Reviewer #3 (Public review):

Summary:

The authors revisit the role of DR6 in axon degeneration following physical injury (Wallerian degeneration), examining both its effects on axons and its role in regulating the Schwann cell response to injury. Surprisingly, and in contrast to previous studies, they find that DR6 deletion does not delay the rate of axon degeneration after injury, suggesting that DR6 is not a mediator of this process.

Overall, this is a valuable study. As the authors note, the current literature on DR6 is inconsistent, and these results provide useful new data and clarification. This work will help other researchers interpret their own data and re-evaluate studies related to DR6 and axon degeneration.

Strengths:

(1) The use of two independent DR6 knockout mouse models strengthens the conclusions, particularly when reporting the absence of a phenotype.

(2) The focus on early time points after injury addresses a key limitation of previous studies. This approach reduces the risk of missing subtle protective phenotypes and avoids confounding results with regenerating axons at later time points after axotomy.

Weaknesses:

(1) The study would benefit from including an additional experimental paradigm in which DR6 deficiency is expected to have a protective effect, to increase confidence in the experimental models, and to better contextualize the findings within different pathways of axon degeneration. For example, DR6 deletion has been shown in more than one study to be partially axon protective in the NGF deprivation model in DRGs in vitro. Incorporating such an experiment could be straightforward and would strengthen the paper, especially if some of the neuroprotective effects previously reported are confirmed.

(2) The quality of some figures could be improved, particularly the EM images in Figure 2. As presented, they make it difficult to discern subtle differences.

Reviewer #1 (Public review):

Disclaimer:

This reviewer is not an expert on MD simulations but has a basic understanding of the findings reported and is well-versed with mycobacterial lipids.

Summary:

In this manuscript titled "Dynamic Architecture of Mycobacterial Outer Membranes Revealed by All-Atom 1 Simulations", Brown et al describe outcomes of all-atom simulation of a model outer membrane of mycobacteria. This compelling study provided three key insights:<br /> (1) The likely conformation of the unusually long chain alpha-branched beta-methoxy fatty acids, mycolic acids in the mycomembrane, to be the extended U or Z type rather than the compacted W-type. (2) Outer leaflet lipids such as PDIM and PAT provide regional vertical heterogeneity and disorder in the mycomembrane that is otherwise prevented in a mycolic acid-only bilayer.<br /> (3) Removal of specific lipid classes from the symmetric membrane systems leads to significant changes in membrane thickness and resilience to high temperatures.

Strengths:

The authors take a step-wise approach in building the complexity of the membrane and highlight the limitations of each of the approaches. A case in point is the use of supraphysiological temperature of 333 K or even higher temperatures for some of the simulations. Overall, this is a very important piece of work for the mycobacterial field, and will help in the development of membrane-disrupting small molecules and provide important insights for lipid-lipid interactions in the mycomembrane.

Weaknesses:

(1) The authors used alpha-mycolic acids only for their models. The ratios of alpha, keto, and methoxy-mycolic acids are known in the literature, and it may be worth including these in their model. Future studies can be aimed at addressing changes in the dynamic behavior of the MOM by altering this ratio, but the inclusion of all three forms in the current model will be important and may alter the other major findings of the current study.

(2) The findings from the 14 different symmetric membrane systems developed with the removal of one complex lipid at a time are very interesting but have not been analysed/discussed at length in the current manuscript. I find many interesting insights from Figures S3 and S5, which I find missing in the manuscript. These are as follows:

a) Loss of PDIM resulted in reduced membrane thickness. This is a very important finding given that loss of PDIM can be a spontaneous phenomenon in Mtb cultures in vitro and that this is driven by increased nutrient uptake by PDIM-deficient bacilli (Domenech and Reed, 2009 Microbiology). While the latter is explained by the enhanced solute uptake by several PE/PPE transporter systems in the absence of PDIM (Wang et al, Science 2020), the findings presented by Brown et al could be very important in this context. A discussion on these aspects would be beneficial for the mycobacterial community.

b) I find it interesting that loss of PAT or DAT does not change membrane thickness (Figure S3). While both PAT and PDIM can migrate to the interleaflet space, loss of PDIM and PAT has a different impact on membrane thickness. It is worth explaining what the likely interactions are that shape membrane thickness in the case of the modelled MOM.

c) Figure S5: Is the presence of SGL driving PDIM and PAT to migrate to the inter-leaflet space? Again, a discussion on major lipid-lipid interactions driving these lipid migrations across the membrane thickness would be useful.

Reviewer #2 (Public review):

Summary:

The manuscript reports all-atom molecular dynamics simulations on the outer membrane of Mycobacterium tuberculosis. This is the first all-atom MD simulation of the MTb outer membrane and complements the earlier studies, which used coarse-grained simulation.

Strengths:

The simulation of the outer membrane consisting of heterogeneous lipids is a challenging task, and the current work is technically very sound.

The observation about membrane heterogeneity and ordered inner leaflets vs disordered outer leaflets is a novel result from the study. This work will also facilitate other groups to work on all-atom models of mycobacterial outer membrane for drug transport, etc.

Weaknesses:

Beyond a challenging simulation study, the current manuscript only provides qualitative explanations on the unusual membrane structure of MTb and does not demonstrate any practical utility of the all-atom membrane simulation. It will be difficult for the general biology community to appreciate the significance of the work, based on the manuscript in its current form, because of the high content of technical details and limited evidence on the utility of the work.

Major Points:

(1) The simulation by Basu et al (Phys Chem Chem Phys 2024) has studied drug transports through mycolic acid monolayers. Since the authors of the current study have all atom models of MTb outer membrane, they should carry out drug transport simulations and compare them to the outer membranes of other bacteria through which drugs can permeate. In the current manuscript, it is only discussed in lines 388-392. Can the disruption of MA cyclopropanation be simulated to show its effect on membrane structure ?

(2) In line 277, the authors mention about 6 simulations which mimic lipid knockout strains. The results of these simulations, specifically the outcomes of in silico knockout of lipids, are not described in detail.

(3) Figure 5 shows PDIM and PAT-driven lipid redistribution, which is a significant novel observation from the study. However, comparison of 3B and 3D shows that at 313K, the movement of the PDIM head group is much less. Since MD simulations are sensitive to random initial seeds, repeated simulations with different random seeds and initial structures may be necessary.

(4) As per Figure 1, in the initial structure, the head group of PAT should be on the membrane surface, similar to TDM and TMM, while PDIM is placed towardsthe interior of the outer membrane. However, Figure 5 shows that at t=0, PAT has the same Z position as PDIM. It will be necessary to provide Z-position Figures for TMM and TDM to understand the difference. Is it really dependent on the chemical structure of the lipid moiety or the initial position of the lipid in the bilayer at the beginning of the simulation?

Minor Point:

In view of the complexity of the system undertaken for the study, the manuscript in its current form may not be informative for readers who are not experts in molecular simulations.

Reviewer #2 (Public review):

This manuscript describes the use of a powerful technique called microfluidics to elucidate the mechanisms explaining how overexpression (OE) of Ssd1 and caloric restriction (CR) in yeast extend replicative lifespan (RLS). Microfluidics measures RLS by trapping cells in chambers mounted to a slide. The chambers hold the mother cell but allow daughters to escape. The slide, with many chambers, is recorded during the entire process, roughly 72 hours, with the video monitored afterwards to count how many daughters each of the trapped mothers produces. The power of the method is what can be done with it. For example, the entire process can be viewed by fluorescence so that GFP and mCherry-tagged proteins can be followed as cells age. The budding yeast is the only model where bona fide replicative aging can be measured, and microfluidics is the only system that allows protein localization and levels to be measured in a single cell while aging. The authors do a wonderful job of showing what this combination of tools can do.

The authors had previously shown that Ssd1, an mRNA-binding protein, extends RLS when overexpressed. This was attributed to Ssd1 sequestering away specific mRNAs under stress, likely leading to reduced ribosomal function. It remained completely unknown how Ssd1 OE extended RLS. The authors observed that overexpressed, but not normally expressed, Ssd1 formed cytoplasmic condensates during mitosis that are resolved by cytokinesis. When the condensates fail to be resolved at the end of mitosis, this signals death.

It has become clear in the literature that iron accumulation increases with age within the cell. The transcriptional programs that activate the iron regulon also become elevated in aging cells. This is thought to be due to impaired mitochondrial function in aging cells, with increased iron accumulation as an attempt at restoring mitochondrial activity. The authors show that Ssd1 OE and CR both reduce the expression of the iron regulon. The data presented indicate that iron accumulation shortens RLS: deletion of iron regulon components extends RLS, and adding iron to WT cells decreases RLS, but not when Ssd1 is overexpressed or when cells are calorically restricted. Interestingly, iron chelation using BPS has no impact on WT RLS, but decreases the elevated RLS in CR cells and cells overexpressing Ssd1. It was not initially clear why iron chelation would inhibit the extended lifespan seen with CR and Ssd1 OE. This was addressed by an experiment where it was shown that the iron regulon is induced (FIT2 induction) when iron is chelated. Thus, the detrimental effects of induction of the iron regulon by BPS and iron accumulation on RLS cannot be tempered by Ssd1 OE and CR once turned on.

I did not find any weaknesses to be addressed in this paper. The draft was well-written, and the extensive experimentation was well-designed, performed, and controlled. However, I did make minor comments that I recommend the authors address:

(1) Why would BPS not reduce RLS in WT cells? The authors could test whether OE of FIT2 reduces RLS in WT cells.

(2) The authors should add a brief explanation for why the GDP1 promoter was chosen for Ssd1 OE.

(3) On page 12, growth to saturation was described as glucose starvation. This is more accurately described as nutrient deprivation. Referring to it as glucose starvation is akin to CR, which growing to saturation is not. Ssd1 OE formed condensates upon saturation but not in CR. Why do the authors think Ssd1 OE did not form condensates upon CR? Too mild a stress?

(4) The authors conclude that the main mechanism for RLS extension in CR and Ssd1 OE is the inhibition of the iron regulon in aging cells. The data certainly supports this. However, this may be an overstatement as other mutations block CR, such as mutations that impair respiration. The authors do note that induction of the iron regulon in aging cells could be a response to impaired mitochondrial function. Thus, it seems that the main goal of CR and Ssd1 OE may be to restore mitochondrial function in aging cells, one way being inactivation of the iron regulon. A discussion of how other mutations impact CR would be of benefit.

(5) The cell cycle regulation of Ssd1 OE condensates is very interesting. There does not appear to be literature linking Ssd1 with proteasome-dependent protein turnover. Many proteins involved in cell cycle regulation and genome stability are regulated through ubiquitination. It is not necessary to do anything here about it, but it would be interesting to address how Ssd1 condensates may be regulated with such precision.

(6) While reading the draft, I kept asking myself what the relevance to human biology was. I was very impressed with the extensive literature review at the end of the discussion, going over how well conserved this strategy is in yeast with humans. I suggest referring to this earlier, perhaps even in the abstract. This would nail down how relevant this model is for understanding human longevity regulation.

In conclusion, I enjoyed reading this manuscript, describing how Ssd1 OE and CR lead to RLS increases, using different mechanisms. However, since the 2 strategies appear to be using redundant mechanisms, I was surprised that synergism was not observed.

Reviewer #3 (Public review):

In this paper, the authors investigate how the RNA-binding protein Ssd1 and calorie restriction (CR) influence yeast replicative lifespan, with a particular focus on age-dependent iron uptake and activation of the iron regulon. For this, they use microfluidics-based single-cell imaging to monitor replicative lifespan, protein localization, and intracellular iron levels across aging cells. They show that both Ssd1 overexpression and CR act through a shared pathway to prevent the nuclear translocation of the iron-regulon regulator Aft1 and the subsequent induction of high-affinity iron transporters. As a result, these interventions block the age-related accumulation of intracellular free iron, which otherwise shortens lifespan. Genetic and chemical epistasis experiments further demonstrate that suppression of iron regulon activation is the key mechanism by which Ssd1 and CR promote replicative longevity.

Overall, the paper is technically rigorous, and the main conclusions are supported by a substantial body of experimental data. The microfluidics-based assays in particular provide compelling single-cell evidence for the dynamics of Ssd1 condensates and iron homeostasis.

My main concern, however, is that the central reasoning of the paper-that Ssd1 overexpression and CR prevent the activation of the iron regulon-appears to be contradicted by previous findings, and the authors may actually be misrepresenting these studies, unless I am mistaken. In the manuscript, the authors state on two occasions:

"Intriguingly, transcripts that had altered abundance in CR vs control media and in SSD1 vs ssd1∆ yeast included the FIT1, FIT2, FIT3, and ARN1 genes of the iron regulon (8)"

"Ssd1 and CR both reduce the levels of mRNAs of genes within the iron regulon: FIT1, FIT2, FIT3 and ARN1 (8)"

However, reference (8) by Kaeberlein et al. actually says the opposite:

"Using RNA derived from three independent experiments, a total of 97 genes were observed to undergo a change in expression >1.5-fold in SSD1-V cells relative to ssd1-d cells (supplemental Table 1 at http://www.genetics.org/supplemental/). Of these 97 genes, only 6 underwent similar transcriptional changes in calorically restricted cells (Table 2). This is only slightly greater than the number of genes expected to overlap between the SSD1-V and CR datasets by chance and is in contrast to the highly significant overlap in transcriptional changes observed between CR and HAP4 overexpression (Lin et al. 2002) or between CR and high external osmolarity (Kaeberlein et al. 2002). Intriguingly, of the 6 genes that show similar transcriptional changes in calorically restricted cells and SSD1-V cells, 4 are involved in iron-siderochrome transport: FIT1, FIT2, FIT3, and ARN1 (supplemental Table 1 at http://www.genetics.org/supplemental/)."

Although the phrasing might be ambiguous at first reading, this interpretation is confirmed upon reviewing Matt Kaeberlein's PhD thesis: https://dspace.mit.edu/handle/1721.1/8318 (page 264 and so on).

Moreover, consistent with this, activation of the iron regulon during calorie restriction (or the diauxic shift) has also been observed in two other articles:

https://doi.org/10.1016/S1016-8478(23)13999-9

https://doi.org/10.1074/jbc.M307447200

Taken together, these contradictory data might blur the proposed model and make it unclear how to reconcile the results.

1. This are the findings or the result in the study of Learner's Reading Strategies and Comprehension Skills

♖🌐 .🌌💬.🎭.📓

Gyuri's Daily Notes - 2025 Sept Week 4

11:59 "wir arbeiten ja mit sprache ... der begriff querdenken wird jetzt geframed ... ein wort das mal extrem positiv besetzt war ... diesen begriff geben wir nicht einfach her ... nicht politisieren lassen ... die sprache gehört den menschen ... großer backlash ... keine promo mehr fürs album weil niemand mit uns sprechen wollte ... überall ausgeladen ... shows abgesagt ... 3 jahre"<br /> spätestens seit "TrauKeinemPromi" (Tilman Knechtel) ist ja bekannt, dass ALLE prominenten "künstler" abhängig sind von der "industrie", und sobald diese "künstler" zu weit abweichen vom mainstream, werden sie gecancelt, verbrannt, ausgestoßen, fallen gelassen.<br /> beispiel: die verzweifelte "entschuldigung" von Xavier Naidoo in seinem "OneLove" video vom 19.04.2022: "Ich habe erkannt, auf welchen Irrwegen ich mich teilweise befunden habe und dass ich in den letzten Jahren viele Fehler gemacht habe ..."<br /> das war naidoo sein "Gang nach Canossa": "Als Gang nach Canossa bezeichnet man den Bitt- und Bußgang des römisch-deutschen Königs Heinrich IV. von Dezember 1076 bis Januar 1077 zu Papst Gregor VII. zur Burg Canossa ... Dies war notwendig geworden, nachdem Heinrich IV. im Zuge seiner Auseinandersetzung mit dem Papst exkommuniziert worden war. Gemäß zeitgenössischen Quellen soll der König drei Tage lang vor den Toren der Burg um die Wiederaufnahme in die Kirche gefleht haben. Schließlich gewährte ihm der Papst Einlass und erteilte ihm die Absolution."

DOI: 10.1038/s41467-025-57164-3

Resource: RRID:BDSC_25102

Curator: @maulamb

SciCrunch record: RRID:BDSC_25102

What is this?

DOI: 10.1038/s41467-025-57164-3

Resource: RRID:BDSC_25101

Curator: @maulamb

SciCrunch record: RRID:BDSC_25101

What is this?

DOI: 10.1038/s41467-025-57164-3

Resource: RRID:BDSC_25107

Curator: @maulamb

SciCrunch record: RRID:BDSC_25107

What is this?

DOI: 10.1038/s41419-020-2580-3

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

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DOI: 10.1038/s41419-020-2580-3

Resource: RRID:Addgene_48139

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DOI: 10.1038/s41419-020-2580-3

Resource: RRID:Addgene_62988

Curator: @scibot

SciCrunch record: RRID:Addgene_62988

What is this?

t was frustrating because I’m from a city in southern China where we don’t make dumplings for New Year, yet I was forced to be the expert on all things Chinese. The author is right: this objectifies minority students. We’re not cultural dictionaries, we’re individuals with our own experiences.

I experienced the importance of cultural codes firsthand when I did a group project with American classmates. We were asked to present our research in a creative way, and I prepared a detailed poster with graphs and quotes, something common in my home country for academic presentations. But my teammates were confused: “Why not make a video or do a skit?” They explained that in U.S. classrooms, creative often means interactive or performative, not just visual.

True safety isn’t about silence; it’s about feeling heard.

Including marginalized perspectives isn’t about checking a bo, it’s about treating their work as seriously as the canon. Literature courses should integrate works from different ethnic groups throughout the semester, not as an afterthought. This way, we learn to see diversity as part of the core, not an add-on.

This fear of shifting paradigms isn’t just about losing control; it’s about the lack of support for teachers to learn new methods. Multicultural education can’t work if educators are left to navigate the change alone, they need structured training, not just pressure to be more inclusive.

The fear of politics interfering with the fundamental of teaching has brainwashed all educators. Teachers often tip toe around to be safe rather than teach to educate the future

There is not a formula that leads us to the promised land. Great teachers can adapt to different learning g types students may have. Their ability to work with the students at different paces is valuable

Reviewer #1 (Public review):

Sinzato et. al. investigated how shear flow in a rheological chamber affects the assembly and fragmentation of cyanobacterial aggregates, with the goal of understanding how such aggregates might form naturally, and/or be destroyed industrially. The authors used a combination of experiments and models to show that cyanobacterial colonies can be difficult to fragment with fluid flows. Additionally, they provide biophysical support for the idea that such aggregates likely form primarily when cells stay together after cell division, rather than coming together from disparate paths.

This work has significant relevance to the field, both practically and naturally. Combatting or preventing toxic cyanobacterial blooms is an active area of environmental research that offers a practical backbone for this manuscript's ideas. Additionally, the formation and behavior of cellular aggregates in general is of widespread interest in many fields, including marine and freshwater ecology, healthcare and antibiotic resistance research, biophysics, and microbial evolution. In this field, there are still outstanding questions regarding how microbial aggregates form into communities, including if and how they come together from separate places. Therefore, I believe that researchers from many distinct fields would find interest in the topic of this paper, and particularly Figure 5, in which a phase space that is meant to represent the different modes of aggregate formation and destruction is suggested, dependent on properties of the fluid flow and particle concentration.

Altogether, the authors were successful in their investigation, and I find their claims to be justified. In particular, the authors achieve strong results from their experiments. Below, I outline key claims of the paper and indicate the level to which they were supported by their data.

• Their first major claim is that fluid flows alone must be quite strong in order to fragment the cyanobacterial aggregates they have studied. With their rheological chamber, they explicitly show that energy dissipation rates must exceed "natural" conditions by multiple orders of magnitude in order to fragment lab strain colonies, and even higher to disrupt natural strains sampled from a nearby freshwater lake. This claim is well-supported by their experiments and data.

• The authors then claim that the fragmentation of aggregates due to fluid flows occurs primarily through erosion of small pieces from larger aggregates. Because their experimental setup does not allow them to directly observe this process (for example, by watching one aggregate break into pieces), they rely on indirect methods to support the claim. Overall, the experimental evidence is generally supportive, but the models leave some gaps. I describe this conclusion in more detail below.

• The strongest evidence for the erosion-dominated process comes from the authors' measurements of transfer of biomass between large and small size classes, as in Figure 2E and Figure 2D. The authors claim that only the erosion model can reproduce this kind of biomass transfer. However, it also seems that the idealized erosion model alone is not fully sufficient to capture the observed behavior. In Figure 2D, there remains a gap between their experiment and the prediction of the erosion model, which grows larger over time (Supplemental Figure S9). While the authors suggest that the erosion model is better than the equal-fragmentation model, it is also true that tracking the mean size (Figure 2B) or small size distribution (Figure S6) cannot distinguish between these models.

• Taken altogether, the experimental evidence favors an erosion-dominated process. However, a few minor questions remain regarding the models. Why does the equal-fragmentation model predict no biomass transfer between size classes? To what extent, quantitatively, does the erosion model outperform the equal fragments model at capturing the biomass size distributions? Finally, why does the idealized erosion fail to capture the size distribution at late stages in Supplemental Figure S9 - would this discrepancy be resolved if the authors considered individual colony variances in cell adhesion (for instance, as hypothesized by the authors in lines 133-137)? I do not believe these questions curb the other results of the paper.

• Their third major claim is that fluid flows only weakly cause cells to collide and adhere in a "coming together" process of aggregate formation. They test this claim in Figure 3, where they suspend single cells in their test chamber and stir them at moderate intensity, monitoring their size histogram. They show that the size histogram changes only slightly, indicating that aggregation is, by-and-large, not occurring at a high rate. Therefore, they lend support to the idea that cell aggregation likely does not initiate group formation in toxic cyanobacterial blooms. Additionally, they show that the median size of large colonies also does not change at moderate turbulent intensities. These results agree with previous studies (their own citation 25) indicating that aggregates in toxic blooms are clonal in nature. This is an important result, and well-supported by their data, but only for this specific particle concentration and stirring intensity. Later, in Figure 5 they show a much broader range of particle concentrations and energy dissipation rates that they leave untested. However, they refer to other literature that does test these regions of the phase map.

• The fourth major result of the manuscript is displayed in Equation 8 and Figure 5, where the authors derive an expression for the ratio between the rate of increase of a colony due to aggregation vs. the rate due to cell division. They then plot this line on a phase map, altering two physical parameters (concentration and fluid turbulence) to show under what conditions aggregation vs. cell division are more important for group formation. Because these results are derived from relatively simple biophysical considerations, they have the potential to be quite powerful and useful, and represent a significant conceptual advance. By combining their experiments with discussions of other experimental investigations of scum formation in cyanobacterial blooms, the authors have investigated the two most relevant zones of this map for the present study (Zones II and III), and have made a strong contribution to the literature in regards to artificial mixing to disrupt cyanobacterial blooms.

Other notes:

The authors rely heavily on size distributions to make the claims of their paper. I was pleased to find the calibration histograms in Supplemental Figure S8, which provide information as to how and why they made corrections to the histograms they observed. From these calibration histograms, it seems that larger colonies are more accurately measured in the cone-and-plate shear setup, while smaller colonies can be missed, presumably due to resolution issues.

Author response:

The following is the authors’ response to the original reviews

Reviewer #1 (Public review):

(1) Their first major claim is that fluid flows alone must be quite strong in order to fragment the cyanobacterial aggregates they have studied. With their rheological chamber, they explicitly show that energy dissipation rates must exceed "natural" conditions by multiple orders of magnitude in order to fragment lab strain colonies, and even higher to disrupt natural strains sampled from a nearby freshwater lake. This claim is well-supported by their experiments and data.

We thank the reviewer for this positive comment. We fully agree, as our fragmentation experiments on division-formed colonies clearly demonstrate their strong mechanical resistance in naturally occurring flows.

(2) The authors then claim that the fragmentation of aggregates due to fluid flows occurs through erosion of small pieces. Because their experimental setup does not allow them to explicitly observe this process (for example, by watching one aggregate break into pieces), they implement an idealized model to show that the nature of the changes to the size histogram agrees with an erosion process. However, in Figure 2C there is a noticeable gap between their experiment and the prediction of their model. Additionally, in a similar experiment shown in Figure S6, the experiment cannot distinguish between an idealized erosion model and an alternative, an idealized binary fission model where aggregates split into equal halves. For these reasons, this claim is weakened.

The two idealized models of colony fragmentation, namely erosion of single cells and fragmentation into equal sizes (or binary fission), lead to distinguishable final size distributions. We believe that our experiments for division-formed colonies support the hypothesis of the erosion mechanism. Specifically, Figure 2E shows that colony fragmentation resulted in a decrease of large colonies and a strong increase of single cells and dimers (two cells). In our view, the strong increase of single cells and dimers provides quite convincing (but indirect) evidence supporting the erosion mechanism. This is described on lines 112-121. To further address the reviewer’s concern, we have included in the revised version of Figure 2 (panels B and D) a direct comparison between these two fragmentation models for large division-formed colonies fragmented at a high dissipation rate of ε = 5.8 m²/s³. Furthermore, we have included the new Supplementary Figure S9, which details the model predictions for the colony size distribution at various time points.

The ideal equal fragments model (i.e., where every fracture event produces two identical fragments with half the original biovolume) does not capture the biovolume transfer from large colonies to single cells, as observed for the experimental results in panel D of Figure 2 and panel E of Figure S9. In contrast, the erosion model, in panel D of Figure 2 and panel D of Figure S9, provides a good prediction of the experimental results within the experimental uncertainty. The different fragmentation models are discussed in lines 226-228 of the revised manuscript and lines 865-873 of the SI.

(3) Their third major claim is that fluid flows only weakly cause cells to collide and adhere in a "coming together" process of aggregate formation. They test this claim in Figure 3, where they suspend single cells in their test chamber and stir them at moderate intensity, monitoring their size histogram. They show that the size histogram changes only slightly, indicating that aggregation is, by and large, not occurring at a high rate. Therefore, they lend support to the idea that cell aggregation likely does not initiate group formation in toxic cyanobacterial blooms. Additionally, they show that the median size of large colonies also does not change at moderate turbulent intensities. These results agree with previous studies (their own citation 25) indicating that aggregates in toxic blooms are clonal in nature. This is an important result and well-supported by their data, but only for this specific particle concentration and stirring intensity. Later, in Figure 5 they show a much broader range of particle concentrations and energy dissipation rates that they leave untested.

We thank the reviewer for this positive comment. We agree that our experimental results show clear evidence that aggregated colonies have a weaker structure in comparison to division-formed colonies, thus supporting the hypothesis that clonal expansion is the main mechanism for colony formation under most natural settings. The range of energy dissipation rates of our experimental setup covers almost entirely the region for which aggregated and division-formed colonies differ in their fragmentation behavior (Zone III of Figure 5). Within this zone, aggregated colonies are fragmented and only the division-formed colonies are able to withstand the hydrodynamic stresses. Furthermore, we show that this fragmentation behavior has a low sensitivity to the total biovolume fraction, as displayed in the Supplementary Figures S2 and S4 and discussed in lines 151-154 and 160-163. We agree that our cone-and-plate setup covers a limited parameter range, and we have added a detailed discussion of these limitations in the revised manuscript, under section Materials and Methods in lines 462-473.

