration. * Le projet LISA France 2030 vise à améliorer le bien-être d'environ 70 000 élèves dans 190 établissements en France et à l'international. * L'objectif principal est de favoriser le bien-être de tous les élèves pour qu'ils puissent apprendre et progresser ensemble. * L'école joue un rôle essentiel dans le bien-être et l'épanouissement des enfants et des adolescents.

      Outils et ressources LISA * Plateforme numérique : Elle comprend un outil de repérage et une base de ressources. * Questionnaire Facettes : Un questionnaire de 12 questions permet aux enseignants de faire une observation structurée de chaque élève. * Tableau de bord individuel : Un tableau de bord est généré à partir des réponses au questionnaire, offrant un résumé des forces et des besoins de chaque élève ainsi que des suggestions pour les enseignants. * Base de ressources Lzapédia : Elle contient des fiches pratiques prêtes à l'emploi et des hyperliens vers des ressources externes pédagogiques et théoriques. * Formations : LISA propose des formations pratiques en ligne et en présentiel adaptées aux besoins des enseignants.

      Le monde digital et les jeunes * Les services digitaux sont variés et chacun propose des interactions différentes. Ils incluent les jeux vidéo, les réseaux sociaux, les sites de rencontre, etc.. * Les réseaux sociaux répondent à un besoin humain essentiel de relations sociales et d'élargissement du réseau. * La métrique (nombre de likes, de suivis) peut changer la relation à soi-même et aux autres. * Les services digitaux sont en constante évolution, s'adaptant aux usages et captant les données des utilisateurs. * Les services digitaux peuvent créer un risque de sollicitation permanente, avec une offre illimitée et des réponses immédiates.

      Usage des écrans par les adolescents * Une étude a montré que la majorité des adolescents ont été exposés à de la pornographie, des réseaux sociaux ou des jeux vidéo. * Certains adolescents passent plus de 20 heures par semaine sur les réseaux sociaux ou les jeux vidéo. * Il y a une inquiétude sur le fait de passer trop de temps sur les écrans, et sur les risques d'addiction

      Addiction aux écrans * La science a repris les critères habituels des addictions pour les appliquer au domaine des écrans. * Les critères comme la préoccupation, le sevrage, la tolérance, la régulation émotionnelle, la perte de contrôle ont été examinés. * L'OMS a reconnu le trouble du jeu vidéo comme une addiction. * Les motivations à jouer incluent l'avancement dans le jeu, la compétition, les relations sociales, l'évasion. * L'évasion est souvent un facteur lié aux conduites addictives.

      Bien-être digital * Le bien-être est une expérience et un fonctionnement psychologique optimaux. * Le bien-être numérique est devenu central, car de nombreuses dimensions du bien-être passent par le numérique. * L'objectif est de trouver un équilibre entre les avantages et les inconvénients de la connectivité, avec un soutien fonctionnel maximal et des difficultés minimales. * Il est important de considérer les difficultés fonctionnelles liées aux interactions digitales, au harcèlement et à la gestion des conflits.

      Régulation et outils * Les smartphones proposent des outils de contrôle du temps d'écran, de limitation des notifications, et de blocage d'applications. * Ces outils sont utilisés par une partie de la population, mais leur efficacité est jugée modérée. * Il est essentiel d'optimiser l'usage des écrans pour qu'ils soient au service du bien-être, en étant conscient des avantages et des inconvénients de chaque usage. * Il faut prendre du recul par rapport à un usage spécifique, en identifiant les bonnes et les mauvaises choses et en décidant des changements possibles pour améliorer son bien-être digital.

      Recommandations pour les enseignants et les parents * Il est important d'échanger avec les élèves et les enfants sur leur usage des écrans. * Les parents doivent aussi être un modèle dans leur propre utilisation des écrans. * Il est crucial de comprendre les motivations des jeunes à utiliser les écrans, comme le plaisir, l'évasion, et le besoin de relations sociales. * Il faut diversifier les sources de satisfaction en proposant d'autres types d'activités. * Il est important d'intégrer la question de l'usage harmonieux des outils numériques dans les compétences transversales à l'école. * Il faut encourager la conscience de l'usage et sa modulation pour ne pas interférer avec les autres priorités. * Il faut adapter les approches en fonction de l'âge de l'enfant, avec une diversification des activités pour les plus jeunes et une approche plus participative pour les plus âgés * Il est suggéré de mettre en balance le temps passé sur les écrans avec les autres aspects de la vie

      En conclusion * Le rapport des jeunes aux écrans est complexe, avec des avantages et des inconvénients. * L'approche doit être individualisée, en considérant les besoins et les motivations de chaque personne. * L'objectif est de favoriser un usage harmonieux et conscient des outils numériques, au service du bien-être et de l'épanouissement. * Il est important de discuter avec les jeunes pour les amener à prendre conscience de leur usage et à moduler leur comportement. * Il faut proposer une diversité d'activités pour qu'ils puissent avoir un équilibre dans leur vie et pour pouvoir faire un choix éclairé * Il faut diversifier les sources de satisfaction * Il ne faut pas oublier les fondamentaux.

      Ce document de synthèse peut servir de base pour un briefing, en soulignant l'importance du bien-être digital et en proposant des pistes d'action pour les enseignants et les parents.

    1. Voici un document de synthèse pour un briefing, basé sur les informations des sources fournies :

      Le Dispositif LISA

      • LISA est un dispositif conçu pour identifier les besoins et les forces des élèves afin de mieux accompagner leur bien-être et leur santé mentale.
      • Il est composé d'une plateforme numérique, d'un parcours de formation pour les enseignants et d'un réseau de collaboration multi-acteur soutenu par l'État.
      • Le projet LISA France 2030 vise à améliorer le bien-être d'environ 70 000 élèves dans 190 établissements en France et à l'international.
      • Le projet s'articule autour de la question de comment favoriser le bien-être des élèves pour leur permettre d'apprendre et de progresser ensemble en s'épanouissant.
      • Chaque élève est unique avec ses propres forces et besoins, et évolue au sein d'un collectif, le groupe classe.
      • L'objectif pour les enseignants est de transmettre des connaissances et de veiller à leur acquisition pour chaque élève au sein d'un groupe.

      Outils et Ressources LISA

      • LISA offre des formations, des outils et des ressources élaborées en collaboration interdisciplinaire.
      • Une plateforme numérique comprend un outil de repérage et une base de ressources avec des interventions concrètes pour la classe.
      • Le questionnaire "facettes" (12 questions) permet aux enseignants de faire une observation structurée de chaque élève.
      • Un tableau de bord individuel est généré, offrant un résumé des forces et des besoins de chaque élève, ainsi que des suggestions pour les enseignants.
      • Les suggestions sont présentées sous forme de fiches simples et pratiques, prêtes à l'emploi.
      • Lzapédia, la base de ressources, contient toutes les fiches pratiques et des hyperliens vers des ressources externes pédagogiques et théoriques.
      • Des formations pratiques en ligne et en présentiel sont proposées, adaptées aux besoins des enseignants.
      • La démarche s'appuie sur un programme de recherche mené par des spécialistes de la santé mentale et du neurodéveloppement, avec un comité éthique scientifique international.

      L'Anxiété : Généralités

      • Les émotions sont des états particuliers de l'organisme, parfois conscients, qui ont été conservés et façonnés pour nous diriger vers des actions adaptatives.
      • L'anxiété est déclenchée par des informations internes et externes, suscitant une chaîne de réactions cérébrales et physiologiques.
      • Les formes d'anxiété et leurs déclencheurs diffèrent selon le patrimoine génétique et l'histoire développementale.
      • Il existe une expérience consciente des émotions et un enregistrement avec des indicateurs physiologiques et des modifications de l'activité cérébrale.
      • L'anxiété est liée à l'activation du système défensif, qui a pour but de nous aider à faire face aux menaces.
      • L'anxiété est ressentie en amont ou après la rencontre avec une menace, tandis que la peur est l'émotion ressentie lors de la confrontation directe avec une menace.

      Le Système Défensif et l'Anxiété

      • Le système défensif nous dirige de manière automatisée vers des réponses comme la fuite, le combat, ou l'immobilisation.
      • Ce système active l'axe hypothalamo-hypophyso-surrénalien (axe du stress), entraînant une cascade de modifications physiologiques (accélération du rythme cardiaque, etc.).
      • Il met en pause les fonctions non essentielles pour faire face à la menace (digestion, système reproductif).
      • L'anxiété est une émotion normale, mais certaines personnes sont plus sensibles en raison de prédispositions génétiques ou d'événements de vie.
      • L'anxiété sociale survient dans des situations sociales, notamment lorsqu'on est observé par autrui.
      • Les personnes anxieuses évitent ces situations, ce qui alimente le trouble.
      • Le mutisme sélectif est un trouble spécifique où les personnes n'arrivent pas à prendre la parole en dehors de la sphère intime.
      • L'anxiété de performance est ressentie lors de la réalisation d'une tâche devant un public.
      • Le trouble d'anxiété sociale est courant, particulièrement entre 13 et 25 ans.
      • Ce trouble est souvent associé à d'autres troubles mentaux (dépression, etc.) et peut avoir des impacts négatifs sur la performance scolaire et le développement.

      Anxiété et Tests

      • L'anxiété durant les tests n'est pas un trouble mental connu, mais elle peut être importante et invalidante.
      • Cette anxiété a un impact négatif sur la performance scolaire.

      Facteurs de Maintien de l'Anxiété

      • L'anxiété sociale est maintenue par l'association des pensées, des émotions et des comportements.
      • Les pensées typiques de l'anxiété sociale incluent la peur du jugement, de l'échec, etc..
      • Les comportements d'évitement (éviter des situations sociales) et de micro-évitement (regarder le sol, utiliser son téléphone) soulagent à court terme, mais alimentent l'anxiété à long terme.
      • Les thérapies cognitives et comportementales (TCC) travaillent sur les pensées, les émotions et les comportements pour briser ce cercle vicieux.

      Prévention de l'Anxiété en Classe

      • La régulation de la classe par les enseignants est cruciale : un climat positif minimise les difficultés de l'ordre de la santé mentale.
      • Des relations positives avec les élèves et des feedbacks positifs sont importants.
      • Les enseignants doivent intégrer des activités pédagogiques dédiées au bien-être émotionnel.
      • Il est essentiel de créer une communauté apprenante où l'acceptation des émotions et l'empathie sont encouragées.
      • Les enseignants peuvent se former aux compétences psychosociales (CPS) et utiliser des programmes comme "École des émotions".
      • Modéliser l'acceptation des erreurs est important pour minimiser l'anxiété de performance.
      • Il faut se concentrer sur les progrès et l'amélioration plutôt que sur les erreurs.
      • Modéliser l'acceptation et la régulation collective des émotions en parlant des émotions et en enseignant des techniques de régulation (respiration calme, etc.).
      • L'apprentissage de la régulation émotionnelle peut se faire en utilisant des personnages de fiction ou des modèles réels pour illustrer que les difficultés émotionnelles peuvent être surmontées.
      • Pour les élèves à risque, les placer en binôme avec des élèves bienveillants et éviter l'évitement sont des stratégies utiles.
      • Pour l'anxiété liée aux tests, donner plus de temps, aider à la préparation, et rendre le cadre d'évaluation clair sont recommandés.
      • Les interventions comme la TCC sont très efficaces pour accompagner ces troubles.
      • Il peut y avoir des groupes d'affirmation de soi ou d'habiletés sociales.
    1. Voici un résumé des points clés concernant le projet LISA, l'apprentissage socio-émotionnel (ASE) et le bien-être, en vue d'un briefing :

      Le projet LISA

      • LISA est un dispositif conçu pour identifier et comprendre les besoins et les forces des élèves, afin de mieux accompagner leur bien-être et leur santé mentale.

      • Il se compose d'une plateforme numérique, d'un parcours de formation pour les enseignants, et d'un réseau de collaboration multi-acteurs soutenu par l'État.

      • Le projet LISA France 2030 vise à améliorer le bien-être d'environ 70 000 élèves dans 190 établissements en France et à l'international.

      • Le projet LISA cherche à favoriser le bien-être des élèves pour leur permettre d'apprendre et de progresser ensemble en s'épanouissant.

      • LISA propose des formations, des outils et des ressources élaborées à partir d'une collaboration interdisciplinaire d'enseignants, de cliniciens, de chercheurs et d'ingénieurs.

      • La plateforme numérique de LISA inclut un outil de repérage (questionnaire "facettes" avec 12 questions) et une base de ressources.

      • Le questionnaire permet aux enseignants de faire une observation structurée de chaque élève.

        • Un tableau de bord individuel est généré, offrant un résumé des forces et des besoins de chaque élève, ainsi que des suggestions pour les enseignants.
      • Les suggestions sont présentées sous forme de fiches pratiques prêtes à l'emploi. La base de ressources "Lzapédia" est composée de fiches pratiques et d'hyperliens vers des ressources externes.

      • LISA propose également des formations pratiques en ligne et en présentiel, adaptées aux besoins des enseignants.

      • La démarche s'appuie sur un programme de recherche mené par des spécialistes de la santé mentale et du neurodéveloppement, avec un comité éthique scientifique international.

      L'importance de l'apprentissage socio-émotionnel (ASE)

      • La santé mentale des jeunes est un problème qui requiert une intervention immédiate, avec une souffrance accrue observée dans les rapports ministériels et internationaux.

      • Les jeunes expriment de plus en plus de colères, de déceptions et de pessimisme, menant à l'anxiété et au manque de motivation.

      • L'école joue un rôle crucial dans le bien-être et l'épanouissement des enfants et des adolescents, passant la majorité de leur temps à l'école.

      • Le développement des compétences socio-émotionnelles (CSE), également appelées compétences psychosociales (CPS), est essentiel.

      • Les compétences psychosociales (CPS) sont un ensemble de compétences non techniques et non académiques, incluant

        • la créativité,
        • le travail en équipe,
        • l'identification et la régulation des émotions.
      • Les troubles de l'apprentissage et de la santé mentale sont de plus en plus présents dans les établissements scolaires, affectant à la fois les individus et l'environnement scolaire et familial.

      • Les enseignants manquent de ressources pour aider les élèves ayant des besoins particuliers, et le projet LISA vise à leur fournir des outils pour accompagner ces jeunes.

      • L'intégration des compétences socio-émotionnelles dans le cursus scolaire est un enjeu important, bien que des réticences persistent.

      Les compétences émotionnelles

      • Plusieurs théories et modèles existent pour définir et comprendre les compétences émotionnelles.

      • Le modèle de l'intelligence émotionnelle de Salovey et Mayer (4 branches) : * perception, * utilisation, * compréhension et régulation des émotions.

      • Le modèle de la compétence émotionnelle (5 branches) : * identification, * compréhension, * expression, * régulation et utilisation des émotions (versants intrapersonnel et interpersonnel).

      • Il existe 3 niveaux d'approfondissement de ces modèles : * les connaissances, * les aptitudes, et * la disposition.

      • L'intelligence émotionnelle n'est pas l'antithèse de l'intelligence générale; les émotions ne sont pas opposées à la pensée.

      • Les compétences émotionnelles peuvent être développées et enseignées, bien que certaines personnes soient naturellement plus compétentes.

      Les bénéfices de l'apprentissage socio-émotionnel

      • Les interventions psychosociales à l'école ont des impacts positifs sur la santé mentale des jeunes.

      • On observe * une réduction de l'entrée dans le tabagisme, de * la consommation d'alcool, * des comportements violents et * des problèmes de comportement en classe.

      • L'ASE réduit également * les problèmes avec la justice, * les tentatives de suicide, * les prises de risques sexuels, * l'anxiété et le stress.

      • Il améliore le contrôle de la colère, la capacité de concentration, la résolution de problèmes et la connaissance en santé mentale.

      • L'ASE favorise une meilleure acceptation des personnes souffrant de problèmes psychiques.

      • Il y a une réduction * des symptômes dépressifs légers, * des comportements à risque, * du harcèlement, et * une amélioration des relations interpersonnelles.

      • L'ASE améliore * les résultats scolaires, * l'attitude générale envers l'école, * et est d'autant plus efficace si l'enseignant est impliqué.

      Les référentiels et modèles de compétences psychosociales

      • Il existe plusieurs référentiels et modèles de compétences psychosociales, chacun avec des approches différentes.
        • UNESCO et ODD (Objectifs de Développement Durable) : focus sur le bien-être.
        • OCDE : basé sur le modèle des "Big Five" de la personnalité.
        • Modèle d'éducation en quatre dimensions (compétences, traits de caractère, apprentissage).
      • CASEL (Collaborative for Academic, Social, and Emotional Learning) : conscience de soi, gestion de soi, conscience sociale, compétences relationnelles et prise de décisions responsables.
      • OMS (life skills) : aptitude à maintenir un état de bien-être mental.
      • Santé Publique France : compétences cognitives, émotionnelles et sociales.

      Bien-être

      • Le bien-être de l'enfant est à la fois subjectif (hédonique) et psychologique (eudémonique).

      • Le bien-être subjectif se concentre sur le plaisir, les émotions positives et la satisfaction de la vie.

      • Le bien-être psychologique se concentre sur la réalisation du potentiel, le sens de la vie et le développement personnel.

      • Plusieurs modèles de bien-être existent (motivations, équilibre défis/habilités, épanouissement PERMA).

      Recherche et études

      • Des études ont été menées sur le bien-être des enfants en France, notamment avec la "Children World Survey".

      • L'approche dialogique en pédagogie développe les émotions positives et la créativité.

      • L'enseignement des émotions par les enseignants améliore leur propre bien-être et leur pratique professionnelle.

      • L'ASE est fondamental pour prévenir le décrochage scolaire.

      • Le sentiment d'efficacité personnelle est déterminant pour la performance scolaire.

      • Un axe de recherche actuel vise à travailler les compétences psychosociales des enseignants pour développer le bien-être enseignant.

      Conclusion

      • La qualité de vie est liée aux compétences psychosociales, à l'apprentissage socio-émotionnel, au bien-être et au sentiment d'efficacité personnelle.

      • La gestion des émotions permet d'améliorer l'empathie, la communication, la résolution de conflits et la prise de décisions responsables.

      • L'ASE favorise la réduction de l'anxiété, des relations positives, un soutien social, la persévérance, la résilience et la réussite, créant une école plus inclusive.

      Ce résumé met en évidence l'importance de l'ASE et du bien-être dans le contexte scolaire et souligne le rôle central du projet LISA pour répondre à ces enjeux.

    1. 3.8.2.1 Android RPC Although RPCs are typically associated with client-server computing in a distributed system, they can also be used as a form of IPC between processes running on the same system. The Android operating system has a rich set of IPC mechanisms contained in its binder framework, including RPCs that allow one process to request services from another process. Android defines an application component as a basic building block that provides utility to an Android application, and an app may combine multiple application components to provide functionality to an app. One such application component is a service, which has no user interface but instead runs in the background while executing long-running operations or performing work for remote processes. Examples of services include playing music in the background and retrieving data over a network connection on behalf of another process, thereby preventing the other process from blocking as the data are being downloaded. When a client app invokes the bindService() method of a service, that service is “bound” and available to provide client-server communication using either message passing or RPCs. A bound service must extend the Android class Service and must implement the method onBind(), which is invoked when a client calls bindService(). In the case of message passing, the onBind() method returns a Messenger service, which is used for sending messages from the client to the service. The Messenger service is only one-way; if the service must send a reply back to the client, the client must also provide a Messenger service, which is contained in the replyTo field of the Message object sent to the service. The service can then send messages back to the client. To provide RPCs, the onBind() method must return an interface representing the methods in the remote object that clients use to interact with the service. This interface is written in regular Java syntax and uses the Android Interface Definition Language—AIDL—to create stub files, which serve as the client interface to remote services. Here, we briefly outline the process required to provide a generic remote service named remoteMethod() using AIDL and the binder service. The interface for the remote service appears as follows: /* RemoteService.aidl */ interface RemoteService {   boolean remoteMethod(int x, double y); { This file is written as RemoteService.aidl. The Android development kit will use it to generate a .java interface from the .aidl file, as well as a stub that serves as the RPC interface for this service. The server must implement the interface generated by the .aidl file, and the implementation of this interface will be called when the client invokes remoteMethod(). When a client calls bindService(), the onBind() method is invoked on the server, and it returns the stub for the RemoteService object to the client. The client can then invoke the remote method as follows: RemoteService service;    . . . service.remoteMethod(3, 0.14); Internally, the Android binder framework handles parameter marshaling, transferring marshaled parameters between processes, and invoking the necessary implementation of the service, as well as sending any return values back to the client process.