(4) The fourth major result of the manuscript is displayed in Equation 8 and Figure 5, where the authors derive an expression for the ratio between the rate of increase of a colony due to aggregation vs. the rate due to cell division. They then plot this line on a phase map, altering two physical parameters (concentration and fluid turbulence) to show under what conditions aggregation vs. cell division are more important for group formation. Because these results are derived from relatively simple biophysical considerations, they have the potential to be quite powerful and useful and represent a significant conceptual advance. However, there is a region of this phase map that the authors have left untested experimentally. The lowest energy dissipation rate that the authors tested in their experiment seemed to be \dot{epsilon}~1e-2 [m^2/s^3], and the highest particle concentration they tested was 5e-4, which means that the authors never tested Zone II of their phase map. Since this seems to be an important zone for toxic blooms (i.e. the "scum formation" zone), it seems the authors have missed an important opportunity to investigate this regime of high particle concentrations and relatively weak turbulent mixing.

We agree with the reviewer that Zone (II) of Figure 5 is of great importance to dense bloom formation under wind mixing and that this parameter range was not covered by our experiments using a cone-and-plate shear flow. The measuring range of our device was motivated by engineering applications such as artificial mixing of eutrophic lakes using bubble plumes, as well as preliminary experiments which demonstrated that high levels of dissipation rate were required to achieve fragmentation. The range of dissipation rates that can be achieved by the cone-and-plate setup is limited at the lower end by the accumulation of colonies near the stagnation point at the conical tip and at the upper end by the spillage of fluid out of the chamber. We now discuss this measuring range in lines 462-473 of the revised manuscript.

Although our setup does not cover Zone (II), we now refer to recent results in the literature for evidence of aggregation-dominance at Zone (II). The experimental study of Wu et al. (2024) (reference number 64 of the revised manuscript) investigated the formation of Microcystis surface scum layers in wind-mixed mesocosms. Their study identified aggregation of colonies in the scum layer, resulting in increases of colony size at rates faster than cell division. These results agree with our model, and the parameters range investigated fall within the Zone II. We have included in the revised version, lines 328-337, a detailed discussion elucidating the parameter range covered in our experiments and the findings of Wu et al. (2024).

Other items that could use more clarity:

(5) The authors rely heavily on size distributions to make the claims of their paper. Yet, how they generated those size distributions is not clearly shown in the text. Of primary concern, the authors used a correction function (Equation S1) to estimate the counts of different size classes in their image analysis pipeline. Yet, it is unclear how well this correction function actually performs, what kinds of errors it might produce, and how well it mapped to the calibration dataset the authors used to find the fit parameters.

We agree with the reviewer that more details of the correction function should be included. We have included in the revised version of the Supporting Information, in lines 785-796, a more detailed explanation of the correction function. Furthermore, a direct comparison of raw and corrected histograms of the size distribution and its associated uncertainty is presented in the new Supplementary Figure S8.

(6) Second, in their models they use a fractal dimension to estimate the number of cells in the group from the group radius, but the agreement between this fractal dimension fit and the data is not shown, so it is not clear how good an approximation this fractal dimension provides. This is especially important for their later derivation of the "aggregation-to-cell division" ratio (Equation 8)

We agree with the reviewer that more details on the estimation of fractal dimension are needed. The revised version, under Materials and Methods in lines 508-515, now includes the detailed estimation procedure, the number of colonies analysed, and the associated uncertainty.

Reviewer #1 (Recommendations For The Authors):

In light of the weak evidence for claim #2 outlined above, I believe the paper would benefit from a more explicit comparison in Figure 2C of the two models - idealized erosion, and idealized binary fission. With such a comparison, the authors would have stronger footing to claim that one process is more important than the other.

As mentioned in our answer above to comment #2 of public review, we have included in the revised version of Figure 2 (panels B and D) a direct comparison between the erosion and equal fragments (binary fission) models for large division-formed colonies fragmented under ε = 5.8 m²/s³. The comparison is further detailed in the new Supplementary Figure S9 for representative time points. Only the erosion models can recover the biovolume transfer from large colonies to single cells, as observed for the experimental results in Figure 2D and further detailed in Figure S9D. We believe that the revised version of Figure 2 and the new Supplementary Figure S9 provide strong evidence in support of the erosion fragmentation model.

Would the authors comment on their chosen range of experimental dissipation rates? For instance, was their goal more to investigate industrial/engineering applications where the goal is to disrupt the cyanobacteria, but not really typical natural conditions under which the groups might form?

The choice of experimental dissipation rates in our experiment was such that it covers engineering applications such as artificial mixing of eutrophic lakes using bubble plumes. We have now clarified in the Introduction, on lines 37-39, that artificial mixing has been successfully applied in several lakes to suppress cyanobacterial blooms. Furthermore, we have now clarified in the caption of Figure 5 that the bars on the right side indicate typical values of dissipation rates induced by natural wind-mixing, bubble plumes in artificially mixed lakes, and laboratory-scale experiments such as cone-and-plate systems and stirred tanks. The dissipation rates induced by the bubble plumes in artificially mixed lakes could potentially fragment aggregated cyanobacterial colonies and thus disrupt bloom formation. However, our preliminary experiments demonstrated that high levels of dissipation rate were required to achieve fragmentation, therefore we’ve focused on the upper range of values (0.01 to 10 m²/s³).

The dissipation rates generated by the cone-and-plate approach are indeed higher than the dissipation rates under typical natural conditions in lakes. We have now added a detailed discussion of the range of dissipation rates generated by the cone-and-plate approach in the revised manuscript, under section Materials and Methods in lines 462-473, where we also explain that these values are higher than the natural dissipation rates generated by wind action in lakes. However, the more generic insights obtained by our study, shown in Figure 5, are relevant for dissipation rates of natural lakes (e.g., Zone II). Therefore, in our discussion of Figure 5 we have now included the recent findings of Wu et al. (2024) (reference number [64] of the revised manuscript), who studied bloom formation of Microcystis in mesocosm experiments at dissipation rates representative of natural conditions; see also our reply to the next comment.

The authors should consider testing the space of Zone II on their phase map, for instance at very high particle concentrations and even lower rotational speeds, in order to show that their derivations match experiments.

Good point. As mentioned in our answer above to comment #4 of the public review, Zone II lies beyond the measuring range of our experimental setup. Instead, we refer to the recent study of Wu et al. (2024) (reference number [64] of the revised manuscript) which demonstrated that dense scum layers of Microcystis colonies are aggregation-dominated. These mesocosm experiments agree with our model predictions and their parameter range falls within Zone II. We have included in the revised version, lines 328-337, a detailed discussion where we elucidate the parameter range covered in our experiments and compare our predictions for Zone II with the recent findings of Wu et al. (2024).

The authors should show their calibration data and fit for the correction function of equation S1. Additionally, you may consider showing "raw" and "corrected" histograms of the size distribution, to demonstrate exactly what corrections are made.

As mentioned in our answer above to comment #5 of the public review, we have included in the revised version of the Supporting Information the new Supplementary Figure S8, which shows the raw and adjusted histograms of the size distribution, including the associated uncertainties. Furthermore, the correction function is now explained in detail in the new Supporting Information Text in lines 785-796.

The authors might consider commenting on Figure S3 a bit more in the main text. Even at very high dissipation rates, the cyanobacterial groups don't plummet to size 1, but stay in an equilibrium around 10-20x the diameter of a single cell. What might this mean for industrial applications trying to break up the groups?

We agree with the reviewer that further discussion of Figure S3, panels E and F, is warranted. In the revised version of the manuscript, under section Fragmentation of Microcystis colonies occurs through erosion in lines 133-137, we have now included a discussion of this figure. Figure S3F shows that more than 90% of the total biovolume ends up in the category “small colonies” (mostly single cells and dimers); hence, most of the initially large colonies do fragment to single cells or dimers. Only about 5-10% of the biovolume remains as “large colonies” of 10-20 cells. Although it is challenging to draw definitive conclusions about the behavior of these remaining large colonies, as they account for only a minor fraction of the suspension, one hypothesis is that variability in mechanical properties between colonies results in a subset of colonies exhibiting exceptional resistance even to very high dissipation rates (see lines 133-137).

Minor comments:

Typo Caption of Figure 2: Should read [m^2/s^3] for units

Thanks for catching this typo. The units in the caption of Figure 2 has been corrected to [m^2/s^3].

There is no Equation 10 in Materials and Methods as indicated in the rheology section.

We thank the reviewer for pointing out the lack of clarity in this algebraic manipulation. In fact, the yield stress has to be substituted in the current Equation 11 (previously Eq.10), from which the critical dissipation rate must be substituted in Equation 3. The result is the critical colony size (l* = 2.8) mentioned in line 243 of the revised manuscript. The correct equation numbers and algebraic substitutions are now indicated in lines 241-243 of the revised version of the manuscript.

<Reviewer #2 (Public review):

Especially the introduction seems to imply that shear force is a very important parameter controlling colony formation. However, if one looks at the results this effect is overall rather modest, especially considering the shear forces that these bacterial colonies may experience in lakes. The main conclusion seems that not shear but bacterial adhesion is the most important factor in determining colony size. As the importance of adhesion had been described elsewhere, it is not clear what this study reveals about cyanobacterial colonies that was not known before.

We would like to emphasize several key findings that our study reveals about the impacts of fluid flow on cyanobacterial colonies:

(I) Quantification of mechanical strength in cyanobacterial colonies: Our results demonstrate the high mechanical strength of cyanobacterial colonies, as evidenced by the requirement of high shear rates to achieve fragmentation. This is new knowledge, that was not known before for cyanobacterial colonies. To this end, our study highlights the resilience of these colonies against naturally occurring flows and bridges the gap between theoretical assumptions about colony strength and experimentally measured mechanical properties.

(II) The discovery that the mechanical strength of colonies differs between colonies formed by cell division and colonies formed by aggregation. This is again new knowledge, that was not known before for cyanobacterial colonies.

(III) Validation of a hypothesis regarding colony formation: Using a fluid-mechanical approach, we confirm the findings of recent genetic studies (references 25 and 67 of the revised version of the manuscript) which indicated that colony formation occurs predominantly via cell division rather than cell aggregation under natural conditions (except in very dense blooms).

(IV) Practical guidelines for cyanobacterial bloom control: Our findings provide valuable insights into the design of artificial mixing systems applied in several lakes. Artificial mixing of lakes is based on fundamentals of fluid flow, aiming at preventing aggregation of buoyant cyanobacteria in scum layers at the water surface. Our results show that the dissipation rates generated by bubble blumes in artificially mixed lakes can fragment cyanobacterial colonies formed by aggregation, but are not intense enough to cause fragmentation of division-formed colonies (see Figure 5 and lines 348-360).

The agreement between model and experiments is impressive, but the role of the fit parameters in achieving this agreement needs to be further clarified.

The influence of the fit parameters (namely the stickiness α1 and the pairs of colony strength parameters S1,q1,S2,q2) is discussed in the sections Dynamical changes in colony size modelled by a two-category distribution in lines 247-253 and Materials and Methods in lines 559-565. We kept the discussion concise to maintain readability. However, we agree with the reviewer that additional details about the importance of the fit parameters and the sensitivity of the results to these parameters could be beneficial. In the revised version of the section Materials and Methods in lines 560-563, we have included a detailed discussion of the fit parameters.

The article may not be very accessible for readers with a biology background. Overall, the presentation of the material can be improved by better describing their new method.

We apologize for the limited readability of the description of the experimental setup and model used. In the revised version of the manuscript and the SI, we have detailed further the new methods presented here. The modifications include a detailed description of the operating range of the cone-and-plate shear setup (subsection Cone-and-plate shear of the section Materials and Methods, in lines 462-473). Furthermore, we think that incorporation of the recent experimental results of Wu et al. (2024), on lines 331-337 of the manuscript, will appeal to readers with a biology background. Their mesocosm experiments support our model prediction that aggregation is the dominant mechanism for colony formation in region (II) of Figure 5.

Reviewer #2 (Recommendations For The Authors):

(1) The authors seem too modest in claiming technological advance. They should describe the technological advance of combining microscopy with rheometry, in such a way that this invites others to apply this or similar approaches on biological samples. Even though I feel that the advancement of knowledge of this system by their method is relatively modest, there may be more advances in other systems.

We appreciate the positive view of the reviewer towards the importance of this technology and we agree that its advantages should be advertised to researchers investigating similar systems. We have now given more attention to the technological advance of combining microscopic imaging with rheometry in the final paragraph of the Conclusions (lines 386400), where we now also briefly discuss an interesting recent study of marine snow (Song et al. 2023, Song and Rau 2022, reference numbers 70 and 71 of the revised manuscript), which used a similar combination of microscopy and rheometry as in our study. Furthermore, in the Methods section, we now briefly explain how the rheometry can be adjusted to investigate other systems (lines 474-480).

(2) It seems reasonable -also based on what we already know about these aggregates - to assume that the main difference in shear sensitivity between field samples and cultures lies in the production of extracellular polysaccharide substance (EPS). To go beyond what is already known, the study could try to provide more direct and quantitative evidence for EPS involvement. For example, using a chemical quantification of EPS levels, or perturbing EPS levels using digestive enzymes.

We agree with the reviewer that further characterization of the EPS is highly relevant to understand the mechanical strength of colonies. However, we believe that chemical quantification and/or degradation of EPS lies beyond the scope of our article and should be addressed by future studies.

(3) Assuming EPS is indeed the reason for the differences in shear resistance: the authors speculate the reason why the field samples have more EPS lies in chemical composition (Calcium/nitrogen levels). In addition, there could be grazing that is known to promote aggregation (possibly increasing EPS), or just inherent genetic differences between strains. I am not necessarily expecting the authors to explore this direction experimentally, but it seems certainly feasible and would make the final result less speculative.

We agree with the reviewer that there are more biotic and abiotic factors that can influence EPS amount and composition. The influence of grazing and other relevant factors on cell adhesion is discussed in references [26-29], cited in our introduction in lines 50-53. As discussed in our answer to recommendation #2, we believe that a quantitative investigation of these various factors is beyond the scope of this work and should be addressed in future studies.

(4) A cool finding seems to be the critical relative diameter (Fig 2E), a colony size that seems invariant under shear. I was slightly surprised that the authors seem to take little effort to understand this critical diameter mechanistically (for example by predicting it, or experimentally perturbing it). Again, not a necessary requirement, but this is where the study could harness its technological advantage to provide a more quantitative understanding of something that goes beyond the existing knowledge of the system.

We apologize to the reviewer if our descriptions and discussions of Figure 2 were unclear. One of the key conclusions from our experiments is that the critical relative diameter depends on the dissipation rate, as shown in Figure 2F. This dependence is also incorporated into the model through the constitutive equation (2). Furthermore, we expect the mechanical resistance of colonies, quantified by the critical relative diameter, to be affected by other biotic and abiotic factors that influence EPS amount and composition.

(5) The jump from 0.019 to 1.1 m²/s³ seems large. What was the reason for not exploring intermediate values? The authors should also define low, modest and intense dissipation rates more clearly. Currently, they seem somewhat arbitrarily defined, i.e. 0.019 m²/s³ is described as low (methods) and moderate (results). In Fig 2, the authors further talk about low dissipation rates without a quantitative description.

We thank the reviewer for pointing out the lack of clarity in the choice of parameter range and the nomenclature. Regarding the former, the suspension of division-formed colonies of Microcystis strain V163 displayed negligible fragmentation for dissipation rates between 0.019 to 1.1 m²/s³, as seen in Figures S2A and S3A. Due to the low sensitivity of the fragmentation results in this region, we don’t expect change in behavior for intermediate values. Regarding the nomenclature, we have corrected the inconsistencies throughout the text. We have chosen to name the dissipation rate values as: low for values typical of windmixing, moderate for values typical of the core of bubble plumes, and intense for values typical of propellers. Whenever mentioned in the text, the numerical value of dissipation rate is also included to avoid doubt.

(6.) The structure and narrative of the paper can be improved. The article first describes all lab culture experiments and then the model, while the first figure already shows model fits. Perhaps it would be better to first describe the aggregation experiments, to constrain the appropriate terms of the model, and then move to fragmentation.

We appreciate the recommendation of the reviewer regarding the structure. We have chosen to describe first the fragmentation experiments (Fig. 2), as these can be understood without introducing the aggregation effects. In contrast, the steady state results in the aggregation experiments (Fig. 3) come from the balance between aggregation and fragmentation. Therefore, we judged the current order to be more appropriate. The model fits are combined with the experimental results in Figures 2 and 3 to have a concise display. We have ensured that all the concepts required to understand each figure panel are explained prior to their discussion.

(7) The number of data points that go into the histogram needs to be indicated. The main reason is that the authors report the distribution in terms of the biovolume fraction, suggesting the numerical counts are converted into volume. This to me seems like the most sensible parameter, but I could not find how this conversion is calculated (my apologies if I missed it). This seems especially relevant because a single large colony can impact this histogram quite considerably.

We apologize for the lack of clarity in the calibration and conversion steps of the size distribution. As discussed above in the answer to comment #5 of the reviewer #1, more details of the calibration process have been added to the revised version of the Supporting Information Text in lines 785-796. Furthermore, the new Supplementary Figure S8 presents examples of the raw and adjusted size distribution, including the total number of counted colonies per histogram and the associated uncertainties in the concentration and biovolume distributions.

(8) Over the timescales measured here, colonies could start sinking (or floating), possibly in a size-dependent manner, that could lead to a bias due to boundary effects. Did the authors consider this potential artifact?

The sinking or floating of colonies is a relevant process which was taken into account in the choice of our parameter range for the dissipation rate. The minimum dissipation rate used in our experiments ensures that the upward inertial velocity near stagnation is sufficient to counteract the sedimentation of colonies. A detailed discussion of the choice of the parameter range is now included in the revised version of the Materials and Methods in lines 462-473.

(9) "On the one hand, sequencing of the genetic diversity within Microcystis colonies supports the hypothesis that colony formation undernatural conditions is primarily driven by cell division [25]. On the other hand, cell aggregation can occur on a shorter time scale and may offer improved protection against high grazing pressure [26]." This appears somewhat constructed, as what is described as "on the other hand" is not evidence against the genetic diversity.

We agree that the suggested dichotomy in this text appeared somewhat constructed, and we have now removed the wording “on the one hand” and “on the other hand”. The studies from reference [25] demonstrated that the genetic diversity between independent Microcystis colonies is much greater than the diversity within colonies. If cell aggregation was the dominant mechanism, a similar genetic diversity would be observed between and within colonies, which contrasts the findings from reference [25]. We have adjusted the text in the revised manuscript, in lines 46-54, to clarify this point.

(10) The phase diagram seems largely based on extrapolations that are made outside of the measurement regime (e.g. dark red bars indicating the dissipation rate, Fig 5 - by the way 1 this color scheme could use some better contrast, by the way 2 Fig S7 suggests a wider dissipation rate range as indicated in Fig 5, why?). Hence there seems to be the need to more clearly lineate experimental results, simulations, and extrapolations in the phase diagram.

We agree with the reviewer that further clarifications should be given about the parameter range covered in our experiments and apologize for the lack of readability in the color scheme of Fig 5. In lines 329-337, 346-347, 353-355, we have highlighted the parameters range covered by our experiments as well as the range covered by previous studies of windmixed mesocosm (namely reference [64] of the revised manuscript). Regarding the color scheme of Figure 5, we have modified the legend of the figure to improve readability. The color contrast was increased and leader lines were added to connect the colored bars with the respective label.

(11) Unfortunately, the manuscript did not contain line numbers.

We apologize to the reviewer for the lack of line numbers in our initial version. The revised version of the manuscript now contains line numbers, both in the main text and the supporting information.

(12) Fig 2D. Caption is too minimal. Y-axis could better be named "Fraction of colonies" as both small and large colonies are plotted.

The caption for Figure 2D was extended to better describe the plot. We have kept the y-axis label as “Fraction of small colonies”, since this is the quantity displayed by the three curves in the plot.

(13) An inset should have axis labels.

All the insets in our plots display the same variables as their respective plots. In order to keep the plots light and preserve readability, we therefore prefer to present the axis labels only along the x-axis and y-axis of the main plots, which implies by convention that the same axis labels also apply to the insets. To the best of our knowledge, this is a common approach.

(14) Page 5, first words. Likely Fig 3A, not 2A was meant.

We thank the reviewer for pointing out this readability issue. We intend to compare both Figures 2A and 3A. The text of the revised manuscript, in lines 146-148, has been adjusted with the correct figure numbers.

(15) Introduction, second last paragraph, third last line. "suspension leaded to a broad distribution" I assume you meant "... led to a ..."

We thank the reviewer for pointing out this typo. It has been corrected (line 122).

The 1920 poem "Mr. Apollinax" centers itself on a group of scholars and students attending a dinner party. The character of Mr. Apollinax himself is heavily inspired by Bertrand Russell, a mentor and friend to Eliot, who was also a famous logician, though his concepts at times were difficult for even well-versed philosophers and scholars to comprehend. In the poem, the descriptions of Mr. Apollinax and his behavior at the dinner party obtains direct parallels to "Death by Water" and the motif of the ocean itself.

1. "His laughter was submarine and profound / Like the old man of the sea’s" (lines 8-9). In these lines, there are two clear connections to water or the ocean: the description of Mr. Apollinax's laugh as being "submarine", and the equating of such laugh to "the old man of the sea's". Beyond his supreme intellect, Mr. Apollinax is portrayed as a man deriving a great amount of pleasure and fulfillment from the world, as conveyed by his "submarine and profound laugh", indicating that unlike the hollow and despairing voices of TWL, Mr. Apollinax acquires a humor rooted in a genuine "inner richness" and vitality.
2. "Where worried bodies of drowned men drift down in the green silence, / Dropping from fingers of surf" (lines 11-12). These lines stood out to me as referencing the general human condition of the time: people are swept away by the chaotic tide of worldly forces (industrialization, war, pursuit of goods or wealth) that have risen in the rapidly changing times of the early 20th century. The world has become so complicated that it has become nearly impossible for the regular individual to properly navigate it, leading them to "drown". This metaphorical drowning could indicate several things: on a broader scale, the fall of humanity into sin and wrongdoing; on a similar vein, humanity becoming separate from the ideals of religion or spirituality; or the succumbing of mankind to the pursuit of worldly things such as wealth, fame, or material goods. In my mind, a good argument could be made for each of these possibilities.
3. "I looked for the head of Mr. Apollinax rolling under a chair / Or grinning over a screen / With seaweed in its hair" (lines 13-15). Now, this is an interesting detail. It is another example of decapitation (the first major instance being the headless corpse of a sex worker in Le Fleurs du Mal). The head, which literally represents reason and intellect, being separated from the body suggests a collapse of rational order and a breakdown of the mind itself. In the context of TWL, such a disjunction reflects the intellect’s estrangement from emotional or spiritual grounding. The description of the head “grinning over a screen” adds an absurd and grotesque dimension to the scene, transforming what might otherwise be horrific into a moment of unsettling fascination. Ultimately, the tension between humor and horror illuminates a key theme that runs through TWL: intellect, when isolated from the fuller spectrum of human experience (emotion, spiritual faith, and vitality) risks devolving into alienation or madness.

Underneath this section, can you add a section about the process? Step 1: request your invite Step 2: Attend a pre-event interview to help me curate the perfect group experience (and make sure you're ready for this experience!) Step 3: 2 Days of action taking Step 4: Attend your Feedback Call to celebrate your wins and cement your aha-moments

brainstorm all possibilities and then narrow your search for your 2-3 most promising ideas

Okay, so the "smart" virtue being about people skills and not actual intelligence kind of blew my mind. I've definitely been on teams before where the smartest person in the group was actually the worst team member because they had no idea how to read social situations. When we did that conflict exercise, or well when my team. I'm realizing I probably need to work on this more myself because I tend to just say what I think without always considering how it's landing with everyone else. Does anyone else struggle with knowing when to speak up versus when to just let something go?

PMID: 40612488

Gene: MECP2

HGNC: 6990

Case: 13.9-year-old female patient

DiseaseAssertion: Rett, (Z-RTT/PSV: Zappella Rett syndrome/preserved speech variant)

FamilyInfo: Tics were also present in the mother and grandmother, de novo MECP2 variant was detected in the proband, a maternally inherited VoUS class 3 in GABBR1 (p. F692S), was identified in the proband, and segregated in the mother and grandmother

ParentalTest: FullPhase

CasePresentingHPOs: HP:0012760, HP:0000817, HP:5200030, HP:0012171, HP:0100035, HP:0100034, HP:0000739, HP:0100716, HP:0030051, HP:0002136, HP:0001276, HP:0002342, HP:0007010, HP:0000717

CaseHPOFreeRext: the case exhibited social withdrawal, difficulties in relationships, limited verbal communication, midline pinching and hand clapping, skin picking, progressive hypertonia with rigidity of the limbs, impaired her fine motor abilities.

CaseNOTHPOs: NR

CaseNOTHPOFreeText: NR

CasePreviousTesting: Whole Genome Array-CGH yielded normal results

PreviouslyPublished: Not previously published

GenotypingMethod: Exome Genome

Variant: NP_004983.1: c.397C>T; p.Arg133Cys

HGVS: NM_001110792.2:c.433C>T

ClinVarID: 11809

CAID: CA211250

gnomAD: NR

MultipleGeneVariants:

DiseaseEntity: Rett

AlleleOrigin: Germline

Variant: NM_001470.4:c.2075T>C

Mutation: Missense

Zygosity: Heterozygous

CAID: CA3689865

gnomAD: 0.000024 (GnomAD v4.1.0)

PreviouslyPublished: No

Tools of the typewriter trade by [[Retrotype]]

Excellent overview of many of the basic tools for typewriter repair. Didn't have the strongest grasp of all the tools' specific names, but good enough for describing their general use cases.

Example of a typewriter toolset including a case made for telephone company repair, but which works with typewriters.