      Android extends the concept of RPCs beyond traditional client-server models by integrating them into its IPC mechanisms using the binder framework. In Android, applications often consist of multiple components, including services that run in the background and perform tasks for other processes. Through the Android Interface Definition Language (AIDL), developers define remote interfaces that allow communication between different application processes. The binder framework manages marshaling and unmarshaling of parameters, ensuring efficient communication. This approach is essential for resource management in mobile systems, as it enables processes to offload work to background services while maintaining system responsiveness. By leveraging RPCs, Android provides a structured way to manage process interactions while abstracting the complexities of low-level data transfer.

    1. Trump justificó el golpe contra su principal socio comercial al asegurar que sus vecinos han sido omisos en el combate al crimen organizado y la crisis migratoria, y señalando que existía una “alianza intolerable” entre los carteles de la droga y las autoridades mexicanas. Las acusaciones no cayeron bien al sur de la frontera. “Rechazamos categóricamente la calumnia que hace la Casa

      nota sobre las noticias de hoy

    1. Briefing Doc: L'éducation aux médias et à l'information

      Source: Extrait d'une présentation vidéo sur l'éducation aux médias et à l'information (EMI) destinée aux parents d'élèves.

      L'intervenante est Mathilde, une enseignante-chercheuse spécialiste des usages numériques des jeunes, membre du Centre pour l'éducation aux médias et à l'information (CLEMI) de l'académie de Toulouse.

      Thèmes principaux:

      L'importance de l'EMI dans un monde saturé d'informations:

      L'intervenante souligne d'emblée la nécessité d'une éducation aux médias et à l'information face à la multiplication des sources et la rapidité de circulation de l'information, en particulier pour les adolescents.

      Elle insiste sur le rôle de l'école dans ce processus, "de donner les clés, les repères, le mode d'emploi, de former à l'usage d'internet, de former à l'usage des réseaux sociaux, à se faire un esprit critique autour de l'information et être en capacité de la décoder" (extrait de la vidéo).

      Les usages numériques des jeunes:

      Contrairement à certaines idées reçues, les jeunes sont conscients de la nécessité de vérifier la fiabilité de l'information.

      Ils s'informent via des plateformes variées comme Instagram, TikTok et Snapchat, et développent leurs propres stratégies de validation des sources.

      L'intervenante mentionne l'influence grandissante des "journalistes influenceurs" comme Hugo Décrypte, qui proposent une approche vulgarisée et accessible de l'actualité.

      Les dangers de la désinformation et les biais cognitifs:

      La présentation met en garde contre les dangers de la désinformation, notamment via les "bulles informationnelles" créées par les algorithmes de recommandation.

      Ces bulles peuvent renforcer les biais cognitifs, en particulier le biais de confirmation qui pousse les individus à ne rechercher que des informations confirmant leurs opinions préexistantes.

      Outils et stratégies pour accompagner les enfants:

      La présentation propose une série de conseils et d'outils pour aider les parents à accompagner leurs enfants dans le développement de leur esprit critique.

      Il est notamment conseillé de:

      • Expliquer la différence entre information, mésinformation et désinformation

      • Apprendre aux enfants à vérifier les sources et les images (en utilisant des outils comme Google Lens et TinEye)

      • Discuter avec eux de l'actualité et des informations qu'ils rencontrent en ligne

      • Les sensibiliser aux dangers des contenus violents et haineux et les encourager à signaler ces contenus aux plateformes

      • Les accompagner dans la construction de leur identité numérique et les sensibiliser à la permanence des informations publiées en ligne

      Idées et faits importants:

      • L'éducation aux médias et à l'information est une thématique transversale qui s'intègre à l'ensemble des disciplines et des cycles scolaires.

      • Le CLEMI propose des formations pour les enseignants et des ressources pour les élèves et les parents.

      • L'importance du dialogue et de la communication entre parents et enfants face aux enjeux de la désinformation est primordiale.

      • Les jeunes développent des stratégies de validation des sources qui leur sont propres et qui ne correspondent pas toujours à celles des adultes.

      • Il est important de sensibiliser les jeunes aux dangers des algorithmes de recommandation et à l'importance de diversifier leurs sources d'information.

      Citations:

      • "Ils ont accès en temps réel à des informations multiples."

      • "On considère que c'est à l'école aussi de donner les clés, les repères, le mode d'emploi, de former à l'usage d'internet, de former à l'usage des réseaux sociaux, à se faire un esprit critique autour de l'information et être en capacité de la décoder."

      • "La préoccupation des jeunes autour de la fiabilité de l'information est est une et est une est vraiment une priorité pour eux."

      • "L'idée, en fait, c'est de se faire une idée de toutes de toutes qu'on nous offre et de piocher, un petit peu, tout ce qui qui est bon."

      • "L'objectif de tik to, c'est toujours de vous, de vous, de vous laisser en tête, de vous laisser naviguer sur la plateforme et que vous y restiez le plus longtemps possible."

      • "Essayer de les écouter, ils n'ont pas forcément les mêmes usages du numérique que vous, mais ils ont des stratégies en fait de sélection d'information, de repérage d'information et ils sont capables de se repérer."

      Conclusion:

      Cette présentation met en lumière la complexité de l'écosystème informationnel actuel et la nécessité d'une éducation aux médias et à l'information dès le plus jeune âge.

      Elle propose des pistes concrètes pour aider les parents à accompagner leurs enfants dans la navigation de cet univers et à développer leur esprit critique face à l'information.

    1. webinaire de la FCPE Nationale, axé sur la manière de remplir efficacement le dossier MDPH pour un enfant en situation de handicap.

      Le webinaire, comprenant des interventions de spécialistes et des membres de la FCPE, détaille les étapes de la demande, du recueil des informations médicales et scolaires à la constitution du dossier administratif, incluant la gestion des recours en cas de refus.

      L'objectif est de guider les parents dans cette démarche souvent complexe, en leur fournissant des conseils pratiques et des explications claires sur le fonctionnement de la MDPH.

      Voici des éléments de synthèse pour un briefing sur le dossier MDPH, basés sur les sources fournies :

      Objectif du dossier MDPH

      • Le dossier MDPH (Maison Départementale des Personnes Handicapées) est crucial pour définir les besoins de chaque enfant et mettre en place les dispositifs d'accompagnement nécessaires.
      • Il permet d'évaluer les besoins et de proposer des aides adaptées.

      Acteurs impliqués

      • La FCPE (Fédération des Conseils de Parents d'Élèves) propose un webinaire pour aider les familles à remplir le dossier MDPH.
      • Des professionnels de la MDPH, des équipes éducatives, des médecins, et des enseignants référents sont impliqués dans le processus d'évaluation.
      • Des assistantes sociales peuvent être sollicitées pour compléter le volet social du dossier.

      Constitution du dossier

      • Le dossier comprend une demande administrative, un certificat médical récent (moins d’un an), et le GEVA-Sco (Guide d'évaluation des besoins de compensation en matière de scolarisation).
      • Des bilans (orthophoniste, psychomotricien, psychométrique) peuvent être joints, ainsi que des devis pour certains professionnels (ergothérapeute).
      • Un complément social est souvent requis, rempli par l’assistante sociale de secteur.
      • Il est important de signer toutes les pages nécessaires, notamment la page quatre, car un oubli de signature peut entraîner un retour du dossier.
      • Si les deux parents ne sont pas d'accord ou si le deuxième parent est absent, il faut le signaler par écrit.
      • Un accusé de réception est important pour prouver que le dossier a été déposé.
      • Il est préférable d’envoyer les documents par internet, via le portail de la MDPH, en créant un compte avec un numéro de dossier mais le dépôt papier reste possible avec accusé de réception.
      • Il est conseillé de ne pas agrafer les documents si le dossier est envoyé en version papier.

      Évaluation et décisions

      • L'équipe pluridisciplinaire de la MDPH étudie le dossier. Elle est composée généralement d'un médecin, d'un enseignant référent, et éventuellement d'un coordonnateur.
      • Elle fait une proposition d’aide ou de compensation.
      • Un taux de handicap est déterminé par le médecin (inférieur à 50%, entre 50 et 79%, ou supérieur à 80%). Un taux supérieur à 50% ouvre droit à l'Allocation d'Éducation de l'Enfant Handicapé (AEEH).
      • La proposition est présentée à la CDAPH (Commission des Droits et de l'Autonomie des Personnes Handicapées), qui valide ou non les propositions faites par l'équipe pluridisciplinaire.
      • La décision est notifiée aux parents dans les 15 jours.

      Types d’aides et de prestations

      • L’AEEH est une prestation financière versée par la CAF (Caisse d'Allocations Familiales) ou la MSA (Mutualité Sociale Agricole).
      • La Prestation de Compensation du Handicap (PCH) est une aide financière pour des besoins spécifiques (matériel, ergothérapie, etc.).
      • Le dossier MDPH peut inclure une demande de matériel informatique ou adapté, ainsi que pour des soins en libéral (orthophonie, psychomotricité) ou en établissement.
      • La MDPH peut financer des interventions de professionnels comme un ergothérapeute.

      Recours

      • En cas de refus ou d'insatisfaction, un recours administratif est possible.
      • Il est nécessaire d'envoyer un recours gracieux à la personne indiquée sur la notification de refus, en y ajoutant des éléments nouveaux.
      • Le recours gracieux peut aboutir à un nouvel examen du dossier, un appel téléphonique du coordonnateur, ou une proposition de PPS (Projet Personnalisé de Scolarisation).
      • En cas de litige, un médiateur de l’Éducation Nationale peut être sollicité.
      • Un avocat peut être consulté pour aider dans les démarches.
      • Il est conseillé de joindre une copie de la décision contestée au recours administratif.

      Points importants

      • Un enfant est considéré comme tel par la MDPH jusqu’à ses 20 ans.
      • L’école est souvent à l’origine de la demande et réunit l’équipe éducative.
      • Le GEVA-Sco est essentiel pour toute demande liée à la scolarisation, et doit être rempli par l’enseignant référent lors d'une demande de renouvellement de dossier.
      • Il faut être précis dans la description des besoins et des difficultés de l'enfant.
      • Les bilans ne sont pas financés par la MDPH, mais des aides peuvent être accordées pour les séances.
      • Les parents doivent être impliqués et faire part de leurs observations.
      • Il existe des centres référents qui peuvent aider à remplir le certificat médical.
      • Le projet de vie est un document qui permet de faire le point sur les besoins et les aspirations de l’enfant ou de l’adulte.
      • Un Livret Parcours Inclusif (LPI) est en cours de développement pour faciliter le suivi des dossiers, mais il n'est pas encore complètement opérationnel.

      Ce briefing devrait permettre de mieux comprendre les étapes et les enjeux liés au dossier MDPH.

    1. Voici des points de synthèse pour un briefing sur Parcoursup, basés sur les sources et notre conversation précédente :

      Fonctionnement et calendrier de Parcoursup :

      • Les lycéens s'inscrivent sur la plateforme Parcoursup et peuvent formuler jusqu'à 10 vœux pour des formations sous statut étudiant et 10 vœux supplémentaires pour des formations en apprentissage. Il est important de noter que ces vœux ne sont pas classés.
      • La date limite pour s'inscrire et formuler des vœux est le 13 mars 2025 à 23h59 (heure de Paris). Il est inutile de se précipiter pour formuler ses vœux au début de la période d'inscription, car la date de soumission n'a pas d'impact sur l'examen des dossiers.
      • Après avoir formulé leurs vœux, les candidats doivent compléter leur dossier et confirmer leurs vœux avant le 2 avril 2025 à 23h59 pour qu'ils soient examinés par les formations. Les candidats recevront des messages de rappel avant ces échéances.

      Types de formations disponibles sur Parcoursup :

      • Parcoursup propose des formations sélectives (classes préparatoires, BTS, BUT, écoles) et non sélectives (licences universitaires). Les formations non sélectives accueillent les candidats jusqu'à leur capacité d'accueil.
      • Il existe également des formations en apprentissage.
      • Certaines formations ne sont pas sur Parcoursup, il faut donc être vigilant.

      Recherche et sélection des formations :

      • Le site de Parcoursup (parcoursup.gouv.fr) offre un accès à la carte des formations et à un guide "les bons réflexes pour choisir sa formation". Il est possible de filtrer les formations par type (apprentissage, public, etc.).
      • Chaque formation a une "carte d'identité" avec des informations importantes. Il y a 6 rubriques: Présentation de la formation, critères d'examen, modalités de candidature et contacts.
      • Les formations sélectives peuvent examiner les dossiers sur dossier, ou en plus sur des épreuves écrites ou orales. Pour les formations non sélectives, l'examen se fait uniquement sur dossier.
      • Les critères d'examen sont regroupés en cinq thématiques : résultats scolaires, méthodes de travail, savoir-être, motivation et engagement. Le poids de chaque critère est indiqué en pourcentage. Les formations peuvent également préciser les éléments spécifiques pris en compte pour chaque critère.
      • Parcoursup met à disposition des rapports d'analyse des candidatures pour chaque formation, signés par le président de l'établissement, avec des données très précises.
      • Les candidats peuvent consulter des données sur l'admission (nombre de candidats, propositions d'admission, etc.) et sur le taux d'accès par type de baccalauréat. Le taux d'accès est la proportion de candidats d'un profil donné ayant reçu une proposition d'admission.
      • Parcoursup affiche des données sur l'insertion professionnelle, telles que le taux d'emploi et le salaire indicatif après les études. Ces données sont calculées au niveau national par les services statistiques ministériels.

      Le dossier Parcoursup :

      • Le dossier Parcoursup comprend les bulletins scolaires (notes et appréciations) de première et terminale. Les notes sont remontées directement par les lycées.
      • Les candidats doivent fournir une attestation PIX.
      • Les formations peuvent demander une lettre de motivation.
      • Les candidats peuvent renseigner une rubrique facultative "activités et centres d'intérêt".
      • Le dossier comprend également une "fiche avenir" remplie par l'équipe pédagogique. Cette fiche contient des appréciations spécifiques des professeurs. Les lycéens n'ont pas accès aux appréciations de la fiche avenir.

      Vœux et sous-vœux :

      • Un vœu correspond à une formation dans un établissement donné.
      • Certaines formations peuvent être regroupées en vœux avec des sous-vœux, notamment les PASS (Parcours d'accès spécifique santé). Un vœu de regroupement compte pour un seul vœu, mais chaque sous-vœu compte dans le nombre total de vœux que le candidat formule.
      • Les formations en soins infirmiers et paramédicales peuvent être regroupées en voeux multiples.

      Accompagnement et soutien :

      • Parcoursup offre un accompagnement spécifique pour les étudiants en situation de handicap.
      • Les candidats peuvent contacter les professionnels de Parcoursup via une messagerie et un numéro de téléphone.
      • Les professeurs principaux des lycées ont également une visibilité sur les dossiers de leurs élèves.

      Points importants à retenir :

      • Parcoursup n'est pas un algorithme qui décide des affectations. Ce sont les enseignants des formations post-bac qui examinent les dossiers.
      • Les formations sont libres de choisir leurs critères d'examen et de décider des modalités d'admission.
      • Il est conseillé de diversifier ses vœux et d'éviter de n'en formuler qu'un seul.
      • Parcoursup vise à favoriser l'égalité des chances et à soutenir les candidats qui en ont le plus besoin.

      Ce briefing peut être complété avec les ressources disponibles sur le site de Parcoursup, notamment les vidéos explicatives.

      Il est important de souligner que la plateforme a fait l'objet de tests avec des lycéens et que les données sont régulièrement mises à jour.

    2. webinaire, sur Parcoursup, la plateforme d'admission post-bac en France.

      L'intervenant explique le fonctionnement de la plateforme, en détaillant les nouvelles fonctionnalités pour l'année 2025, notamment un comparateur de formations et une meilleure visualisation des données statistiques d'admission des années précédentes.

      Il met l'accent sur la transparence de l'information et la collaboration avec les lycées, les fédérations de parents d'élèves, et les étudiants pour améliorer l'expérience utilisateur.

      Le but principal est de rassurer les lycéens et leurs parents sur le processus d'admission et de leur fournir des outils pour faire des choix éclairés.

      Voici un sommaire minuté des idées fortes de la transcription,

      • [00:00:07] Présentation de l'événement, 2600 inscrits en France et dans les établissements d'enseignement français à l'étranger.
      • [00:01:07] Les candidats s'inscrivent sur la plateforme et formulent leurs vœux.
      • [00:02:51] Rappel des formations disponibles sur Parcoursup, important pour la réflexion des lycéens sur leurs vœux.
      • [00:03:38] On distingue les formations sélectives (classes préparatoires, BTS, etc.) et les formations non sélectives (licences). Il y a plus de places proposées dans les licences non sélectives.
      • [00:03:58] Il y a aussi des formations en apprentissage sur Parcoursup.
      • [00:04:08] Certaines formations ne sont pas sur Parcoursup.
      • [00:04:15] Un guide "les bons réflexes pour choisir sa formation" est disponible sur le site de Parcoursup.
      • [00:04:44] 10 000 formations en apprentissage sont proposées, principalement en BTS et BUT.
      • [00:06:19] Le site parcoursup.gouv.fr et la carte des formations sont mis en avant.
      • [00:06:42] On peut rechercher des formations par mots-clés, zone géographique, type de formation, et en utilisant des filtres (apprentissage, public).
      • [00:07:28] Il existe une rubrique "formations similaires" sur la plateforme.
      • [00:08:29] Il est possible de se faire des annotations progressives sur la plateforme.
      • [00:09:02] Il est possible de comparer jusqu'à cinq formations sur la plateforme.
      • [00:10:49] Les informations utiles sur chaque formation sont : la présentation, les critères, les modalités de candidature.
      • [00:11:29] Les modalités d'examen sont indiquées pour chaque formation. Les formations sélectives peuvent examiner les candidatures sur dossier, ou dossier plus épreuves écrites ou orales. Les universités pour les formations non sélectives examinent sur dossier.
      • [00:13:57] Les critères d'examen sont regroupés en cinq thématiques : résultats scolaires, méthodes de travail, savoir-être, motivation, engagement.
      • [00:14:47] Chaque formation précise les éléments pris en compte pour chaque critère.
      • [00:15:38] Chaque formation publie un rapport d'analyse des candidatures.
      • [00:16:52] Les candidats peuvent consulter les taux d'accès par type de baccalauréat.
      • [00:19:42] Il est possible de consulter les réponses obtenues les années précédentes.
      • [00:23:26] Les enseignements de spécialité sont pris en compte dans l'examen des candidatures.
      • [00:24:06] Les données sur les taux d'accès doivent servir au dialogue et ne sont pas des garanties.
      • [00:24:31] Les recherches montrent que les jeunes filles ont tendance à l'autocensure pour l'accès aux formations sélectives.
      • [00:27:46] 75% des formations ont des statistiques sur l'insertion professionnelle.
      • [00:28:15] Les données de salaire sont disponibles depuis le 15 janvier.
      • [00:28:47] Les données sont calculées nationalement par les services statistiques ministériels.
      • [00:30:33] Les informations disponibles sur les formations sont importantes pour la réflexion du lycéen et de sa famille.
      • [00:30:45] Comment vérifier le sérieux d'une formation ?
      • [00:32:37] Le ministère de l'enseignement supérieur travaille à améliorer l'appréciation de la qualité des formations.
      • [00:33:02] Le guide "les bons réflexes" est disponible sur parcoursup.gouv.fr.
      • [00:33:24] Il faut s'interroger sur les modalités pédagogiques de la formation.
      • [00:33:53] Les formations sur Parcoursup doivent respecter une charte.
      • [00:34:42] Les formations sur Parcoursup ne peuvent pas demander de frais de scolarité.
      • [00:37:46] Il est possible d'échanger avec les professionnels de Parcoursup.
      • [00:38:00] Les favoris créés avant la candidature sont importés.
      • [00:38:54] L'apprentissage est financé par l'état.
      • [00:39:55] Les bulletins scolaires et les appréciations des enseignants donnent des indices sur la capacité de travail de l'élève.
      • [00:40:27] La progression de l'élève entre la première et la terminale est prise en compte.
      • [00:41:23] Le taux d'accès est calculé par formation et par type de baccalauréat.
      • [00:44:36] Les données sont calculées par Parcoursup et sont fiables et neutres.
      • [00:47:27] Il faut d'abord se créer un compte sur Parcoursup avant de formuler des vœux.
      • [00:49:14] Les parents peuvent accompagner leur enfant à tout moment de la procédure.
      • [00:51:50] Une lettre de motivation est parfois demandée.
      • [00:52:03] Les vœux ne doivent pas être classés pour éviter l'autocensure.
      • [00:55:03] Il est possible de faire des regroupements de vœux multiples dans les formations en soins infirmiers et paramédicales.
      • [00:55:14] Le sous-vœu correspond à un établissement dans une formation donnée.
      • [00:56:24] Il est possible de faire 20 sous-vœux au global.
      • [01:01:12] Il y a une grande diversité de formations sur Parcoursup.
      • [01:01:40] Pour les formations sélectives, il n'y a pas de sectorisation géographique.
      • [01:02:59] On peut être prioritaire dans une formation en fonction de son secteur géographique.
      • [01:05:39] Il n'y a pas de sous-vœux pour les formations en apprentissage.
      • [01:06:30] Dernier jour pour formuler des vœux : 13 mars 2025.
      • [01:07:27] Les notes sont prises en compte par semestre ou trimestre selon les établissements.
      • [01:08:42] La rubrique "éléments liés à ma scolarité" est une reprise d'informations du lycée.
      • [01:10:38] Les spécificités des sous-vœux à l'internat sont expliquées.
      • [01:13:52] Rappel de la date limite pour formuler les vœux : 13 mars 2025.
      • [01:15:17] Un récapitulatif des vœux sera envoyé par message et SMS.
      • [01:16:54] L'inscription sur Parcoursup est un moment de réflexion pour les lycéens.
      • [01:17:02] La lettre de motivation n'est pas obligatoire pour toutes les formations.
      • [01:21:55] Les expériences extra-scolaires et les engagements sont pris en compte.
      • [01:24:39] L'attestation PIX est à fournir.
      • [01:24:45] Les activités et centres d'intérêt sont à renseigner dans le dossier.
      • [01:25:40] Les bulletins scolaires remontent la moyenne, le positionnement et les appréciations des enseignants.
      • [01:25:58] Parcoursup ne remonte pas le taux d'absentéisme.
      • [01:26:50] Les notes sont remontées par les lycées.
      • [01:27:08] Les élèves doivent vérifier les notes saisies par leur établissement.
      • [01:28:03] La "fiche avenir" comprend une appréciation littérale du professeur.
      • [01:29:01] Les lycéens n'ont pas accès aux appréciations de la fiche avenir avant le 2 juin.
      • [01:30:04] Il existe des dispositifs pour les étudiants en situation de handicap.
      • [01:32:18] Les étudiants en situation de handicap peuvent demander un réexamen de leur dossier.
      • [01:33:45] Il faut citer des informations plutôt que rédiger dans la rubrique "activités et centres d'intérêt".
      • [01:35:22] Il faut être sincère dans la description de ses activités et de ses centres d'intérêt.
      • [01:36:17] La lettre de motivation est souvent demandée par vœu et par sous-vœu.
      • [01:36:52] La lettre de motivation doit être spécifique à la formation visée.
      • [01:37:24] Il faut relire la lettre de motivation, mais ne pas la rédiger à la place de l'élève.
      • [01:38:16] Les lycéens doivent parler d'eux dans la lettre de motivation.
      • [01:38:25] Parcoursup ne décide pas de l'affectation.
      • [01:39:33] Parcoursup soutient les étudiants qui en ont le plus besoin.
      • [01:39:51] Il faut prendre connaissance du calendrier et des modalités de Parcoursup.

      Ce sommaire permet de rapidement identifier les éléments clés de la procédure Parcoursup.

    1. Voici un sommaire minuté de la transcription, mettant en évidence les idées fortes :

      • 0:05-0:27 : Introduction par Benoit Le Corre, le journaliste, qui exprime son questionnement sur le but de filmer Jérémie et présente son approche de raconter la vie des gens. Il mentionne que c'est son deuxième reportage et demande aux spectateurs de s'abonner à sa chaîne.
      • 0:39-1:27 : Benoit anticipe que la vidéo ne sera pas facile à regarder et qu'elle n'a pas été facile à tourner. Il décrit un faux départ, expliquant qu'il n'a pas tout de suite sorti la caméra parce qu'il ne le sentait pas. Il souligne que Jérémie n'a pas l'habitude de la caméra, et il décide d'y aller doucement.
      • 1:29-2:35 : Jérémie se montre peu confiant quant à ce que Benoit pourra tirer de lui, soulignant le désordre de sa maison et son manque d'argent. Il décrit sa chambre comme une "chambre d'alcc**lo". Il révèle qu'il a consommé deux bouteilles de pastis et plusieurs bières la veille.
      • 2:37-3:05 : Jérémie révèle qu'il a lui-même demandé à être filmé. Benoit explique qu'il a rencontré Jérémie grâce à son voisin Jacques, un abstinent qui essaie d'aider Jérémie. Il se demande si faire cette vidéo est une bonne idée, mais il espère que ça pourra l'aider.
      • 3:09-3:35 : Jérémie se prépare pour le tournage, en soulignant ses habitudes de coiffure et son choix de vêtements. Il mentionne ne pas aimer sa tête avec des lunettes. Il révèle qu'il a "bouffé l'héritage de son père" décédé en 2011.
      • 3:38-4:06 : Jérémie montre à Benoit l'ancienne salle de jeux de sa fille, remplie de Playmobil. Il hésite à les vendre tout de suite, mais envisage de le faire le lendemain.
      • 4:16-4:49 : Jérémie avoue que cette vidéo est aussi pour sa fille. Il explique qu'il est plus facile pour lui de parler à Benoit qu'à sa fille. Il exprime la difficulté d'expliquer à sa fille pourquoi il est ainsi.
      • 4:52-5:27 : Jérémie se souvient de la dernière fois qu'il a vu sa fille dans un magasin. Il est ému en évoquant comment sa fille tient compte de son budget.
      • 5:30-5:51 : Benoit remarque que les journalistes dissimulent parfois des choses, et que les gestes sont importants. Il dit qu'il retourne voir Jérémie le lendemain avec un peu d'appréhension.
      • 6:02-6:30 : Jérémie explique qu'il est toujours fatigué le matin tant qu'il n'a pas bu. Il est interrogé sur ce qu'il ferait avec 150 000 euros, et il mentionne partir en voyage avec sa fille.
      • 6:33-6:56 : Jérémie explique qu'il va chez sa mère. Il révèle qu'il n'a pas encore bu ce matin-là. Il explique qu'il a un décollement de la rétine qui affecte sa vision.
      • 6:59-7:17 : Jérémie révèle qu'il a été chauffeur. Il explique qu'il ne veut pas être filmé pour le moment avec sa mère.
      • 8:09-8:36 : La mère de Jérémie exprime son inquiétude face à l'addiction de son fils. Elle se sent coupable et pense que Jérémie a un chagrin. Jérémie demande à Benoit s'il veut boire un verre.
      • 8:40-8:53 : Jérémie admet qu'il n'a pas fait grand chose pour arrêter de boire parce qu'il ne le faisait pas pour lui. Il mentionne avoir tenté trois cures, mais sans succès.
      • 9:04-9:12 : Jérémie exprime son souhait d'aller skier avec sa mère et sa fille s'il gagnait 150 000 euros.
      • 9:13-9:48 : La mère de Jérémie avoue qu'elle a eu peur de lui quand il était sous l'influence de l'alcool. Jérémie révèle qu'il ne se rendait pas compte de ce qu'il lui a fait subir.
      • 9:50-10:14 : Jérémie va à son épicerie habituelle. Il commande un pastis, comme d'habitude. Benoit remarque la familiarité entre Jérémie et le commerçant.
      • 10:31-10:39 : Le commerçant exprime sa tristesse de voir Jérémie détruire sa vie à chaque fois qu'il passe en caisse.
      • 10:47-11:17 : Jérémie et Benoit partagent un repas. Jérémie dit qu'il a été touché par les propos de sa mère, et il a réalisé qu'elle a eu peur de lui.
      • 11:17-11:29 : Benoit explique qu'il s'est mis en retrait le premier soir car il était difficile de communiquer avec Jérémie.
      • 11:29-11:54 : Une chanson sur l'amour est jouée, et il y a une réflexion sur le thème de l'amour.
      • 12:24-12:39 : Benoit exprime son optimisme quant au journalisme et à la possibilité de changement pour Jérémie et sa famille. Il espère mener une interview formelle avec Jérémie.
      • 13:14-13:41 : Benoit propose de faire une interview, mais cela s'avère compliqué. Jérémie révèle qu'il n'a pas dormi de la nuit et qu'il a bu avec un ami. Benoit explique qu'il va reprendre des questions qu'il a déjà posées.
      • 13:41-14:27 : Jérémie admet qu'il est alcoolique et qu'il a commencé à boire à l'âge de 11-12 ans. Il explique qu'il accepte d'être filmé pour sa mère, qu'il a vue triste et dont il admire l'amour inconditionnel. Il avoue avoir été difficile avec ses beaux-pères.
      • 14:31-15:03 : Jérémie reconnait que son corps lui dit stop. Il est confronté à l'idée que sa mort pourrait attrister ses proches. Il comprend qu'il risque de laisser des gens tristes s'il meurt.
      • 15:05-15:54 : Jérémie exprime son désir d'être là pour sa fille, qu'il pense rendre triste à cause de son problème d'alcool. Il imagine le futur dans cinq ans où il serait "beau gosse" et irait chercher sa fille à l'école.
      • 15:57-16:22 : Benoit arrête le tournage car il sent qu'il a envie de sauver Jérémie, ce qui n'est pas son rôle. Il comprend que sa présence n'aide pas Jérémie et que le changement ne peut venir que de lui-même. Il conclut que l'avenir de Jérémie est flou, et qu'il ne faut pas le juger.
      • 16:35-17:07 : Benoit mentionne qu'il a lui-même mis dix ans à se remettre de la mort de son père. Il propose à Jérémie d'arrêter le tournage s'il en a besoin. Jérémie accepte de continuer, disant que ces 15 minutes sont comme deux verres.
    1. Voici un sommaire minuté avec les idées fortes du texte, en gras:

      • Introduction (0:00-1:50)

        • Héloïse Junier, psychologue spécialisée dans la petite enfance (0-6 ans), présente son parcours et son intérêt pour le lien entre la recherche scientifique et la pratique sur le terrain.
        • Elle a écrit une BD sur les émotions de l'enfant pour les adultes et anime des formations sur le sujet.
        • Elle introduit la question des besoins fondamentaux de l'enfant pour son bien-être émotionnel.
      • Les Besoins de l'Enfant (1:50-4:40)

        • Les besoins physiques (manger, boire, dormir) sont importants, et leur non-satisfaction impacte la régulation émotionnelle.
        • Le besoin de sécurité est un méta-besoin qui englobe tous les autres et doit être satisfait pour que l'enfant puisse explorer, jouer, et apprendre.
        • Un enfant en insécurité peut avoir du mal à manger, dormir, et explorer son environnement, même si ses autres besoins sont satisfaits.
        • Il est essentiel de combler les besoins physiques et psychologiques pour un bon fonctionnement de l'enfant.
        • Les besoins psychologiques sont souvent oubliés car ils ne sont pas toujours visibles.
      • Conséquences des Besoins Non Satisfaits (4:40-6:30)

        • Les besoins non satisfaits entraînent un état d'alerte et un déséquilibre hormonal.
        • Cela génère du stress, des émotions désagréables (colère, frustration, peur) et des comportements inadaptés (pleurs, cris, etc.).
        • L'image de l'iceberg : les émotions désagréables visibles sont la partie émergée, et les besoins non satisfaits sont la partie immergée.
        • Il faut se demander "pourquoi" l'enfant réagit ainsi, plutôt que de se concentrer sur le "comment" le faire arrêter.
      • L'importance de l'Empathie (6:30-7:30)

        • Se poser la question des besoins de l'enfant ouvre la porte à l'empathie.
        • Cela permet d'éviter un rapport de force et de comprendre que le comportement de l'enfant est lié à un manque interne.
        • Il est difficile de rester rationnel face à la colère de l'enfant, surtout en cas de fatigue ou de stress.
        • L'absence de rationalité peut entraîner de la violence envers l'enfant.
      • Le Décalage entre la Théorie et la Pratique (7:30-8:30)

        • Il existe un décalage entre la théorie et la pratique.
        • Il est impossible d'appliquer les conseils à 100%, mais il est important de tendre vers cet idéal.
        • Il faut déculpabiliser les parents ou les professionnels qui ont des difficultés.
      • Les Émotions : Utiles et Adaptatives (8:30-9:10)

        • Toutes les émotions, même la colère, sont adaptatives et utiles pour comprendre que quelque chose ne va pas.
        • Les émotions permettent de retrouver un état de bien-être et d'optimiser les chances de survie.
        • Il faut rationaliser que les émotions sont essentielles.
      • Combler les Besoins : Clé du Bien-être (9:10-9:45)

        • Pour un enfant coopératif et tolérant à la frustration, il faut combler ses besoins physiques et psychologiques, notamment le besoin de relation.
        • Un environnement sécurisant et affectueux favorise un bon état émotionnel chez l'enfant.
      • Idées Reçues sur la Colère et le "Caprice" (9:45-12:00)

        • La colère est l'émotion la moins bien supportée et a une mauvaise réputation.
        • Le terme de "caprice" est un abus de langage et n'est pas scientifique.
        • Le caprice est une interprétation adulte d'une émotion non comprise.
        • Il y a une confusion entre l'élément déclencheur (l'envie) et l'élément inducteur (les besoins insatisfaits).
      • Le Cerveau de l'Enfant en Colère (12:00-13:40)

        • L'élément déclencheur met le cerveau en état d'alerte et déclenche une réaction en chaîne.
        • L'amygdale cérébrale se met en alerte, comme si la survie de l'enfant était menacée.
        • Les hormones de stress se libèrent, entraînant une explosion de colère.
        • Le cortex préfrontal de l'enfant n'est pas mature pour réguler ses impulsions et ses émotions.
      • L'enfant n'a pas le choix d'exprimer sa colère, comme le vomi.

      • Comment Réagir à la Colère de l'Enfant (13:40-16:00)

        • Sécuriser l'environnement.
        • Se mettre à hauteur de l'enfant.
        • Ne pas parler pendant le pic de colère, mais proposer un temps de câlin quand l'enfant redescend.
        • Mettre des mots sur ce que l'enfant a ressenti et sur ce que vous avez ressenti aussi.
        • Contenir l'enfant s'il se met en danger ou s'il agresse autrui, en privilégiant un contact oculaire.
      • Que Ne Pas Faire en Cas de Colère (16:00-17:00)

        • Ne pas réagir sous l'effet de ses propres émotions.
        • Faire une pause et sentir les signes de stress chez soi.
        • Utiliser des mantras pour rationaliser la situation.
        • La respiration permet de se détendre.
        • Renouveler l'air ambiant.
        • Passer le relais si possible.
        • Boire pour éviter la déshydratation.
      • Conclusion (18:00-18:45)

        • Devenir l'adulte que l'on aurait aimé avoir quand on était enfant.
        • Se demander comment on aurait aimé que l'on réagisse face à notre propre colère enfantine.
        • C'est un bon point de départ pour bien agir face à la colère des enfants.
    2. Voici des documents de synthèse pour un briefing, basés sur les informations de la source :

      Thème Central : La Gestion des Émotions de l'Enfant

      • L'importance des besoins fondamentaux : Pour qu'un enfant se sente bien et coopère, il est crucial de répondre à ses besoins, qu'ils soient physiques (manger, boire, dormir) ou psychologiques.
      • Le besoin de sécurité comme méta-besoin : Le besoin de sécurité englobe tous les autres besoins et doit être comblé en priorité. Un enfant qui ne se sent pas en sécurité aura du mal à explorer, jouer, et apprendre. Un enfant qui sursaute au moindre bruit, qui a du mal à dormir ou qui est constamment accroché à son adulte de référence est probablement en état de vigilance par manque de sécurité.
      • Les conséquences des besoins non satisfaits : Le manque de satisfaction des besoins engendre un état d'alerte, du stress, des émotions désagréables et des comportements inadaptés.
      • L'image de l'iceberg : Les comportements (pleurs, cris, colère) sont la partie visible de l'iceberg, tandis que les besoins insatisfaits en sont la partie immergée. Il faut chercher le "pourquoi" derrière le comportement, et non seulement le "comment" le faire cesser.
      • L'empathie comme clé : Se demander de quoi l'enfant a besoin permet d'ouvrir une porte vers l'empathie et d'éviter les rapports de force.

      La Colère de l'Enfant

      • La colère est une émotion adaptative : Toutes les émotions, y compris la colère, sont utiles et adaptatives. Elles signalent que quelque chose ne va pas et permettent de retrouver un état de bien-être.
      • Le mythe du "caprice" : Le terme de "caprice" est un abus de langage non scientifique. Il s'agit d'une interprétation adulte d'une émotion non comprise.
      • Confusion entre déclencheur et inducteur : Il ne faut pas confondre l'élément déclencheur (l'envie, comme un jouet refusé) et l'élément inducteur (les besoins non satisfaits, comme le manque de sommeil ou de calme).
      • Le cerveau de l'enfant en colère : Face à un élément déclencheur, l'amygdale cérébrale se met en alerte et le corps produit des hormones de stress qui mènent à une explosion émotionnelle. Le cortex préfrontal de l'enfant, qui permet de réguler les émotions, n'est pas encore mature. La colère est un débordement émotionnel involontaire.

      Comment Réagir Face à la Colère

      • Sécuriser l'environnement : S'assurer que l'enfant ne se blesse pas.
      • Rester calme et se mettre à hauteur de l'enfant : Éviter de parler pendant le pic de colère.
      • Proposer un câlin et mettre des mots : Après le pic de colère, proposer un câlin et mettre des mots sur ce que l'enfant a ressenti, et aussi sur vos propres émotions.
      • Contenir si nécessaire : Contenir l'enfant uniquement s'il se met en danger ou agresse autrui, en privilégiant un contact visuel.
      • Ne pas réagir sous l'effet de ses émotions : Faire une pause, respirer, utiliser des mantras, boire de l'eau, et passer le relais si possible.