• Shore A durometer gauge 2:22
• nylon fishing/picture hanging wire spec to 25kg (for drawband replacement)
• thick waxed string/yarn for repairing fishing nets (for drawbands)
• nitrile gloves (to prevent staining, issues with mineral spirits, and other caustic chemicals)
• XPower pressure blower for blowing out dust/dirt and mineral spirits. (smaller than an air compressor)
• nail grooming set with tweezers, picks, etc. (not technically necessary, but sometimes useful)
• dental tools (for use as spring hooks)
• Renaissance micro-crystalline wax (non-corrosive, made for British Museum, good on marble, wood, leather, etc. Good on bare metal for treating previously rusted metal. (It's recommended to use an abrasive polish for improving the shine of glossy paint however)
• Pouch and set of precision screwdrivers (he only uses the flatheads though the set includes other) Prefer hollow ground tips which are squared off rather than wedges.
• Chapman bit set of screwdrivers (with hollow ground tips) He prefers these for hard to remove screws. Issue that it's a bit thicker at the tip.
• Liquid wrench penetrating oil for helping to loosen screws (he likes this better than WD-40)
• brash wire brushes
• steel wire brushes (uses less frequently as they're more abrasive)
• pouch of precision wrenches (imperial and metric) his are bladed, Moody tools wrenches (mfg.) prefer the thinnest tips
• microfiber cloths
• jig for soldering typeslugs on typearms
• pouch with various typewriter specific pliers:
  • 3 prong pliers (total of 9 prongs) for making bends/forming typebars (especially making bends in the middle of bars rather than the end.;
  • peening bend pliers;
  • bending pliers for sideways bends esp. with thinner typebars;
  • vertical adjustment pliers (with rollers) not good for making adjustments of 3mm or more;
  • forming pliers with screws on the end to rotate heads for bending, peening and cutting;
  • peening pliers (bending by metal displacement)
• Magnetized screwdrivers
• forceps
• screw grabber (active capture)
• spring hooks (push/pull)
• nylon brushes for dusting
• needle nose pliers
• t-bender with slotted head for forming metal
• small bottles for mineral spirits and sewing machine oil. They have small metal tips for precision application.

Observation: They met 3 men in a canoe who had hunted for a year and made $900 from furs. The men ran out of food and powder. It rained that night.

Interpretation: This shows how hunters worked for a long time in the wilderness and lived off trading furs.

Connection: It connects to the importance of hunting and trading in the early West.

I learned that fur trading was one of the main ways people survived and made money in the early West. This adds to my connection because it shows how important nature and animals were to the economy before cities and factories grew. It’s important because it helps me see how life and work changed over time from depending on hunting and trading to building towns and using new jobs and trades.

Change over time: Back then, selling furs was one of the main ways to earn money. Later, hunting slowed down because there were fewer animals, new towns were built, and people started finding other jobs instead of trading animal skins.

Observation: They left early, went past Big Monitu Creek, saw buffalo tracks, and Lewis checked out a salt spring with some men.

Interpretation: This shows they were looking at animals and natural things like salt while they traveled.

Connection: It links to how the trip was about learning what the land had, not just moving through it.

I learned that Lewis and his team studied everything around them, like animals and natural resources such as salt. This adds to my connection because it shows the expedition was about discovery and learning what the new land could offer. It’s important because their findings helped the U.S. understand the land’s value, resources, and how people could live there. It shows how exploration helped the country grow and use its new land wisely.

Context: In 1804, the U.S. had just bought this land in the Louisiana Purchase. People didn’t know what was there, so the expedition was sent to study the land, animals, and resources.

1. What Marx is criticizing

Marx is talking about a mistake in how people understand history:

Some thinkers separate dominant ideas from the people and social conditions that created them.

In other words, they treat ideas as if they exist independently, not as a product of society and its material conditions.

1. What happens when you do that

People start to believe that history is driven by ideas themselves, rather than by the economic and social realities behind them.

Philosophers like Hegel did this: they treated ideas as if they were self-developing forces shaping history.

1. Consequence of this way of thinking

If you focus only on “thought” or “the Idea,” you can end up thinking that all human relations and society come from ideas — like “Man” or “the essence of Man” — rather than from material conditions like class, work, or property.

so there's a body of knowledge that it's a structured knowledge that's

each of those is represented by NFP that can also be transferred as a means of transferring ownership .4 - Universal Resource Name

Can be connected to any number of other Knowledge Assets

Reviewer #3 (Public review):

The manuscript "Theory of active self-organization of dense nematic structures in the actin cytoskeleton" analysis self-organized pattern formation within a two-dimensional nematic liquid crystal theory and uses microscopic simulations to test the plausibility of some of the conclusions drawn from that analysis. After performing an analytic linear stability analysis that indicates the possibility of patterning instabilities, the authors perform fully non-linear numerical simulations and identify the emergence of stripe-like patterning when anisotropic active stresses are present. Following a range of qualitative numerical observations on how parameter changes affect these patterns, the authors identify, besides isotropic and nematic stress, also active self-alignment as an important ingredient to form the observed patterns. Finally, microscopic simulations are used to test the plausibility of some of the most crucial assumptions underlying continuum simulations.

The paper is well written, figures are mostly clear, and the theoretical analysis presented in both, main text and supplement, is rigorous. Mechano-chemical coupling has emerged in recent years as a crucial element of cell cortex and tissue organization and it is plausible to think that both, isotropic and anisotropic active stresses, are present within such effectively compressible structures. Even though not explicitly stated this way by the authors, I would argue that combining these two is one of the key ingredients that distinguishes this theoretical paper from similar ones.

The diversity of patterning processes experimentally observed and theoretically described is nicely elaborated on in the introduction of the paper. The theory development and discussion of the continuum model itself is also well-embedded in a review of the relevant broad literature on active liquid crystals and active nematics, which includes plenty of previous results by the authors themselves. Interestingly, several of the patterns identified in the present work, such as 2D hexagonal and pulsatory patterns (Kumar et al, PRL, 2014), as well as contractile patches (Mietke et al, PRL 2019) have been observed previously in different, but related, active isotropic fluid models. In light of this crowded literature, the authors do good job in delineating key results obtained in the present manuscript from existing work.

The results of numerical simulations are well-presented. The discussion of numerical observations is comprehensive, but also at many times qualitative. Some of the observations resonate with recent discussions in the field, for example the observation of effectively extensile dynamics in a contractile system, which is interesting and reminiscent of ambiguities about extensile/contractile properties discussed in recent preprints (Nejad et al, Nat Comm 2024). It is convincingly concluded that, besides nematic stress on top of isotropic one, active self-alignment is a key ingredient to produce the observed patterns.

The authors must be complimented for trying to gain further mechanistic insights into their conclusions using microscopic filament simulations that were diligently performed. It is rightfully stated that these simulations only provide plausibility tests about key assumptions underlying the hydrodynamic theory. Within this scope, I would say the authors are successful. At the same time, it leaves open questions that could have been discussed more carefully. For example, I wonder what can be said about the regime \kappa>0 microscopically, in which the continuum theory does also predict the formation of stripe patterns? How does the spatial inhomogeneous organization the continuum theory predicts fit in the presented, microscopic picture and vice versa? The authors clearly explain the scope and limitations of the microscopic model, which suggests that questions like these will be interesting directions of future investigations.

Overall, the paper represents a valuable contribution to the field of active matter that should provide a fruitful basis to develop new hypothesis about the dynamic self-organisation and mechanics of dense filamentous bundles in biological systems.

Author response:

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

eLife assessment

In this study, the authors offer a theoretical explanation for the emergence of nematic bundles in the actin cortex, carrying implications for the assembly of actomyosin stress fibers. As such, the study is a valuable contribution to the field actomyosin organization in the actin cortex. While the theoretical work is solid, experimental evidence in support of the model assumptions remains incomplete. The presentation could be improved to enhance accessibility for readers without a strong background in hydrodynamic and nematic theories.

To address the weaknesses identified in this assessment, we have expanded the motivation and description of the theoretical model, specifically insisting on the experimental evidence supporting its rationale and assumptions. These changes in the revised manuscript are implemented in the two first paragraphs of Section “Theoretical model” and in a more detailed description and justification of the different mathematical terms that appear in that section. We have made an effort to map in our narrative different terms to mechanistic processes in the actomyosin network. Even if the nature of the manuscript is inevitably theoretical, we think that the revised manuscript will be more accessible to a broader spectrum of readers.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

In this article, Mirza et al developed a continuum active gel model of actomyosin cytoskeleton that account for nematic order and density variations in actomyosin. Using this model, they identify the requirements for the formation of dense nematic structures. In particular, they show that self-organization into nematic bundles requires both flow-induced alignment and active tension anisotropy in the system. By varying model parameters that control active tension and nematic alignment, the authors show that their model reproduces a rich variety of actomyosin structures, including tactoids, fibres, asters as well as crystalline networks. Additionally, discrete simulations are employed to calculate the activity parameters in the continuum model, providing a microscopic perspective on the conditions driving the formation of fibrillar patterns.

Strengths:

The strength of the work lies in its delineation of the parameter ranges that generate distinct types of nematic organization within actomyosin networks. The authors pinpoint the physical mechanisms behind the formation of fibrillar patterns, which may offer valuable insights into stress fiber assembly. Another strength of the work is connecting activity parameters in the continuum theory with microscopic simulations.

We thank the referee for these comments.

Weaknesses:

(A) This paper is a very difficult read for nonspecialists, especially if you are not well-versed in continuum hydrodynamic theories. Efforts should be made to connect various elements of theory with biological mechanisms, which is mostly lacking in this paper. The comparison with experiments is predominantly qualitative.

We understand the point of the referee. While it is unavoidable to present the continuum hydrodynamic theory behind our results, we have made an effort in the revised manuscript to (1) motivate the essential features required from a theoretical model of the actomyosin cytoskeleton capable of describing its nematic self organization (two first paragraphs of Section “Theoretical model”), and to (2) explicitly explain the physical meaning of each of the mathematical terms in the theory, and when appropriate, relate them to molecular mechanisms in the cytoskeleton. We hope that the revised manuscript addresses the concern of the referee.

Regarding the comparison with experiments, they are indeed qualitative because the main point of the paper is to establish a physical basis for the self-organization of dense nematic structures in actomyosin gels. Somewhat surprisingly, we argue that a compelling mechanism explaining the tendency of actomyosin gels to form patterns of dense nematic bundles has been lacking. As we review in the introduction, these patterns are qualitatively diverse across cell types and organisms in terms of geometry and dynamics, and for this reason, our goal is to show that the same material in different parameter regimes can exhibit such qualitative diversity. A quantitative comparison is difficult for several reasons. First, many of the parameters in our theory have not been measured and are expected to vary wildly between cell types. In fact, estimates in the literature often rely on comparison with hydrodynamic models such as ours. For this reason, we chose to delineate regimes leading to qualitatively different emerging architectures and dynamics. Second, the patterns of nematic bundles found across cell types depend on the interaction between (1) the intrinsic tendency of actomyosin gels to form such structures studied here and (2) other elements of the cellular context. For instance, polymerization and retrograde flow from the lamellipodium, the physical barrier of the nucleus, and the interaction with the focal adhesion machinery are essential to understand the emergence of stress fibers in adherent cells. Cell shape and curvature anisotropy control the orientation of actin bundles in parallel patterns in the wings and trachea of insects. Nuclear positions guide the actin bundles organizing the cellularization of Sphaeroforma arctica [11]. Here, we focus on establishing that actomyosin gels have an intrinsic ability to self organize into dense nematic bundles, and leave how this property enables the morphogenesis of specific structures for future work. We have emphasized this point in the revised section of conclusions.

(B) It is unclear if the theory is suited for in vitro or in vivo actomyosin systems. The justification for various model assumptions, especially concerning their applicability to actomyosin networks, requires a more thorough examination.

We thank the referee for this comment. Our theory is applicable to actomyosin gels originating from living cells. To our knowledge, the ability of reconstituted actomyosin gels from purified proteins to sustain the kind of contractile dynamical steady-states observed in living cells is very limited. In the revised manuscript, we cite a very recent preprint presenting very exciting but partial results in this direction [49]. Instead, reconstituted in vitro systems encapsulating actomyosin cell extracts robustly recapitulate contractile steady-states. This point has been clarified in the first paragraph of Section “Theoretical model”.

(C) The classification of different structures demands further justification. For example, the rationale behind categorizing structures as sarcomeric remains unclear when nematic order is perpendicular to the axis of the bands. Sarcomeres traditionally exhibit a specific ordering of actin filaments with alternating polarity patterns.

We agree with the referee and in the revised manuscript we have avoided the term “sarcomeric” because it refers to very specific organizations in cells. What we previously called “sarcomeric patterns”, where bands of high density exhibit nematic order perpendicular to the axis of the bands, is not a structure observed to our knowledge in cells. It is introduced to delimit the relevant region in parameter space. In the revised manuscript, we refer to this pattern as “banded pattern with perpendicular nematic organization” or “banded pattern” in short.

(D) Similarly, the criteria for distinguishing between contractile and extensile structures need clarification, as one would expect extensile structures to be under tension contrary to the authors' claim.

We thank the referee for raising this point, which was not sufficiently clarified in the original manuscript. We first note that in incompressible active nematic models, active tension is deviatoric (traceless and anisotropic) because an isotropic component would simply get absorbed by the pressure field enforcing incompressibility. Being compressible, our model admits an active tension tensor with deviatoric and isotropic components. We consider always a contractile (positive) isotropic component of active tension, but the deviatoric component can be either contractile (𝜅 > 0) or extensile (𝜅 < 0), where we follow the common terminology according to which in contractile/extensile active nematics the active stress is proportional to q with a positive/negative proportionality constant [see e.g. https://doi.org/10.1038/s41467018-05666-8]. Furthermore, as clarified in the revised manuscript, total active stresses accounting for the deviatoric and isotropic components are always contractile (positive) in all directions, as enforced by the condition |𝜅| < 1.

For fibrillar patterns, we need 𝜅 < 0, and therefore active stresses are larger perpendicular to the nematic direction. This means that the anisotropic component of the active tension is extensile, although, accounting for the isotropic component, total active tension is contractile (see Fig. 1c). This is now clarified in the text following Eq. 7 and in Fig. 1.

However, following fibrillar pattern formation and as a result of the interplay between active and viscous stresses, the total stress can be larger along the emergent dense nematic structures (“contractile structures”) or perpendicular to them (“extensile structures”). To clarify this point, in the revised Fig. 4 and the text referring to it, we have expanded our explanation and plotted the difference between the total stress component parallel to the nematic direction (𝜎∥) and the component perpendicular to the nematic direction (𝜎⊥), with contractile structures satisfying 𝜎∥ − 𝜎⊥ > 0 and extensile structures satisfying 𝜎∥ − 𝜎⊥ < 0. See lines 280 to 303. This is consistent with the common notion of contractile/extensile systems in incompressible nematic systems [see e.g. https://doi.org/10.1038/s41467-018-05666-8].

(E) Additionally, its unclear if the model's predictions for fiber dynamics align with observations in cells, as stress fibers exhibit a high degree of dynamism and tend to coalesce with neighboring fibers during their assembly phase.

In the present work, we focus on the self-organization of a periodic patch of actomyosin gel. However, in adherent cells boundary conditions play an essential role, as discussed in our response to comment (A) by this referee. In ongoing work, we are studying with the present model the dynamics of assembly and reconfiguration of dense nematic structures in domains with boundary conditions mimicking in adherent cells, possibly interacting with the adhesion machinery, finding dynamical interactions as those suggested by the referee. As an example, we show a video of a simulation where at the edge of the circular domain, there is an actin influx modeling the lamellipodium, and in four small regions friction is higher simulating focal adhesions. Under these boundary conditions, the model presented in the paper exhibits the kind of dynamical reorganizations alluded by the referee.

Author response video 1.

We would like to note, however, that the prominent stress fibers in cells adhered to stiff substrates, so abundantly reported in the literature, are not the only instance of dense nematic actin bundles. In the present manuscript, we emphasize the relation of the predicted organizations with those found in different in vivo contexts not related to stress fibers, such as the aligned patterns of bundles in insects (trachea, scales in butterfly wings), in hydra, or in reproductive organs of C elegans; the highly dynamical network of bundles observed in C elegans early embryos; or the labyrinth patters of micro-ridges in the apical surface of epidermal cells in fish.

(F) Finally, it seems that the microscopic model is unable to recapitulate the density patterns predicted by the continuum theory, raising questions about the suitability of the simulation model.

We thank the referee for raising this question, which needs further clarification. The goal of the microscopic model is not to reproduce the self-organized patterns predicted by the active gel theory. The microscopic model lacks essential ingredients, notably a realistic description of hydrodynamics and turnover. Our goal with the agent-based simulations is to extract the relation between nematic order and active stresses for a small homogeneous sample of the network. This small domain is meant to represent the homogeneous active gel prior to pattern formation, and it allows us to substantiate key assumptions of the continuum model leading to pattern formation, notably the dependence of isotropic and deviatoric components of the active stress on density and nematic order (Eq. 7) and the active generalized stress promoting ordering.

We should mention that reproducing the range of out-of-equilibrium mesoscale architectures predicted by our active gel model with agent-based simulations seems at present not possible, or at least significantly beyond the state-of-the-art. To our knowledge, these models have not been able to reproduce the heterogeneous nonequilibrium contractile states involving sustained self-reinforcing flows underlying the pattern formation mechanism studied in our work. The scope of the discrete network simulations has been clarified in lines 340 to 349 in the revised manuscript.

While agent-based cytoskeletal simulations are very attractive because they directly connect with molecular mechanisms, active gel continuum models are better suited to describe out-of-equilibrium emergent hydrodynamics at a mesoscale. We believe that these two complementary modeling frameworks are rather disconnected in the literature, and for this reason, we have attempted substantiate some aspects of our continuum modeling with discrete simulations. We have emphasized the complementarity of the two approaches in the conclusions.

Reviewer #1 (Recommendations For The Authors):

Questions on the theory:

Does rho describe the density of actin or myosin? The authors say that they are modeling actomyosin material as a whole, but the actin and myosin should be modeled separately. Along, similar lines, does Q define the ordering of actin or myosin?

Active gel models of the actomyosin cytoskeleton have been formulated with independent densities for actin and for myosin or using a single density field, implicitly assuming a fixed stoichiometry. Super-resolution imaging of the actomyosin cytoskeleton also suggest that in principle it makes sense to consider different nematic fields for actin and for myosin filaments. In the revised manuscript, we now explicitly mention that our density and nematic field are effective descriptions of the entire actomyosin gel (lines 82-84).

A more detailed model would entail additional material parameters, not available experimentally, which may help reproduce specific experiments but that would make the systematic study of the different behaviors much more difficult. Our approach has been to keep the model minimal meeting the fundamental requirements outlined in the first paragraphs of Section “Theoretical model”.

Should the active stress depend on material density? It seems strange (from Eq. 3) that active stress could be non-zero even where density is zero, since sigma_act does not depend on rho.

Yes, active stress is assumed to be proportional to density. Eq. 3 in the original manuscript was misleading (it was multiplied by rho in Eq. 2). In the revised manuscript, we have explained with a bit more detail the theoretical model, clarifying this point.

The authors should clearly explain their rationale for retaining certain types of nonlinear terms while ignoring others in theory. For instance, the nonlinearities in the equations of motion are sometimes quadratic in the fields, while there are also some cubic terms. Please remark up to what order in the fields the various interactions are modeled.

We thank the referee for raising this point. The nonlinearities in the theory are easily explained on the basis of a small number of choices. We have added a new paragraph towards the end of Section “Theoretical model” (lines 145 to 152) providing a rationale for the origin and underlying assumptions leading to different nonlinearities.

To connect with experiments and the biological context, please explain the biological origin of various terms in the model: (1) L-dependent terms in Eq. 2 and 4, (2) Flowalignment of nematic order and experimental evidence in support of it, (3) densitydependent susceptibility terms in Eq. 4

(1) Unfortunately, the L-dependent terms are very bulky, but are very standard in nematic theories. The best way to understand their physical significance is through the expression of the nematic free-energy, which is now given and explained in the revised manuscript (Eq. 3). The resulting complicated expression for the molecular field and the nematic stress (Eqs. 4 and 5) are mathematical consequences of the choice of nematic free energy. In the revised manuscript, we also attempt to provide a basis for these terms in the context of the actin cytoskeleton. (2) To our knowledge, the best reference supporting this term from experiments is Reymann et al, eLife (2016). In the revised manuscript, we have provided a physical interpretation. (3) We have expanded the motivation and plausible microscopic justification of this term.

There are different 'activity' terms in the model. Their biophysical origin is not made clear. For example, the authors should make clear if these activities arise from filament or motor activity. Relatedly, the authors should provide a comprehensive discussion of the signs of the different active parameters and their physical interpretations.

In an active gel model, activity parameters are phenomenological and how they map to molecular mechanisms is not precisely known, although conventionally contractile active tension is ascribed to the mechanical transduction of chemical power by myosin motors. The fact is that, besides myosin activity, there are many nonequilibrium processes in the actomyosin cytoskeleton that may lead to active stresses including (de)polymerization of filaments or (un)binding of crosslinkers. In the revised manuscript, we have added sentences illustrating how different terms may result from microscopic mechanisms, but providing a precise mapping between our model and nonequilibrium dynamics of proteins is beyond the scope of our work, although our discrete network simulations address this issue to a certain degree.

Following the suggestion of the referee, our description of the theory now discusses much more extensively the signs of activity parameters and their physical interpretations, e.g. the text following Eq. 7.

Throughout the paper, various activity terms are varied independently of each other. Is that a reasonable assumption given that activities should depend on ATP and are thus not independent of one another?

We agree that, ultimately, all active process depend on the conversion of chemical energy into mechanical energy. However, recent work has highlighted how active tension also depends on the microscopic architecture of the network controlled by multiple regulators of the actomyosin cytoskeleton (e.g. Chug et al, Nat Cell Biol, 2017). It is reasonable to expect that, for a given rate of ATP consumption, chemical power will be converted into mechanical power in different ways depending on the micro-architecture of the cytoskeleton, e.g. the stoichiometry of filaments, crosslinkers, myosins, or the length distribution of filaments (very long filaments crosslinked by myosins may be difficult to reorient but may contract efficiently).

We have added a paragraph in Section “Theoretical model” with a discussion, lines 153 to 156.

Sarcomeres are muscle fibers that exhibit alternating polarity pattern. Such patterning is not evident in what the authors call 'sarcomeres' in Fig. 2. I believe the authors should revise their terminology and not loosely interpret existing classifications in the field.

We thank the referee for raising this point. We have changed the terminology.

Fig 2a: Is the cartoon for filament alignment incorrect for kappa>0?

The cartoon is correct. In the revised manuscript we have explained more clearly the physical meaning of kappa in the text following Eq. 7. In the caption of Fig. 1 and of Fig. 2a, we have also clarified that when the absolute value of kappa is <1, then active tension is positive in all directions.

Within the section "Requirements for fibrillar and banded patterns", it will be useful to show the figures for varying the different active parameters in the main figures.

We have followed the referee’s suggestion and moved Supp. Fig. 1 of the original manuscript to the main figures.

How do the authors decide if bundles are contractile or extensile? Why are contractile bundles under tension while extensile bundles are under compression? I would expect the opposite.

We agree that this point deserves a more detailed explanation. In the revised manuscript and in the new Figure 4, we further develop this point. The fibrillar pattern forms when kappa<0. We further assume that -1<kappa<0, so that active tension is positive in all directions. In this regime, the deviatoric (anisotropic) part of active tension is extensile. However, following pattern formation and because of the interplay between active and viscous stresses, the total stress in the emerging bundles may become extensile or contractile, depending on whether the largest component of stress is perpendicular or along the bundle axis. This is now presented in the updated figure, with new panels presenting maps of the total tension. The text discussing this point has been rewritten and we hope that the new version is much clearer (lines 280 to 303).

A contractile bundle tends to shorten, but it cannot do it because of boundary conditions or the interaction with other bundles. As a result they are in tension. Conversely, an extensile bundle tries to elongate, but being constrained, it becomes compressed. As an analogy, consider the cortex of a suspended cell. The cortex is contractile, but it cannot contract because of volume regulation in th cell, which is typically pressurized. As a result, tension in the cortex is positive, as shown by Laplace’s law [10.1016/j.tcb.2020.03.005]. We have tried to clarify this point in the revised manuscript.

Can the authors reproduce alternating density patterns using the cytosim simulations? This is an important step in establishing the correspondence between the continuum theory and the agent-based model.

We have addressed this point in our response to public comment (F) of this referee.

The authors do not provide code or data.

The finite element code with an input file require to run a representative simulation in the paper is now made available, see Ref. [74].

The customizations of Cytosim needed to account for nematic order in our discrete network simulations are available, see Ref. [98].

Reviewer #2 (Public Review):

Summary:

The article by Waleed et al discusses the self organization of actin cytoskeleton using the theory of active nematics. Linear stability analysis of the governing equations and computer simulations show that the system is unstable to density fluctuations and self organized structures can emerge. While the context is interesting, I am not sure whether the physics is new. Hence I have reservations about recommending this article.

We thank the referee for these comments. In the revised manuscript, we have highlighted the novelty, particularly in the last paragraph of the introduction, the first two paragraphs of Section “Theoretical model”, and in the conclusions. Despite a very large literature on theoretical models of stress fibers, actin rings, and active nematics, we argue that the active self-organization of dense nematic structures from an isotropic and low-density gel has not been compellingly explained so far. Many models assume from the outset the presence of actin bundles, or explain their formation using localized activity gradients. The literature of active nematics has extensively studied symmetry breaking and the self-organization. However, most of the works assume initial orientational order. Only a few works study the emergence of nematic order from a uniform isotropic state, but consider dry systems lacking hydrodynamic interactions or incompressible and density-independent systems [37,38]. Yet, pattern formation in actomyosin gels is characterized by large density variations, and by highly compressible flows, which coordinate in a mechanism relying on an advective instability and self-reinforcing flows.

Our theoretical model is not particularly novel, and as we mention in the manuscript, it can be particularized to different models used in the literature. However, we argue that it has the right minimal features to capture nematic self-organization in actomyosin gels. To our knowledge, no previous study explains the emergence of dense and nematic structures from a low-density isotropic gel as a result of activity and involving the advective instability typical of symmetry-breaking and patterning in the actomyosin cytoskeleton. These are important qualitative features of our results that resonate with a large experimental record, and as such, we believe that our work provides a new and compelling mechanism relying on self-organization to explain the prominence and diversity of patterns involving dense nematic bundles in the actomyosin cytoskeleton across species.

Strengths:

(i) Analytical calculations complemented with simulations (ii) Theory for cytoskeletal network

Weaknesses:

Not placed in the context or literature on active nematics.

We agree with the referee that this was a weakness of the original manuscript. In the revised manuscript, within reasonable space constraints given the size and dynamism of the field of active nematics, we have placed our work in the context of this field (end of introduction and first two paragraphs of Section “Theoretical model”). The published version of our companion manuscript [45] also contributes to providing a clear context to our theoretical model within the field.

Reviewer #2 (Recommendations For The Authors):

The article by Waleed et al discusses the self organization of actin cytoskeleton using the theory of active nematics. Linear stability analysis of the governing equations and computer simulations show that the system is unstable to density fluctuations and self organized structures can emerge. While the context is interesting, I am not sure whether the physics is new. Hence I have reservations about recommending this article. I explain my questions comments below.

We have responded to this comment above.

(i) Active nematics including density variations have been dealt quite extensively in the literature. For example, the works of Sriram Ramaswami have dealt with this system including linear stability analysis, simulations etc. In what way is the present work different from the system that they have considered?