      Conclusion

      • Tendre vers l'idéal : Il est difficile d'appliquer ces conseils à 100%, mais il faut tendre vers cet idéal et déculpabiliser.
      • Devenir l'adulte que l'on aurait aimé avoir : Se demander comment on aurait aimé être traité quand on était enfant est une bonne façon de réagir de manière adaptée face à la colère des enfants.
  2. Jan 2025
    1. Voici un sommaire minuté des idées fortes de la transcription, avec les éléments clés en gras:

      • 0:09-0:29 : Introduction du live par Catherine Jubot, psychologue de l'éducation nationale et directrice du CIO de Bourgoin, et présentation du thème : les ressources et l'accompagnement pour l'orientation post-lycée.
      • 0:35-1:30 : Présentation d'Estelle de Moisson, psychologue de l'éducation nationale au CIO de Grenoble. L'accent est mis sur le fait que l'orientation est un processus qui prend du temps et qu'il est normal d'avoir des hésitations ou de changer d'avis. Il est important de ne pas rester seul et de demander de l'aide.
      • 1:30-2:01 : Information sur l'enregistrement du live et possibilité de poser des questions via le chat. Présentation du rôle des CIO, lieux d'accueil pour accompagner la réflexion sur l'orientation.
      • 2:01-3:23 : Les CIO offrent un accueil personnalisé, des entretiens, des ateliers, et de la documentation (ONISEP, CIDJ). Ils accueillent tous les publics (jeunes scolarisés, adultes, décrocheurs). Les psychologues de l'éducation nationale (anciennement conseillers d'orientation psychologues) sont rattachés aux CIO et interviennent dans les établissements scolaires.
      • 3:23-4:01 : Rôle des psychologues de l'éducation nationale : écoute bienveillante, accompagnement sur les parcours, les passerelles, les voies de formation et les secteurs professionnels.
      • 4:01-5:00 : La période de Parcoursup est un moment stressant. Les psychologues accompagnent les jeunes dans la connaissance de soi (points forts, centres d'intérêt, personnalité) et dans l'identification de secteurs professionnels potentiels.
      • 5:00-6:10 : Importance de s'informer sur les formations (BTS, BUT, licences, classes préparatoires, etc.). Se faire accompagner pour comprendre le jargon de Parcoursup et les différents types d'études (courtes, longues, professionnalisantes, théoriques).
      • 6:10-7:17 : Les psychologues aident à s'informer sur les métiers et à utiliser des questionnaires d'intérêt, qui ne sont pas des "boules de cristal" mais des outils de discussion. Il ne faut pas hésiter à les consulter, même sans question précise.
      • 7:17-8:15 : Les psychologues sont disponibles dans les établissements scolaires (sur rendez-vous) et dans les CIO (avec et sans rendez-vous, y compris pendant les vacances scolaires).
      • 8:15-9:04 : Les sites des CIO proposent des informations sur les journées portes ouvertes et les ateliers. Certains CIO proposent des ateliers spécifiques, par exemple sur Parcoursup ou les études à l'international.
      • 9:04-9:35 : Des ateliers peuvent aussi être proposés pour les parents, car l'orientation est une source de stress pour eux aussi.
      • 9:35-10:15 : L'orientation est une question qui peut se poser tout au long de la vie, y compris à l'âge adulte. La réflexion sur l'orientation est un processus continu.
      • 10:15-11:09 : L'orientation n'est pas un enjeu majeur unique, il est normal de se tromper et de se réorienter. Il existe des passerelles entre les formations.
      • 11:09-12:03 : Il est possible de se réorienter, y compris en cours d'année universitaire (semestre 2). Le dossier Parcoursup est conservé d'une année sur l'autre. Se tromper une fois permet d'être plus motivé la deuxième année.
      • 12:03-13:15 : Importance d'aller à la pêche aux informations, en particulier les journées portes ouvertes (JPO). Anticiper l'orientation réduit le stress. Il faut se familiariser avec les lieux, discuter avec les étudiants, et les professeurs.
      • 13:15-14:46 : Plus on s'informe, plus le choix sera pertinent. On est là pour aider même après les JPO. Ne pas rester seul et se rappeler que se tromper n'est pas grave, car on apprend de ces expériences.
      • 14:46-15:22 : Les changements font partie de la vie et permettent de se construire. Se réorienter est possible et ne doit pas être vu comme un échec.
      • 15:22-16:03 : Ressources en ligne pour préparer l'orientation : plateforme Avenir, site de l'ONISEP (vidéos sur ONISEP TV), ressources régionales (côté formation).
      • 16:03-17:50 : Parcoursup est une mine d'or d'informations, avec des cartes de visite pour chaque formation, les critères d'analyse des candidatures, et les formations similaires. On peut y accéder même sans candidater, et contacter les responsables pédagogiques et les étudiants ambassadeurs.
      • 17:50-18:25 : Il est possible de créer un compte Parcoursup dès la seconde et de mettre des formations en favori.
      • 18:25-19:33 : Importance de se renseigner pour sortir du flou, des clichés et des idées reçues. Les conseils sont utiles mais il faut vérifier par soi-même. La décision finale revient à l'élève.
      • 19:33-20:10 : Ne pas prendre pour argent comptant tous les conseils. L'orientation est un choix personnel. Il faut explorer et ne pas s'autocensurer.
      • 20:10-20:17 : Transition vers les questions du public.
      • 20:17-20:55 : Réponses aux questions :
        • Possibilité de prendre rendez-vous pendant les heures de cours, avec un justificatif
        • Possibilité de venir avec ses parents
      • 20:55-22:20 : Réponses aux questions:
        • Les questionnaires en ligne existent mais ne sont pas des "boules de cristal". Ils sont plus utiles comme base de discussion avec un professionnel.
        • Les questionnaires sont une photo de soi à un instant T.
        • L'important est de travailler la connaissance de soi avec un professionnel
      • 22:20-23:11 : Les jeunes ont rarement "envie de tout faire". L'orientation est une question d'élimination et de découverte des 180 secteurs professionnels.
      • 23:11-24:20 : Les filières générales ne sont pas prioritaires en BTS. Il existe des quotas. Il est important de vérifier sur Parcoursup les profils des élèves recrutés. L'apprentissage peut être une solution pour les élèves de filière générale.
      • 24:20-25:07 : Il ne faut pas s'autocensurer et il faut expliquer sa motivation pour intégrer un BTS.
      • 25:07-26:35 : Déroulement d'un rendez-vous avec un psychologue d'orientation. L'entretien s'adapte à la demande de chacun, avec un temps de consultation plus long dans les CIO. Il n'y a pas de bonnes ou de mauvaises questions ou de mauvais projets, le meilleur projet est celui qui vous convient.
      • 26:35-27:35 : Passage d'une seconde professionnelle à une seconde générale et technologique : possible en fin d'année si l'avis d'orientation est favorable.
      • 27:35-28:18 : Après un bac pro, il est possible d'accéder aux mêmes études qu'un bac général ou technologique avec un bon dossier scolaire.
      • 28:18-30:22 : La pression sur le choix des spécialités est à relativiser. Certaines formations très scientifiques nécessitent des spécialités scientifiques, mais il existe des remises à niveau à l'université.
      • 30:22-31:14 : Il faut enlever la pression sur le choix des spécialités. Vérifier les informations sur parcoursup et les doublettes de spécialités les plus prises.
      • 31:14-31:38 : Il n'est pas possible de refaire le même bac pour améliorer ses résultats.
      • 31:38-32:05 : Conclusion du live : ne restez pas seul, contactez les CIO ou les psychologues scolaires..

      Ce sommaire met en évidence les principaux points abordés dans le live, en soulignant l'importance de l'accompagnement, de l'information, et du droit à l'erreur dans le processus d'orientation.

    1. Reviewer #1 (Public review):

      Summary:

      The study examines human biases in a regime-change task, in which participants have to report the probability of a regime change in the face of noisy data. The behavioral results indicate that humans display systematic biases, in particular, overreaction in stable but noisy environments and underreaction in volatile settings with more certain signals. fMRI results suggest that a frontoparietal brain network is selectively involved in representing subjective sensitivity to noise, while the vmPFC selectively represents sensitivity to the rate of change.

      Strengths:

      (1) The study relies on a task that measures regime-change detection primarily based on descriptive information about the noisiness and rate of change. This distinguishes the study from prior work using reversal-learning or change-point tasks in which participants are required to learn these parameters from experiences. The authors discuss these differences comprehensively.

      (2) The study uses a simple Bayes-optimal model combined with model fitting, which seems to describe the data well.

      (3) The authors apply model-based fMRI analyses that provide a close link to behavioral results, offering an elegant way to examine individual biases.

      Weaknesses:

      My major concern is about the correlational analysis in the section "Under- and overreactions are associated with selectivity and sensitivity of neural responses to system parameters", shown in Figures 5c and d (and similarly in Figure 6). The authors argue that a frontoparietal network selectively represents sensitivity to signal diagnosticity, while the vmPFC selectively represents transition probabilities. This claim is based on separate correlational analyses for red and blue across different brain areas. The authors interpret the finding of a significant correlation in one case (blue) and an insignificant correlation (red) as evidence of a difference in correlations (between blue and red) but don't test this directly. This has been referred to as the "interaction fallacy" (Niewenhuis et al., 2011; Makin & Orban de Xivry 2019). Not directly testing the difference in correlations (but only the differences to zero for each case) can lead to wrong conclusions. For example, in Figure 5c, the correlation for red is r = 0.32 (not significantly different from zero) and r = 0.48 (different from zero). However, the difference between the two is 0.1, and it is likely that this difference itself is not significant. From a statistical perspective, this corresponds to an interaction effect that has to be tested directly. It is my understanding that analyses in Figure 6 follow the same approach.

      Relevant literature on this point is:

      Nieuwenhuis, S, Forstmann, B & Wagenmakers, EJ (2011). Erroneous analyses of interactions in neuroscience: a problem of significance. Nat Neurosci 14, 1105-1107. https://doi.org/10.1038/nn.2886

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

      There is also a blog post on simulation-based comparisons, which the authors could check out: https://garstats.wordpress.com/2017/03/01/comp2dcorr/

      I recommend that the authors carefully consider what approach works best for their purposes. It is sometimes recommended to directly compare correlations based on Monte-Carlo simulations (cf Makin & Orban). It might also be appropriate to run a regression with the dependent variable brain activity (Y) and predictors brain area (X) and the model-based term of interest (Z). In this case, they could include an interaction term in the model:

      Y = \beta_0 + \beta_1 \cdot X + \beta_2 \cdot Z + \beta_3 \cdot X \cdot Z

      The interaction term reflects if the relationship between the model term Z and brain activity Y is conditional on the brain area of interest X.

      Another potential concern is that some important details about the parameter estimation for the system-neglect model are missing. In the respective section in the methods, the authors mention a nonlinear regression using Matlab's "fitnlm" function, but it remains unclear how the model was parameterized exactly. In particular, what are the properties of this nonlinear function, and what are the assumptions about the subject's motor noise? I could imagine that by using the inbuild function, the assumption was that residuals are Gaussian and homoscedastic, but it is possible that the assumption of homoscedasticity is violated, and residuals are systematically larger around p=0.5 compared to p=0 and p=1.

      Relatedly, in the parameter recovery analyses, the authors assume different levels of motor noise. Are these values representative of empirical values?

      The main study is based on N=30 subjects, as are the two control studies. Since this work is about individual differences (in particular w.r.t. to neural representations of noise and transition probabilities in the frontoparietal network and the vmPFC), I'm wondering how robust the results are. Is it likely that the results would replicate with a larger number of subjects? Can the two control studies be leveraged to address this concern to some extent?

      It seems that the authors have not counterbalanced the colors and that subjects always reported the probability of the blue regime. If so, I'm wondering why this was not counterbalanced.

    1. Voici un document de synthèse pour un briefing, basé sur les informations des sources et notre conversation :

      Introduction et présentation du problème * Le documentaire audio "Mon enfant terrible" explore les défis de la parentalité à travers l'histoire d'Oscar, un enfant de 2 ans et 8 mois qui fait des crises de colère intenses et fréquentes. * Ces colères se manifestent par des hurlements, des coups, des morsures, et un comportement difficile à gérer pour ses parents. * La mère se sent dépassée, perdue et coupable face à ces réactions, tandis que le père est démuni. Le couple se dispute souvent sur la manière de gérer les crises.

      Les colères d'Oscar : un quotidien difficile * Les crises d'Oscar sont soudaines, immenses et se produisent à tout moment de la journée et de la nuit. * Elles surviennent dans divers contextes : à la maison, à la crèche (au début), au parc, dans les transports en commun, au supermarché. * La mère tient un journal des colères pour essayer de comprendre leur fréquence et leur intensité. Elles sont notées sur une échelle de 1 à 5. * Les colères sont souvent déclenchées par des refus, des frustrations ou des désirs non satisfaits (ne pas vouloir rester dans la poussette, ne pas vouloir sortir du bain, ne pas aimer les haricots, ne pas vouloir aller à la crèche avec son père, etc.).

      Les réactions des parents * Les parents essaient différentes méthodes pour calmer Oscar : parlementer, utiliser des casques anti-bruit, l'enfermer dans sa chambre. * Cependant, ils sont souvent en désaccord sur la marche à suivre, ce qui engendre des disputes et augmente leur stress. * La mère avoue avoir parfois cédé pour éviter une crise, mais elle a également eu des réactions de colère, allant jusqu'à crier plus fort que son enfant ou même le taper. * Les parents se sentent jugés par les autres, en particulier lors des colères en public.

      Le témoignage d'amies et la culpabilité maternelle * Des amies ayant également des enfants "difficiles" témoignent de leurs propres difficultés. Elles évoquent notamment la culpabilité maternelle. * Elles révèlent que leurs enfants ont fait ressortir des aspects de leur personnalité qu'elles ne soupçonnaient pas, comme une violence ou une colère qu'elles ne pensaient pas avoir. * L'une d'elle décrit comment elle en est venue à anticiper les colères de sa fille pour les éviter, allant jusqu'à céder à ses caprices.

      Recherche de solutions : l'éducation bienveillante et la psychothérapie * Les parents cherchent des solutions dans les livres sur l'éducation bienveillante et la parentalité positive. * Ils consultent les conseils d'une experte en parentalité positive, Charlotte Ducharme, qui prône l'empathie et la compréhension des émotions de l'enfant. * Néanmoins, ils ont du mal à rester cohérents dans cette approche et alternent entre bienveillance et autoritarisme. * Face à l'échec de ces méthodes, ils consultent une pédopsychiatre et la psychologue de la crèche.

      Les conseils des professionnels et une prise de conscience * La pédopsychiatre explique qu'Oscar exprime par la colère des émotions qu'il ne sait pas exprimer autrement. * Elle souligne qu'il y a des bénéfices secondaires à son comportement, notamment l'attention de ses parents et une relation exclusive avec eux. * La psychologue encourage les parents à faire front ensemble, à établir des règles, et à prendre du temps pour eux. * Après ces consultations, un changement inattendu se produit : les colères d'Oscar disparaissent soudainement. * La mère réalise qu'en traquant chaque colère d'Oscar, elle avait fait de lui le centre de son attention. En cessant de se focaliser uniquement sur ses crises, elle lui permet de se détacher de son rôle de "vilain petit canard". * Elle prend conscience qu'il ne faut pas enfermer les enfants dans un rôle, car ils finissent par y ressembler. Elle apprend qu'Oscar est aussi un enfant affectueux, joueur et plein d'énergie.

      Conclusion * Le documentaire met en lumière la complexité de la parentalité et les difficultés rencontrées face aux colères d'un enfant. * Il souligne l'importance de la cohérence, de la compréhension et de l'écoute pour accompagner les enfants dans leur développement émotionnel. * Il suggère que les colères peuvent être une manière pour l'enfant d'exprimer un mal-être et de rechercher l'attention de ses parents. * Il est essentiel de ne pas enfermer l'enfant dans un rôle et de le considérer comme un être unique avec ses propres émotions et besoins. * Il y a une dimension universelle dans ce récit, les parents ont tendance à se sentir coupable et à s'interroger sur leurs compétences.

      Points clés à retenir :

      • Les colères des enfants sont une forme d'expression
      • La cohérence parentale est essentielle
      • Il est crucial de ne pas enfermer l'enfant dans un rôle
      • La culpabilité des parents est un sentiment fréquent
      • L'importance de l'écoute et de la compréhension dans l'éducation
    2. Voici un résumé minuté avec les idées fortes en gras, basé sur la transcription fournie :

      • [0:00-0:55] Introduction et présentation d'Oscar : La narratrice ironise sur le bonheur d'être mère, présentant Oscar, son fils de 2 ans et 8 mois, comme étant tout sauf calme et gentil. La version courte de son quotidien est idyllique, mais la version longue inclut des hurlements, des portes claquées et des morsures.

      • [0:55-1:39] Les colères d'Oscar et le désarroi de la mère : Oscar fait des colères soudaines et intenses, frappant, tapant des pieds et devenant rouge. La mère se sent perdue face à ces réactions. L'histoire d'Oscar commence bien avant sa naissance, avec la rencontre de ses parents.

      • [1:39-2:50] Rencontre des parents et désaccord initial sur les enfants : La narratrice raconte sa rencontre avec le père d'Oscar, Alex. Au début, Alex ne voulait pas d'enfants, ayant une mauvaise image des enfants colériques dans les supermarchés. Il ne voulait pas non plus discipliner un enfant.

      • [2:50-3:31] L'arrivée du premier enfant et l'acceptation du second : La narratrice finit par convaincre Alex d'avoir un enfant. Leur premier fils, Emile, est calme et patient. Alex accepte plus facilement l'idée d'un deuxième enfant.

      • [3:31-4:45] La naissance d'Oscar et ses premières colères : Oscar naît par césarienne et sa première colère survient avant même son premier anniversaire. Sa nounou, Mary, décrit une crise de pleurs intense et inexplicable.

      • [4:45-5:34] Un journal des colères et des descriptions à la crèche : La mère commence à tenir un journal des colères d'Oscar. Les colères se produisent de plus en plus souvent et de plus en plus fort. Le personnel de la crèche décrit les colères d'Oscar comme étant impressionnantes et sonores.

      • [5:34-6:42] Les difficultés du père face aux colères : Le père d'Oscar se sent démuni face à ses colères matinales. Partir en laissant son enfant dans cet état est horrible pour lui.

      • [6:42-7:40] Le comportement d'Oscar à la crèche : À la crèche, Oscar joue avec ses amis, écoute des histoires, chante joyeusement et fait des concessions. Il reçoit même une médaille de bonne conduite de sa référente.

      • [7:40-8:32] Les colères à la maison : À la maison, c'est la "castagne" pour chaque activité : bain, repas, sommeil. Les parents essaient différentes méthodes, mais sont en désaccord sur la marche à suivre.

      • [8:32-9:26] Le désespoir des parents et les explications de l'entourage : La narratrice craque, surtout quand Alex dit que c'est ce qu'il craignait. L'entourage donne des explications variées (place du deuxième enfant, signe astrologique, genre de l'enfant, âge), auxquelles la mère s'accroche temporairement.

      • [9:26-10:12] Témoignage d'une amie : Une amie, Elise, raconte les colères de son propre enfant, Louis, à partir de 2 ans, qui sont devenues ingérables vers 3 ans et demi-4 ans.

      • [10:12-11:16] La violence révélée par les enfants : Elise révèle que son fils l'a poussée dans ses retranchements et lui a fait découvrir un aspect monstrueux de sa personnalité. Elle se voit déformée par la colère dans le miroir. Ces accès de colère peuvent monter puis disparaître en un instant.

      • [11:16-12:07] La culpabilité : Les mères se sentent coupables de ne pas savoir gérer les colères de leurs enfants et d'être elles-mêmes débordées par leurs émotions. Le père d'Oscar a découvert un pan inconnu de la personnalité de sa femme, marqué par le stress et la colère.

      • [12:07-12:53] Stratégies d'évitement et peur : La mère cède souvent pour éviter les colères. Les réactions de son enfant lui font peur, et les siennes aussi. Une autre amie, Rachel, raconte qu'elle anticipait les colères de sa fille pour les éviter.

      • [12:53-14:01] Les colères en public et le jugement des autres : Les colères dans la rue sont particulièrement difficiles à gérer. La mère décrit des épisodes de colères dans le métro et dans une aire de jeux. Elle se sent jugée par les autres.

      • [14:01-15:34] Un témoignage d'Elise : Elise raconte une scène de colère de son fils au supermarché, où elle a été publiquement critiquée par d'autres clients. Elle se sent une mauvaise mère.

      • [15:34-16:48] Besoin d'aide et recours aux livres : Les parents se rendent compte qu'ils ont besoin d'aide. Ils cherchent des conseils dans des livres sur l'éducation bienveillante et la parentalité positive.

      • [16:48-17:41] Conseils de Charlotte Ducharme : Une experte, Charlotte Ducharme, donne des conseils sur YouTube : changer ses croyances sur la colère, faire preuve d'empathie et aider l'enfant à revenir au calme.

      • [17:41-18:59] Application des conseils et retour à la réalité : La mère essaie d'appliquer les conseils pour aider son enfant à contrôler ses émotions. Un livre pour enfant est utilisé comme support. Cependant les crises reprennent.

      • [18:59-19:33] Le manque de cohérence des parents : Les parents ont du mal à rester cohérents dans leur approche éducative et alternent entre bienveillance et autoritarisme. Ils n'arrivent pas à tenir la distance.

      • [19:33-21:21] Recours à la psychothérapie : La famille décide de consulter un pédopsychiatre. La première séance est chaotique, Oscar ayant mis le cabinet sens dessus dessous. La psy explique que c'est une manière pour lui de se défouler.

      • [21:21-22:23] Suite des colères et consultations : Les colères d'Oscar continuent, avec des épisodes au centre de loisirs et pendant le bain. La mère consulte la psychologue de la crèche.

      • [22:23-23:31] Séance avec la psychologue de la crèche : Les parents décrivent les colères, leur propre incompréhension et leurs disputes.

      • [23:31-25:22] Explications de la psychologue : La psy explique qu'Oscar n'arrive pas à exprimer ce qu'il ressent autrement que par la colère. Il cherche l'attention et une relation exclusive avec ses parents. Son comportement est une manière d'être avec eux, même si cette manière n'est pas satisfaisante.

      • [25:22-26:37] Pistes de solutions : Il est important que les parents fassent front ensemble, qu'ils définissent des règles, qu'ils trouvent du temps pour eux sans les enfants et qu'ils identifient ce sur quoi ils peuvent lâcher prise. La situation va finir par s'améliorer.