(ii) Active flows leading to self organization has been a topic of discussion in many works. For example: (i) Annual Review of Fluid Mechanics, Vol. 43:637-659, 2010, https://doi.org/10.1146/annurev-fluid-121108-145434 (ii) S Santhosh, MR Nejad, A Doostmohammadi, JM Yeomans, SP Thampi, Journal of Statistical Physics 180, 699-709 (iii) M. G. Giordano1, F. Bonelli2, L. N. Carenza1,3, G. Gonnella1 and G. Negro1, Europhysics Letters, Volume 133, Number 5. In what way this work is different from any of these?

(iii) I am confused about the models used in the paper. There is significant literature from Prof. Mike Cates group, Prof. Julia Yeomans group, Prof. Marchetti's group who all use similar governing equations. In the present paper, I find it hard to understand whether the model used is similar to the existing ones in literature or are there significant differences. It should be clarified.

Response to (i), (ii) and (iii).

We completely agree with this referee (and also the previous referee), that the contextualization of our work in the field of active nematics was very insufficient. In the revised manuscript, the last paragraph of the introduction and the first two paragraphs of Section “Theoretical model” now address this point. In short, previous active nematic models predicting patterns with density variations have been either for dry active matter (disregarding hydrodynamic interactions), or for suspensions of active particles moving in an incompressible flow. None of these previous works predict nematic pattern formation as a result of activity relying on the advective instability and self-reinforcing compressible flows, leading to high density and high order bundles surrounded by an isotropic low density phase. Yet, these are fundamental features observed in actomyosin gels. Many works deal with symmetry-breaking of a system with pre-existing order, but very few address how order emerges actively from an isotropic state. We thank the referee for pointing at the paper by Santhosh et al, who nicely make this argument and is now cited. Our mechanism is fundamentally different from that in Santhosh, whose model is incompressible and ignores density variations.

We hope that the revised manuscript addresses this important concern.

(i) >(iv) Below Eqn 6, it starts by saying that the “...origin..is clear...” Its not. I don't understand the physical origin of the instability, and this should be clarified, may be with some illustrations.

We apologize for this unfortunate sentence, which we have rewritten in the revised manuscript (lines 181 to 185).

Reviewer #3 (Public Review):

The manuscript "Theory of active self-organization of dense nematic structures in the actin cytoskeleton" analysis self-organized pattern formation within a two-dimensional nematic liquid crystal theory and uses microscopic simulations to test the plausibility of some of the conclusions drawn from that analysis. After performing an analytic linear stability analysis that indicates the possibility of patterning instabilities, the authors perform fully non-linear numerical simulations and identify the emergence of stripelike patterning when anisotropic active stresses are present. Following a range of qualitative numerical observations on how parameter changes affect these patterns, the authors identify, besides isotropic and nematic stress, also active self-alignment as an important ingredient to form the observed patterns. Finally, microscopic simulations are used to test the plausibility of some of the conclusions drawn from continuum simulations.

The paper is well written, figures are mostly clear and the theoretical analysis presented in both, main text and supplement, is rigorous. Mechano-chemical coupling has emerged in recent years as a crucial element of cell cortex and tissue organization and it is plausible to think that both, isotropic and anisotropic active stresses, are present within such effectively compressible structures. Even though not yet stated this way by the authors, I would argue that combining these two is of the key ingredients that distinguishes this theoretical paper from similar ones. The diversity of patterning processes experimentally observed is nicely elaborated on in the introduction of the paper, though other closely related previous work could also have been included in these references (see below for examples).

We thank the referee for these comments and for the suggestion to emphasize the interplay of isotropic and anisotropic active tension, which is possible only in a compressible gel, as mentioned in the revised manuscript. We have emphasized this point in different places in the revised manuscript. We thank the suggestions of the referee to better connect with existing literature.

To introduce the continuum model, the authors exclusively cite their own, unpublished pre-print, even though the final equations take the same form as previously derived and used by other groups working in the field of active hydrodynamics (a certainly incomplete list: Marenduzzo et al (PRL, 2007), Salbreux et al (PRL, 2009, cited elsewhere in the paper), Jülicher et al (Rep Prog Phys, 2018), Giomi (PRX, 2015),...). To make better contact with the broad active liquid crystal community and to delineate the present work more compellingly from existing results, it would be helpful to include a more comprehensive discussion of the background of the existing theoretical understanding on active nematics. In fact, I found it often agrees nicely with the observations made in the present work, an opportunity to consolidate the results that is sometimes currently missed out on. For example, it is known that self-organised active isotropic fluids form in 2D hexagonal and pulsatory patterns (Kumar et al, PRL, 2014), as well as contractile patches (Mietke et al, PRL 2019), just as shown and discussed in Fig. 2. It is also known that extensile nematics, \kappa<0 here, draw in material laterally of the nematic axis and expel it along the nematic axis (the other way around for \kappa>0, see e.g. Doostmohammadi et al, Nat Comm, 2018 "Active Nematics" for a review that makes this point), consistent with all relative nematic director/flow orientations shown in Figs. 2 and 3 of the present work.

We thank the referee for these suggestions. Indeed, in the original submission we had outsourced much of the justification of the model and the relevant literature to a related pre-print, but this is not reasonable. The companion publication has now been accepted in the New Journal of Physics, with significant changes to better connect the work to the field of active nematics. A preprint reflecting those changes is available in Ref. [64], but we hope to reference the published paper that will come out soon.

In the revised manuscript, we have significantly rewritten the Section “Theoretical model” to frame the continuum model in the context of the field of active nematics. While our model and results have commonalities with previous work, there are also important differences. We have highlighted the novelty of the present work along with the relation with previous studies and theoretical models in the last paragraph of the introduction and the first two paragraphs of Section “Theoretical model”. Furthermore, as suggested by the referee, we have made an effort to connect our results with previous work by Kumar, Mietke, Doostmohammadi and others.

Regarding the last point alluded by the referee (“extensile nematics, \kappa<0 here, draw in material laterally of the nematic axis and expel it along the nematic axis”), the picture raised by the referee would be nuanced for our compressible system as compared to the incompressible systems discussed in that reference. As we have elaborated in our response to point (D) of Referee #1, our systems are overall contractile (with positive active tension in all directions), but the deviatoric component of the active tension can be either extensile or contractile. In our “extensile” models (left in Fig. 2c), material is drawn to laterally to the nematic axis but it is not expelled along this axis. Instead, it is “expelled” by turnover. In the revised manuscript, we have added a comment about this.

The results of numerical simulations are well-presented. Large parts of the discussion of numerical observations - specifically around Fig. 3 - are qualitative and it is not clear why the analysis is restricted to \kappa<0. Some of the observations resonate with recent discussions in the field, for example the observation of effectively extensile dynamics in a contractile system is interesting and reminiscent of ambiguities about extensile/contractile properties discussed in recent preprints (https://arxiv.org/abs/2309.04224). It is convincingly concluded that, besides nematic stress on top of isotropic one, active self-alignment is a key ingredient to produce the observed patterns.

We thank the referee for these comments. We are reluctant to extend the detailed analysis of emergent architectures and dynamics to the case \kappa > 0 as it leads to architectures not observed, to our knowledge, in actin networks. In the revised manuscript, we have expanded and clarified the characterization of emergent contractile/extensile networks by reporting the relative magnitude of stress along and perpendicular to the nematic direction. Our revised manuscript clearly shows that even though all of our simulations describe locally contractile systems with extensile anisotropic active tension, the emergent meso-structures can be either extensile or contractile, with the extensile ones exhibiting the usual bend-type instability (a secondary instability in our system) described classically for extensile active nematic systems. We have rewritten the text discussing this (lines 280 to 303), where we have placed these results in the context of recent work reporting the nontrivial relation between the contractility/extensibility of the local units vs the nematic pattern.

I compliment the authors for trying to gain further mechanistic insights into this conclusion with microscopic filament simulations that are diligently performed. It is rightfully stated that these simulations only provide plausibility tests and, within this scope, I would say the authors are successful. At the same time, it leaves open questions that could have been discussed more carefully. For example, I wonder what can be said about the regime \kappa>0 (which is dropped ad-hoc from Fig. 3 onward) microscopically, in which the continuum theory does also predict the formation of stripe patterns - besides the short comment at the very end? How does the spatial inhomogeneous organization the continuum theory predicts fit in the presented, microscopic picture and vice versa?

We thank the referee for this compliment. We think that the point raised by the referee is very interesting. It is reasonable to expect that the sign of \kappa may not be a constant but rather depend on S and \rho. Indeed, for a sparse network with low order, the progressive bundling by crosslinkers acting on nearby filaments is likely to produce a large active stress perpendicular to the nematic direction, whereas in a dense and highly ordered region, myosin motors are more likely to effectively contract along the nematic direction whereas there is little room for additional lateral contraction by additional bundling. As discussed in our response to referee #1, we believe that studying the formation of patterns using the discrete network simulations is far beyond the scope of our work. We discuss in lines 332 to 341, as well as in the last paragraph of the conclusions, the scope and limitations of our discrete network simulations.

Overall, the paper represents a valuable contribution to the field of active matter and, if strengthened further, might provide a fruitful basis to develop new hypothesis about the dynamic self-organisation of dense filamentous bundles in biological systems.

Reviewer #3 (Recommendations For The Authors):

• The statement "the porous actin cytoskeleton is not a nematic liquid-crystal because it can adopt extended isotropic/low-order phases" is difficult to understand and should be clarified, as the next paragraph starts formulating a nematic active liquid crystal theory. Do the authors mean a crystal that "Tends to be in a disordered phase?", according to its equilibrium properties? It would still be a "nematic liquid crystal", only its ground state is not a nematic phase.

We agree with the referee, and we hope that changes in the introduction and in Section “Theoretical model” address this comment.

• I could not find what Frank energy is precisely used, that would be helpful information.

In the revised manuscript, we have provided the expression for the nematic free energy in Eq. 3.

• The Significance of green/purple arrows in Fig 2a sketch unclear, green arrows also in b,c, do they represent the same quantity? From the simulations images it is overall it is very difficult to see how the flows are oriented near the high-density regions (i.e. if they are towards / away from the strip).

We thank the referee for bringing this up. The colorcodings of the sketches were confusing. The modified figures (Fig. 1(c) and Fig. 2(a)) present now a clearer and unified representation of anisotropic tension. The green arrows in Fig. 2(c) represent the out-of-equilibrium flows in the steady state. We agree that the zoom is insufficient to resolve the flow structure. For this reason, in the revised Fig. 2, we have added additional panels showing the flow with higher resolution.

• It is currently unclear how the linear stability results - beyond identification of the parameter \delta - inform any of the remaining manuscript. Quantitative comparisons of the various length scales seen in simulated patterns (e.g. Fig. 2b, 3c etc) with linear predictions and known characteristic length scales would be instructive mechanistically, would make the overall presentation more compelling and probes limitations of linear results.

In the revised manuscript, we have provided further information so that the readers can appreciate the predictions and limitations of the linear stability results. We have added a sentence and a Figure to show that, in addition to the critical activity, the linear theory provides a good prediction of the wavelengh of the pattern. See lines 199 to 201.

• It is not clear what is meant by "[bundle-formation] requires that active tension perpendicular to nematic orientation is larger than along this direction", and therefore also not why that would be "counter-intuitive". If interpreted naively, I would say that a large tension brings in more filaments into the bundle, so that may well be an obviously helpful feature for bundle formation and maintenance. In any case, it would be helpful if clarity is improved throughout when arguments about "directions of tensions" are made.

We have significantly rewritten the first paragraphs of section “Microscopic origin…” to clarify this point (lines 330 to 339). This paragraph, along with other changes in the manuscript such as the explanation of Eq. 7 or the discussion about the stress anisotropy in the new version of Fig. 4 (see lines 280 to 303), provide a better explanation of this important point.

• All density color bars: Shouldn't they rather be labelled \rho/\rho_0?

Yes! We have corrected this typo.

• Scalar product missing in caption definition of order parameter Fig. 2

We have corrected this typo.

• Fig. 3a: I suggest to put the expression for q0 in the caption

We have changed q_0 by S_0 and clarified its meaning in the caption of what now is Fig 4.

• Paragraph on bottom right of page 6 should several times probably refer to Fig. 3c(...), instead of Fig. 3b

We have corrected this typo.

I found this page 6 years ago because I did a search for the phrase "metaphysics of adjacency" as the meaning/intent-full hash/name for what I've been out-tuiting towards at the time. That one post was the only one web page out of petazillions that had this three word combination on it I would call it today metanexialitic intenional tacit awareness

From the ones adjacents its the ones that are portals to the longest trails well worth blazing and expolring

all driven from future orientated awareness of possibilities

or even impossibiities that desrve to be rendered inevitable and present

How ridiculous LLMs look from this perspective?

not to mention bogus notions like the manyverse?

all its apparent plausibikity is lost if we consider new trails spanninn to the future starting at every moment

https://via.hypothes.is/https://hyperpost.peergos.me/%F0%9F%8E%AD/gyuri/do/web/snarf/%F0%9F%93%85/2025/10/2/Metaphysics.of.Adjacency.html

Reviewer #3 (Public review):

Summary:

Using latest knock-in technology, the authors generated a set of five mouse lines with expression of recombinases in striatal projection neurons and dopaminergic neurons for public use. They rigorously characterize the expression of the recombinases by intersectional crossing with reporter lines to demonstrate that these lines are faithful, and they perform electrophysiological experiments in slices to provide evidence that the respective neurons show the expected features in these assays.

Strengths:

The characterization of the new mouse lines is exceptional, and these will be widely used by the community. The mouse lines are openly available for the community to use.

Weaknesses:

No weaknesses were identified by this Reviewer.

Author response:

We thank all three reviewers for their thoughtful and constructive evaluations of our manuscript, “Generation of knock-in Cre and FlpO mouse lines for precise targeting of striatal projection neurons and dopaminergic neurons.” We are encouraged that the reviewers recognize the value, specificity, and utility of these new lines for the basal ganglia and dopamine research communities. Below, we summarize our planned revisions and clarifications in response to the reviewers’ comments.

(1) Novelty and comparison with existing lines

We appreciate Reviewer 1’s point regarding the existence of previously generated Cre and Flp lines targeting similar neuronal populations. Our project was initiated six years ago, and during the course of generating and characterizing all five lines, we became aware that similar individual lines have since been developed by other groups. Nevertheless, our study provides a coordinated and independently validated set of lines created using a standardized knock-in (KI) strategy and distributed through Jackson Laboratories for unrestricted community use. Importantly, whereas previous BAC transgenic approaches rely on random insertion, which can lead to position effects and ectopic expression, our design places the recombinase coding sequence immediately downstream of the endogenous stop codon using a self-cleaving T2A peptide. This ensures expression under native promoter and regulatory control, preserving physiological gene regulation.

To address the Reviewers’ points, we will (i) expand the Introduction and Discussion to clarify the rationale and advantages of endogenous promoter–driven recombinase expression over BAC-based systems, emphasizing that our lines provide a uniform, promoter-controlled, and publicly accessible toolkit for the community, (ii) and explore including a comparative table summarizing differences in construct design, expression fidelity, and recombination efficiency across published lines (e.g., PMID 33979604, 38965445).

(2) Quantification, validation, and comparison of Cre vs FlpO

We agree with Reviewers 1 and 2 that further quantification and discussion of Cre versus FlpO fidelity will strengthen the manuscript. The observed difference in expression breadth between Cre and FlpO lines likely reflects a fundamental property of the recombinases themselves rather than a discrepancy in targeting. Cre recombinase is significantly more enzymatically efficient than FlpO, meaning that even very low endogenous levels of gene expression (e.g., Drd1a or Adora2a) can drive Cre-dependent recombination, whereas FlpO requires higher expression thresholds. Consequently, reporter-based readouts will inherently appear broader for Cre lines, despite both being driven by the same endogenous promoters.

To address these points, we will (i) provide quantitative co-labeling analyses for the DAT-FlpO line with TH immunostaining to assess efficiency and specificity, (ii) clarify in the Results and Discussion that differences between Cre and FlpO expression patterns largely stem from differences in recombinase kinetics and sensitivity, not mismatched promoter activity, (iii) and include representative high-resolution images and relevant statistics in the revised figures. Importantly, we would like to note that RNAscope may not be an ideal validation approach in this context, as in situ transcript detection cannot capture the enzymatic threshold differences that determine reporter recombination and thus will not help address observed differences between Cre and FlpO lines. Finally, we are actively performing electrophysiological comparisons between Cre and FlpO lines to rigorously quantify potential physiological differences between them. Updated analyses will be incorporated as available or described as ongoing future work.

(3) Discussion of scope and interpretation

We appreciate the reviewers’ suggestions to better contextualize the scope of this resource. We will revise the Discussion to (i) highlight that the Cre–FlpO pairings enable powerful intersectional and cross-line strategies for dissecting basal ganglia and midbrain circuitry, (ii) and clarify that our goal was to generate a rigorously validated foundational resource, with detailed functional comparisons and manipulation studies to be explored in subsequent work.

In summary, we thank the reviewers for their insightful feedback. The planned revisions and clarifications will underscore the unique strengths of our knock-in design, explore potential Cre–FlpO differences, and highlight the value of this standardized and accessible toolkit for the neuroscience community.

Reviewer #3 (Public review):

Summary:

In this paper, Kumar et al investigated the role of two decapping activators, Edc3 and Scd6, in regulating mRNA decay and translation in yeast. Using a variety of approaches including RNA-seq, ribosome profiling, proteomics, polysome analysis, and metabolomics the authors demonstrate that whereas single deletions of Edc3 or Scd6 have modest effects, the double mutant leads to increased abundance of mRNAs, many of which overlap with those targeted by the decapping activators Dhh1 and Pat1. The data suggest that Edc3 and Scd6 function redundantly to recruit Dhh1 to the Dcp2 decapping complex, thereby promoting mRNA turnover and translational repression. The authors show that these factors cooperate with Dhh1/Pat1 to repress transcripts involved in respiration, mitochondrial function, and alternative carbon source utilization, linking post-transcriptional regulation to nutrient responses. The study establishes Edc3 and Scd6 as important, but redundant regulators that fine-tune gene expression and metabolic adaptation in response to nutrient availability.

Strengths:

The paper has several strengths, including the comprehensive approach taken by the authors using multiple experimental techniques (RNA-seq, ribosome profiling, Western blotting, TMT-MS, polysome profiling, and metabolomics) to provide multiple lines of evidence to support their conclusions. The authors demonstrate clear redundancy of the factors by using single and double mutants for Edc3 and Scd6 and their global approach enables an understanding of these factors' roles across the yeast transcriptome. The work connects post-transcriptional processes to nutrient-dependent gene regulation, providing insights into how cells adapt to changes in their environment. The authors demonstrate the redundant roles of Edc3 and Scd6 in mRNA decapping and translation repression. Their RNA-seq and ribosome profiling results convincingly show that many mRNAs are derepressed only in the double mutants, confirming their hypothesis of redundancy. Furthermore, the functional cooperation between Edc3/Scd6 and Dhh1/Pat1 in regulating specific metabolic pathways, including mitochondrial function and carbon source utilization, is supported by the metabolomic data.

Weaknesses:

The study uses indirect evidence to support claims about the effect on mRNA stability rather than directly measuring mRNA stability. However, the combination of Pol II occupancy and RNA abundance measurements is consistent with the claims regarding mRNA stability. The addition of new experiments in the revision co-IPing Dhh1 and Dcp2 strengthens the argument that Edc3 and Scd6 recruit these factors.

Author response:

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

Reviewer #1 (Public review): 

Strengths: 

Overall, this manuscript is well-written and contains a large amount of high-quality data and analyses. At its core, it helps to shed light on the overlapping roles of Edc3 and Scd6 in sculpting the yeast transcriptome. 

Weaknesses: 

(1) While the data presented makes conclusions about mRNA stability based on corresponding ChIP-Seq analyses and analyzing other mutants (e.g. Dcp2 knockout), at no point is mRNA stability actually ever directly assessed. This direct assessment, even for select transcripts, would further strengthen their conclusions. 

We appreciate the reviewer’s concern but wish to emphasize that we conducted ChIP-Seq analysis of RNA Polymerase II occupancies in the CDSs of all genes, known to be a reliable indicator of transcription rate, and found only small increases in Pol II occupancies that cannot account for the increased transcript levels of the cohort of mRNAs up-regulated in the scd∆6edc3∆ double mutant (Fig. 3E). This provides strong evidence that increased transcription is not the main driver of increased mRNA abundance in this mutant.  Bolstering this conclusion, we showed that the Hap2/Hap3/Hap4/Hap5 complex of transcription factors responsible for induction of Ox. Phos. genes was not activated in scd6Δedc3Δ cells in glucose medium (Fig. 6F(ii)); nor was the Adr1 activator of CCR genes activated (Fig. S9C(i)), ruling out transcriptional induction of their target genes in glucose-replete scd6Δ/edc3Δ cells and instead favoring reduced degradation as the mechanism underlying derepression of Ox. Phos. and CCR gene transcripts in this mutant. In Fig. 3B, we further showed that the majority of mRNAs up-regulated in the scd6Δedc3Δ double mutant are also derepressed by dcp2Δ, and in Fig. 3D that the mRNAs up-regulated in scd∆6edc3∆ cells exhibit a higher than average codon protection index (CPI) indicating a heightened involvement of decapping and co-translational degradation by Xrn1 in their decay. To provide additional support for our conclusion, we have conducted new experiments to measure the abundance of capped mRNAs genome-wide by CAGE sequencing of total mRNA in both WT and scd∆6edc3∆ cells.  As established previously, normalizing CAGE TPMs to total mRNA TPMs determined by RNA-Seq, dubbed the C/T ratio, provides a reliable measure of the capped proportion of each transcript.  The new data presented in Fig. 3C indicate that the mRNAs up-regulated in the scd∆6edc3∆ mutant have significantly lower than average C/T ratios in WT cells, whereas the C/T ratios for the down-regulated transcripts are higher than average, and that these differences between the two groups and all expressed mRNAs are diminished in the scd∆6edc3∆ double mutant. These are the results expected if the up-regulated mRNAs are selectively targeted for decapping in WT cells dependent on Edc3/Scd6, whereas the downregulated mRNAs are targeted by Edc3/Scd6 less than the average transcript. In the original version of the paper, we came to the same conclusion by analyzing our previous CAGE data for the dhh1∆ mutant for the same transcripts dysregulated scd∆6edc3∆ cells, now presented as supportive data in Fig. S3F. Finally, we added the fact that among all four Dhh1 target mRNAs examined in the previous study of He et al. (2022) and found here to be up-regulated selectively in the scd6∆edc3∆ double mutant (Fig. S10), two of them (SDS23 and HXT6) were shown directly to have longer half-lives in dhh1∆ vs. WT cells by He et al. (2018). Hence, the combined evidence is compelling that selective up-regulation of particular mRNAs in the scd∆6edc3∆ mutant results from diminished decapping/decay rather than enhanced transcription; and we feel that the additional supporting evidence that would be provided by measuring half-lives of a small group of up-regulated transcripts would not justify the considerable effort required to do so.  Moreover, the standard approach for such experiments of impairing transcription with an inhibitor of Pol II or a Pol II Ts^- mutation has been criticized because of the known buffering (suppression) of mRNA decay rates in response to impaired transcription.

(2) Scd6 and Edc3 show a high level of functional redundancy, as demonstrated by the double mutant. As these proteins form complexes with other decapping factors/activators, I'm curious if depleting both proteins in the double mutant destabilizes any of these other factors. Have the authors ever assessed the levels of other key decapping factors in the double mutants (i.e. Dhh1, Pat1, Dcp2...etc)? I wonder if depleting both proteins leads to a general destabilization of key complexes. It would also be interesting to see if depleting Edc3 or Scd6 leads to a concomitant increase in the other protein as a compensatory mechanism. 

We thank the reviewer for this insight.  Examining our Ribo-Seq and TMT-MS data revealed that Dhh1 expression and steady-state abundance are increased ~2-fold in the scd6∆edc3∆ strain, indicating that the up-regulation of many of the same mRNAs by scd6∆edc3∆ and dhh1∆ does not result indirectly from reduced levels of Dhh1 in the scd6∆edc3∆ mutant. The predicted increased in Dhh1 expression might signify a compensatory response to the absence of Scd6/Edc3.  We also observed an ~40% reduction in Dcp2 translation (RPFs) and mRNA abundance in the scd6∆edc3∆ strain, which might contribute to the up-regulation of mRNAs dysregulated in this mutant. However, our new immunoblot analyses revealed no significant reduction in steady-state Dcp2 levels in scd6∆edc3∆ cells (Input lanes in Figs. 3F and S4C(i)-(ii)). Moreover, our previous finding that the majority of mRNAs subject to NMD, up-regulated by both upf1∆ and dcp2∆, are not upregulated by scd6∆edc3∆ implies that Dcp2 abundance in scd6∆edc3∆ cells is adequate for normal levels of NMD and favors a direct role for Scd6/Edc3 in accelerating degradation of most transcripts up-regulated in this mutant. We have added these points to the DISCUSSION.

(3) While not essential, it would be interesting if the authors carried out add-back experiments to determine which domain within Scd6/Edce3 plays a critical role in enforcing the regulation that they see. Their double mutant now puts them in a perfect position to carry out such experiments. 

We agree with the reviewer that our scd6∆edc3∆ strain provides an opportunity to dissect the Scd6 and Edc3 proteins to determine which domains and motifs of each protein are most critically required for their functions in activating mRNA decay. However, if conducted thoroughly, this would entail an extensive analysis requiring a combination of genetics, biochemistry and genomics.  Considering the large amount of data already presented in 43 and 34 panels of main and supplementary figures, respectively, we feel that these additional experiments would be conducted more appropriately as a stand-alone follow-up study.

Reviewer #2 (Public review): 

Weaknesses: 

The authors show very nicely in Figure S1A that growth phenotypes from scd6Δedc3∆ can be rescued by transformation of EDC3 (pLfz614-7) or SCD6 (pLfz615-5). The manuscript might benefit from using these rescue strategies in the analysis performed (e.g. RNA-seq, ribosome occupancies, and translational efficiencies). Also, these rescue assays could provide a good platform to further characterise the protein-protein interactions between Edc3, Scd6, and Dhh1. 

We responded to this point immediately above in responding to Rev. #1.

Reviewer #3 (Public review): 

Weaknesses: 

The limitations of the study include the use of indirect evidence to support claims that Edc3 and Scd6 recruit Dhh1 to the Dcp2 complex, which is inferred from correlations in mRNA abundance and ribosome profiling data rather than direct biochemical evidence. 

While the reviewer makes a valid point, it is important to note that the greater correlations between effects of scd6∆edc3∆ with those conferred by dhh1∆ vs. pat1∆ also extended to changes in metabolites (Fig. 7A-C). To provide more direct evidence that Edc3 and Scd6 recruit Dhh1 to the Dcp2 complex, we have now conducted co-immunoprecipitation experiments (presented in new Figs. 3F and S5) demonstrating that association of Dhh1 with Dcp2 is diminished in the scd6∆edc3∆ double mutant but not in either scd6∆ or edc3∆ single mutant, thus providing biochemical support for our proposal.

Also, there is limited exploration of other signals as the study is focused on glucose availability, and it is unclear whether the findings would apply broadly across different environmental stresses or metabolic pathways. Nonetheless, the study provides new insights into how mRNA decapping and degradation are tightly linked to metabolic regulation and nutrient responses in yeast. The RNA-seq and ribosome profiling datasets are valuable resources for the scientific community, providing quantitative information on the role of decapping activators in mRNA stability and translation control. 

While not disputing the facts of this comment, we think it is unjustified to label as a weakness that our study focused on glucose-grown cells considering the large amount of new data and insights made possible by our multi-omics approach, presented in >70 separate figure panels and nine supplementary datafiles, which the reviewer has characterized as being valuable to the scientific community.  Parallel studies in non-preferred carbon or nitrogen sources are underway and represent large-scale investigations in their own right, for which the current dataset in glucose-replete cells provides the critical reference condition.

Reviewer #1 (Recommendations for the authors): 

The authors made a note that a set of 37 mRNAs is repressed exclusively by Edc3 with little contribution by Scd6, a list that includes the RPS28B mRNA. Edc3 has been previously reported to promote the decay of this mRNA in a deadenylation-independent fashion by binding to an element in its 3'UTR (PMIDs 15225544, 24492965). Can the authors comment on whether Edc3 may be binding to similar elements in the 3'UTRs of these transcripts in their shortlist? This could be an interesting topic matter for discussion as well. 

While an interesting idea, this seems unlikely because the 3’UTR sequence in RPS28B mRNA was shown to bind Rps28 protein itself to confer heightened decapping and decay dependent on Edc3 in a negative autoregulatory loop that exerts tight control over Rps28 protein levels.  It would be surprising if Edc3mediated repression of the other 36 mRNAs would involve Rps28 as none of them encode cytoplasmic ribosomal proteins. Nevertheless, we searched for a conserved motif among the 3’UTRs of the 37 mRNAs using the MEME suite and found enrichment for motifs identified for RNA binding proteins Hrp1 and Nab2 and two novel motifs, but none of these motifs could be recognized within in the Rps28 autoregulatory loop.  We have chosen not to comment on these findings in the revised manuscript to avoid lengthening it unnecessarily with inconclusive observations.

Reviewer #2 (Recommendations for the authors): 

The authors show very nicely in Figure S1A that growth phenotypes from scd6Δedc3∆ can be rescued by the transformation of EDC3 (pLfz614-7) or SCD6 (pLfz615-5). The manuscript might benefit from using these rescue strategies on the analysis performed (e.g. RNA-seq, ribosome occupancies, and translational efficiencies); or expressing truncated mutants of EDC3 (pLfz614-7) or SCD6 (pLfz615-5), to show that they can act as dominant negative competitors, either on the binding to Dhh1 and Dcp2. 

We addressed this comment above in our response to this Reviewer.

Reviewer #3 (Recommendations for the authors): 

(1) Labels such as "mRNA_up_s6,e3" are not defined in figures or the text. I suggest clearer sample labeling throughout. 

The labels had been defined at first mention in the RESULTS but are now indicated there more explicitly, as well as in the legend to Fig. 1.

(2) In Figure 1D it is surprising that the mRNA profile has a peak in the 5' UTR. I would expect to see such a peak in ribosome footprinting data. Is it possible these are incorrectly labeled?

The figure is correctly labeled. Generally, one does not expect to see RPFs in the 5’UTR region unless there is an efficiently translated uORF, which appears not to be the case for MDH2.

In general, the information in this panel and C is inadequate. None of the numbers are clearly explained in the figure legend or in the figure. 

We had cited the legend to Fig. S3C for details of all such gene browser images but have now inserted this information into the Fig. 1D legend, at the first occurrence of such data in the regular figures. 

(3) Figures 1C and 1D are in the wrong order.

Corrected.

(4) Figure 2D is a very complicated Venn Diagram. I suggest using UpSet plots as an alternative to Venn diagrams to more clearly convey overlaps between sets.  

We provided additional explanatory text in the Fig. 2D legend to facilitate understanding.

(5) The use of the same color scheme to represent different sets in panels of the same figure is a source of confusion. E.g. the cyan in Figures 2A, 2D, and 2E indicates unrelated categories, but one would think they are related.

The use of the same cyan color in these three figure panels actually does designate results for the same set of 591 mRNAs up-regulated in the three mutants.  The application of the color schemes is now mentioned explicitly in Figs. 1, 2, and S3.

(6) Reporting of p-values = 0 in figures is not useful.

Corrected.

(7) The whole manuscript is extremely long which reduces the overall impact. For example, the introduction is six pages long. I suggest reducing redundant text and being more concise to enhance readability. 

We tried to streamline the text wherever possible, in particular shortening the Introduction by two pages.

(8) Many abbreviations are used throughout the text that are not introduced the first time they are used. 

Corrected throughout.

(9) The ERCC normalization is unclear. Were the spike-ins added before cell lysis to allow estimation of per-cell RNA counts or to the extracted RNA? If added to extracted RNA rather than cells it is not clear to me how the claim can be made regarding increased mRNA abundance in the mutants. 

We thank the reviewer for this comment. As we explained in the Methods, 2.4 µl of 1:100 diluted ERCC RNA Spike-In Control Mix 1 was added to 1.2 µg of each total RNA sample prior to cDNA library preparation.  Because the majority of total mRNA is comprised of rRNA, this normalization yields the abundance of each mRNA relative to rRNA. Owing to repression of rESR mRNAs encoding ribosomal proteins and biogenesis factors in the scd6∆edc3∆ strain (Fig. S3D), the ribosome content per cell is expected to be reduced in this mutant vs. WT. We showed previously that the isogenic dcp2∆ mutant that elicits an ESR response of similar magnitude, showed a 30% reduction in bulk ribosomal subunits per cell compared to same WT strain examined here {Vijjamarri, 2023 #7866}.  Assuming a similar reduction in ribosome abundance in the scd6∆edc3∆ mutant, the changes in mRNA per cell conferred by the scd6∆edc3∆ mutation are expected to be 0.7-fold of the ERCCnormalized values given in Fig. 3E, yielding fold-changes of 2.00 and 0.62 for the mRNA_up and mRNA_dn, groups, respectively, which still differ substantially from the corresponding changes in normalized Rpb1 occupancies of 1.2 and 0.93, respectively.  We have added this new analysis to the text of RESULTS.

(10) The use of the terms "up-regulated" and "derepressed" throughout is confusing. Both refer to observed increased abundance of mRNAs, but they imply different causes which are never clearly defined. 

We changed all occurrences of “derepressed” to “up-regulated”.

Reviewer #3 (Public review):

Summary:

By expressing protein in a strain that is unable to phosphorylate KdpFABC, the authors achieve structures of the active wildtype protein, capturing a new intermediate state, in which the terminal phosphoryl group of ATP has been transferred to a nearby Asp, and ADP remains covalently bound. The manuscript examines the coupling of potassium transport and ATP hydrolysis by a comprehensive set of mutants. The most interesting proposal revolves around the proposed binding site for K+ as it exits the channel near T75. Nearby mutations to charged residues cause interesting phenotypes, such as constitutive uncoupled ATPase activity, leading to a model in which lysine residues can occupy/compete with K+ for binding sites along the transport pathway.

Strengths:

The high resolution (2.1 Å) of the current structure is impressive, and allows many new densities in the potassium transport pathway to be resolved. The authors are judicious about assigning these as potassium ions or water molecules, and explain their structural interpretations clearly. In addition to the nice structural work, the mechanistic work is thorough. A series of thoughtful experiments involving ATP hydrolysis/transport coupling under various pH and potassium concentrations bolsters the structural interpretations and lends convincing support to the mechanistic proposal. The SSME experiments are generally rigorous.

Weaknesses:

The present SSME experiments do not support quantitative comparisons of different mutants, as in Figures 4D and 5E. Only qualitative inferences can be drawn among different mutant constructs.

Author response:

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

Reviewer #1 (Public review): 

Summary: 

This study on potassium ion transport by the protein complex KdpFABC from E. coli reveals a 2.1 Å cryo-EM structure of the nanodisc-embedded transporter under turnover conditions. The results confirm that K+ ions pass through a previously identified tunnel that connects the channel-like subunit with the P-type ATPase-type subunit. 

Strengths: 

The excellent resolution of the structure and the thorough analysis of mutants using ATPase and ion transport measurements help to strengthen new and previous interpretations. The evidence supporting the conclusions is solid, including biochemical assays and analysis of mutants. The work will be of interest to the membrane transporter and channel communities and to microbiologists interested in osmoregulation and potassium homeostasis. 

Weaknesses: 

There is insufficient credit and citation of previous work. 

The manuscript has been thoroughly revised with special attention to acknowledging all past work relevant to the study.

Reviewer #2 (Public review): 

Summary: 

The paper describes the high-resolution structure of KdpFABC, a bacterial pump regulating intracellular potassium concentrations. The pump consists of a subunit with an overall structure similar to that of a canonical potassium channel and a subunit with a structure similar to a canonical ATP-driven ion pump. The ions enter through the channel subunit and then traverse the subunit interface via a long channel that lies parallel to the membrane to enter the pump, followed by their release into the cytoplasm. 

Strengths: 

The work builds on the previous structural and mechanistic studies from the authors' and other labs. While the overall architecture and mechanism have already been established, a detailed understanding was lacking. The study provides a 2.1 Å resolution structure of the E1-P state of the transport cycle, which precedes the transition to the E2 state, assumed to be the ratelimiting step. It clearly shows a single K+ ion in the selectivity filter of the channel and in the canonical ion binding site in the pump, resolving how ions bind to these key regions of the transporter. It also resolves the details of water molecules filling the tunnel that connects the subunits, suggesting that K+ ions move through the tunnel transiently without occupying welldefined binding sites. The authors further propose how the ions are released into the cytoplasm in the E2 state. The authors support the structural findings through mutagenesis and measurements of ATPase activity and ion transport by surface-supported membrane (SSM) electrophysiology. 

Weaknesses: 

While the results are overall compelling, several aspects of the work raised questions. First, the authors determined the structure of the pump in nanodiscs under turnover conditions and observed several structural classes, including E1-P, which is detailed in the paper. Two other structural classes were identified, including one corresponding to E2. It is unclear why they are not described in the paper. Notably, the paper considers in some detail what might occur during the E1-P to E2 state transition, but does not describe the 3.1 Å resolution map for the E2 state that has already been obtained. Does the map support the proposed structural changes? 

As was seen in previous work by Silberberg et at. (2022), imaging KdpFABC under turnover conditions can produce multiple enzymatic states. We focus on the E1~P state and associated biophysical analyses to provide a clear and concise story that is focused on the conduction pathway for K⁺ ions. We continue to work with the cryo-EM data as well as other supporting methodologies and datasets with the goal of producing an additional manuscript that will describe other conformations. The class of particles producing the 3.1 Å structure shown in Fig. 1 – figure suppl. 2 is heterogeneous and thus requires further classification to elucidate conformational changes, as is apparent from the downstream processing of the E1 classes also shown in that figure. We cannot therefore derive any conclusions about the configuration of side chains at the CBS based on this structure. Nevertheless, two previous structures of the E2.Pi state - 7BGY and 7BH2 which were stabilized MgF₄ and BeF_x, respectively – show the structural change that is described in the paragraph discussing D583A. Given the consistency and relatively high resolution (2.9 and 3.0 Å, respectively) of these two independent structures, we believe that they provide strong support for our proposal for Lys586 acting as a built-in counter ion.

The paper relies on the quantitative activity comparisons between mutants measured using SSM electrophysiology. Such comparisons are notoriously tricky due to variability between SSM chips and reconstitution efficiencies. The authors should include raw traces for all experiments in the supplementary materials, explain how the replicates were performed, and describe the reproducibility of the results. Related to this point above, size exclusion chromatography profiles and reconstitution efficiencies for mutants should be shown to facilitate comparison between measured activities. For example, could it be that the inactive V496R mutant is misfolded and unstable? 

Similarly, are the reduced activities of V496W and V496H (and many other mutants) due to changes in the tunnel or poor biochemical properties of these variants? Without these data, the validity of the ion transport measurements is difficult to assess. 

To address this concern, we have generated a series of supplementary figures for Figs. 2, 4, 5, and 6, which show all of the raw traces underlying our SSME data (Figure 2 - figure supplements 2-4, Figure 4 - figure supplement 1,Figure 5 - figure supplement 3, Figure 6 - figure supplement 2). We have also included further detail about the experimental protocols, including number and type of replicates, in an expanded "Activity Assays" section of Methods.

In addition, we have included SEC profiles for each of the V496 mutants, which show that they are all well behaved in detergent solution prior to reconstitution (Fig. 4 - figure supplement 1). We are not able to directly document reconstitution efficiencies as it is not practical to separate proteoliposomes from unincorporated protein prior to preparing the sensors used for SSME. Binding currents are seen for several of the inactive mutants (e.g., Q116R in Rb and NH₄ in Fig. 2 - figure supplement 3 and V496R in Fig. 4 - figure supplement 1), which demonstrate that protein is indeed present in the corresponding proteoliposomes even though no sustained transport current is observed.

The authors propose that the tunnel connecting the subunits is filled with water and lacks potassium ions. This is an important mechanistic point that has been debated in the field. It would be interesting to calculate the volume of the tunnel and estimate the number of ions that might be expected in it, given their concentration in bulk. It may also be helpful to provide additional discussion on whether some of the observed densities correspond to bound ions with low occupancy.  

As suggested, we calculated the internal volume of the tunnel within KdpA (from the S4 K⁺ site to the KdpA/KdpB subunit interface) based on the profile derived from Caver. Based on this volume (4.9 x 10^-25 L), a single K⁺ ion within this cavity would correspond to 3.4 M, which is near saturation for a solution of KCl. We added this information together with an acknowledgment of low-occupancy K⁺ to the fourth paragraph of the Discussion:

" Fourth, based on the volume of the cavity in KdpA, a single K⁺ ion would correspond to a concentration of 3.4 M, suggesting that multiple ions would exceed the solubility limit especially in the absence of counterions. Finally, map densities within the tunnel were either of comparable strength or weaker than surrounding side chain atoms, unlike at S3 and canonical binding sites. Although it is possible that weaker density could represent low occupancy K⁺ ions, we favor a mechanism whereby individual K⁺ ions occupy the tunnel transiently as they transit between the selectivity filter and the canonical binding site."

In order to make this analysis, we developed a python script to calculate the volume of the tunnel as defined by the Caver software (this software is available via github.com/dls4n/tunnel). In turn, this enabled us to distinguish water molecules that were actually in the tunnel rather than bound more deeply within the structure of KdpA. As a result, we updated the water distribution plot in Fig. 4b. Notably, the 17 water molecules within this cavity would correspond to 57.8 M, which is reasonably near the expected 55 M for an aqueous solution.

Reviewer #3 (Public review): 

Summary: 

By expressing protein in a strain that is unable to phosphorylate KdpFABC, the authors achieve structures of the active wild-type protein, capturing a new intermediate state, in which the terminal phosphoryl group of ATP has been transferred to a nearby Asp, and ADP remains covalently bound. The manuscript examines the coupling of potassium transport and ATP hydrolysis by a comprehensive set of mutants. The most interesting proposal revolves around the proposed binding site for K+ as it exits the channel near T75. Nearby mutations to charged residues cause interesting phenotypes, such as constitutive uncoupled ATPase activity, leading to a model in which lysine residues can occupy/compete with K+ for binding sites along the transport pathway. 

Strengths:  

Although this structure is not so different from previous structures, its high resolution (2.1 Å) is impressive and allows the resolution of many new densities in the potassium transport pathway. The authors are judicious about assigning these as potassium ions or water molecules, and explain their structural interpretations clearly. In addition to the nice structural work, the mechanistic work is thorough. A series of thoughtful experiments involving ATP hydrolysis/transport coupling under various pH and potassium concentrations bolsters the structural interpretations and lends convincing support to the mechanistic proposal. 

Weaknesses: 

The structures are supported by solid membrane electrophysiology. These data exhibit some weaknesses, including a lack of information to assess the rigor and reproducibility (i.e., the number of replicates, the number of sensors used, controls to assess proteoliposome reconstitution efficiency, and the stability of proteoliposome absorption to the sensor). 

To address this concern, we have generated a series of supplementary figures for Figs. 2, 4, 5, and 6, which show all of the raw traces underlying our SSME data (Figure 2 - figure supplements 2-4, Figure 4 - figure supplement 1,Figure 5 - figure supplement 3, Figure 6 - figure supplement 2). We have also included further detail about the experimental protocols, including number and type of replicates, in the "Activity Assays" section of Methods.

Reviewing Editor Comments

After discussing the evaluations, the Reviewers and Reviewing Editor have identified the following essential revisions that would need to be addressed to improve the eLife assessment:

(1) Work from others in the field should be adequately described and acknowledged: 

(a) Page 2: " A series of X-ray and cryo-EM structures of KdpFABC from E. coli have led to proposals of a novel transport mechanism befitting the unprecedented partnership of these two superfamilies within a single protein complex." 

The authors must give credit where credit is due (namely, the Haenelt/Paulino groups having discovered the transport pathway). Why don't they cite Stock et al., where this pathway was described first? The Stokes group proposed an entirely different pathway initially. 

Explicit reference to this work has been added to as follows:

“A series of X-ray and cryo-EM structures of KdpFABC from E. coli (Huang et al., 2017; Silberberg et al., 2022, 2021; Stock et al., 2018; Sweet et al., 2021) indicate a novel transport mechanism befitting the unprecedented partnership of these two superfamilies within a single protein complex. As first proposed by Stock et al. (Stock et al., 2018), there is now a consensus that K⁺ enters the complex from the extracellular side of the membrane through the selectivity filter of KdpA, but is blocked from crossing the membrane.”

(b) Page 4 " As a result, many previous structures (Huang et al., 2017; Silberberg et al., 2021; Stock et al., 2018; Sweet et al., 2021) feature the S162A mutation to avoid inhibition rather than the fully WT protein used for the current work." 

This is not correct. At least the work by Huang et al 2017 and Stock et al 2021 was done without the mutation. This is why the structures also captured the off-cycle state when no E2 inhibitor was used. But in Silberberg et al 2022 the mutant was used, but this is not mentioned 

The Q116R mutant was used by Huang et al., but indeed not used for the Stock et al paper. We have replaced the sentence in the manuscript with the following:

“Use of the KdpD knockout strain allowed us to produce WT and mutant protein free from Ser162 phosphorylation.”

(c) Page 4: " In the paper, we report on the most highly populated state (44% of particles)". Exactly the same was also seen in detergent solution, which should be mentioned. 

Reference to the Silberberg 2022 paper, where E1~P was the most highly populated state, has been added. The percentage of particles was removed as we are still processing data from the other states, which will we hope will be described in a future manuscript.

(d) Page 7 "Asp583 and Lys586 are two conserved residues on M5 that have previously been shown......indicating that this particular mutation interfered with energy coupling."  The lack of discussion of the Haenelt/Paulino 2021 paper, where they have analyzed the coupling in detail and described a proximal binding site where K+ is coordinated by D583 and the neighbouring Phe is very concerning. 

To correct this oversight, we made the following changes to the text: 

On pg. 7 in the Results section, we refer to the 2005 paper from Bramkamp & Altendorf:

“Consistent with earlier work on this mutant (Bramkamp and Altendorf, 2005), the D583A mutant displayed substantial ATPase activity (30% of WT) but no transport, indicating that this particular mutation interfered with energy coupling.”

At the end of pg. 10 in the Discussion, we revised the paragraph discussing D583 and Lys586 to explicitly refer to the mechanism of transport described in the 2021 paper from Silberberg et al, including proximal and distal binding sites as well as uncoupling due to the D583A mutation.

“Similar to the Glu370/Arg493 charge pair in KdpA, Asp583 and Lys586 are the only charged residues in the membrane core of KdpB. Although they are not seen to interact directly in our structure, they coordinate accessory waters associated with the canonical binding site. Previous molecular dynamics simulations (Silberberg et al., 2021) indicate that Asp583 couples with Phe232 to form a “proximal binding site” for K⁺ ions. Based on these simulations, these authors proposed a mechanism whereby neutralization of this site either by ion binding or by D583A substitution served to stimulate ATPase activity. Indeed, earlier work on D583A (Bramkamp and Altendorf, 2005) as well as current data demonstrate uncoupling, in which K⁺ independent ATPase activity was observed even though transport was abolished. A plausible explanation for this stimulation is seen in the behavior of Lys586 in previous structures of the E2·Pi state (7BGY and 7BH2) (Sweet et al., 2021). In these structures, M5 undergoes a conformational change that pushes the side chain of Lys586 into the CBS. As a consequence of the D583A mutation, this Lys could be freed to act as a built-in counter ion as in related P-type ATPases ZntA (Wang et al., 2014) and AHA2 (Pedersen et al., 2007). In regard to the proximal binding site and the partnering “distal binding site” on the KdpA-side of the subunit interface, our structure does not show densities at either site and thus does not provide any support for the related mechanism. In any case, in the WT complex it seems likely that Asp583 exerts allosteric control over Lys586 and ensures that its movement into the binding site is coordinated with the transition from E1~P to E2·Pi, thus leading to displacement of K⁺ from the CBS and release to the cytoplasm. “

(e) Page 8 " The intersubunit tunnel is arguably one of the most intriguing elements of the KdpFABC complex. Although it has been postulated to conduct K+, experimental evidence has been lacking. " 

Incorrect, see Silberberg 2021. 

On this point, we beg to differ. Although this 2021 paper shows densities in experimental cryo-EM maps and effects of mutations to residues at the KdpA and KdpB interface, the intra-tunnel transport mechanism is based on computational analysis (MD simulations) and not experimental evidence. We softened the statement to read as follows:

“Although it has been postulated to conduct K⁺, direct experimental evidence has been hard to come by.”

(f) In this context, also f232 is not mentioned anywhere in the text, although depicted in almost all figures. 

Phe232 is shown as a point of reference for the KdpA/KdpB subunit interface. We added a reference to Phe232 in the Results section labeled “Intersubunit tunnel” as well as the paragraph in the Discussion addressed in point d) above.

" These densities, which we have modeled as water, are most prevalent near the vestibule, which is the wider part of the tunnel, but then disappear completely at the subunit interface near Phe232, which is the narrowest part of the tunnel and also distinctly hydrophobic (Fig. 4)."

" Previous molecular dynamics simulations (Silberberg et al., 2021) indicate that Asp583 couples with Phe232 to form a “proximal binding site” for K⁺ ions."

(g) Page 2 "Later, it was recognized that KdpA belongs to the Superfamily of K+ Transporters (SKT superfamily), which also includes bona fide K+ channels such as KcsA, TrkH and KtrB (Durell et al., 2000). " 

KcsA is not a member of the SKT superfamily. 

Thanks. This is correct, although the SKT superfamily is believed to have evolved from KcsA. KcsA has been removed from the sentence and a reference added to a review of the SKT superfamily:

“which also includes bona fide K⁺ channels such as TrkH and KtrB (Diskowski et al., 2015; Durell et al., 2000).”

(2) Two other structural classes were identified, including one corresponding to E2. It is unclear why they are not described in the paper. Notably, the paper considers in some detail what might occur during the E1-P to E2 state transition, but does not describe the 3.1 Å resolution map for the E2 state that has already been obtained. Does the map support the proposed structural changes? 

As was seen in previous work by Silberberg et at. (2022), imaging KdpFABC under turnover conditions can produce multiple enzymatic states. We focus on the E1~P state and associated biophysical analyses to provide a clear and concise story. We continue to work with the cryo-EM data as well as other supporting methodologies and datasets with the goal of producing an additional manuscript that will describe other conformations. The class of particles producing the 3.1 Å structure shown in Fig. 1 – figure suppl. 2 is heterogeneous and thus requires further classification to elucidate conformational changes, as is apparent from the downstream processing of the E1 classes also shown in that figure. We cannot therefore derive any conclusions about the configuration of side chains at the CBS based on this structure. Nevertheless, two previous structures of the E2.Pi state - 7BGY and 7BH2 which were stabilized MgF₄ and BeF_x, respectively – show the structural change that is described in the paragraph discussing D583A. Given the consistency and relatively high resolution (2.9 and 3.0 Å, respectively) of these two independent structures, we believe that they provide strong support for our proposal for Lys586 acting as a built-in counter ion.

(3) The paper relies on the quantitative activity comparisons between mutants measured using SSM electrophysiology. Such comparisons are notoriously tricky due to variability between SSM chips and reconstitution efficiencies. The authors should include raw traces for all experiments in the supplementary materials, explain how the replicates were performed, and describe the reproducibility of the results. 

To address this concern, we have generated supplementary figures for Figs. 2, 4, 5, and 6, which show all of the raw traces underlying our SSME data (Figure 2 - figure supplements 2-4, Figure 4 - figure supplement 1,Figure 5 - figure supplement 3, Figure 6 - figure supplement 2). We have also added a detailed description of replicates, sensor stability and the experimental protocols in the "Activity Assays" section of Methods. In addition, we have highlighted observations of pre-steady state binding currents that were seen for some mutants (e.g., Q116R assayed with Rb⁺, NH₄⁺ and Na⁺), in which an initial, transient current response was observed without an ensuing transport current. The depiction of this raw data has allowed us to explain our use of the current response at 1.25 s, after decay of this binding current, as a measure of transport rate. This approach is consistent with recommendations by the manufacturer, as documented in their 2023 publication (Bazzone et al. https://doi.org/10.3389/fphys.2023.1058583).

(4) Related to this point above, size exclusion chromatography profiles and reconstitution efficiencies for mutants should be shown to facilitate comparison between measured activities. For example, could it be that the inactive V496R mutant is misfolded and unstable? Similarly, are the reduced activities of V496W and V496H (and many other mutants) due to changes in the tunnel or poor biochemical properties of these variants? Without these data, the validity of the ion transport measurements is difficult to assess. 

We have included SEC profiles for each of the V496 mutants, which show that they are all well behaved in detergent solution prior to reconstitution (Fig. 4 - figure supplement 1). We are not able to directly document reconstitution efficiencies as it is not practical to separate proteoliposomes from unincorporated protein prior to preparing the sensors used for SSME. Binding currents are seen for several of the inactive mutants (e.g., Q116R in Rb and NH₄ in Fig. 2 - figure supplement 3 and V496R in Fig. 4 - figure supplement 1), which demonstrate that protein is indeed present in the corresponding proteoliposomes even though no sustained transport current is observed.

(5) What are the different lines in Figure 1 - Supplement 1, panel G? 

This panel depicted a series of SSME traces as an example of the raw data, but has been removed from the revised version given the inclusion of all the raw traces. These new figures include a legend explaining the conditions for each trace.