      • [26:37-27:39] Un changement inattendu : Après avoir appliqué les conseils de la psychologue, un changement étrange se produit : les colères d'Oscar disparaissent d'un coup. Il apprend même à dire "oui".

      • [27:39-28:54] L'explication du changement : La mère réalise qu'Oscar était devenu le centre de son attention à cause de ses colères. En étant moins le seul à hurler, Oscar se détache de l'image de "vilain canard" que sa mère avait de lui.

      • [28:54-29:19] Ne pas enfermer les enfants dans un rôle : Il ne faut pas enfermer les enfants dans un rôle car ils finissent par y ressembler. Il est important de se souvenir qu'Oscar est aussi un enfant affectueux, joueur et plein d'énergie.

    1. Voici un sommaire minuté avec les idées fortes de la conférence, tel que demandé :

      • Introduction (0:00-2:30)
        • Présentation de Kévin Jean, épidémiologiste, et de son parcours.
        • L'importance de la transition écologique et son lien avec la santé sont soulignés.
        • Le vélo est présenté comme un sujet central, reliant transition écologique et santé.
      • Le vélo et ses transitions (2:30-6:00)
        • Histoire du vélo, de la drésienne au vélo moderne, en passant par le vélocipède.
        • Les innovations clés : pédales, chaîne de transmission.
        • L'efficacité énergétique du vélo est mise en avant : il est le mode de déplacement le plus efficace, consommant 5 fois moins d'énergie qu'un homme à pied.
      • Évolution sociale du vélo (6:00-8:00)
        • Le vélo passe d'un objet de loisir aristocratique à un moyen de transport populaire.
        • L’essor de la voiture dans les Trente Glorieuses a conduit à un déclin de l'utilisation du vélo.
        • La voiture devient le mode de déplacement majoritaire, y compris pour les courtes distances.
      • Enjeux environnementaux et sanitaires (8:00-10:00)
        • Le secteur des transports est le premier émetteur de gaz à effet de serre en France, la voiture étant le principal contributeur.
        • Les externalités négatives de la voiture sont soulignées : pollution, accidents, sédentarité, coûts pour la santé.
        • Le concept de « motonormativité » est introduit : on tolère des nuisances de la voiture qu'on n'accepterait pas pour d'autres choses.
      • Liens entre mobilité et santé : manque d'activité physique (10:00-13:00)
        • L’OMS recommande 150 minutes d'activité physique modérée par semaine pour les adultes.
        • Un adulte sur trois et 70-80% des jeunes ont une activité physique insuffisante.
        • En France, 95% de la population adulte manque d'activité physique selon l'ANS.
        • Il est essentiel de sortir d'une vision individuelle de l'activité physique et de prendre en compte l'environnement et les modes de vie.
      • Activité physique et santé (13:00-16:00)
        • Les études épidémiologiques montrent un lien entre activité physique et réduction du risque de maladies cardiovasculaires.
        • Une étude sur les employés des bus londoniens a mis en évidence le rôle de l'activité physique dans la santé.
        • L’activité physique a un effet sur de nombreux paramètres biologiques (système cardiovasculaire, métabolisme, santé mentale).
        • La marche et le vélo réduisent le risque de mortalité.
      • Potentiel du vélo (16:00-23:00)
        • Une étude évalue les bénéfices sanitaires et climatiques du vélo en France en 2019.
        • L’étude montre que le vélo permet d'éviter 2400 décès et 6000 cas de maladies chroniques par an.
        • Chaque kilomètre parcouru à vélo permet d'économiser 1€ de coûts sociaux de santé.
        • Une simulation montre que si 25% des trajets courts en voiture étaient faits à vélo, 1800 décès supplémentaires pourraient être évités.
        • Malgré des études montrant les bénéfices, la part modale du vélo est restée stable entre 2008 et 2019.
      • Neutralité carbone et scénarios (23:00-28:00)
        • Les scénarios de neutralité carbone (Négawatt, ADEME) incluent une augmentation de l'usage du vélo.
        • Dans le scénario Négawatt, le vélo est multiplié par 7, permettant d'éviter 10 000 décès par an dès 2040.
        • Les scénarios de l'ADEME, qui reposent sur différentes hypothèses, montrent que les impacts sanitaires peuvent varier fortement, soulignant l'importance d'intégrer la santé dans les débats sur la neutralité carbone.
      • Conclusion (28:00-31:00)
        • Ce qui est bon pour le climat est bon pour la santé publique, et les bénéfices sont immédiats.
        • Les politiques de transition bas carbone sont aussi des politiques de santé publique.
        • Les aménagements urbains doivent être repensés pour favoriser la mobilité active et les espaces verts.
      • Questions et réponses (31:00-38:00)
        • Le vélo électrique est une forme d'activité physique et permet d'allonger les distances parcourues à vélo.
        • Les infrastructures induisent la demande de mobilité active.
        • Le bénéfice de faire du vélo dépasse largement les risques.
        • La faible pratique du vélo chez les jeunes est liée à un manque de sécurité dans les villes.
        • La pollution atmosphérique peut être intégrée aux modèles mais l'impact de l'activité physique est plus important pour la santé que la réduction de la pollution atmosphérique.
        • Les aides à l'achat et à la réparation de vélos ont tendance à être réduites dans le contexte actuel.
    1. Voici un document de synthèse pour un briefing, basé sur la conférence de Sonia Lavadinho et les échanges qui ont suivi, en mettant en évidence les points clés :

      Titre : La Ville Relationnelle : Un Nouveau Paradigme pour la Santé et le Bien-être Urbain

      Introduction

      • Sonia Lavadinho, anthropologue urbaine et fondatrice du cabinet Bfluid, a présenté une vision novatrice de la ville, en mettant l'accent sur la nécessité de repenser nos environnements urbains pour favoriser la santé physique et mentale.
      • La ville relationnelle propose de remplacer le paradigme de la ville fonctionnelle, héritée du 20e siècle, par un modèle plus adapté aux défis du 21e siècle.
      • L'objectif est de créer des espaces qui encouragent le mouvement, les interactions sociales et le lien avec le vivant.

      Problématiques et Constats

      • Déconnexion du corps biologique : Nous passons trop de temps assis, devant des écrans, et en voiture, ce qui nous déconnecte de nos corps et de nos besoins biologiques.
      • Manque de mouvement : Les enfants passent moins de temps à l'extérieur que les prisonniers, et le contact avec la nature est de plus en plus rare.
      • Solitude et manque d'empathie : Ces problèmes sont directement liés au manque de contact avec son propre corps et avec les autres.
      • Zoning intergénérationnel : Les différentes générations sont séparées, ce qui entrave la mixité sociale et le développement d'une ville pour tous.
      • Sédentarité : Le temps passé en voiture contribue à la sédentarité, et les enfants sont de plus en plus dépendants de la voiture pour leurs déplacements.
      • Isolement et solitude: Ces phénomènes touchent toutes les catégories d'âge et augmentent les risques de maladies.

      Concepts Clés

      • Espaces "mielleux" : Des espaces publics où les gens ont envie de rester plus longtemps et de revenir plus souvent, favorisant les interactions sociales et le bien-être.
      • La ville du dehors : Le contact avec l'eau, le vivant, et la nature est essentiel pour le bien-être.
      • Ville amie de toutes les générations : Une ville qui permet de se connecter avec les personnes d'âges différents.
      • Biodiversité des publics : La diversité des âges, des genres et des origines dans les espaces publics est un signe de santé et de vitalité.
      • Ville comestible et communale: Une ville qui permet aux habitants de renouer avec leur alimentation et les circuits courts.
      • Ville du temps libre : Une ville qui valorise le temps libre et notamment la nuit comme source d'activité et de bien-être.

      Les Sept Figures de la Ville Relationnelle

      • La ville du dehors.
      • La ville amie de toutes les générations.
      • La ville du faire et du tiers solidaire.
      • La ville de la surprise.
      • La ville comestible et communale.
      • La ville du temps libre.

      Cinq Leviers d'Action pour l'Existant

      1. Armature des rues : Transformer les grandes rues et avenues en "ruisseaux de fraîcheur" avec plus de végétation.
      2. Parcs et leur "deuxième peau" : Agir sur les rues qui entourent les parcs pour augmenter leur accessibilité et leur attractivité.
      3. Bandes ludiques : Intégrer le jeu dans l'espace public pour que les enfants puissent jouer librement.
      4. Carrefours vivants : Transformer les carrefours, souvent sources de stress, en lieux de vie en augmentant la convivialité et en y intégrant une diversité d'usages.
      5. Parkings prévenants : Améliorer les parkings pour que le premier pas hors de la voiture soit agréable.

      Stratégies et Méthodes

      • Double rue : Activer des boucles itinérantes pour les piétons afin d'animer un quartier et de favoriser le commerce.
      • Balades croquées : Impliquer les citoyens en leur prêtant les crayons des dessinateurs pour qu'ils expriment leurs idées.
      • Atelier "on se marie" : Un espace de discussion pour que tous soient d'accord avec le projet avant sa validation.
      • Monitoring et évaluation : Suivre les indicateurs de qualité de vie dans les espaces publics pour mesurer l'impact des projets.

      Recommandations

      • Agir à grande échelle : Ne pas travailler rue par rue ou école par école, mais avoir une approche systémique.
      • Travailler avec les "ninzagers" : Inclure les personnes qui bloquent les projets (architectes, urbanistes, etc.) dès le début, en les mettant sur le terrain avec les citoyens.
      • Rapprocher le "soft" et le "hard" de la ville : Faire collaborer les acteurs de la santé, de la culture et du social avec ceux de l'urbanisme.
      • Prioriser l'espace public : Aménager l'espace public avant de construire les bâtiments pour que les habitants puissent en profiter dès leur arrivée.
      • La nuit comme levier : Investir le premier et le quatrième temps de la nuit pour amener plus de sécurité et de convivialité.
      • Mettre l'accent sur la biodiversité des publics : Amener une variété de personnes dans la rue pour créer une surveillance naturelle et réduire la violence.

      Conclusion

      • La ville relationnelle est un concept essentiel pour améliorer la santé et le bien-être des populations urbaines.
      • Les cinq leviers d'action proposés par Sonia Lavadinho offrent des pistes concrètes pour transformer nos villes.
      • Il est crucial d'impliquer les citoyens dans les projets et de suivre leur impact.
      • La ville de demain sera relationnelle ou ne sera pas.
    2. Voici un résumé minuté de la transcription de la conférence de Sonia Lavadinho, avec les idées fortes mises en évidence en gras :

      • 0:00-0:21 : Introduction de la 2e journée des JASP, avec un accueil des participants.
      • 0:31-1:50 : Remerciements et présentation de l'équipe organisatrice des Journées annuelles de santé publique.
      • 1:50-2:46 : Reconnaissance du territoire autochtone ancestral sur lequel se déroule l'événement, et mention des 11 nations autochtones du Québec.
      • 2:46-3:49 : Présentation des JASP comme événement de formation accrédité, et importance du réseautage, soulignant la collaboration au sein du réseau de santé publique.
      • 3:55-4:32 : Annonce d'une plénière avec le Docteur Luc Boileau et Pierre Gerlier, axée sur les transformations en cours.
      • 4:32-5:33 : Informations sur la traduction simultanée par IA, et sur des activités en marge comme une conférence sur les points de services locaux.
      • 5:33-6:11 : Présentation d'un 5 à 7 organisé par une équipe de santé au travail, avec un jeu coopératif basé sur le thème de la pandémie.
      • 6:11-7:06 : Remerciements à l'équipe de l'Institut, aux responsables d'activités, et mention des partenaires du consortium pour la formation continue en santé publique.
      • 7:06-7:53 : Annonce du concours de la relève en 3 minutes, des présentations par affiche, et mention des gagnants dans les catégories nouvelles connaissances et pratiques inspirantes.
      • 7:53-9:24 : Annonce d'une mention d'honneur catégorie nouvelle connaissance et pratique inspirante et du prix étudiant dans la même catégorie.
      • 9:24-11:29 : Introduction de Sonia Lavadinho et de sa conférence sur la ville relationnelle. Ses objectifs sont d'ouvrir une réflexion sur les innovations urbanistiques favorables à la santé et d'illustrer les processus et résultats de ces innovations. Il est aussi précisé que Sonia Lavadinho est une anthropologue urbaine et géographe, fondatrice du cabinet Bfluid.
      • 11:29-12:23 : Sonia Lavadinho commence sa conférence en insistant sur le lien entre l'environnement urbain et la santé, soulignant que nos environnements urbains peuvent nous rendre malades ou plus sains.
      • 12:23-13:01 : Elle partage des données sur l'augmentation du taux d'obésité chez les migrants aux États-Unis, mettant en évidence l'impact du mode de vie urbain sur l'alimentation.
      • 13:01-14:16 : Elle souligne que le manque de mouvement et la déconnexion de nos corps biologiques sont des problèmes majeurs, citant une étude anglaise sur le temps passé à l'extérieur par les enfants. Elle ajoute que le contact avec le vivant est aussi essentiel.
      • 14:16-15:27 : Elle aborde la question de la solitude et du manque d'empathie, liés au manque de contact avec son propre corps et avec les autres, et met en avant une approche d'espaces "mielleux".
      • 15:27-16:15 : Elle présente son livre "La ville relationnelle", qui insiste sur sept figures de la ville relationnelle.
      • 16:15-17:31 : Elle souligne l'importance de la ville du dehors, de la ville amie de toutes les générations et le problème du zoning intergénérationnel.
      • 17:31-19:04 : Elle parle du phénomène inédit de la cohabitation de toutes les générations sur terre, soulignant la nécessité d'une ville pour tous.
      • 19:04-20:26 : Elle aborde des concepts comme la ville du faire, du tiers solidaire, de la surprise, la ville comestible, et la ville du temps libre, soulignant l'importance de la nuit pour lutter contre l'obésité.
      • 20:26-21:22 : Elle met l'accent sur les cycles de vie, en particulier les enfants dès le ventre de la mère, et l'importance de l'accompagnement en cas de situation handicapante et de vieillissement.
      • 21:22-22:20 : Elle propose trois manières de faire, notamment la balade nocturne, les dynamiques de proximité et le premier pas actif hors de chez soi en privilégiant le lien avec le vivant.
      • 22:20-23:47 : Elle prend comme exemple un quartier de Bordeaux, pour montrer le lien entre la capacité à produire la ville du dehors et les espaces verts, la présence de l'eau, de l'animalité, et les rencontres informelles. Elle souligne l'importance de la biodiversité des publics et de liens de voisinage.
      • 23:47-24:47 : Elle introduit cinq leviers pour agir dans l'existant, en vue de créer une ville relationnelle, active et en bonne santé, et mentionne le plan d'urbanisme favorable à la santé de la ville de Paris.
      • 24:47-26:16 : Elle détaille le premier levier : l'armature des rues, en mentionnant l'exemple de Lyon et de Barcelone qui a lancé une refonte totale de ses grandes avenues.
      • 26:16-27:57 : Elle illustre avec l'exemple de Buenos Aires, avec une carte mentale des problèmes de marchabilité, et une stratégie de relier les parcs aux gares et les gares entre elles. Elle attire l'attention sur le fait qu'il est possible d'avoir un impact sur la moitié de la population en travaillant seulement 3% des rues.
      • 27:57-29:43 : Elle parle du second levier, qui est de travailler sur les parcs déjà existants et sur leur deuxième peau (les rues qui les entourent), pour répondre à des questions de vulnérabilité, de fragilité, d'isolement et de fraîcheur.
      • 29:43-30:55 : Elle présente le troisième levier : les bandes ludiques, qui consistent à mettre le jeu à portée des enfants sans qu'ils aient à demander la permission. Elle mentionne l'exemple du Superkilen à Copenhague, qui relie trois quartiers populaires.
      • 30:55-32:28 : Elle évoque l'opération "rue aux écoles" à Paris, pour amener plus de végétalisation et de jeux, et pour que les rues deviennent utilisables par tous. Elle souligne la nécessité d'agir massivement et pas école par école.
      • 32:28-33:10 : Elle met en avant les bénéfices rétroactifs de ces actions pour les ainés, les actifs, et pour lutter contre le zoning intergénérationnel et la solitude.
      • 33:10-34:47 : Elle parle du quatrième levier : les carrefours, en mentionnant un exemple à Mendrizio, puis à Bâle, où l'approche est d'apaiser les carrefours, car ils sont source de stress. Elle parle de l'impact cumulé du stress lié à la traversée des carrefours.
      • 34:47-36:21 : Elle explique comment transformer les carrefours en lieux de vie, en libérant l'espace, en mettant des zones de rencontre, et en créant une situation d'attention mutuelle. Elle souligne qu'agir sur un carrefour a un impact exponentiel car il est à la croisée de plusieurs quartiers.
      • 36:21-37:41 : Elle introduit le cinquième levier : les parkings prévenants, en s'inspirant de ce qui a été fait à Montréal pendant la pandémie, pour faire en sorte que le premier pas hors de la voiture soit agréable.
      • 37:41-38:59 : Elle donne des exemples de parkings à Hambourg et à Copenhague qui incitent les automobilistes à quitter leur voiture et à être plus actif. Elle ajoute que le temps passé en voiture contribue à la sédentarité.
      • 38:59-40:44 : Elle aborde la question de l'isolement et de la solitude, qui touchent toutes les catégories d'âge et qui augmentent les risques de maladies cardiaques ou d'AVC.
      • 40:44-42:08 : Elle mentionne l'exemple de Bilbao qui a travaillé sur les coupures entre les quartiers en agissant notamment sur les ponts, et en créant des espaces confortables pour les piétons.
      • 42:08-42:54 : Elle souligne l'importance de faire l'espace public avant les immeubles pour que les habitants puissent profiter de l'espace dès le premier jour.
      • 42:54-43:57 : Elle présente les indicateurs mis en place pour la ville de Paris, comme la biodiversité, les sociabilités, les ralentissements, et la latéralisation, afin de déterminer si un espace public est réellement relationnel. Elle termine en disant que la ville de demain sera relationnelle ou ne sera pas.
      • 43:57-44:33 : Elle pose la question aux participants sur ce qu'ils veulent emporter comme type d'actions et sur ce qu'ils ont retenu de sa conférence.
      • 44:33-45:25 : Une participante de la Direction de santé publique prend la parole et pose deux questions, l'une concernant la gentrification et l'autre la préservation du patrimoine.
      • 45:25-46:58 : Sonia Lavadinho explique que pour résoudre le problème de la gentrification, elle utilise un système de double rue, ce qui permet d'activer plusieurs rues à la fois et donc de mieux contrôler les prix.
      • 46:58-48:33 : Concernant le patrimoine, elle admet que c'est un problème car il y a souvent des conflits avec les architectes des bâtiments de France. Elle recommande d'associer les acteurs du patrimoine dès le départ du projet.
      • 48:33-50:05 : Elle mentionne les "ninzagers" (les personnes qui bloquent les projets) et recommande de les faire participer dès le début en les mettant sur le terrain avec les citoyens. Elle souligne la nécessité de rapprocher ceux qui font le "soft" de la ville (santé, culture, petite enfance, etc.) de ceux qui font le "hard" de la ville (voirie, espaces verts, etc.).
      • 50:05-50:58 : Une participante de l'Office de participation publique de Longueuil questionne sur l'implication des citoyens dans les projets.
      • 50:58-52:30 : Sonia Lavadinho explique comment elle implique les citoyens en allant sur le terrain, en filmant ce que les gens font et ne font pas, et en identifiant les comportements latents. Elle parle aussi du taux d'évaporation piéton, comme indicateur.
      • 52:30-54:14 : Elle détaille la méthode des "balades croquées", où les dessinateurs prêtent leurs crayons aux gens pour qu'ils donnent leurs idées. Elle explique aussi l'atelier "on se marie" pour que les gens soient d'accord avec le projet.
      • 54:14-55:40 : Elle parle de l'importance du monitoring et de l'évaluation des projets, avec des exemples de villes comme Barcelone et Zurich. Elle souligne le rôle du monde de la santé dans la mise en place d'indicateurs simples.
      • 55:40-56:13 : Un participant questionne sur l'impact de l'insécurité et de l'itinérance sur la fréquentation des espaces publics.
      • 56:13-58:29 : Sonia Lavadinho répond qu'en général, c'est l'inverse, car les espaces verts et la présence d'enfants augmentent la sécurité. Elle insiste sur l'importance d'amener une grande biodiversité de publics dans la rue pour créer un effet de surveillance.
      • 58:29-1:00:05 : Elle ajoute que la ville de la nuit est aussi un levier, surtout en agissant sur le premier temps de la nuit (18h-21h) et sur le quatrième temps (5h-7h30). Elle recommande de travailler de façon contextuelle et non pas de manière défensive.
      • 1:00:05-1:00:31 : Fin de la conférence et remerciements.
    1. Voici un sommaire minuté du podcast "Les pensées suicidaires", mettant en évidence les idées fortes en gras :