(6) How was the 44 % population of the single-occupancy E1 state estimated (it does not correspond to the number of particles in Figure 1 - Supplement 2. 

The calculation of 44% for the E1~P state was premature, given that we are still analyzing the data from the turnover conditions. The revised manuscript simply states that E1~P represented the largest population of particles, which is consistent with this state preceding the rate limiting step of the PostAlbers cycle. Reference is made to the Silberberg 2022 paper, which made a similar observation in a detergent-solubilized sample.

(7) The text states that Km for Q116E is "<10 uM". However, the fitted value is 90 µM in Figure 2e. 

This was a typographical error. The text now states that Km for Q116E is <100 M.

(8) The Km values for Rb, NH4, and Na in Figures 2g and h, and Na in Figure 2i do not make sense. They should be removed. 

The values for Km were determined by fitting the Michaelis-Menton equation to the data as detailed in the Methods section. Although the curves visually appear rather flat relative to other ions, the fitting generated respectable confidence limits and are therefore defensible in a statistical context. Furthermore, the curves that are shown are based on those values of Km and it would be inappropriate not to cite them.

(9) Figure 3 would benefit from a slice through the protein to orient the viewer. 

Thanks for the suggestion. We have added panels to Figs. 3, 5 and 6 in an effort to orient the reader to the site that is depicted.

(10) The differences between R493E, Q, and M do not appear to be significant. 

The y-axis is logarithmic which makes a visual comparison difficult. To alleviate this, P values were calculated based on one-way ANOVA analysis are results are indicated in Fig. 3c and 3d. They show that all of the Arg493 mutations have Km significantly higher than WT. Differences between R493E orR493Q and R493Q orR493M are not significant at the p<0.01 level, while the difference between R493E and R493M is highly significant (p<0.001).  The associated text on pg. 6 has been slightly modified as follows:

“Changes to Arg493 generally increase Km (lower apparent affinity) without affecting Vmax, with Met substitution having greater effect than charge reversal (R493E).”

(11) Page 5, paragraph 2. Q116R and G232D don't seem like the world's most intuitive mutations. It appears there is a historical reason for looking at these. Could the rationale be explained in the text? (Why R and D specifically?) 

These mutations have historical significance, having been generated by random mutagenesis during early characterization of the Kdp system by Epstein and colleagues. A sentence containing relevant references has been added to this paragraph to provide this context:

“Specifically, Q116R and G232D substitutions were initially discovered by random mutagenesis during early characterization of the Kdp system (Buurman et al., 1995; Epstein et al., 1978) and have featured in many follow-up studies (Dorus et al., 2001; Schrader et al., 2000; Silberberg et al., 2021; Sweet et al., 2020; van der Laan et al., 2002).”

Below are the recommendations from each of the reviewers, some of which were not included as essential revisions, but that can also be helpful to further strengthen the manuscript. 

Reviewer #1 (Recommendations for the authors): 

It is essential that the authors correct their selective, incomplete, and in places inappropriate references to work from others in the field. 

Specific points: 

(1) Page 2: " A series of X-ray and cryo-EM structures of KdpFABC from E. coli have led to proposals of a novel transport mechanism befitting the unprecedented partnership of these two superfamilies within a single protein complex." 

The authors must give credit where credit is due (namely, the Haenelt/Paulino groups having discovered the transport pathway). Why don't they cite Stock et al., where this pathway was described first? The Stokes group proposed an entirely different pathway initially. 

(2) Page 4 " As a result, many previous structures (Huang et al., 2017; Silberberg et al., 2021; Stock et al., 2018; Sweet et al., 2021) feature the S162A mutation to avoid inhibition rather than the fully WT protein used for the current work." 

This is not correct. At least the work by Huang et al 2017 and Stock et al 2021 was done without the mutation. This is why the structures also captured the off-cycle state when no E2 inhibitor was used. But in Silberberg et al 2022 the mutant was used, but this is not mentioned 

(3) Page 4: " In the paper, we report on the most highly populated state (44% of particles)". Exactly the same was also seen in detergent solution, which should be mentioned. 

(4) Page 7 "Asp583 and Lys586 are two conserved residues on M5 that have previously been shown......indicating that this particular mutation interfered with energy coupling."  The lack of discussion of the Haenelt/Paulino 2021 paper, where they have analyzed the coupling in detail and described a proximal binding site where K+ is coordinated by D583 and the neighbouring Phe is very concerning. 

(5) Page 8 " The intersubunit tunnel is arguably one of the most intriguing elements of the KdpFABC complex. Although it has been postulated to conduct K+, experimental evidence has been lacking. " 

Incorrect, see Silberberg 2021. 

(6) In this context, also f232 is not mentioned anywhere in the text, although depicted in almost all figures. 

References have been added to address all of these points. See item 1) under Reviewing Editor’s Comments above.

Other points: 

(7) Page 2 "Later, it was recognized that KdpA belongs to the Superfamily of K+ Transporters (SKT superfamily), which also includes bona fide K+ channels such as KcsA, TrkH and KtrB (Durell et al., 2000). " 

KcsA is not a member of the SKT superfamily. 

KcsA has been removed from the sentence and a reference added to a review of the SKT family:

“which also includes bona fide K⁺ channels such as TrkH and KtrB (Diskowski et al., 2015; Durell et al., 2000).”

(8) Page 9 " Our demonstration of coupled transport of NH4+ and Rb+ G232D not only confirms that the selectivity filter governs ion selection, but that the pump subunit, KdpB, is relatively promiscuous."  Check grammar. 

This sentence has been updated as follows:

“Our observation that G232D is capable of coupled transport for NH₄<sup>+</sup and Rb⁺ confirms not only that the selectivity filter governs ion selection, but that the pump subunit, KdpB, is relatively promiscuous.

Reviewer #2 (Recommendations for the authors): 

(1) From an editorial point of view, I suggest a few changes to enhance readability and clarity for non-specialists. A description of the overall transport cycle at the start of the paper (perhaps as a supplementary figure) could help put the work into perspective for general readers who may not be familiar with P-type ATPase mechanisms. It is unclear what "single" and "double" occupancy refer to in the structural classes description. Why is only one structural class described in detail? I would suggest moving the discussion of what is going on with the Nterminus of KdpB to the Results section, where it is described, and shortening the corresponding paragraph in the Discussion. I would furthermore suggest adding a figure that illustrates the proposed regulatory role of the terminus and how phosphorylation might affect it. Otherwise, this section of the results reads very hollow. 

A diagram showing the Post-Albers cycle is shown as part of Fig. 1 and is described at the end of the second paragraph. This sentence only mentioned KdpB, which may have caused confusion. We therefore changed the sentence to read as follows:

“Like other P-type ATPases, KdpFABC employs the Post-Albers reaction cycle (Fig. 1) involving two main conformations (E1 and E2) and their phosphorylated states (E1~P and E2-P) to drive transport (Albers, 1967; Post et al., 1969).”

Single and double occupancy was meant to refer to the number of KdpFABC complexes residing in a nanodisc. This can be seen in the class averages in Fig. 1 - figure supplement 2. The legends to Fig. 1 figure supplements 1 and 2 have been revised to explain this observation more explicitly:

"Slight asymmetry of the main peak is consistent with a subpopulation of nanodiscs containing two KdpFABC complexes (Fig. 1 - figure supplement 2)."

and

"A subset of these particles were further classified to generate four main classes representing nanodiscs with a single copy of KdpFABC in either E1 or E2 conformations, nanodiscs with two copies of KdpFABC which were mainly E1 conformation, and junk."

As stated above, the class of particles producing the 3.1 Å structure shown in Fig. 1 – figure suppl. 2 is heterogeneous and requires further classification to elucidate conformational changes, as is apparent from the downstream processing of the E1 classes also shown in that figure. We continue to analyze the cryo-EM data and aim to produce a second manuscript that will include descriptions of other conformations together with the additional biophysical analysis related to their function.

With regard to the N-terminus, we have gone on to generate a truncation of residues 2-9 in KdpB. After expression and purification, this construct remained coupled with ATPase and transport activities similar to WT, which makes proposals of a regulatory effect less compelling. Because of the novelty of observing the N-terminus and the possibility that it plays a subtle role in the kinetics of the cycle not revealed under the current assay conditions, we have retained a brief discussion of this structural observation, but moved it into the Results section as suggested.

"Given the regulatory roles played by N- and C-termini of a variety of other P-type ATPases (Bitter et al., 2022; Cali et al., 2017; Lev et al., 2023; Timcenko et al., 2019; Zhao et al., 2021), we generated a construct in which residues 2-9 of the N-terminus of KdpB were truncated. However, ATPase and transport activities remained coupled at levels similar to WT, indicating that any functional role of the N-terminus is relatively subtle and not manifested under current assay conditions."

(2) The wording "exceedingly strong densities" seems ambiguous. 

We have changed this to “strong” in the Abstract and "exceptionally strong" in the Discussion. The precise values for these densities are shown in density histograms in Fig. 2 – figure supplement 1 and Fig. 5 – figure supplement 2. In the text, the densities are described as follows:

Results sections describing the selectivity filter:

"In fact, this S3 site contains the strongest densities in the entire map, measuring 7.9x higher than the threshold used for Fig. 2a (Fig. 2 – figure suppl. 1a)."

Results section describing the CBS:

"Given that this is the strongest density in KdpB, measuring 5.6x higher than the map densities shown in Fig. 5 (Fig. 5 – figure suppl 2b), we have modeled it as K⁺."

(3) What are the different lines in Figure 1 - Supplement 1, panel G? 

This panel depicted a series of SSME traces as an example of the raw data, but has been removed from the revised version given the inclusion of all the raw traces. These new figures include a legend explaining the conditions for each trace.

(4) How was the 44 % population of the single-occupancy E1 state estimated (it does not correspond to the number of particles in Figure 1 - Supplement 2. 

The calculation of 44% for the E1~P state was premature, given that we are still analyzing the data from the turnover conditions. We will consider citing an updated value in a future publication once this analysis is complete. The revised manuscript simply states that E1~P represented the largest population of particles, which is consistent with this state preceding the rate limiting step of the Post-Albers cycle. Reference was made to the Silberberg 2022 paper, where a similar observation was made.

(5) Panel 1d is called out of order after panel 1e. Please label Ser 162 in the panel. 

The order of these panels have been switched and Ser162 has been labelled as suggested.

(6) Several panels in Figure 1- Supplement 1 are neither referenced nor described. 

This figure supplement is referred to multiple times in the Results and the Methods sections of the text as well as in the figure legends. Although each panel is not individually referenced, all of this information is relevant at different points in the manuscript and is explained in the legend.

(7) Is the coordinating geometry for the S3 site consistent with what was previously observed for KcsA and relatives? 

The general arrangement of carbonyl atoms in the S3 site is the same in KcsA and KdpA, described by the MacKinnon group as a square antiprism. However, KcsA has strict four-fold symmetry and KdpA does not. As a result, there are small discrepancies between the coordinating geometries in the two structures. This point was made graphically in our original report on the X-ray structure of KdpFABC (Huang et al. 2007, Extended Data Fig. 3), though the positions of the carbonyls are more accurately determined in the current structure due to increased resolution. We added a sentence to the Selectivity Filter section of the Results stating the following:

"This coordination geometry is also consistent with that seen in the K⁺ channel KcsA, though the strict four-fold symmetry of that homo-tetramer produces a more regular structure, as indicated by the smaller variance in liganding distance (2.77 Å with s.d. 0.075 Å in 1K4C) and as depicted by Huang et al. in Extended Data Fig. 3 (Huang et al., 2017)."

(8) Label G232D in Figure 2a. 

G232 is out of the plane shown in Fig. 2a. However, we have added a label for Cys344 to help identify the selectivity filter strands that are shown. Note, however, that G232 is visible and labeled in Fig. 2 - figure suppl. 1. This has now been noted in the legend for Fig. 2.

(9) The text states that Km for Q116E is "<10 uM". However, the fitted value is 90 uµ in Figure 2e. 

This was a typographical error. The text now states that Km for Q116E is <100 M.

(10) The Km values for Rb, NH4, and Na in Figures 2g and h, and Na in Figure 2i do not make sense. They should be removed. 

The values for Km were determined by fitting the Michaelis-Menton equation to the data as detailed in the Methods section. Although the curves visually appear rather flat relative to other ions, the fitting generated respectable confidence limits and are therefore defensible in a statistical context. Furthermore, the curves that are shown are based on those values of Km and it would be inappropriate not to cite them.

(11) Figure 3 would benefit from a slice through the protein to orient the viewer. 

Thank you for the suggestion. We have added panels to Figs. 3, 5 and 6 in an effort to orient the reader to the site that is depicted.

(12) The differences between R493E, Q, and M do not appear to be significant. 

The y-axis is logarithmic which makes a visual comparison difficult. To alleviate this, P values were calculated based on one-way ANOVA analysis are results are indicated in Fig. 3c and 3d. They show that all of the Arg493 mutations have Km significantly higher than WT. Differences between R493E orR493Q and R493Q orR493M are not significant at the p<0.01 level, while the difference between R493E and R493M is highly significant (p<0.001).  The associated text on pg. 6 has been slightly modified as follows:

“Changes to Arg493 generally increase Km (lower apparent affinity) without affecting Vmax, with Met substitution having greater effect than charge reversal (R493E).”

Reviewer #3 (Recommendations for the authors): 

Overall, the text was very clear, experiments were rationalized well, and conclusions were justified. A few small comments: 

(1) Page 5, paragraph 2. Q116R and G232D don't seem like the world's most intuitive mutations. It appears there is a historical reason for looking at these. Could the rationale be explained in the text? (Why R and D specifically?) 

These mutations are of historical importance, having been generated by random mutagenesis during early characterization of the Kdp system. A sentence containing relevant references has been added to this paragraph to provide this information as context:

“Specifically, Q116R and G232D substitutions were initially discovered by random mutagenesis during early characterization of the Kdp system (Buurman et al., 1995; Epstein et al., 1978) and have featured in many follow-up studies (Dorus et al., 2001; Schrader et al., 2000; Silberberg et al., 2021; Sweet et al., 2020; van der Laan et al., 2002).”

(2) Typo: page 14, "diluted" 

This typo has been corrected.

(3) The Methods section for SSM electrophysiology could use some additional description of how the data/statistics were collected. How many replicates? Were all replicates from a single sensor/ were multiple sensors examined? Were controls done to test whether the same number of liposomes remain absorbed by the sensor over the length of the experiment? 

We have extended our description of experimental protocols in the "Activity Assays" section of Methods. This includes the number and type of replicates as well as a discussion of binding currents that were seen for some mutants. Furthermore, a new series of supplementary figures for Figs. 2, 4, 5, and 6 show all of the raw traces for the SSME measurements (Figure 2 - figure supplements 2-4, Figure 4 - figure supplement 1, Figure 5 - figure supplement 3, Figure 6 - figure supplement 2).

We have included SEC profiles for each of the V496 mutants, which show that they are all well behaved in detergent solution prior to reconstitution (Fig. 4 - figure supplement 1). We are not able to directly document reconstitution efficiencies as it is not practical to separate proteoliposomes from unincorporated protein prior to preparing the sensors used for SSME. Binding currents are seen for several of the inactive mutants (e.g., Q116R in Rb and NH₄ in Fig. 2 - figure supplement 3 and V496R in Fig. 4 - figure supplement 1), which demonstrate that protein is indeed present in the corresponding proteoliposomes even though no sustained transport current is observed.

Oorschelp → Gehoorgang → Trommelvlies → Hamer → Aambeeld → Stijgbeugel → Ovaal venster → Basilair membraan → Haarcellen → Gehoorzenuw

Onze Grote Tantes Hebben Altijd Stoere Oude Blauwe Hoeden Gekocht.

Hoera Kleine Pinguins Leren Glijden Naar Finland

Author response:

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

Reviewer #2 (Public review):

(1)  The sharpening model of expectation can predict surround suppression. The authors could further clarify how the cancellation model predicts a monotonic profile of expectation (Figure 1C) with the highest response at the expected orientation, while the cancellation model suggests a suppression of neurons tuned toward the expected stimulus.

We thank the reviewer for the comment. We would like to emphasize that as the expected signal is suppressed, the relative weight or salience of unexpected inputs increases. We have clarified this interpretation in the manuscript as follows:

“Here, given these two mechanisms making opposite predictions about how expectation changes the neural responses of unexpected stimuli, thereby displaying different profiles of expectation, we speculated that if expectation operates by the sharpening model with suppressing unexpected information, we should observe an inhibitory zone surrounding the focus of expectation, and its profile then should display as a center-surround inhibition (Fig. 1c, left). If, however, expectation operates as suggested by the cancelation model with highlighting unexpected information, the inhibitory zone surrounding the focus of expectation should be eliminated, and the profile should instead display a monotonic gradient (Fig. 1c, right).”

(2) I'm a bit concerned about whether the profile solely arises from modulation of expectation. The two auditory cues are each associated with a fixed orientation, which may be confounded by other cognitive processes like visual working memory or attention (which I think the authors also discussed). Although the authors tried to use SFD task to render orientation task-irrelevant, luminance edges (i.e., orientation) and spatial frequency in gratings are highly intertwined and orientation of the gratings may help recall the first grating's SF (fixed at 0.9 c/{degree sign}), especially given the first and second grating's orientations are not very different (4.8{degree sign}).

We agree that dissociating expectation from attention and other top-down processes remains a key challenge in visual expectation research (see Summerfield & Egner, 2009; Summerfield & de Lange, 2014; de Lange et al., 2018). As is generally acknowledged, expectation reflects the probability of a sensory event, while selective attention relates to its behavioral relevance. To minimize attentional influences, our task design ensured that grating orientation was not taskrelevant: on each trial, participants discriminated either orientation or spatial frequency difference, such that orientation itself did not require attentional allocation, a point already discussed in the manuscript.

Regarding visual working memory, we argue that even if participants recalled the first grating’s spatial frequency in the SFD task, they were not required to retain its precise spatial frequency (or orientation), as their task was simply to judge whether the second grating appeared denser or sparser. In other words, orientation (or spatial frequency) itself was not task-relevant. Moreover, although not included in the manuscript, we conducted a post-experiment debriefing in which participants were asked whether they noticed any association between the auditory tone and the grating orientation. None of the participants reported this relationship correctly, suggesting that the tone-orientation mapping remained implicit and was unlikely to be driven by strategic attention or memory.

However, we acknowledge that certain confounding processes such as statistical learning or implicit mapping acquisition cannot be fully ruled out given the current paradigm. Future studies using methods with higher temporal resolution (e.g., EEG/MEG) may help to dissociate these mechanisms more precisely.

(3) For each of the expected orientations (20{degree sign} or 70{degree sign}), the unexpected ones are linearly separable (i.e., all unexpected ones lie on one side of the expected angle). This might further encourage people to shift their attended or expected orientation, according to the optimal tuning hypothesis. Would this provide an alternative explanation to the tuning shift that the authors found?

We thank the reviewer for pointing out the relevance of the optimal tuning hypothesis. We acknowledge that the optimal tuning theory (Navalpakkam & Itti, 2007) is an important framework, particularly in visual search paradigms, where attentional templates may shift away from non-target features to enhance discriminability.

In our task, this hypothesis would predict a shift of expectation toward <20° in E20° trials and >70° in E70° trials, given that all unexpected orientations lie on one side of the expected angle. Importantly, the optimal tuning hypothesis predicts such shifts not only in Δ20°, Δ25°, and Δ30° trials but also in the Δ0° trials. In this regard, the observed shift in Δ20° and Δ30° (Experiment 2) and Δ25° (Experiment 3) trials is broadly consistent with the predictions of the optimal tuning account. However, we did not observe a corresponding shift away from nontarget features in the Δ0° condition, suggesting limited behavioral evidence for optimal tuning effects under our current task settings.

It is important to note that most previous studies supporting optimal tuning (e.g., Navalpakkam & Itti, 2007; Scolari & Serences, 2009; Geng, DiQuattro, & Helm, 2017; Yu & Geng, 2019) have used visual search paradigms that differ from our design in several critical ways, including the number of stimuli presented, their spatial arrangement (eccentricity), task demands, and so on. Therefore, it is difficult to determine whether the optimal tuning hypothesis could serve as an alternative explanation within the context of our current study. We agree that future studies could further examine how such task parameters influence the presence or absence of optimal tuning.

(4) It is great that the authors conducted computational modeling to elucidate the potential neuronal mechanisms of expectation. But I think the sharpening hypothesis (e.g., reviewed in de Lange, Heilbron & Kok, 2018) focuses on the neural population level, i.e., narrowing of population tuning profile, while the authors conducted the sharpening at the neuronal tuning level. However, the sharpening of population does not necessarily rely on the sharpening of individual neuronal tuning. For example, neuronal gain modulation can also account for such population sharpening. I think similar logic applies to the orientation adjustment experiment. The behavioral level shift does not necessarily suggest a similar shift at the neuronal level. I would recommend that the authors comment on this.

We thank the reviewer for this to-the-point comment. As de Lange et al. (2018) noted, “there is not always a direct correspondence between neural-level and voxel-level selectivity patterns.” That is, neuronal tuning, population-level tuning, voxel-level selectivity, and behavioral adaptive outcomes may reflect different underlying mechanisms and do not necessarily align in a one-toone fashion. We fully acknowledge that population-level tuning effects may also result from various neuronal mechanisms such as gain modulation (for review, see Salinas & Thier, 2000), shifts in preferred orientation (Ringach, et al., 1997; Jeyabalaratnam et al., 2013), asymmetric broadening of tuning curves (Schumacher et al., 2022), or tuning curve sharpening (Ringach, et al., 1997; Schoups et al., 2001).  

In our modeling, we implemented sharpening and shifts of neuronal tuning curves as a conceptual model simplification, intended to explore potential mechanisms underlying expectation-related center-surround suppression effects. While sharpening-based accounts (e.g., Kok et al. 2012) have often been emphasized, we stress that other mechanisms, such as gain modulation or tuning shifts, may also contribute. Our goal is not to provide a definitive account, but to highlight such plausible mechanisms and encourage future investigation. We have revised the Discussion to emphasize that multiple mechanisms may underlie the observed effects.

“We note that our implementation of sharpening and shifts at the neuronal level serves as a conceptual model simplification, as population-level tuning, voxel-level selectivity, and behavioral adaptive outcomes may reflect different underlying neuronal mechanisms and do not necessarily align in a one-to-one fashion. Here, we stress that other potential mechanisms beyond sharpening, such as tuning shifts, may also contribute to visual expectation.” 

(5) If the orientation adjustment experiment suggests that both sharpening and shifting are present at the same time, have the authors tried combining both in their computational model?

We agree with the reviewer that it is necessary to consider the combined model. Accordingly, we implemented a computational model incorporating sharpening of the expected orientation channel together with shifting of the unexpected orientation channels. This model

successfully captured the sharpening of the expected-orientation channel and the shift of the unexpectedorientation channels (Supplementary Fig. 3). For the expected orientation (Δ0°) , results showed that the amplitude change was significantly higher than zero on both OD (t(23) = 2.582, p = 0.017, Cohen’s d = 0.527) and SFD (t(23) = 2.078, p = 0.049, Cohen’s d = 0.424) tasks (Supplementary Fig. 3e, vertical stripes); the width change was significantly lower than zero on both OD (t(23) = -2.438, p = 0.023, Cohen’s d = 0.498) and SFD (t(23) = -2.578, p = 0.017, Cohen’s d = 0.526) tasks (Supplementary Fig. 3e, diagonal stripes). For unexpected orientations (Δ10°-Δ40°), however, the amplitude and width changes were not significant with zero on either OD (amplitude change: t(23) = 0.443, p = 0.662, Cohen’s d = 0.091; width change: t(23) = -1.819, p = 0.082, Cohen’s d = 0.371) or SFD (amplitude change: t(23) = 1.130, p = 0.270, Cohen’s d = 0.231; width change: t(23) = -1.710, p = 0.101, Cohen’s d = 0.349) tasks (Supplementary Fig. 3f). In the meantime, the location shift was significantly different than zero for unexpected orientations (Δ10°-Δ40°, OD task: t(23) = 3.611, p = 0.001, Cohen’s d = 0.737; SFD task: t(23) = 2.418, p = 0.024, Cohen’s d = 0.493 (Supplementary Fig. 3g). These results provided further evidence that tuning sharpening and tuning shift jointly contribute to center– surround inhibition in expectation.  

Reviewer#1 (Recommendation for the Author):

(1) A direct comparison between tasks (baseline vs. expectation conditions) would have strengthened the findings. Specifically, contrasting performance in the orientation discrimination task with the spatial frequency discrimination task could have provided clearer evidence that participants actually used the auditory cues to attend to the expected orientation. This comparison would be particularly important for validating cue manipulation in the orientation discrimination task.

We agree that a direct comparison between the orientation discrimination (OD) and spatial frequency discrimination (SFD) tasks could further clarify how expectation (auditory cues) differentially modulates orientation relevance. However, the primary goal of the current study was to examine expectation effects within each task separately and to demonstrate that such effects are independent of attentional modulation driven by the task-relevance of orientation.

In addition, the OD and SFD tasks differ not only in the relevant task features (orientation vs. spatial frequency discrimination), but also in stimulus properties and difficulty, for example, the arbitrary use of 20–70° as the orientation range and ~0.9 cycles/° as the spatial frequency setting, a direct comparison could introduce confounding factors unrelated to expectation.

Importantly, Previous studies (e.g., Kok et al., 2012, 2017; Aitken et al., 2020) and our current results show that participants performed significantly better when the auditory cue matched the expected orientation, supporting the validity of our expectation manipulation.

(2) An interesting consideration is why the center-surround inhibition profile of expectation was independent of the task-relevance of orientation. Previous studies (e.g., Kok et al., 2012) have found that orientation discrimination patterns differ depending on whether orientation is taskrelevant or irrelevant. This could be useful to discuss the possible discrepancies.

We thank the reviewer for this inspiring comment. Kok et al. (2012) showed that both orientation and contrast tasks elicited similar fMRI decoding results, regardless of task relevance, suggesting neural mechanisms of expectation operate independently of whether orientation is task relevant. Behaviorally, they reported better performance for expected versus unexpected trials in the orientation task (3.4° vs. 3.8°, t(17) = 2.8, p = 0.013), and a marginal trend (although not significant) in the contrast task (4.3% vs. 5.0%, t(17) = 1.9, p = 0.075). If any differences between the two tasks exist, they may lie in the correlation between behavioral and fMRI effects, a question that goes beyond the scope of the current study. Therefore, it is hard to strongly conclude that orientation discrimination patterns differ depending on whether orientation is taskrelevant or irrelevant in their paper.

Our study differs from theirs in at least two important ways, which may account for the clearer expectation facilitatory effect we observed in the expectation (Δ0°) condition. First, in our study, the orientation-irrelevant task involved spatial frequency discrimination (SFD) rather than contrast discrimination. Compared to contrast, spatial frequency has been shown to exhibit a clear cueing effect, as reported in Fang & Liu (2019). Second, our design included a baseline condition, which was absent in their study. We computed discrimination sensitivity (DS) to quantify how much the discrimination threshold (DT) changed relative to baseline. By using this baseline-referenced approach, we observed a significant facilitatory expectation effect in the Δ0° condition, an effect that shifted from marginal significance in their orientation-irrelevant task to clear significance in our study.

(3) The authors might consider briefly explaining how the orientation adjustment paradigm used in this study is particularly effective for examining the potential co-existence of tuning sharpening and tuning shift computations, and how this approach complements traditional orientation discrimination tasks in characterizing expectation-related mechanisms.

We thank the reviewer for this valuable suggestion. We agree that further clarification is needed to better connect the two experiments. To explain this, we have elaborated further in the manuscript.

“To further explore the co-existence of both Tuning sharpening and Tuning shift computations in center-surround inhibition profile of expectation, participants were asked to perform a classic orientation adjustment experiment. Unlike profile experiment (discrimination tasks), the adjustment experiment provides a direct, trial-by-trial measure of participants’ perceived orientation, capturing the full distribution of responses. This enables the construction of orientation-specific tuning curves, allowing us to detect both tuning sharpening and tuning shifts, thereby offering a more nuanced understanding of the computational mechanisms underlying expectation.”

(4) These interesting findings raise important questions about their relationship to existing hybrid models of attentional modulation. Could the authors discuss how their results might align with or extend previous work demonstrating combined feature-similarity gain and surround suppression effects for orientation (e.g., Fang & Liu, 2019)? Could a hybrid model potentially provide a better account of these data than the pure surround suppression model?

We thank the reviewer for this valuable comment. We agree that hybrid model should be mentioned in the manuscript and we have elaborated further in the Discussion.

“For example, within the orientation space, the inhibitory zone was about 20°, 45°, and 54° for expectation evident here, feature-based attention[21], and visual perceptual learning[35], respectively; within the feature-based attention, it was about 30° and 45° in color [77] and motion direction [53] spaces, respectively These variations hint at the exciting possibility that the width of the inhibitory surround may flexibly adapt to stimulus context and task demands, ultimately facilitating our perception and behavior in a changing environment. This principle is consistent with the hybrid model of feature-based attention [53,54,75], where attention is deployed adaptively to prioritize task-relevant information through feature-similarity gain which filters out the most distinctive distractors, and surround suppression which inhibits similar and confusable ones, thereby jointly shaping the attentional tuning profile.”

(5) On page 19, there appears to be a missing symbol in the description of the Tuning Sharpening model. The text states: 'the tuning width of each channel's tuning function is parameterized by ??', where the question marks seem to indicate a missing parameter symbol.

We appreciate the reviewer’s careful attention. Yes, the "ơ" is missing, which was likely caused by a formatting issue. We have corrected it.

Reviewer #3 (Public review):

Summary:

This paper investigates the Matthew effect, where early success in funding peer review can translate into potentially unwarranted later success. It also investigated the previously found "setback" effect for those who narrowly miss out on funding.

Strengths:

The study used data from six funding agencies, which increases the generalisability, and was able to link bibliographic data for around 95% of applicants. The authors nicely illustrate how the previously found "setback" effect for near-miss applicants could be a collider bias due to those who chose to apply sometime later. This is a good explanation for the counter-intuitive effect and is nicely shown in Figure 5.

Weaknesses:

Most of the methods were clearly presented, but I have a few questions and comments, as outlined below.

In Figure 4(a) why are the "post" means much lower than the "pre"? This contradicts the expected research trajectory of researchers. Or is this simply due to less follow-up time? But doesn't the field citation ratio control for follow-up time?

The choice of the log-normal distribution for latent quality was not entirely clear to me. This would create some skew, rather than a symmetric distribution, which may be reasonable but log-normal distributions can have a very long tail which might not mimic reality, as I would not expect a small number of researchers to be extremely above the crowd. However, then the skew was potentially dampened by using percentile scores. Some further reasoning and plots of the priors would help.

Can the authors confirm the results of Figure S9 which show no visible effect of altering the standard deviation for the review parameter or the mean citations? Is this just because the prior for quality is dominated by the data? Could it be that the width of the distribution for quality does not matter, as it's the relative difference/ranking that counts? So the beta in equation 6 changes to adjust to the different quality scale?

The contrary result for the FWF is not explained (Table S3). Does this funder have different rules around re-applicants or many other competing funders?

The outlined qualitative research sounds worthwhile. Another potential mechanism (based on anecdote) is that some researchers react irrationally to rejection or acceptance, tending to think that the whole agency likes or hates their work based on one experience. Many researchers do not appreciate that it was a somewhat random selection of reviewers who viewed their work, and it will unlikely be the same reviewers next time.

"A key implication is the importance of encouraging promising, but initially unsuccessful applicants to reapply." Yes, A policy implication is to give people multiple chances to be lucky, perhaps by giving fewer grants to more people, which could be achieved by shortening the funding period (e.g., 4 year fellowships instead of 5 years). Although this will have some costs as applicants would need to spend more time on applications and suffer increased stress of shorter-term contracts. The bridge grants is potentially an ideal half-way house between many short-term and few long-term awards. Giving more grants to fewer people is supported by this analysis showing a diminishing returns in research outputs with more funding, DOI: 10.1371/journal.pone.0065263.

Making more room for re-applicants also made me wonder if there should be an upper cap on funding, potentially for people who have been incredibly successful. Of course, funders generally want to award successful researchers, but people who've won over some limit, for example $50 million, could likely be expected to win funding from other sources such as philanthropy and business. Graded caps could occur by career stage.

https://www.assemblee-nationale.fr/dyn/17/dossiers/effets_psychologiques_tiktok_mineurs

Note d'Information : Synthèse du Rapport d'Enquête sur TikTok et les Mineurs

Synthèse

Ce document de synthèse présente les conclusions et recommandations du rapport de la commission d'enquête parlementaire sur les effets psychologiques de l'application TikTok sur les mineurs.

Le rapport dresse un constat accablant : le modèle économique de TikTok, basé sur la captation de l'attention à des fins commerciales, expose les jeunes utilisateurs à des contenus dangereux et addictifs, avec des conséquences graves pour leur santé mentale et physique.

L'algorithme de la plateforme, par sa conception même, tend à amplifier les contenus négatifs et à enfermer les jeunes vulnérables dans des spirales de souffrance psychologique.

Face à ce diagnostic, la commission a formulé 43 recommandations articulées autour de plusieurs axes majeurs :

1. Une régulation renforcée des plateformes à l'échelle européenne, en s'appuyant sur le Digital Services Act (DSA), et au niveau national, par des obligations de modération humaine et de transparence algorithmique.

2. Des mesures de protection directes pour les mineurs, notamment la proposition phare d'interdire l'accès aux réseaux sociaux avant l'âge de 15 ans et de généraliser la "pause portable" dans les lycées.

3. Une politique de sensibilisation et d'éducation massive de l'ensemble de la société, visant les enfants, les parents, les professionnels de santé et le corps enseignant.

Le rapport met également en lumière des nuances d'approche entre ses co-auteurs, notamment sur la priorité à donner entre l'interdiction pour les moins de 15 ans (soutenue par la rapporteure Laure Miller) et une régulation prioritaire des plateformes pour créer des espaces sécurisés (privilégiée par le président Arthur de la Porte).

Enfin, une action judiciaire a été engagée par le président de la commission, qui a saisi le parquet de Paris pour "négligence coupable" de la part de TikTok.

I. Contexte et Genèse de la Commission d'Enquête

Le Constat Alarmant de l'Exposition des Mineurs

La commission d'enquête s'inscrit dans un contexte d'exposition précoce et intensive des mineurs aux écrans et aux réseaux sociaux, étayé par plusieurs études :

• Usage Précoce : Selon un rapport de l'ARCOM (septembre 2025), une majorité de jeunes de 10 à 14 ans utilise les réseaux sociaux avant l'âge minimal légal de 13 ans. L'âge moyen de la première utilisation est de 11 ans pour les plateformes vidéo et de 12 ans pour les réseaux sociaux.

• Exposition aux Contenus Choquants : Les trois quarts des 11-17 ans déclarent avoir déjà été exposés à des contenus qu'ils jugent choquants. TikTok joue un rôle particulièrement préoccupant, avec 38 % de ses utilisateurs gravement exposés à des contenus violents.

• Faiblesse de la Vérification de l'Âge : Seuls 10 % des utilisateurs de TikTok interrogés par l'ARCOM ont fait l'objet d'une demande de vérification d'âge ou ont vu leur compte bloqué.

• Temps d'Écran Élevé : Une étude de Santé publique France montre que les enfants de 9 à 11 ans passent en moyenne 2h33 par jour devant les écrans. Sur TikTok spécifiquement, le temps moyen pour les jeunes utilisateurs est de 1h43, pouvant atteindre 7 à 8 heures par jour selon les témoignages recueillis.

La Démarche de la Commission

Lancée en avril sur la base d'une proposition de résolution transpartisane, la commission a mené un travail intensif pour "ouvrir le capot de la machine TikTok".

• Périmètre : Le focus a été mis sur TikTok, considéré comme le réseau social le plus utilisé par les jeunes et dont le modèle (vidéos en plein écran défilant à l'infini) est imité par les autres plateformes.

• Méthodologie :

◦ 67 réunions tenues en 84 jours.    ◦ 163 personnes auditionnées pendant 95 heures, incluant des pédopsychiatres, des sociologues, des associations familiales, des familles de victimes (regroupées dans le collectif Algos), des influenceurs (positifs comme Hugo Travers et d'autres plus controversés), ainsi que des représentants des plateformes, dont une audition de 8 heures pour TikTok.    ◦ Une consultation publique sur le site de l'Assemblée nationale ayant recueilli près de 30 000 participations, dont 19 000 lycéens.

II. Diagnostic : Les Effets Psychologiques et les Mécanismes de TikTok

Le constat de la commission est qualifié de "sans appel" et "accablant". Il met en cause directement le modèle économique et algorithmique de la plateforme.

Un Algorithme Conçu pour l'Addiction et la Rétention

L'algorithme et le design de TikTok sont décrits comme une "machine à sous de vidéo" ou des "sucreries mentales" conçues pour maximiser le temps passé sur l'application.

• Rétention d'Attention : L'algorithme est extrêmement efficace pour retenir l'attention des utilisateurs, ce qui a pour premier effet une privation du temps de sommeil, impactant la santé mentale, l'attention et la capacité de suivi scolaire.

• Logique Commerciale : L'objectif est un "business de la data et du temps de cerveau disponible", où les mineurs constituent une cible stratégique majeure.

• Renforcement de la Souffrance : L'algorithme détecte rapidement les vulnérabilités. Un jeune en situation de mal-être se verra proposer de plus en plus de contenus violents, choquants ou liés à sa souffrance (suicide, anorexie), l'enfermant dans un cercle vicieux.

• Responsabilité Quasi-Éditoriale : Bien que les plateformes n'aient pas de responsabilité éditoriale au sens juridique, la commission estime que par la sélection et la mise en avant des contenus ("curialisation"), elles ont une responsabilité de fait, qui les soumet à des obligations de protection des mineurs au titre du DSA.

La Nature des Contenus Préjudiciables

La commission a identifié une large gamme de contenus néfastes, bénéficiant d'une large audience car les émotions négatives retiennent davantage l'attention.

• Promotion du suicide, de l'automutilation et des troubles du comportement alimentaire.

• Conseils de santé dangereux.

• Exposition à la violence sous toutes ses formes, au racisme, à l'antisémitisme et à la pédocriminalité.

• Banalisation de contenus radicaux et extrémistes, menant à une fragmentation identitaire et à la création de "bulles" qui altèrent le rapport au monde et menacent la cohésion nationale.

Impacts sur la Santé Physique et Mentale des Mineurs

Les auditions de professionnels de santé ont confirmé que si un lien de causalité direct est difficile à prouver, l'utilisation des réseaux sociaux amplifie avec certitude les vulnérabilités et le mal-être préexistants.

Catégorie d'Impact

Description des Effets Observés

Santé Physique

Privation de sommeil.

Développement Cognitif

Réduction de la capacité de concentration et de la tolérance à l'ennui.

Construction Sociale

Pression sur l'image corporelle, notamment pour les jeunes filles, avec la promotion d'un idéal de corps féminin irréaliste.

Rapport à la Violence

Banalisation de la violence, cyberharcèlement.

Santé Mentale

Amplification des troubles anxieux, de la dépression et des idéations suicidaires chez les jeunes vulnérables.

III. Les 43 Recommandations : Axes d'Action et Points de Débat

Axe 1 : La Régulation des Plateformes (Niveau Européen et National)

La commission préconise une double approche : poursuivre le combat à l'échelle de l'UE, tout en agissant rapidement au niveau national.

• Le Levier Européen : Huit recommandations visent à renforcer le rôle de l'Europe. Le Digital Services Act (DSA) est identifié comme l'outil principal, mais son application est lente. Les travaux de la commission ont déjà contribué à consolider la position française à Bruxelles.

• Régulation Algorithmique : Une proposition vise à imposer que le flux de contenus par défaut soit celui des abonnements de l'utilisateur ("suivi") et non le flux "Pour Toi", jugé plus dangereux car entièrement piloté par l'algorithme.

• Renforcement de la Modération : Le rapport souligne la réduction des moyens de modération humaine par TikTok et insiste sur la nécessité de les renforcer.

• Soutien aux Signaleurs de Confiance : Il est proposé de créer un fonds de dotation, financé par les plateformes sur le principe du "pollueur-payeur", pour garantir l'indépendance et les moyens des associations qui signalent les contenus illicites (ex: e-Enfance, Stop Fisha).

Axe 2 : La Protection des Mineurs et la Sensibilisation de la Société

• L'Interdiction d'Accès avant 15 ans : C'est une des propositions les plus débattues. Elle vise à protéger les plus jeunes durant une période cruciale de leur développement, en attendant une régulation efficace des plateformes.

• La "Pause Portable" dans les Établissements Scolaires : Le rapport recommande d'étendre aux lycées l'interdiction du téléphone portable déjà en vigueur dans les collèges, soulignant les bénéfices observés sur la sérénité des élèves et la socialisation.

• Sensibilisation Massive : Le rapport insiste sur le fait qu'une interdiction seule est insuffisante. Il appelle à :

◦ Des campagnes d'information "choc" grand public.    ◦ Le renforcement des messages de prévention dans les carnets de santé.    ◦ La formation des professionnels de santé et du personnel de l'Éducation nationale, souvent démunis face à ces enjeux.    ◦ L'accompagnement des parents, en luttant contre l'injonction contradictoire entre le "tout numérique" scolaire et les appels à limiter les écrans.

Axe 3 : Les Points de Divergence et de Nuance

Le rapport reflète un consensus sur le diagnostic, mais des approches différentes sur certaines solutions.

• Débat sur l'Interdiction vs. la Régulation :

◦ Mme Laure Miller défend l'interdiction pour les moins de 15 ans comme une mesure de protection immédiate, arguant que les droits fondamentaux des enfants (santé, sécurité) sont actuellement bafoués.    ◦ M. Arthur de la Porte privilégie une approche axée sur la régulation des plateformes pour rendre les espaces numériques sécurisés, afin de permettre un accès contrôlé aux 13-15 ans dans l'esprit de la loi Marcangeli.

• La Question de la Responsabilité Parentale :

◦ Mme Miller a proposé de créer un "délit de négligence numérique" pour les cas d'exposition excessive et manifeste, après une période de sensibilisation de trois ans, afin d'inscrire la protection numérique dans les devoirs parentaux.    ◦ M. de la Porte exprime une nuance, considérant que l'enjeu principal est de combattre les plateformes et non de pénaliser les parents.

IV. Enjeux Transversaux et Suites Envisagées

La Vérification de l'Âge : Défis Techniques et Protection des Données

La mise en œuvre de toute limite d'âge repose sur une vérification efficace. Des solutions respectueuses de la vie privée sont en développement, basées sur le principe du "double anonymat" où un tiers de confiance valide la majorité sans transmettre de données personnelles à la plateforme. La France participera à une expérimentation européenne d'un tel outil à partir du printemps 2025.

Le Rôle des Autres Acteurs

La discussion a souligné la nécessité d'impliquer l'ensemble de la chaîne de responsabilité, incluant non seulement les plateformes, mais aussi les opérateurs télécoms et les constructeurs de terminaux (smartphones).

L'Action Judiciaire et la Pression sur les Plateformes

En conclusion de ses travaux, le président Arthur de la Porte a saisi le parquet de Paris sur la "négligence coupable" de TikTok.

Cette démarche vise à engager la responsabilité pénale de la plateforme, considérée comme un levier essentiel pour contraindre les géants du numérique à modifier leurs pratiques, en complément des sanctions administratives prévues par le DSA.

Reviewer #2 (Public review):

Summary:

The authors have developed a behavioral paradigm to experimentally manipulate the sense of control experienced by participants by varying the level of difficulty in a wheel-stopping task. In the first study, this manipulation is tested by administering the task in a factorial design with two levels of controllability and two levels of stressor intensity to a large number of participants online, while simultaneously recording subjective ratings of perceived control, anxiety, and stress. In a second study, the authors employed the wheel stopping task to induce a high sense of controllability and investigate whether this manipulation buffers the response to a subsequent stress induction when compared to a neutral task, such as watching pleasant videos.

Strengths:

(1) The authors validate a method to manipulate stress.

(2) The authors use an experimental manipulation to induce an enhanced sense of controllability to test its impact on the response to stress induction.\

(3) The studies involved big sample sizes.

Weaknesses:

(1) The study was not preregistered.

(2) The control manipulation is conflated with task difficulty and, therefore, the reward rate. In the revised version of the manuscript, the authors perform statistical analysis to demonstrate that the relationship between perceived level of control and subjective stress remains robust after the inclusion of win rate in the model. This analysis strengthens the authors's claims, but the evidence would more substantial if the design did not conflate reward rate and control. The authors properly discuss this issue in the revised manuscript.

This study will be of interest to psychologists and cognitive scientists who are interested in understanding how controllability and its subjective perception influence how people respond to stress exposure. The demonstration that an increased sense of control buffers/protects against subsequent stress is important and may trigger further studies to characterize this phenomenon better. However, beyond the highlighted weaknesses, the current study only studied the effect of stress induction consequent to the performance of the WS task on the same day, and its generalizability is not warranted.

Reviewer #3 (Public review):

Summary:

This is an interesting investigation on the benefits of perceiving control and its impact on the subjective experience of stress. To assess the subjective sense of control, the authors introduce a novel wheel stopping (WS) task where control is manipulated via size and speed to induce conditions of low and high control. The authors demonstrate that the subjective sense of control is associated with experienced subjective stress and individual differences related to mental health measures. In a second experiment, they further demonstrate that an increased sense of control buffers subjective stress induced by a trier social stress manipulation, more so than a typical stress-buffering mechanism of watching neutral/calming videos.

Strengths:

Several strengths of the manuscript can be highlighted. For instance, the paper introduces a new paradigm and a clever manipulation to test a significant and important question. Additionally, it is a well-powered investigation that allows for confidence in replicability and demonstrate both high internal consistency and high external validity, along with an interesting set of individual difference analyses. Finally, the results are quite interesting and support prior literature, while also making a significant contribution to the field in understanding the benefits of perceiving control.

Weaknesses:

The authors have addressed all my queries, and I believe the revised paper has been improved and will make an important contribution to the literature.

Author response:

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

Reviewer #1 (Public review):

Summary:

This work investigated how the sense of control influences perceptions of stress. In a novel "Wheel Stopping" task, the authors used task variations in difficulty and controllability to measure and manipulate perceived control in two large cohorts of online participants. The authors first show that their behavioral task has good internal consistency and external validity, showing that perceived control during the task was linked to relevant measures of anxiety, depression, and locus of control. Most importantly, manipulating controllability in the task led to reduced subjective stress, showing a direct impact of control on stress perception. However, this work has minor limitations due to the design of the stressor manipulations/measurements and the necessary logistics associated with online versus in-person stress studies.

Nevertheless, this research adds to our understanding of when and how control can influence the effects of stress and is particularly relevant to mental health interventions.

We thank the reviewer for their clear and accurate summary of the findings. 

Strengths:

The primary strength of this research is the development of a unique and clever task design that can reliably and validly elicit variations in beliefs about control. Impressively, higher subjective control in the task was associated with decreased psychopathology measures such an anxiety and depression in a non-clinical sample of participants. In addition, the authors found that lower control and higher difficulty in the task led to higher perceived stress, suggesting that the task can reliably manipulate perceptions of stress. Prior tasks have not included both controllability and difficulty in this manner and have not directly tested the direct influence of these factors on incidental stress, making this work both novel and important for the field.

We thank the reviewer for their positive comments.

Weaknesses:

One minor weakness of this research is the validity of the online stress measurements and manipulations. In this study, the authors measure subjective stress via self-report both during the task and also after either a Trier Social Stress Test (high-stress condition) or a memory test (low-stress condition). One concern is that these stress manipulations were really "threats" of stress, where participants never had to complete the stress tasks (i.e., recording a speech for judgment). While this is not unusual for an in-lab study and can reliably elicit substantial stress/anxiety, in an online study, there is a possibility for communication between participants (via online forums dedicated to such communication), which could weaken the stress effects. That said, the authors did find sensible increases and decreases of perceived stress between relevant time points, but future work could improve upon this design by including more complete stress manipulations and measuring implicit physiological signs of stress.

We thank the reviewer for urging us to expand on this point. The reviewer is right that stress was merely anticipatory and is in that sense different to the canonical TSST. However, there are ample demonstrations that such anticipatory stress inductions are effective at reliably eliciting physiological and psychological stress responses (e.g. Nasso et al., 2019; Schlatter et al., 2021; Steinbeis et al., 2015). Further, there is evidence that online versions of the TSST are also effective (DuPont et al., 2022; Meier et al., 2022), including evidence that the speech preparation phase conducted online was related to increases in heart rate and blood pressure (DuPont et al., 2022). Importantly, and as the reviewer notes in relation to our study specifically, the anticipatory TSST had a significant impact on subjective stress in the expected direction demonstrating that it was effective at eliciting subjective stress. We have elaborated further on this in our manuscript (pages 8 and 9) as follows: 

“Prior research has found TSST anticipation to elicit both psychological and physiological stress responses [37-39], suggesting that the task anticipation would be a valid stress induction despite participants not performing the speech task. Moreover, prior research has validated the use of remote TSST in online settings [40, 41], including evidence that the speech preparation phase (online) was related to increased heart rate and blood pressure compared to controls [40].”

Reviewer #2 (Public review):

Summary:

The authors have developed a behavioral paradigm to experimentally manipulate the sense of control experienced by the participants by changing the level of difficulty of a wheel-stopping task. In the first study, this manipulation is tested by administering the task in a factorial design with two levels of controllability and two levels of stressor intensity to a large number of participants online while simultaneously recording subjective ratings on perceived control, anxiety, and stress. In the second study, the authors used the wheel-stopping task to induce a high sense of controllability and test whether this manipulation buffers the response to a subsequent stress induction when compared to a neutral task, like looking at pleasant videos.

We thank the reviewer for their accurate summary.

Strengths:

(1) The authors validate a method to manipulate stress.

(2) The authors use an experimental manipulation to induce an enhanced sense of controllability to test its impact on the response to stress induction.

(3) The studies involved big sample sizes.

We thank the reviewer for noting these positive aspects of our study. 

Weaknesses:

(1) The study was not preregistered.

This is correct.

(2) The control manipulation is conflated with task difficulty, and, therefore the reward rate. Although the authors acknowledge this limitation at the end of the discussion, it is a very important limitation, and its implications are not properly discussed. The discussion states that this is a common limitation with previous studies of control but omits that many studies have controlled for it using yoking.

We agree that these are very important issues to consider in the interpretation of our findings. It is important to note, that while our task design does not separate these constructs, we are able to do so in our statistical analyses. For example, our measure of perceived difficulty was included in analyses assessing the fluctuations in stress and control in which subjective control still had a unique effect on the experience of stress over and above perceived difficulty, suggesting that subjective control explains variance in stress beyond what is accounted for by perceived difficulty. Similarly, we have also included additional analyses in which we include the win rate (i.e. percentage of trials won) as a covariate when assessing the relationship between subjective control, perceived difficulty and subjective stress, in which subjective control and perceived difficulty still uniquely predict subjective stress when controlling for win rate. This suggests that there is unique variance in subjective control, separate from perceived task difficulty and win rate that is relevant to stress. We have included these analyses (page 16 of manuscript) as follows:

“To further isolate the relationship between subjective control and stress separate from perceived task difficulty or objective task performance, we also included the overall win rate (percentage of trials won during the WS task) in the models. In Study 1, lower feelings of control were related to higher levels of subjective stress (β= -0.12, p<.001) even when controlling for both  win rate (β= -0.06, p=.220) and perceived task difficulty (β= 0.37, p<.001, Table S10). This also replicated in Study 2, where lower subjective control was associated with higher feelings of stress (β= -0.32, p<.001) when controlling for perceived task difficulty (β= 0.31, p<.001) and win rate (β= -0.11, p=.428, Table S11). This suggests that there is unique variance in subjective feelings of control, separate from task performance, relevant to subjective stress.”

As well as expanding on this in the Discussion (pages 27 and 28) as follows:

“While our task design does not separate control from obtained reward, we are able to do so in the statistical analyses. Like with perceived difficulty, we statistically accounted for reward rate and showed that the relationship between subjective control and stress was not accounted for by reward rate, for example. Similarly, participants received feedback after every trial, and thus feedback valence may contribute to stress perception. However, given that overall win rate (which captures the feedback received during the task) did not predict stress over and above perceived difficulty or subjective control, it suggests that feedback is unlikely to relate to stress over and above difficulty. Future work will need to disentangle this further to rule out such potential confounds.”

Further, in terms of the wider literature on these issues, we have added more to this point in our discussion, especially in relation to previous literature that also varies control by reward rate (e.g. Dorfman & Gershman, 2019, who use a reward rate of 80% in high control conditions and 50% in low control conditions). This can be found in the manuscript on page 27 as follows: 

“Previous research typically accounts for different outcomes (e.g. punishment) by yoking controllable and uncontrollable conditions [3] though other work has manipulated the controllability of rewards by changing the reward rate [for example 30] where a decoy stimulus is rewarded 50% of the time in the low control condition but 80% in the high control condition).”

(3) The methods are not always clear enough, and it is difficult to know whether all the manipulations are done within-subjects or some key manipulations are done between subjects.

We have added more information in the methods section (page 8) clarifying withinsubject manipulations (WS task parameters) and between-subject manipulations (stressor intensity task, WS task version in Study 1, and WS task/video task in Study 2). Additionally, as recommended by Reviewer 1, we have provided more information in the methods section and Table S3 regarding the details of on-screen written feedback provided to participants after each trial of the WS Task.