      • 0:00-0:21: Introduction au podcast "Apprendre à aider" et avertissement sur le contenu, qui traite de troubles de santé mentale et de situations réelles. Il est crucial de contacter les numéros d'urgence (112, 15, 18 ou le 3114) en cas de détresse psychique ou de pensées suicidaires.
      • 0:21-0:58: Présentation du podcast sur le secourisme en santé mentale, avec l'objectif de briser les tabous autour des troubles psychiques. L'animatrice, Oriana, souligne l'importance d'engager la conversation.
      • 0:58-1:44: L'association d'idées "t'as un problème = t'es fou" est abordée, soulignant comment les capacités de résilience peuvent être débordées. Il est mentionné que selon l'Organisation Mondiale de la Santé, plus de 700 000 personnes meurent chaque année par suicide dans le monde. En France, il y a 9 000 décès par suicide et 200 000 tentatives chaque année. Le suicide peut être évité, et les personnes suicidaires ne veulent pas mourir, mais ne plus souffrir. Parler ouvertement des pensées suicidaires peut sauver une vie.
      • 1:44-16:20: Témoignage de Stéphanie, secouriste en santé mentale, qui raconte comment elle a aidé une amie ayant des pensées suicidaires. Elle explique comment sa formation PSSM lui a permis d'écouter et d'accueillir les paroles de son amie sans jugement. Elle a proposé des ressources à son amie, comme le 3114 et un thérapeute. Elle souligne l'importance d'être disponible et d'écouter. La formation lui a apporté une structure et une capacité à réagir plus efficacement. Elle explique que son amie va bien aujourd'hui après avoir eu un espace pour partager sa souffrance.
      • 16:20-23:46: Stéphanie évoque une intervention antérieure où elle n'était pas formée, où elle a posé une question directe sur les idées suicidaires à un client, ce qu'elle n'aurait pas fait après sa formation. Elle souligne l'importance de l'information et du 3114, qu'elle n'avait pas à l'époque. La formation PSSM lui a permis de mieux comprendre les troubles psychiques et d'aller au-delà des apparences. Elle insiste sur l'importance d'aller questionner et de ne pas avoir peur d'aborder ces sujets.
      • 23:46-36:20: Intervention de Christophe Debien, psychiatre, qui donne des définitions de la crise suicidaire, du passage à l'acte et de la tentative de suicide. Il explique que la crise suicidaire est un processus qui prend du temps, souvent six mois. La souffrance psychique s'intensifie lorsque les solutions ne fonctionnent pas. Le passage à l'acte est un moment où la personne cherche à s'extraire de sa souffrance. Il précise que les personnes suicidaires ne veulent pas mourir, mais que leur douleur s'arrête. Il explique que la tentative de suicide n'est pas un chantage, mais un appel à l'aide qu'il faut valoriser. Il explique qu'il faut aller au-delà de l'expression pour comprendre la souffrance. Il identifie les populations les plus touchées : les jeunes (15-25 ans) qui font plus de tentatives de suicide, et les hommes de 45-50 ans et les personnes âgées (plus de 70 ans) qui meurent plus.
      • 36:20-47:35: Le psychiatre aborde les facteurs de vulnérabilité. La crise suicidaire s'installe sur une accumulation de problèmes, tels que ruptures, deuils, dettes. L'histoire de la personne et les événements de vie précoces, comme les violences infantiles, ont un impact sur la vulnérabilité. Les signes d'alerte sont une rupture avec le fonctionnement antérieur, un changement de comportement et des perturbations du sommeil. Il explique que les proches ne peuvent pas toujours voir ces signes et qu'il ne faut pas culpabiliser. Les perturbations du sommeil peuvent être un marqueur important. Face à une personne ayant des pensées suicidaires, il faut d'abord s'assurer de sa propre disponibilité et de sa capacité à aider. Il faut se former pour sortir de la peur liée à l'ignorance. Il faut oser aller vers l'autre et dialoguer. La formation permet d'acquérir le vocabulaire et les outils nécessaires. L'objectif est d'orienter vers des professionnels de santé et de ne pas se substituer à eux.
      • 47:35-54:57: Le psychiatre souligne l'effet positif de poser clairement la question des intentions suicidaires, notant que cela peut être anxiolytique et que les personnes expriment alors leurs idées plus en détail. Il rappelle que les personnes en souffrance ont souvent du mal à communiquer et que le fait de verbaliser les idées suicidaires est un soulagement.
      • 54:57-58:40: Il aborde les relais possibles lors de pensées suicidaires, notant les difficultés du système de santé. Le premier relais de proximité est le médecin généraliste. Il mentionne également l'infirmière scolaire. Il insiste sur le rôle du 3114 pour évaluer le risque suicidaire et apaiser la personne. Le 3114 peut conseiller des ressources au plus proche de celui qui appelle.
      • 58:40-65:30: Témoignage d'Anne, secouriste, qui raconte comment elle a été appelée par un proche pour aider un jeune homme inquiet pour sa compagne. Elle a conseillé au jeune homme d'appeler le 3114, ce qui a permis d'aborder le sujet avec sa compagne. Anne souligne l'importance d'écouter et de poser des questions pour aider. On peut appeler le 3114 même si l'on est inquiet pour quelqu'un d'autre.
      • 65:30-72:00: Anne raconte une autre intervention professionnelle en tant que sage-femme, où elle a contacté le SAMU pour une femme enceinte ayant des idées suicidaires. Elle explique qu'elle a appelé le SAMU car il y avait un danger imminent. La femme a ensuite été prise en charge à l'hôpital et a rencontré une psychiatre.
      • 72:00-76:00: Avec le recul, Anne souligne l'importance du travail en réseau et de ne pas rester seul avec ces situations. Elle note que son regard sur les troubles psychiques a changé grâce à sa formation PSSM. Elle recommande la formation à tous les citoyens.
      • 76:00-fin: Rappel des ressources disponibles : 3114, carnet du secouriste, site infosucide.org, podcast "Les mots bleus". Il est souligné qu'il est possible d'apprendre à aider une personne ayant des idées suicidaires sans devenir un professionnel. La formation aux premiers secours en santé mentale est mise en avant. Le podcast est une production de PSSM France.
    1. synthèse détaillé basé sur les sources que vous avez fournies, en français, incluant des citations pertinentes :

      Document de Briefing : "Comment aider un ado ?"

      Introduction

      Ce document synthétise les principaux thèmes et idées clés abordés dans le podcast "Apprendre à aider" de PSSM France, en particulier l'épisode consacré à la santé mentale des adolescents.

      L'objectif de ce podcast est de sensibiliser, informer et former sur les premiers secours en santé mentale, en particulier pour accompagner les jeunes en difficulté.

      Le podcast explore les défis spécifiques que les adolescents peuvent rencontrer, l'importance de l'intervention précoce et les outils pratiques disponibles pour les soutenir.

      Thèmes Principaux et Idées Clés

      • La Santé Mentale des Adolescents : Un Enjeu Majeur

      • Prévalence élevée : "Un jeune sur sept dans le monde est susceptible de développer un trouble psychique durant cette période."

      • Manifestations fréquentes : Troubles anxieux, troubles des conduites alimentaires, troubles liés à l'usage de substances, addictions comportementales.

      • Risque de suicide : Le suicide est la deuxième cause de mortalité chez les jeunes en France. "Dans certains cas, ces difficultés psychiques associées à un sentiment d'incapacité à s'en sortir peuvent conduire à des pensées suicidaires, voire même être à l'origine d'un passage à l'acte."

      • Aggravation post-crise sanitaire : Plusieurs enquêtes récentes montrent une dégradation de la santé mentale des jeunes depuis la crise sanitaire.

      L'Importance d'une Intervention Précoce et de la Conversation

      • Briser les tabous : "Chez PSSM, Premiers Secours en Santé Mentale, nous sommes convaincus qu'engager une conversation peut tout changer."

      • Identifier les signes : Être attentif aux changements de comportement, aux signaux de détresse, etc. "On est plus attentif aussi, c'est-à-dire qu'on est plus attentif aux changements de comportement, aux signaux de détresse ou de mal-être."

      • Lutter contre la stigmatisation : L'association "t'as un problème, l'association d'idées c'est 't'es fou, t'es folle, y'a quelque chose qui va pas chez toi, t'es dysfonctionnel' en fait."

      • Écoute active et non-jugement : "On doit accueillir la souffrance telle qu'elle est, sans essayer de la sous-estimer. L'idée, on n'est pas là pour y mettre un quelconque regard ou jugement."

      Le Rôle du Secouriste en Santé Mentale

      • Objectif : Ne pas remplacer les professionnels, mais être un premier maillon dans la chaîne de l'aide. "Sans pour autant se substituer aux professionnels, sans pour autant devenir un soignant."

      • Formation PSSM : La formation donne des outils et un cadre pour agir efficacement. "On sort de la formation déjà avec des nouveaux outils, ça donne un cadre, c'est sécurisant parce qu'on se dit qu'on ne va pas faire n'importe quoi justement, parce qu'on sait qu'on va trouver les bons mots, la bonne manière."

      Application de la méthode AÉRER :

      • Accueil : Créer un espace sécurisant et calme pour parler.

      • Écoute active : Poser des questions ouvertes sans jugement et reformuler pour s'assurer de la bonne compréhension.

      • Reconnaître : Valider les émotions et la souffrance de l'adolescent.

      • Évaluer : Déterminer le niveau d'urgence (pensées suicidaires, tentatives).

      • Relayer : Orienter vers les professionnels (3114, urgences, médecin traitant).

      Le Témoignage de Julien : Un Cas Concret

      • Signes précurseurs : Refus de travailler, repli sur soi, fatigue, etc.

      • Déclencheur : Un refus d'aller aux toilettes en classe.

      • Auto-mutilation : Découverte de nombreuses scarifications. "Il a juste soulevé ses manches de son pull et j'ai vu d'innombrables scarifications."

      • Pensées suicidaires : Révélation de pensées et de tentatives de suicide. "Grâce à la formation, j'ai pu poser une question vraiment très claire, c'est-à-dire est-ce que tu as déjà pensé à te suicider ? Il m'a répondu oui. Je lui ai dit est-ce que tu as tenté ? Il m'a répondu oui."

      • Passage de relais : Implication de plusieurs personnes : Sarah (enseignante), Christelle (secouriste), infirmière. L'importance du travail d'équipe : "On sait qu'il y a une dimension humaine dans l'échange et dans le secours. On sait que certains jeunes ne vont pas se confier à un tel. On ne sait pas pour quelles raisons, mais on en est conscient."

      • Appel au 3114 : Prise de contact avec la ligne de prévention du suicide.

      • Difficulté à impliquer les parents : La peur du jugement et de blesser la mère.

      • Hospitalisation : Suite à une nouvelle tentative de suicide.

      • Le sentiment d'utilité des secouristes : "Mais je pense que oui, parce que là, même si effectivement, il a refait une tentative de suicide, vous voyez, les parents ont pu l'accompagner."

      Le Témoignage de Léa : L'Expérience d'une Secourue

      • Période difficile : Problèmes familiaux, stress lié au bac.

      • Repli sur soi : "Je n'étais pas forcément bien dans ma peau et tout ça a fait que j'étais vraiment mal et qu'au lycée je ne faisais plus rien."

      • Approche de la secouriste : Écoute et confiance. "Elles ont réussi à trouver les mots, à me faire comprendre que j'étais écoutée, que j'étais comprise."

      • Sentiment de sécurité : "Ça m'a fait du bien de parler à quelqu'un d'autre de mon entourage en sachant que rien n'allait sortir d'entre nous trois et que j'allais pouvoir dire ce que je voulais."

      • L'envie d'aider à son tour : "Oui, pour pouvoir aider à mon tour des gens qui ont besoin."

      L'Analyse d'Olivier Canceil : Expert en Psychiatrie

      • Troubles spécifiques à l'adolescence : TCA, automutilation, refus scolaire anxieux, expérimentations avec des toxiques, dépendance aux écrans.

      • Vulnérabilité de l'adolescence : Transformations physiques et psychiques, exigences scolaires et sociales, émancipation, etc.

      • Influence des évènements extérieurs : Covid-19 et son impact.

      • Rôle des réseaux sociaux : Effets positifs et négatifs (harcèlement en ligne).

      • Comment gagner la confiance des adolescents : Écoute active, reformulation, absence de jugement, ne pas banaliser.

      • Conséquences d'une non-intervention : Exclusion sociale, stigmatisation, difficultés scolaires, comportements à risque.

      • Module Jeunes de PSSM : Un module pour les adultes interagissant avec des jeunes de 12 à 18 ans.

      • Module Ado : Un module où les jeunes sont formés à aider d'autres jeunes (pair-aidance). "C'est même le plus intéressant à mes yeux parce qu'il repose beaucoup sur la pair-aidance puisqu'il s'agit de former des jeunes de 12 à 18 ans à prendre en charge leurs camarades de 12 à 18 ans."

      Conclusion

      Le podcast "Apprendre à aider" met en lumière l'importance cruciale de la sensibilisation et de la formation en santé mentale pour les adolescents.

      Il souligne le rôle essentiel que chacun peut jouer, en tant que secouriste en santé mentale, pour accompagner les jeunes en difficulté.

      La formation PSSM, les témoignages et l'analyse d'experts offrent des outils pratiques et une perspective éclairante pour agir de manière efficace et bienveillante.

      Il est essentiel de se souvenir de l'importance d'une écoute active, d'une approche sans jugement et de l'orientation vers des professionnels si nécessaire.

    1. synthèse détaillé basé sur les sources que vous avez fournies.

      Document de Synthèse : Automutilations Non Suicidaires

      Introduction

      Ce document de synthèse examine les principaux thèmes et informations clés concernant les automutilations non suicidaires (NSSI), tirés de l'épisode de podcast "Apprendre à aider" de Premiers Secours en Santé Mentale (PSSM) France.

      Il explore la nature des NSSI, leurs causes, les approches d'aide et de soutien, et l'importance de la formation en secourisme en santé mentale.

      1. Définition et Types d'Automutilations Non Suicidaires

      Définition: Les NSSI sont des blessures physiques intentionnelles infligées à soi-même sans l'intention de mourir. Elles sont une forme de comportement autoagressif.

      "L'automutilation se caractérise par des blessures physiques directes plus ou moins sévères qu'une personne s'inflige à elle-même avec ou sans intention suicidaire."

      Types fréquents :

      • Scarifications (incisions superficielles de la peau)
      • Égratignures
      • Coups intentionnels contre une surface dure
      • Coups de poing et claques
      • Morsures
      • Brûlures

      « il existe différents types d'automutilation la plus fréquente c'est se couper avec un objet quelconque aigu une lame de rasoir ou tout autre objet tranchant mais il y a tout un ensemble de automutilation possi comme se frapper se mordre se tirer les cheveux se gratter des plai se tirer des petx donc toute stratégie qui permet de d'infliger volontairement une blessure au corps »

      2. Prévalence et Facteurs de Risque

      Âge d'apparition: Les NSSI apparaissent le plus souvent à l'adolescence (autour de 13-14 ans), avec un pic autour de 18 ans, et peuvent persister à l'âge adulte.

      "globalement les automatisations apparaissent autour de 13 ou 14 ans avec un pic autour de 18 ans mais on peut dire que globalement les automutilations sont observées dans la période entre 13 et 20 25 ans 28 ans"

      • Prévalence chez les jeunes : On estime que 12 à 35% des jeunes sont concernés par des comportements auto-punitifs comme les scarifications.

      • Augmentation chez les filles et jeunes femmes : Un rapport de 2024 indique une augmentation notable des actes d'automutilation chez les filles et les jeunes femmes.

      • Troubles associés : Les personnes qui s'automutilent ont cinq fois plus de risque d'être concernées par un trouble dépressif.

      "les personnes qui pratiquent l'automutilation ont cinq fois plus de risque d'être concerné par un trouble dépressif"

      • Facteurs de risque: Faible estime de soi, difficultés à réguler les émotions, sentiment de désespoir, difficultés à trouver des solutions, image corporelle négative et culpabilité.

      3. Raisons et Fonctions des Automutilations

      • Régulation émotionnelle: Les NSSI sont souvent utilisées pour soulager une souffrance émotionnelle intense et des ressources limitées pour y faire face. Elles peuvent être perçues comme un exutoire.

      • Prévention du suicide: Dans certains cas, l'automutilation peut prévenir un acte suicidaire en évacuant l'idée de suicide.

      "parfois chez les mêmes sujets la il peut y avoir aussi des sujets suicidaires mais dans un certain nombre de situations les automutilisations protègent permettent d'évacuer l'idée de suicide"

      • Sentiment d'être vivant: La douleur peut ramener une personne à la conscience de son corps, se sentir vivant et présent (en particulier chez les personnes avec des sentiments de dissociation).

      • Communication: Les NSSI peuvent être une manière d'exprimer sa détresse et d'attirer l'attention, souvent de manière non intentionnelle.

      • Évitement: Elles peuvent être utilisées pour éviter des situations stressantes ou difficiles.

      4. Le Lien entre Automutilation et Suicide

      • Pas une intention suicidaire: Il est important de souligner que les NSSI ne sont pas motivées par l'intention de se donner la mort, bien qu'il puisse y avoir une continuité entre les pensées suicidaires et les passages à l'acte pour certaines personnes.

      • Facteurs de risques communs : Les facteurs de risque pour l'automutilation et le suicide sont très proches (faible estime de soi, difficultés à réguler ses émotions, etc.).

      • "les raisons enfin les facteurs de risque présents dans les autromutilations sont quasiment les mêmes que ceux qui sont présents dans les comportements suicidaires les suicidaires"

      • Différenciation nécessaire: Malgré les similitudes, la majorité des adolescents qui s'automutilent ne tenteront jamais de se suicider.

      5. Approches d'Aide et d'Intervention

      • Posture du secouriste:

      Réguler ses propres émotions pour éviter la sur-implication ou la banalisation. "la première chose je dirais c'est réguler sa propre expérience de secouriste c'est-à-dire garder un niveau émotionnel qui permette de ne pas être ni dans la surinchè nous ni dans la banalisation"

      • Faire preuve de curiosité et d'empathie pour comprendre l'expérience de l'adolescent sans jugement.

      • Écoute active: Explorer avec la personne la fonction des automutilations, les émotions qui précèdent l'acte, les déclencheurs, le contexte, etc.

      • Stratégies d'atténuation

      La règle des 15 minutes : aider l'adolescent à passer les périodes de crise émotionnelle intense qui peuvent durer 10-15 minutes.

      • Techniques de respiration, utilisation du froid (glace sur les bras, eau sur le visage), rappels d'images positives, etc.

      • Recourir à des activités alternatives : écouter de la musique, faire de l'exercice physique intense, caresser un animal, dessiner, etc.

      • Importance de la verbalisation: Encourager la personne à exprimer ses émotions et ses pensées pour pouvoir les analyser et les changer.

      • "on travaille beaucoup sur la verbalisation avec Sarah qui élabore très très bien et et qui en plus a besoin d'élaborer mettre des mots sortir les mots et les déposer en l'occurrence à moi puisque c'est moi qui étais là lui fait énormément de bien et lui permet de bah de relativiser un petit peu"

      • Orientation vers des professionnels: Diriger la personne vers des médecins, psychologues, ou d'autres professionnels de santé mentale pour un suivi plus approfondi.

      • "Le rôle du secouriste c'est bien de toute façon de renvoyer ses adolescents vers des professionnels de santé"

      6. Témoignages et Études de Cas

      Le témoignage de Laurence: Laurence Roux-Fouillet, sophrologue et secouriste en santé mentale, a accompagné une jeune fille qui se scarifiait. Elle a mis l'accent sur l'écoute active, la mise en confiance, l'identification des alternatives à l'automutilation et l'orientation vers des professionnels.

      Le témoignage croisé de Bérangère et Sarah :

      Bérangère, éducatrice et secouriste en santé mentale, a aidé Sarah, une jeune femme souffrant de TOC et d'automutilations.

      La prise en charge immédiate de Bérangère, son approche calme et empathique et l'utilisation d'outils d'auto-aide (mur d'outils d'auto-aide avec des billets cognitifs, etc.) ont permis de stabiliser Sarah pendant un moment de crise.

      Importance des kits d'auto-aide: Permettent de donner des alternatives lorsque la personne se sent dépassée.

      7. Rôle de la Formation PSSM

      • Apprentissage des gestes et des bons mots: La formation PSSM fournit un cadre et des outils pour aborder et aider les personnes en souffrance psychique, notamment celles qui s'automutilent.

      • Approche structurée et rassurante: La formation PSSM est très structurée, ce qui rassure les secouristes et les aide à ne pas faire d'erreurs.

      • "moi je la conseillerais parce que c'est extrêmement structuré donc c'est très rassurant"

      • Développement du vocabulaire précis et non stigmatisant: Permet de gagner du temps dans la prise en charge des situations et de se positionner dans un rôle qui aide et qui rassure.

      • Accès à des ressources précieuses: Le site de PSSM France propose des ressources, des adresses et des contacts qui sont précieux pour l'orientation et le soutien des personnes en souffrance.

      • "le site de pssm France quand on est secouriste regorge d'expérience d'adresse moi je trouve par exemple le rissaut associatif est extrêmement fourni très dense"

      • Renforcement du rôle du secouriste : Renforce et soutient les pratiques existantes en offrant un cadre structuré et des compétences plus précises.

      Conclusion

      Les automutilations non suicidaires sont des comportements complexes qui nécessitent une approche empathique, attentive et bienveillante.

      Les formations en secourisme en santé mentale, comme celles proposées par PSSM France, jouent un rôle essentiel dans la préparation des citoyens et des professionnels pour intervenir de manière efficace et appropriée auprès des personnes en souffrance.

    1. Author response:

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

      Reviewer #1 (Public Review):

      Summary:

      In this study, Zhao and colleagues investigate inflammasome activation by E. tarda infections. They show that E. tarda induces the activation of the NLRC4 inflammasome as well as the non-canonical pathway in human THP1 macrophages. Further dissecting NLRC4 activation, they find that T3SS translocon components eseB, eseC and eseD are necessary for NLRC4 activation and that delivery of purified eseB is sufficient to trigger NAIP-dependent NLRC4 activation. Sequence analysis reveals that eseB shares homology within the C-terminus with T3SS needle and rod proteins, leading the authors to test if this region is necessary for inflammasome activation. They show that the eseB CT is required and that it mediates interaction with NAIP. Finally, they that homologs of eseB in other bacteria also share the same sequence and that they can activate NLRC4 in a HEK293T cell overexpression system.

      Strengths:

      This is a very nice study that convincingly shows that eseB and its homologs can be recognized by the human NAIP/NLRC4 inflammasome. The experiments are well designed, controlled and described, and the papers is convincing as a whole.

      Weaknesses:

      The authors need to discuss their study in the context of previous papers that have shown an important role for E. tarda flagellin in inflammasome activation and test whether flagellin and/or E. tarda T3SSs needle or rod can activate NLRC4.

      The authors show that eseB and its homologs can activate NLRC4, but there are also other translocon proteins that are very different such as YopB or PopB. and share little homology with eseB. It would be nice to include a section comparing the different type 3 secretion systems. are there 2 different families of T3SSs, those that feature translocon components that are recognized by NAIP-NLRC4 and those that cannot be recognized?

      (1) The authors need to discuss their study in the context of previous papers that have shown an important role for E. tarda flagellin in inflammasome activation and test whether flagellin and/or E. tarda T3SSs needle or rod can activate NLRC4.

      According to the reviewer’s suggestion, we added the relevant discussion (lines 326-334) and carried out additional experiments to examine whether E. tarda flagellin, needle, and rod could activate NLRC4. The relevant results are shown in Figure S3, Figure S5, and lines 226-230 and 269-274.

      (2) The authors show that eseB and its homologs can activate NLRC4, but there are also other translocon proteins that are very different such as YopB or PopB. and share little homology with eseB. It would be nice to include a section comparing the different type 3 secretion systems. are there 2 different families of T3SSs, those that feature translocon components that are recognized by NAIP-NLRC4 and those that cannot be recognized?

      According to the reviewer’s suggestion, additional experiments were performed to examine the NLRC4-activating potentials of 14 translocator proteins that share low sequence identities with EseB. The relevant results and discussion are shown in Figure S8 and lines 289-301; 364-372, and 377-379.

      Reviewer #2 (Public Review):

      Summary:

      This work by Zhao et al. demonstrates the role of the Edwardsiella tarda type 3 secretion system translocon in activating human macrophage inflammation and pyroptosis. The authors show the requirement of both the bacterial translocon proteins and particular host inflammasome components for E. tarda-induced pyroptosis. In addition, the authors show that the C-terminal region of the translocon protein, EseB, is both necessary and sufficient to induce pyroptosis when present in the cytoplasm. The most terminal region of EseB was determined to be highly conserved among other T3SS-encoding pathogenic bacteria and a subset of these exhibited functionally similar effects on inflammasome activation. Overall, the data support the conclusions and interpretations and provide interesting insights into interactions between bacterial T3SS components and the host immune system.

      Strengths:

      The authors use established and reliable molecular biology and bacterial genetics strategies to characterize the roles of the bacterial T3SS translocon and host inflammasome pathways to E. tarda-induced pyroptosis in human macrophages. These observations are naturally expanded upon by demonstrating the specific regions of EseB that are required for inflammasome activation and the conservation of this sequence among other pathogenic bacteria.

      Weaknesses:

      The functional assessment of EseB homologues is limited to inflammasome activation at the protein level but does not include the effects on cell viability as shown for E. tarda EseB. Confirmation that EseB homologues have similar effects on cell death would strengthen this portion of the manuscript.

      According to the reviewer’s suggestion, the effects of representative EseB homologs on cell death were examined in the revised manuscripts (Figure 5D, Figure S7 and line 289).

      Recommendations for the authors:

      Reviewer #1 (Recommendations For The Authors):

      I only have a few suggestions on how to improve the study:

      Activation of caspase-4 requires entry into the host cytosol. Can this be observed with E. tarda and is it T3SS dependent? The fact that deleting the translocon components abrogates all GSDMD activation (see Fig. 2D) suggests that also Casp4 activation requires an active T3SS. It would be useful for the reader to include some more information on the cellular biology of E. tarda.

      In our study, we found that E. tarda could enter THP-1 cells (Figure S1), and host cell entry was not affected by deletion of eseB-D (Δ_eseB-D_) in the T3SS system (Figure 2B, C). Additional experiments showed that Δ_eseB-D_ abolished the ability of E. tarda to activate Casp4 (Figure S2), implying that Casp4 activation required an active T3SS. Relevant changes in the revised manuscript: lines 223 and 224, 341-342.

      The data presented by the authors suggest that escB is sensed by NLRC4 when overexpressed, they do however not prove that during an infection escB is the main factor that drives NLRC4 activation, since deficiency in escB also abrogated translocation of other potential activators of NLRC4, e.g. flagellin and T3SS needle and rod subunits. I would thus find it essential to properly test if E. tarda flagellin can activate NLRC4 by comparing a WT and flagellin deficient strain, and/or by transfecting or expressing E.t. flagellin in these cells, as well as testing whether E.t. rod and needle subunits act as NLRC4 activators. This is important as previous studies suggested that flagellin is the main activator of cytotoxicity during E. tarda infection.

      Previous studies have shown that flagellin is required for E. tarda-induced macrophage death in fish [1] but not in mice [2]. In the revised manuscript, we performed additional experiments to examine whether E. tarda flagellin, needle, and rod could activate NLRC4. The relevant results are shown in Figure S3, Figure S5, and lines 226-230 and 269-274, and 326-334.

      References

      (1) Xie HX, Lu JF, Rolhion N, Holden DW, Nie P, Zhou Y, et al. Edwardsiella tarda-induced cytotoxicity depends on its type III secretion system and flagellin. Infect Immun. 2014;82(8):3436-45. doi: 10.1128/IAI.01065-13.

      (2) Chen H, Yang D, Han F, Tan J, Zhang L, Xiao J, et al. The bacterial T6SS effector EvpP prevents NLRP3 inflammasome activation by inhibiting the Ca<sup>2+</sup>-dependent MAPK-JNK pathway. Cell Host Microbe. 2017;21(1):47-58. doi: 10.1016/j.chom.2016.12.004.

      Figure 5/S4, please list the names of the eseB homologs. It is cumbersome to have to access GenBank with the accession number to be able to understand what proteins the authors define as homologs of eseB.

      The names were added to the revised Table S2, Figure 5 and Figure S6 (the original Figure S4).

      The authors mention that other translocon proteins, such as YopB/D and PopB/D, were suggested to cause inflammasome activation. How do these compare to eseB and its homologs? Do they share the CT motif?

      Additional experiments were performed to compare the inflammasome activation abilities of EseB and other translocator proteins including YopD and PopD. The relevant results and discussion are shown in Figure S8 and lines 289-301, 364-372, and 377-379.

      It would be nice to show that there are potentially two groups of translocon proteins, one group sharing homology to needle subunits within the CT region and another that is different. A quick look at the sequence of these proteins suggests that they are quite different and much larger than eseB.

      In our study, additional experiments with more translocator proteins indicated that the possession of EseB T6R-like terminal residues does not necessarily guarantee the protein to activate the NLRC4 inflammasome. Relevant results and discussion are shown in lines 289-301, 364-372, and 377-379.

    1. The specific area of the initial problem matters less because, in the end, any situation should be addressable through custom tools. It's more important for the problem to mean something to you.

      Similar a lo descrito antes frente a la escogencia de problemas en las Data Weeks y Data Rodas, que motiva las prácticas de aprendizaje entre pares allí.

    2. At the same time, it is the only environment today in which to learn and practice Moldable Development.

      Esto ya no es cierto. Sistemas como Clerk han adoptado explícitamente el término amoldable para describir la visión, citando a Tudor y su grupo.

      En Grafoscopio, nos hemos venido decantando por Sistemas Maleables, pues siento que describe mejor nuestra intención distinta no de "iniciar una conversación" (entre desarrolladores) como afirma Tudor, sino de enmarcarnos en una conversación más antigua, diversa y amplia, que por ejemplo recoge ideas de Maturana y Varela (autopoiesis), Freire (alfabetismos críticos), Kay (Dynabook), la comunidad de Pharo, la de GToolkit, Wenger (comunidades de prácticas) y las propias en diálogo con ellas (infraestructuras de bolsillo, computación convivial) etc.

    3. Of course, the specific needs will not the same for different stakeholders. But the process to get to the answer can be. That's the magic of Moldable Development.

      Me recuerda lo que decía Markus Denker en 2014 en Nomads do not build Cathedrals respecto a que si la herramienta no cambia con el problema, el salto en complejidad se hace imposible para abordar problemas futuros, cosa que traje en mi tesis, explicando las resonancias de Grafoscopio con estas dos gráficas, tomadas de la presentación de Markus.

      e indicando que, dado que "el artefacto es el currículo" en el caso de los sistemas basados en Smalltalk, como diría Alan Kay, si bien la complejidad de la plataforma crece con el proyecto, la forma de navegarla se mantiene uniforme.

      La idea de cambiar la herramienta junto al problema ha estado en la comunidad de Pharo, sobre el que se construyó GToolkit, hace mucho tiempo. Aunque, con marcadas diferencias respecto a cómo se practica en cada entorno/comunidad: Pharo / GToolkit

    4. Moldable Development involves two distinct roles, each with its own set of skills. The facilitator (in blue on the map) is a technical role that is concerned with the technical part of building tools. But that alone is not enough. The stakeholder (in red) is at least as important. Tools are only meaningful when the relate to a question or hypothesis that is tied to value. That's the job of the stakeholder.

      En la comunidad de Grafoscopio, también hay roles con mayor experticia técnica que otros, a pesar de que todos estamos involucrados en la solución del problema. En nuestro caso, no hay una toma de decisiones, retos y problemas específicos que son decididos por el público no técnico (las que están en rojo en el mapa de Wardley), sino y otras acciones técnicas (en azul) sólo referidas a los desarrolladores, sino que hay un difuminado de color (quizás morado) que se torna más azul entre más técnica es la acción, a pesar de que participan de ella también personas no técnicas, y que se hace más roja entre más administrativa, a pesar de que en la administración participan también los desarrolladores. El lugar de encuentro de estas dos experiencia y la mezcla de colores ocurre particularmente en los talleres como las Data Weeks y las Data Rodas

    5. The principle is that whenever a problem is not comfortable enough, you build a tool that makes it comfortable. This can happen multiple times a day even for a single developer. To put it in perspective, in a typical IDE today, you might have dozens of extensions. When practicing Moldable Development, you can easily get to thousands of tools per system.

      Hemos construido metaherramientas así en Grafoscopio desde hace rato, para hacer problemas abordables e ir abordando la pregunta existencial detrás de la comunidad/metaherramienta Grafoscopio:

      ¿Cómo cambiamos las herramientas digitales que nos cambian?

      El proceso de apropiación inicial fue lento, pero luego hemos tenido un auge grande de metaherramientas y prácticas de aprendizaje alrededor de las mismas, que es reflejado portafolios como este.

      Si bien comparo Grafoscopio a un sistema de plugins, construido sobre GToolkit, que a la vez fue construido sobre Pharo y digo que es similar a cómo un navegador web como Brave es construido sobre la variante de código abierto de Chromium, la forma en que creo pequeños trozos de código en el cotidiano para atender problemas pequeños y hipercontextuales, esta flexibilidad no se parece a nada a la rigidez de lo que se pueda hacer con sólo agregrar plugins predeterminados en un navegador web, que no hablan entre sí y no so programables/extensibles en el contexto cotidiano de uso.

    6. For quite some time we had an issue in the text editor related to the cursor getting stuck when it was at the end of a paragraph. It was annoying, but we did not know how to address it and it stayed open for half a year. That part of the code depended on Pharo objects that were wrapping Rust objects. It was only when we added the ability to inspect Rust objects that we found problem.

      Quizás es por esto que aún se necesita cambiar de ventanas y volver al GToolkit cuando el cursor deja de moverse.

    7. Indeed, we do not go to the Jenkins interface for answering questions about our build systems. We get the Jenkins views in our environment.

      Dado que las vistas de GToolkit son limitadas en comparación con lo que hay disponible en visualizaciones web, como lo hemos experimentando varias veces en proyectos como #CandidatosEnDatos (por ejemplo con la ausencia de histogramas horizontales), en Grafoscopio/mutabiT lo que hacemos es integrar los sistemas en vistas HTML/Web y PDF/Impresas. Si bien se pierde mucho de la interactividad en las segundas, tecnologías como HTMX harían posible tener vistas web con alta interactividad y versatilidad, sin estar limitados por GToolkit. Esa es parte de las exploraciones venideras de este año.

      Futuras vistas de Socialmetrica usarán esta combinación, así como las futuras libretas interactivas web de Grafoscopio desde las que iniciaremos esta exploración, agregando esta funcionalidad progresivamente. Esto brindaría versatilidad web también a las vistas de NOSTR y Mastodon que son las redes sociales alternas que esperamos explorar prontamente (en contraste con BlueSky en el ejemplo arriba mencionado).

    1. Author response:

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

      Public Reviews:

      Reviewer #1 (Public Review):

      Summary:

      The manuscript is dedicated heavily to cell type mapping and identification of sub-type markers in the human testis but does not present enough results from cross-investigation between NOA cases versus control. Their findings are mostly based on transcriptome and the authors do not make enough use of the scATAC-seq data in their analyses as they put forward in the title. Overall, the authors should do more to include the differential profile of NOA cases at the molecular level - specific gene expression, chromatin accessibility, TF binding, pathway, and signaling that are perturbed in NOA patients that may be associated with azoospermia.

      Strengths:

      (1) The establishment of single-cell data (both RNA and ATAC) from the human testicular tissues is noteworthy.

      (2) The manuscript includes extensive mapping of sub-cell populations with some claimed as novel, and reports marker gene expression.

      (3) The authors present inter-cellular cross-talks in human testicular tissues that may be important in adequate sperm cell differentiation.

      Weaknesses:

      (1) A low sample size (2 OA and 3 NOA cases). There are no control samples from healthy individuals.

      Thank you for your comments. We recognize that the small sample size in this study somewhat limits its generalizability. However, in transcriptomic research, limited sample sizes are a common issue due to the complexities involved in acquiring samples, particularly in studies about the reproductive system. Healthy testicular tissue samples are difficult to obtain, and studies (doi: 10.18632/aging.203675) have used obstructive azoospermia as a control group in which spermatogenesis and development are normal.

      (2) Their argument about interactions between germ and Sertoli cells is not based on statistical testing.

      Thank you for your comments. Due to limited funding, we have not yet fully and deeply conducted validation experiments, but we plan to carry out related experiments in the later stage. We hope that the publication of this study will help to obtain more financial support to further investigate the interactions between germ cells and Sertoli cells.

      (3) Rationale/logic of the study. This study, in its present form, seems to be more about the role of sub-Sertoli population interactions in sperm cell development and does not provide enough insights about NOA.

      Thank you for your comments. In Figure 6, we conducted an in-depth analysis and comparison of the differences between the Sertoli cell subtypes and the germ cell subtypes involved in spermatogenesis in the OA and NOA groups. The results revealed that in the NOA group, especially in the NOA3 group, which has a lower sperm count compared to NOA2 and NOA1, there is a significant loss of Sertoli cell subtypes including SC3, SC4, SC5, SC6, and SC8. The NOA1 group, with a sperm count close to that of the OA group, also had a Sertoli cell profile similar to the OA group. The NOA2 group, with a sperm count between that of NOA1 and NOA3, also exhibited an intermediate profile of Sertoli cell subtypes. Therefore, we suggest that change in Sertoli cell subtypes is a key factor affecting sperm count, rather than just the total number of Sertoli cells. We believe that through these analyses, we can provide in-depth insights into NOA, and we hope that the publication of this study will help obtain more funding support to further validate and expand on these findings.

      (4) The authors do not make full use of the scATAC-seq data.

      Thank you for your comments.We have added analysis of the scATAC-seq data and shown in the revised manuscript.

      Reviewer #2 (Public Review):

      Summary:

      Shimin Wang et al. investigated the role of Sertoli cells in mediating spermatogenesis disorders in non-obstructive azoospermia (NOA) through stage-specific communications. The authors utilized scRNA-seq and scATAC-seq to analyze the molecular and epigenetic profiles of germ cells and Sertoli cells at different stages of spermatogenesis.

      Strengths:

      By understanding the gene expression patterns and chromatin accessibility changes in Sertoli cells, the authors sought to uncover key regulatory mechanisms underlying male infertility and identify potential targets for therapeutic interventions. They emphasized that the absence of the SC3 subtype would be a major factor contributing to NOA.

      Weaknesses:

      Although the authors used cutting-edge techniques to support their arguments, it is difficult to find conceptual and scientific advances compared to Zeng S et al.'s paper (Zeng S, Chen L, Liu X, Tang H, Wu H, and Liu C (2023) Single-cell multi-omics analysis reveals dysfunctional Wnt signaling of spermatogonia in non-obstructive azoospermia. Front. Endocrinol. 14:1138386.). Overall, the authors need to improve their manuscript to demonstrate the novelty of their findings in a more logical way.

      Thank you for your detailed review of our work. We greatly appreciate your feedback and have made revisions to our manuscript accordingly.

      Regarding the novelty of our research, we believe our study offers conceptual and scientific advances in several ways:

      We have systematically revealed the stage-specific roles of Sertoli cell subtypes in different stages of spermatogenesis, particularly emphasizing the crucial role of the SC3 subtype in non-obstructive azoospermia (NOA). Additionally, we identified that other Sertoli cell subtypes (SC1, SC2, SC3...SC8, etc.) also collaborate in a stage-specific manner with different subpopulations of spermatogenic cells (SSC0, SSC1/SSC2/Diffed, Pa...SPT3). These findings provide new insights into the understanding of spermatogenesis disorders.

      Compared to the study by Zeng S et al., our research not only focuses on the functional alterations in Sertoli cells but also comprehensively analyzes the interaction patterns between Sertoli cells and spermatogenic cells using scRNA-seq and scATAC-seq technologies. We uncovered several novel regulatory networks that could serve as potential targets for the diagnosis and treatment of NOA.

      We sincerely appreciate your constructive comments and will continue to explore this area further, aiming to make a more significant contribution to the understanding of NOA mechanisms.

      Reviewer #3 (Public Review):

      Summary:

      This study profiled the single-cell transcriptome of human spermatogenesis and provided many potential molecular markers for developing testicular puncture-specific marker kits for NOA patients.

      Strengths:

      Perform single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) on testicular tissues from two OA patients and three NOA patients.

      Weaknesses:

      Most results are analytical and lack specific experiments to support these analytical results and hypotheses.

      Thank you for your thorough review of our work. We highly value your feedback and have made revisions to our manuscript accordingly. Indeed, we have conducted immunofluorescence (IF) experiments to validate the data obtained from single-cell sequencing and have expanded the sample size to enhance the reliability of our results. To better present these validation experiments, we have reorganized and renamed the sample information, making it easier for you to understand which samples were used in the specific experiments. Following the publication of this paper, we plan to secure additional funding to deepen our research, particularly in the area of experimental validation. We sincerely appreciate your support and insightful suggestions, which have greatly helped guide our future research directions.

      Reviewer #1 (Recommendations For The Authors):

      (1) The authors should include results from cross-investigation comparing NOA/OA patients versus controls.

      Thank you for your comments. In this study, OA was the control group. Healthy testicular tissue samples are difficult to obtain, and studies (doi: 10.18632/aging.203675) have used OA as a control group in which spermatogenesis and development are normal.

      (2) In Table S1, the authors should also include the metric for scATAC-seq, and do more to show the findings the authors obtained in RNA is replicated with chromatin accessibility.

      Thank you for your comments. We have added Table S2, which includes the metric for scATAC-seq.

      (3) A single sample from each OA and NOA group may not be enough to confirm colocalization. The authors should include results from all available samples and use quantitative measures.

      Thank you for your comments. I apologize that the sample size in this study was less than three and we could not conduct quantitative analysis. We will increase the sample size and conduct corresponding experiments in subsequent research.

      (4) The Methods section does not include enough description to follow how the analyses were carried out, and is missing information on some of the key procedures such as velocity and cell cycle analyses.

      Thank you for your comments. The method about velocity and cell cycle analyses was added in the revised manuscript. The description is as follows:

      “Velocity analysis

      RNA velocity analysis was conducted using scVelo's (version 0.2.1) generalized dynamical model. The spliced and unspliced mRNA was quantified by Velocity (version 0.17.17).”

      “Cell cycle analysis

      To quantify the cell cycle phases for individual cell, we employed the CellCycleScoring function from the Seurat package. This function computes cell cycle scores using established marker genes for cell cycle phases as described in a previous study by Nestorowa et al. (2016). Cells showing a strong expression of G2/M-phase or S-phase markers were designated as G2/M-phase or S-phase cells, respectively. Cells that did not exhibit significant expression of markers from either category were classified as G1-phase cells.”

      (5) For the purpose of transparency, the authors should upload codes used for analyses so that each figure can be reproduced. All raw and processed data should be made publicly available.

      Thank you for your comments. We have deposited scRNA-seq and scATAC-seq data in NCBI. ScRNA-seq data have been deposited in the NCBI Gene Expression Omnibus with the accession number GSE202647, and scATAC-seq data have been deposited in the NCBI database with the accession number PRJNA1177103.

      Reviewer #2 (Recommendations For The Authors):

      The detailed points the authors need to improve are attached below.

      The results presented in the study have several weaknesses:

      In Figure 1A, it's required to show HE staining results of all patients who underwent single-cell analysis were provided.

      Thank you very much for your valuable suggestions. In Figure 1, we present the HE staining results paired with the single-cell data, covering all patients involved in the single-cell analysis.

      - Saying "identification of novel potential molecular markers for distinct cell types" seems unsupported by the data.

      Thank you for your comments. I'm sorry for the inaccuracy of my description. We have revised this sentence. The description is as follows: These findings indicate that the scRNA-seq data from this study can serve for cellular classification.

      - The methods suggest an integrated analysis of scRNA-seq and scATAC-seq, but from the figures, it seems like separate analyses were performed. It's necessary to have data showing the integrated analysis.

      Thank you for your comments. We have added an integrated analysis of scRNA-seq and scATAC-seq. The results were shown in Figure S2.

      Figure 2 does not seem to well cover the diversity of germ cell subtypes. The main content appears to be about the differentiation process, and it seems more focused on SSCs (stem cell types), but the intended message is not clearly conveyed.

      Thank you for your comments. Figure S1 revealed the diversity of germ cell subtypes. The second part of the results described the integrated findings from Figures 2 and S1.

      - In Figure 2B, pseudotime could be shown, and I wonder if the pseudotime in this analysis shows a similar pattern as in Figure 2D.

      Thank you for your comments. Figure 2B revealed the pseudotime analysis of 12 germ cell subpopulation. Figure 2D revealed RNA velocity of 12 germ cell subpopulation. The two methods are both used for cell trajectory analysis. The pseudotime in Figure 2B showed a similar pattern as in Figure 2D.

      - While staining occurs within one tissue, saying they are co-expressed seems inaccurate as the staining locations are clearly distinct. For example, the staining patterns of A2M and DDX4 (a classical marker) are quite different, so it's hard to claim A2M as a new potential marker just because it's expressed. Also, TSSK6 was separately described as having a similar expression pattern to DDX4, but from the IF results, it doesn't seem similar.

      Thank you for your comments. We have revised the Figure.

      - It was described that A2M (expressed in SSC0-1), and ASB9 (expressed in SSC2) have open promoter sites in SSC0, SSC2, and Diffing_SPG, but it doesn't seem like they are only open in the promoters of those cell types. For example, there doesn't seem to be a peak in Diffing for either gene. The promoter region of the tracks is not very clear, so overall figure modification seems necessary.

      Thank you for your comments. We have revised the Figure.

      - The ATAC signal scale for each genomic region should be included, and clear markings for the TSS location and direction of the genes are needed.

      Thank you for your comments. We have revised the figure and shown in the revised manuscript.

      Figure 3A mostly shows the SSC2 in the G2/M phase, so it seems questionable to call SSC0/1 quiescent. Also, I wonder if the expression of EOMES and GFRA1 is well distinguished in the SSC subtypes as expected.

      Thank you for your comments. We will validate in subsequent experiments whether the expression of EOMES and GFRA1 is clearly distinguished in the SSC subtypes.

      - In Figure 3C, it would be good to have labels indicating what the x and y axes represent. The figure seems complex, and the description does not seem to fully support it.

      Thank you for your comments. We have added labels indicating what the x and y axes represent in the Figure 3C. The x and y axes represent spliced and unspliced mRNA ratios, respectively.

      - While TFs are the central focus, it's disappointing that scATAC-seq was not used.

      Thank you for your comments. TFs analysis using scATAC-seq will be carried out in the future.

      Figure 4: It would be good to have a more detailed discussion of the differences between subtypes, such as through GO analysis. The track images need modification like marking the peaks of interest and focusing more on the promoter region, similar to the previous figures.

      Thank you for your comments. GO analysis results were put in Figure S5. The description is as follows:

      As shown in Figure S5, SC1 were mainly involved in cell differentiation, cell adhesion and cell communication; SC2 were involved in cell migration, and cell adhesion; SC3 were involved in spermatogenesis, and meiotic cell cycle; SC4 were involved in meiotic cell cycle, and positive regulation of stem cell proliferation; SC5 were involved in cell cycle, and cell division; SC6 were involved in obsolete oxidation−reduction process, and glutathione derivative biosynthetic process; SC7 were involved in viral transcription and translational initiation; SC8 were involved in spermatogenesis and sperm capacitation.

      In Figure 5, it would be good to have criteria for the novel Sertoli cell subtype presented. CCDC62 is presented as a representative marker for the SC8 cluster, but from Figure 4C, it seems to be quite expressed in the SC3 cluster as well. Therefore, in Figure 5E's protein-level check, it's unclear if this truly represents a novel SC8 subtype.

      Thank you for your comments. CCDC62 expression was higher in SC8 cluster than in SC3. Since some molecular markers were not commercially available in the market, CCDC62 was selected as SC8 marker for immunofluorescence verification. Immunofluorescence results showed that CCDC62 is a novel SC8 marker.

      - It might have been more meaningful to use SOX9 as a control and show that markers in the same subtype are expressed in the same location.

      Thank you for your comments. To determine PRAP1, BST2, and CCDC62 as new markers for the SC subtype, we co-stained them with SOX9 (a well-known SC marker).

      - Figures 4 and 5 could potentially be combined into one figure.

      Thank you for your comments. Since combining Figures 4 and 5 into a single image would cause the image to be unclear, two images are used to show it.

      In Figure 6, it would be good to support the results with more NOA patient data.

      Thank you for your comments. Patient clinical and laboratory characteristics has been presented in Table 1.

      - Rather than claiming the importance of SC3 based on 3 single-cell patient data, it would be better to validate using public data with SC3 signature genes (e.g., showing the correlation between germ cell and SC3 ratios).

      Thank you for your comments. I'm sorry I didn't find public data with SC3 signature genes. In the future, we will verify the importance of SC3 through in vivo and in vitro experiments.

      - 462: It seems to be referring to Figure 6G, not 6D.

      Thank you for your comments. We have revised it. The description is as follows: As shown in Figure 6G, State 1 SC3/4/5 were tended to associated with PreLep, SSC0/1/2, and Diffing and Diffed-SPG sperm cells (R > 0.72).

      In Figure 7, the spermatogenesis process is basically well-known, so it would be better to emphasize what novel content is being conveyed here. Additionally, emphasizing the importance of SC3 in the overall process based on GO results leaves room for a better approach.

      Thank you for your valuable suggestions. Regarding Figure 7, we recognize that the spermatogenesis process is well-known, and we will focus on highlighting the novel content, particularly the role and significance of the SC3 subtype in spermatogenesis disorders. As for the importance of SC3 in the overall process based on GO results, we have validated this in Figure 8 through co-staining experiments between Sertoli cells and spermatogenic cells in OA and NOA groups. The results demonstrate a significant correlation between the number of SC3-positive cells and SPT3 spermatogenic cells, particularly in the NOA5-P8 group, where both SC3 and SPT3 cell counts are notably lower than in the NOA4-P7 group. This further supports the critical role of SC3 in the spermatogenesis process. Your suggestions have prompted us to refine our data presentation and more clearly emphasize the novel aspects of our research. We will continue to strive to ensure that every part of our research contributes meaningfully to the academic community. Thank you again for your guidance.

      In Figure 8, only the contents of the IF-stained proteins are listed, which seems slightly insufficient to constitute a subsection on its own. It might have been better to conclude by emphasizing some subtypes.

      Thank you for your comments. We have combined this part of the results with other results into one section. The description is as follows:

      “Co-localization of subpopulations of Sertoli cells and germ cells

      To determine the interaction between Sertoli cells and spermatogenesis, we applied Cell-PhoneDB to infer cellular interactions according to ligand-receptor signalling database. As shown in Figure 6G, compared with other cell types, germ cells were mainly interacted with Sertoli cells. We futher performed Spearman correlation analysis to determine the relationship between Sertoli cells and germ cells. As shown in Figure 6H, State 1 SC3/4/5 were tended to be associated with PreLep, SSC0/1/2, and Diffing and Diffed-SPG sperm cells (R > 0.72). Interestingly, SC3 was significantly positively correlated with all sperm subpopulations (R > 0.5), suggesting an important role for SC3 in spermatogenesis and that SC3 is involved in the entire process of spermatogenesis. Subsequently, to understand whether the functions of germ cells and Sertoli cells correspond to each other, GO term enrichment analysis of germ cells and sertoli cells was carried out (Figure S3, S4). We found that the functions could be divided into 8 categories, namely, material energy metabolism, cell cycle activity, the final stage of sperm cell formation, chemical reaction, signal communication, cell adhesion and migration, stem cells and sex differentiation activity, and stress reaction. These different events were labeled with different colors in order to quickly capture the important events occurring in the cells at each stage. As shown in Figure S3, we discovered that SSC0/1/2 was involved in SRP-dependent cotranslational protein targeting to membrane, and cytoplasmic translation; Diffing SPG was involved in cell division and cell cycle; Diffied SPG was involved in cell cycle and RNA splicing; Pre-Leptotene was involved in cell cycle and meiotic cell cycle; Leptotene_Zygotene was involved in cell cycle and meiotic cell cycle; Pachytene was involved in cilium assembly and spermatogenesis; Diplotene was involved in spermatogenesis and cilium assembly; SPT1 was involved in cilium assembly and flagellated sperm motility; SPT2 was involved in spermatid development and flagellated sperm motility; SPT3 was involved in spermatid development and spermatogenesis. As shown in Figure S4, SC1 were mainly involved in cell differentiation, cell adhesion and cell communication; SC2 were involved in cell migration, and cell adhesion; SC3 were involved in spermatogenesis, and meiotic cell cycle; SC4 were involved in meiotic cell cycle, and positive regulation of stem cell proliferation; SC5 were involved in cell cycle, and cell division; SC6 were involved in obsolete oxidation−reduction process, and glutathione derivative biosynthetic process; SC7 were involved in viral transcription and translational initiation; SC8 were involved in spermatogenesis and sperm capacitation. The above analysis indicated that the functions of 8 Sertoli cell subtypes and 12 germ cell subtypes were closely related.

      To further verify that Sertoli cell subtypes have "stage specificity" for each stage of sperm development, we firstly performed HE staining using testicular tissues from OA3-P6, NOA4-P7 and NOA5-P8 samples. The results showed that the OA3-P6 group showed some sperm, with reduced spermatogenesis, thickened basement membranes, and a high number of sertoli cells without spermatogenic cells. The NOA4-P7 group had no sperm initially, but a few malformed sperm were observed after sampling, leading to the removal of affected seminiferous tubules. The NOA5-P8 group showed no sperm in situ (Figure 7A). Immunofluorescence staining in Figure 7B was performed using these tissues for validation. ASB9 (SSC2) was primarily expressed in a wreath-like pattern around the basement membrane of testicular tissue, particularly in the OA group, while ASB9 was barely detectable in the NOA group. SOX2 (SC2) was scattered around SSC2 (ASB9), with nuclear staining, while TF (SC1) expression was not prominent. In NOA patients, SPATS1 (SC3) expression was significantly reduced. C9orf57 (Pa) showed nuclear expression in testicular tissues, primarily extending along the basement membrane toward the spermatogenic center, and was positioned closer to the center than DDX4, suggesting its involvement in germ cell development or differentiation. BEND4, identified as a marker fo SC5, showed a developmental trajectory from the basement membrane toward the spermatogenic center. ST3GAL4 was expressed in the nucleus, forming a circular pattern around the basement membrane, similar to A2M (SSC1), though A2M was more concentrated around the outer edge of the basement membrane, creating a more distinct wreath-like arrangement. In cases of impaired spermatogenesis, this arrangement becomes disorganized and loses its original structure. SMCP (SC6) was concentrated in the midpiece region of the bright blue sperm cell tail. In the OA group, SSC1 (A2M) was sparsely arranged in a rosette pattern around the basement membrane, but in the NOA group, it appeared more scattered. SSC2 (ASB9) expression was not prominent. BST2 (SC7) was a transmembrane protein primarily localized on the cell membrane. In the OA group, A2M (SSC1) was distinctly arranged in a wreath-like pattern around the basement membrane, with expression levels significantly higher than ASB9 (SSC2). TSSK6 (SPT3) was primarily expressed in OA3-P6, while CCDC62 (SC8) was more abundantly expressed in NOA4-P7, with ASB9 (SCC2) showing minimal expression. Taken together, germ cells of a particular stage tended to co-localize with Sertoli cells of the corresponding stages. Germ cells and sertoli cells at each differentiation stage were functionally heterogeneous and stage-specific (Figure 8). This suggests that each stage of sperm development requires the assistance of sertoli cells to complete the corresponding stage of sperm development.”

      Reviewer #3 (Recommendations For The Authors):

      The authors revealed 11 germ cell subtypes and 8 Sertoli cell subtypes through single-cell analysis of two OA patients and three NOA patients. And found that the Sertoli cell SC3 subtype (marked by SPATS1) plays an important role in spermatogenesis. It also suggests that Notch1/2/3 signaling and integrins are involved in germ cell-Stotoli cell interactions. This is an interesting and useful article that at least gives us a comprehensive understanding of human spermatogenesis. It provides a powerful tool for further research on NOA. However, there are still some issues and questions that need to be addressed.<br /> (1) How to collect testicular tissue, please explain in detail. Extract which part of testicular tissue. It's better to make a schematic diagram.

      Thank you for your comments. The process is as follows: Testicular tissues were obtained from two OA patients (OA1-P1 and OA2-P2) and three NOA patients (NOA1-P3, NOA2-P4, NOA3-P5) using micro-dissection of testicular sperm extraction separately.

      (2) Whether the tissues of these patients are extracted simultaneously or separately, separated into single cells, and stored, and then single cell analysis is performed simultaneously. Please be specific.

      Thank you for your comments. The testicular tissues of these patients were extracted separately, then separated into single cells, and single cell analysis was performed simultaneously.

      (3) When performing single-cell analysis, cells from two OA patients were analyzed individually or combined. The same problem occurred in the cells of three NOA patients.

      Thank you for your comments. Cells from two OA patients and three NOA patients were analyzed individually.

      (4) Can you specifically point out the histological differences between OA and NOA in Figure 1A? This makes it easier for readers to understand the structure change between OA and NOA. Please also label representative supporting cells.

      Thank you for your comments. We have revised the description and it was shown in the revised manuscript.

      (5) The authors demonstrate that "We speculate that this lack of differentiation may be due to the intense morphological changes occurring in the sperm cells during this period, resulting in relatively minor differences in gene expression." Please provide some verification of this hypothesis? For example, use immunofluorescence staining to observe morphological changes in sperm cells.

      Thank you for your comments. Due to limited funds, we will verified this hypothesis in future studies.

      (6) The authors demonstrate that " As shown in Figure 5E, we discovered that PRAP1, BST2, and CCDC62 were co-expressed with SOX9 in testes tissues." The staining in Figure 5D is unclear, and it is difficult to explain that SOX9 is co-expressed with PRAP1 BST2 CCDC62 based on the current staining results. The staining patterns of SOX9 (green) and SOX9 (red) are also different. (SOX9 (red) appears as dots, while the background for SOX9 (green) is too dark to tell whether its staining is also in the form of dots.) In summary, increasing the clarity of the staining makes it more convincing. Alternatively, use high magnification to display these results.

      Thank you for your comments. I have redyed and updated this part of the immunofluorescence staining results. Please refer to the files named Figure 1, Figure 2, Figure 5, and Figure 8.

      (7) In Figure 8, the author emphasized the co-localization of Sertoli cells and Germ cells at corresponding stages and did a lot of staining, but it was difficult to distinguish the specific locations of co-localization, which was similar to Figure 5E. If possible, please mark specific colocalizations with arrows or use high magnification to display these results, in order to facilitate readers to better understand.

      Thank you for your comments. We have re-stained and updated this part of the data. Please refer to the immunofluorescence staining data in the updated Figure 8.

      (8) The authors emphasize that macrophages may play an important role in spermatogenesis. Therefore, adding relevant macrophage staining to observe the differences in macrophage expression between NOA and OA should better support this idea.

      Thank you for your comments. Macrophage-related experiments will be further explored in the future.

      (9) Notch1/2/3 signaling and integrin were discovered to be involved in germ cell-Sertoli cell interaction. However there are currently no concrete experiments to support this hypothesis. At least simple verification experiments are needed.

      Thank you for your comments. Due to limited funding, studies will be carried out in the future.

      (10) Data availability statements should not be limited to the corresponding author, especially for big data analysis. This is crucial to the credibility of this data (Have the scRNA-seq and scATAC-seq in this study been deposited in GEO or other databases, and when will they be released to the public?) The data for such big data analysis needs to be saved in GEO or other databases in advance so that more research can use it.

      Thank you for your comments. We have deposited scRNA-seq and scATAC-seq data in NCBI. “ScRNA-seq data have been deposited in the NCBI Gene Expression Omnibus with the accession number GSE202647, and scATAC-seq data have been deposited in the NCBI database with the accession number PRJNA1177103.”