(4) The analysis of internal consistency is based on splitting the data into odd/even sliders. This choice of data parcellation may cause missed drifts in task performance due to learning, practice effects, or tiredness, thus potentially inflating internal consistency.

We agree that this can indeed be an issue, though drift is likely to be present in any task including even in mood in resting-state (Jangraw et al., 2023). To respond to this specific point, we parcellated the timepoints into a 1^st/2^nd half split and report the ICC in the supplementary information. While values are lower, indeed likely due to systematic drifts in task performance as participants learn to perform the task (especially for Study 2 since the order of parameters were designed to get easier throughout the experiment), the ICC values are still high. Control sliders: Study 1 = 0.82, Study 2: = 0.68; Difficulty sliders: Study 1: = 0.84, Study 2 = 0.57; Stress sliders: Study 1 = 0.45, Study 2 = 0.71. As seen, the lowest ICC is for stress sliders in Study 1. This may be because the first 3 sliders (included in the 1^st half split) were all related to the stress task (initial, post-stress, task, post-debrief) and the final 4 sliders (in the 2^nd half split) were the three sliders during the WS task and shortly afterwards. 

(5) Study 2 manipulates the effect of domain (win versus loss WS task), but the interaction of this factor with stressor intensity is not included in the analysis.

We agree that this would be a valuable analysis to include. We have run additional analyses (section Sensitivity and Exploratory Analyses, pages 24 and 25), testing the interaction of Domain (win or loss) with stressor intensity (and time) when predicting the stress buffering and stress relief effects. This revealed no significant main effects of domain or interactions including domain, suggesting that domain did not impact the stress induction or relief differently depending on whether it was followed by the high or low stressor intensity condition. While the control by time interaction (our main effect of interest) still held for stress induction in this more complex model, the control by time interaction did not hold for the stress relief. However, this more complex model did not provide a better fit for the data, motivating us to continue to draw conclusions from the original model specification with domain as a covariate (rather than an interaction).

We outline these analyses on page 24 of the manuscript, as follows:

“Third, we included the interaction of domain with stressor intensity and with time, to test whether the win or loss domain in the WS task significantly impacted stress induction or stress relief differently depending on stressor intensity. There were no significant effects or interactions of domain (Table S14) for stress induction or stress relief, and the main effect of interest (the interaction between time and control) still held for the stress induction (β= 10.20, SE=4.99 p=.041, Table S14), though was no longer significant for the stress relief  (β= 6.72, SE=4.28, p=.117, Table S14). This more complex model did not significantly improve model fit (χ^²(3)= 1.46, p=.691) compared to our original specification (with domain as a covariate rather than an interaction) and had slightly worse fit (higher AIC and BIC) than the original model (AIC = 5477.2 versus 5472.7, BIC = 5538.5 versus 5520.8).”

This study will be of interest to psychologists and cognitive scientists interested in understanding how controllability and its subjective perception impact how people respond to stress exposure. Demonstrating that an increased sense of control buffers/protects against subsequent stress is important and may trigger further studies to characterize this phenomenon better. However, beyond the highlighted weaknesses, the current study only studied the effect of stress induction consecutive to the performance of the WS task on the same day and its generalizability is not warranted.

We thank the reviewer for this assessment and agree that we cannot assume these findings would generalise to more prolonged effects on stress responses.

Reviewer #3 (Public review):

Summary:

This is an interesting investigation of the benefits of perceiving control and its impact on the subjective experience of stress. To assess a subjective sense of control, the authors introduce a novel wheel-stopping (WS) task where control is manipulated via size and speed to induce low and high control conditions. The authors demonstrate that the subjective sense of control is associated with experienced subjective stress and individual differences related to mental health measures. In a second experiment, they further show that an increased sense of control buffers subjective stress induced by a trier social stress manipulation, more so than a more typical stress buffering mechanism of watching neutral/calming videos.

We agree with this accurate summary of our study. 

Strengths:

There are several strengths to the manuscript that can be highlighted. For instance, the paper introduces a new paradigm and a clever manipulation to test an important and significant question. Additionally, it is a well-powered investigation that allows for confidence in replicability and the ability to show both high internal consistency and high external validity with an interesting set of individual difference analyses. Finally, the results are quite interesting and support prior literature while also providing a significant contribution to the field with respect to understanding the benefits of perceiving control.

We thank the reviewer for this positive assessment. 

Weaknesses:

There are also some questions that, if addressed, could help our readership.

(1) A key manipulation was the high-intensity stressor (Anticipatory TSST signal), which was measured via subjective ratings recorded on a sliding scale at different intervals during testing. Typically, the TSST conducted in the lab is associated with increases in cortisol assessments and physiological responses (e.g., skin conductance and heart rate). The current study is limited to subjective measures of stress, given the online nature of the study. Since TSST online may also yield psychologically different results than in the lab (i.e., presumably in a comfortable environment, not facing a panel of judges), it would be helpful for the authors to briefly discuss how the subjective results compare with other examples from the literature (either online or in the lab). The question is whether the experienced stress was sufficiently stressful given that it was online and measured via subjective reports. The control condition (low intensity via reading recipes) is helpful, but the low-intensity stress does not seem to differ from baseline readings at the beginning of the experiment.

We agree that it would be helpful to expand on this further. Similar to the comment made by Reviewer 1, we wish to point out that there are ample demonstrations that such anticipatory stress inductions are effective at reliably eliciting physiological and psychological stress responses (e.g. Nasso et al., 2019; Schlatter et al., 2021; Steinbeis et al., 2015). Further, there is evidence that online versions of the TSST are also effective (DuPont et al., 2022; Meier et al., 2022), including evidence that the speech preparation phase conducted online was related to increases in heart rate and blood pressure (DuPont et al., 2022). We have elaborated further on this in our manuscript on pages 8 and 9 as follows:

“Prior research has found TSST anticipation to elicit both psychological and physiological stress responses [37-39], suggesting that the task anticipation would be a valid stress induction despite participants not performing the speech task. Moreover, prior research has validated the use of remote TSST in online settings [40, 41], including evidence that the speech preparation phase (online) was related to increased heart rate and blood pressure compared to controls [40].”

(2) The neutral videos represent an important condition to contrast with WS, but it raises two questions. First, the conditions are quite different in terms of experience, and it is interesting to consider what another more active (but not controlled per se) condition would be in comparison to the WS performance. That is, there is no instrumental action during the neutral video viewing (even passive ratings about the video), and the active demands could be an important component of the ability to mitigate stress. Second, the subjective ratings of the stress of the neutral video appear equivalent to the win condition. Would it have been useful to have a high arousal video (akin to the loss condition) to test the idea that experience of control will buffer against stress? That way, the subjective stress experience of stress would start at equivalent points after WS3.

We agree with the reviewer that this is an important issue to clarify. In our deliberations when designing this study, we considered that that any task with actionoutcome contingencies would have a degree of controllability. To better distinguish experiences of control (WS task) to an experience of no/neutral control (i.e., neither high nor low controllability), we decided to use a task in which no actions were required during the task itself. Importantly, however, there was an active demand and concentration was still required in order to perform the attention checks regarding the content of the videos and ratings of the videos. 

Thank you for the suggestion of having a high arousal video condition. This would indeed be interesting to test how experiencing ‘neutral’ control and high(er) stress levels preceding the stressor task influences stress buffering and stress relief, and we have included this suggestion for future research in the discussion section (page 28) as below:

“Another avenue for future research would be to test how control buffers against stress when compared to a neutral control scenario of higher stress levels, akin to the loss domain in the WS Task, given that participants found the video condition generally relaxing. However, given that we found no differences dependent on domain for the stress induction in the WS Task conditions, it is possible that different versions of a neutral control condition would not impact the stress induction.”

(3) For the stress relief analysis, the authors included time points 2 and 3 (after the stressor and debrief) but not a baseline reading before stress. Given the potential baseline differences across conditions, can this decision be justified in the manuscript?

We thank the reviewer for raising this. Regarding the stress relief analyses (timepoints 2 and 3) and not including timepoint 1 (after the WS/video task) stress in the model, we have added to the manuscript that there was no significant difference in stress ratings between the high control and neutral control (collapsed across stress and domain) at timepoint 1 (hence why we do not think it’s necessary to include in the stress relief model). Nevertheless, we have now included a sensitivity analysis to test the Timepoint*Control interaction of stress relief when including timepoint 1 stress as a covariate. The timepoint by control interaction still holds, suggesting that the initial stress level prior to the stress induction does not impact our results of interest. The details of this analysis are included in the Sensitivity and Exploratory Analyses section on page 24:

“Although there were no significant differences between control groups in subjective stress immediately after the WS/video task (t(175.6)=1.17, p=.244), we included participants’ stress level after the WS/video task as a covariate in the stress relief analyses (Table S12). The results revealed a main effect of initial stress (β= 0.643, SE=0.040, p<.001, Table S12) on the stress relief after the stressor debrief. Compared to excluding initial stress as in the original analyses (Table 4), there was now no longer a main effect of domain (β= 0.236, SE=2.60, p=.093, Table S12), but the inference of all other effects remained the same. Importantly, there was still a significant time by control interaction (β= 9.65, SE=3.74, p=.010, Table S12) showing that the decrease in stress after the debrief was greater in the highly controllable WS condition than the neutral control video condition, even when accounting for the initial stress level.”

(4) Is the increased control experience during the losses condition more valuable in mitigating experienced stress than the win condition?

We agree that this would be helpful to clarify. To test whether the loss domain was more valuable at mitigating experiences of stress than the win condition, we ran additional analyses with just the high control condition (WS task) to test for a Domain*Time interaction. This revealed no significant Domain*Time interaction, suggesting that the stress buffering or stress relief effect was not dependent on domain in the high control conditions. These analyses are outlined in the Sensitivity and Exploratory Analyses section on page 25:

“Finally, to test whether the loss domain was more valuable at mitigating experiences of stress than the win condition, we ran additional analyses with just the high control condition (WS task) for the stress induction and stress relief to test for an interaction of domain and time. For the stress induction, there was no significant two-way interaction of domain and time (β= -1.45, SE=4.80, p=.763), nor a significant three-way interaction of domain by time by stressor intensity (β= -3.96, SE=6.74, p=.557, Table S15), suggesting that there were no differences in the stress induction dependent on domain. Similarly for the stress relief, there was no significant two-way interaction of domain and time (β= -5.92, SE=4.42, p=.182), nor a significant three-way interaction of domain by time by stressor intensity interaction (β= 8.86, SE=6.21, p=.154, Table S15), suggesting that there were no differences in the stress relief dependent on the WS Task domain.

(5) The subjective measure of control ("how in control do you feel right now") tends to follow a successful or failed attempt at the WS task. How much is the experience of control mediated by the degree of experienced success/schedule of reinforcement? Is it an assessment of control or, an evaluation of how well they are doing and/or resolution of uncertainty? An interesting paper by Cockburn et al. 2014 highlights the potential for positive prediction errors to enhance the desire for control.

We thank the reviewer for this comment. Similar to comments regarding reward rate, our task does not allow us to fully separate control from success/reinforcement because of the manipulation of difficulty. However, we did undertake sensitivity analyses and the inclusion of overall win rate accounted for limited variance when predicting stress over and above subjective control and difficulty (page 16). 

“To further isolate the relationship between subjective control and stress separate from perceived task difficulty or objective task performance, we also included the overall win rate (percentage of trials won during the WS task) in the models. In Study 1, lower feelings of control were related to higher levels of subjective stress (β= -0.12, p<.001) even when controlling for both  win rate (β= -0.06, p=.220) and perceived task difficulty (β= 0.37, p<.001, Table S10). This also replicated in Study 2, where lower subjective control was associated with higher feelings of stress (β= -0.32, p<.001) when controlling for perceived task difficulty (β= 0.31, p<.001) and win rate (β= -0.11, p=.428, Table S11). This suggests that there is unique variance in subjective feelings of control, separate from task performance, relevant to subjective stress.” 

(6) While the authors do a very good job in their inclusion and synthesis of the relevant literature, they could also amplify some discussion in specific areas. For example, operationalizing task controllability via task difficulty is an interesting approach. It would be useful to discuss their approach (along with any others in the literature that have used it) and compare it to other typically used paradigms measuring control via presence or absence of choice, as mentioned by the authors briefly in the introduction.

We are delighted to expand on this particular point and have done so in the Discussion on page 27:

“Previous research typically accounts for different outcomes (e.g. punishment) by yoking controllable and uncontrollable conditions [3] though other work has manipulated the controllability of rewards by changing the reward rate [for example 30] where a decoy stimulus is rewarded 50% of the time in the low control condition but 80% in the high control condition). While our task design does not separate control from obtained reward, we are able to do so in the statistical analyses.” 

(7) The paper is well-written. However, it would be useful to expand on Figure 1 to include a) separate figures for study 1 (currently not included) and 2, and b) a timeline that includes the measurements of subjective stress (incorporated in Figure 1). It would also be helpful to include Figure S4 in the manuscript.

We have expanded Figure 1 to include both Studies 1 and 2 and a timeline of when subjective stress was assessed throughout the experiment as well as adding Figure S4 to the main manuscript (now top panel within Figure 4). 

Reviewer #1 (Recommendations for the authors):

(1) Study 2 shows a greater decrease in subjective stress after the high-control task manipulation than after the pleasant video. One possible confound is whether the amount of time to complete the WS task and the video differ. It could be helpful to look at the average completion time for the WS task and compare that to the length of the videos. Alternatively, in future studies, control for this by dynamically adjusting the video play length to each participant based on how long they took to complete the WS task.

This is an interesting suggestion. As a result, we have included the time taken as a covariate in the stress induction and stress relief analyses to ensure that any differences in time between the WS task and video task were not accounting for any of the stress induction or relief analyses. Controlling for the total time taken did not impact the stress induction or relief results. This is included in the Sensitivity and Exploratory Analyses section on page 24:

“Our second sensitivity analyses was conducted because the experiment took longer to complete for the video condition (mean = 54.3 minutes, SD = 12.4 minutes) than the WS task condition (mean = 39.7 minutes, SD = 12.8 minutes, t(186.19)=-9.32, p<.001). We therefore included the total time (in ms) as a covariate in the stress induction and stress relief analyses for Study 2. This showed that accounting for total time did not change the results of interest (Table S13), further highlighting that the time by control interactions were robust.”

(2) Because participants received feedback about their success/failure in the WS task, a confounding factor could be that they received positive feedback on highly controllable trials and negative feedback on low control trials (and/or highly difficult trials). This would suggest that it is not controllability per se that contributes to stress perception but rather feedback valence. The authors show that this is a likely factor in their results in Study 2, which shows significant effects of the loss domain on perceived control and stress. Was a similar analysis done in Study 1? Do participants receive feedback in Study 1? It would be helpful to include this information somewhere in the manuscript. I would be curious to know whether *any* feedback at all influences controllability/stress perceptions.

We thank the reviewer for this interesting suggestion. It is an interesting question as to whether feedback valence is related to stress in Study 1, and we have added this point to the Discussion on pages 27 and 28. To speak to this point, when we include the overall win rate (which captures the subsequent feedback received) when predicting subjective stress, win rate is not a significant predictor of stress over and above perceived difficulty and subjective control, suggesting that overall feedback valence may not be related to stress in Study 1. We take this as evidence that feedback may not be as important in terms of accounting for the relationship between stress and control. However, we unfortunately do not have any data in which there was no feedback provided to speak to this conclusively. This would be an interesting future study. The excerpt below is added to pages 27 and 28 of the discussion section:

“Like with perceived difficulty, we statistically accounted for reward rate and showed that the relationship between subjective control and stress was not accounted for by reward rate, for example. Similarly, participants received feedback after every trial, and thus feedback valence may contribute to stress perception. However, given that overall win rate (which captures the feedback received during the task) did not predict stress over and above perceived difficulty or subjective control, it suggests that feedback is unlikely to relate to stress over and above difficulty. Future work will need to disentangle this further to rule out such potential confounds.”

To respond specifically to the reviewer’s question about the feedback given to participants, written feedback was provided on screen to participants on a trial-bytrial basis also in Study 1 (i.e. for both studies), and we have provided more clarity about this in the manuscript on page 8 as well as providing additional details in Table S3:

“After each trial, participants were shown written feedback on screen as to whether the segment had successfully stopped on the red zone (or not), and the associated reward (or lack of). See Table S3 for details.”

(3) I'm not sure how to interpret the fact that in Figure S1, the BICs are all essentially the same. Does this mean that you don't really need all of these varying aspects of the task to achieve the same effects? Could the task be made simpler?

The similarity of BIC values suggests that a simpler WS task would have produced a worse account of the data approximately in keeping with the extent to which it is a simpler model. Here, the BIC scores for the models are similar, suggesting that adding these parameters adds explanatory power in keeping with what would have been expected from adding a parameter, but not more. We do note that the BIC is a relatively strict and conservative comparison. The fact that the most complex model overall narrowly improves parsimony; combined with the interpretable parameter values and the prior expectations given the task setup led us to focus on this most complex model.  

(4) A minor point, but the authors refer to their sample as "neurotypical." Were they assessed for prior/current psychopathology/medications? If not, I might use a different term here (perhaps "non-clinical sample"), since some prior work has shown that online samples actually have higher instances of psychopathology compared to community samples.

We have changed the phrasing of ‘neurotypical’ to a ‘non-clinical sample’ as recommended.

Reviewer #2 (Recommendations for the authors):

Figure 4S is very informative and could be presented in the main text.

We have expanded Figure 1 to include both Studies 1 and 2 and a timeline of when subjective stress was assessed throughout the experiment as well as adding Figure S4 to the main manuscript (top panel of Figure 4). 

References:

Dorfman, H. M., & Gershman, S. J. (2019). Controllability governs the balance between Pavlovian and instrumental action selection. Nature Communications, 10(1), 5826. https://doi.org/10.1038/s41467-019-13737-7

DuPont, C. M., Pressman, S. D., Reed, R. G., Manuck, S. B., Marsland, A. L., & Gianaros, P. J. (2022). An online Trier social stress paradigm to evoke affective and cardiovascular responses. Psychophysiology, 59(10), e14067. https://doi.org/10.1111/psyp.14067

Jangraw, D. C., Keren, H., Sun, H., Bedder, R. L., Rutledge, R. B., Pereira, F., Thomas, A. G., Pine, D. S., Zheng, C., Nielson, D. M., & Stringaris, A. (2023). A highly replicable decline in mood during rest and simple tasks. Nature Human Behaviour, 7(4), 596–610. https://doi.org/10.1038/s41562-023-015197

Meier, M., Haub, K., Schramm, M.-L., Hamma, M., Bentele, U. U., Dimitroff, S. J., Gärtner, R., Denk, B. F., Benz, A. B. E., Unternaehrer, E., & Pruessner, J. C. (2022). Validation of an online version of the trier social stress test in adult men and women. Psychoneuroendocrinology, 142, 105818. https://doi.org/10.1016/j.psyneuen.2022.105818

Nasso, S., Vanderhasselt, M.-A., Demeyer, I., & De Raedt, R. (2019). Autonomic regulation in response to stress: The influence of anticipatory emotion regulation strategies and trait rumination. Emotion, 19(3), 443–454. https://doi.org/10.1037/emo0000448

Schlatter, S., Schmidt, L., Lilot, M., Guillot, A., & Debarnot, U. (2021). Implementing biofeedback as a proactive coping strategy: Psychological and physiological effects on anticipatory stress. Behaviour Research and Therapy, 140, 103834. https://doi.org/10.1016/j.brat.2021.103834

Steinbeis, N., Engert, V., Linz, R., & Singer, T. (2015). The effects of stress and affiliation on social decision-making: Investigating the tend-and-befriend pattern. Psychoneuroendocrinology, 62, 138–148. https://doi.org/10.1016/j.psyneuen.2015.08.003

Stap 1: 200.000 - Heffingsvrij vermogen van 57.684 euro = 142.316 euro. Dan kijken welk percentage uit art. 5.2 lid 2 Wet IB 2001 van toepassing is (banktegoeden, overige bezittingen, schulden). Het vakantiehuis valt onder overige bezittingen van Ronald dus wordt 142.316 euro x 5,88% = 8.368 belastbaar vermogen en dat nog vermenigvuldigen met het tarief uit box 3, 36%. 8.368 x 36% = 3.012,5 euro aan verschuldigde belasting.

• ARE 1523820 AgR-terceiro
• Órgão julgador: Primeira Turma
• Relator(a): Min. CRISTIANO ZANIN
• Redator(a) do acórdão: Min. ALEXANDRE DE MORAES
• Julgamento: 25/06/2025
• Publicação: 18/07/2025

AGRAVO INTERNO. RECURSO EXTRAORDINÁRIO COM AGRAVO. PODER GERAL DE CAUTELA DOS TRIBUNAIS DE CONTAS. ART. 71, IX E ART. 75 DA CONSTITUIÇÃO FEDERAL. TEORIA DOS PODERES IMPLÍCITOS. COMPETÊNCIA PARA DETERMINAR À AUTORIDADE ADMINISTRATIVA QUE PROMOVA ANULAÇÃO DE CONTRATO E, SE FOR O CASO, DA LICITAÇÃO DE QUE SE ORIGINOU. PRECEDENTES. AGRAVO INTERNO A QUE SE DÁ PROVIMENTO. - 1. O Plenário do SUPREMO TRIBUNAL FEDERAL já se pronunciou no sentido da validade do <u>poder geral de cautela</u> dos Tribunais de Contas dos Estados (SS 5658 AgR, Rel. Min. LUÍS ROBERTO BARROSO (Presidente), Tribunal Pleno, DJe 04-04-2024).

• 2. Incorporou-se em nosso ordenamento jurídico a pacífica doutrina constitucional norte-americana sobre a teoria dos poderes implícitos - inherent powers -, pela qual no exercício de sua missão constitucional enumerada, o órgão executivo deveria dispor de todas as funções necessárias, ainda que implícitas, <u>desde que não expressamente limitadas</u> (Myers v. Estados Unidos US 272 52, 118), consagrando-se, dessa forma, e entre nós aplicável ao Tribunal de Contas da União e, por simetria, aos Tribunais de Contas dos Estados, o reconhecimento de competências genéricas implícitas que possibilitem o exercício de sua missão constitucional, apenas sujeitas às proibições e limites estruturais da Constituição Federal.

• 1. O “Tribunal de Contas da União – embora não tenha poder para anular ou sustar contratos administrativos – tem competência, conforme o art. 71, IX, para determinar à autoridade administrativa que promova a anulação do contrato e, se for o caso, da licitação de que se originou” (MS 23.550, Red. p/o acórdão o Min. Sepúlveda Pertence). Igual competência é atribuída ao Tribunal de Contas do Estado do Ceará, na forma do art. 75 da Constituição.

• 4. Não configura ilegalidade ou abuso de poder o ato do Tribunal de Contas que aplique medidas cautelares, porque relacionada com a competência constitucional implícita para cumprimento de suas atribuições.

• 5. Agravo Interno a que se dá provimento.

Observação - Acórdão(s) citado(s): (TRIBUNAL DE CONTAS, PRERROGATIVA, PODER GERAL DE CAUTELA, TEORIA DOS PODERES IMPLÍCITOS) MS 23550 (1ªT), MS 24510 (TP), SS 5658 AgR (TP). (FUNDAMENTAÇÃO, DECISÃO JUDICIAL) AI 791292 QO-RG (TP). (RE, DEVIDO PROCESSO LEGAL, AMPLA DEFESA, CONTRADITÓRIO, LIMITES DA COISA JULGADA) ARE 748371 RG (TP). (RE, INDEFERIMENTO, PRODUÇÃO DE PROVA, OFENSA INDIRETA) ARE 639228 RG (TP). (RE, PRINCÍPIO DA INAFASTABILIDADE DA JURISDIÇÃO, OFENSA INDIRETA, REEXAME, FATO, PROVA) RE 956302 RG (TP). - Decisão estrangeira citada: Caso Myers vs. Estados Unidos, US 272 52, 118, da Suprema Corte dos Estados Unidos. Número de páginas: 16. Análise: 11/08/2025, MAV.

Partes AGTE.(S) : TRIBUNAL DE CONTAS DO CEARA PROC.(A/S)(ES) : PROCURADOR-GERAL DO TRIBUNAL DE CONTAS DO ESTADO DO CEARÁ PROC.(A/S)(ES) : LÍLIAN DE CASTRO E SILVA MENEZES DO VALE (15518/CE) PROC.(A/S)(ES) : MAYSA CORTEZ CORTEZ (32431/CE) PROC.(A/S)(ES) : TALLITA FALKENSTINS GOIS MENDES CORDEIRO (31661/CE)

• MS 35920 ED
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. GILMAR MENDES
• Julgamento: 30/10/2023
• Publicação: 14/11/2023

EMBARGOS DE DECLARAÇÃO NO MANDADO DE SEGURANÇA. TRIBUNAL DE CONTAS DA UNIÃO. TOMADA DE CONTAS ESPECIAL. DESCONSIDERAÇÃO DA PERSONALIDADE JURÍDICA. POSSIBILIDADE. REQUISITOS LEGAIS OBSERVADOS. AUSÊNCIA DE DIREITO LÍQUIDO E CERTO. DENEGAÇÃO DA SEGURANÇA. AUSÊNCIA DE OMISSÃO, CONTRADIÇÃO, OBSCURIDADE OU ERRO MATERIAL.

• 1. Os embargos de declaração são cabíveis para sanar a ocorrência de omissão, contradição, obscuridade ou erro material, hipóteses não verificadas no caso dos autos.
• 2. É legal e constitucionalmente fundada a <u>desconsideração da pessoa jurídica</u> pelo TCU, de modo a alcançar o patrimônio de pessoas físicas ou jurídicas envolvidas na prática de atos lesivos ao erário público, observados o contraditório e a ampla defesa.
• 3. TCU assentou a existência de indícios de que a impetrante praticou condutas ilegais que consubstanciariam a desconsideração da personalidade jurídica.
• 4. Feito se encontra em etapa preambular cabendo o exercício exauriente do direito de defesa pelos entes integrados à relação processual, em procedimento sujeito a controle jurisdicional.
• 5. Embargos de declaração rejeitados.

Observação - Acórdão(s) citado(s): (TRIBUNAL DE CONTAS, DESCONSIDERAÇÃO DA PERSONALIDADE JURÍDICA) MS 35506 (1ªT). (TEORIA DO ATO ULTRA VIRES) MS 35555 AgR (2ªT). Número de páginas: 16. Análise: 15/04/2024, SOF.

Indexação TRAMITAÇÃO, TRIBUNAL DE CONTAS DA UNIÃO (TCU), POSSIBILIDADE, APLICAÇÃO, TEORIA DO ATO ULTRA VIRES, RESPONSABILIZAÇÃO, FORMA DIRETA, PESSOA FÍSICA, RESPONSABILIDADE, COMETIMENTO, ATO ILÍCITO. DESCONSIDERAÇÃO DA PERSONALIDADE JURÍDICA, RESERVA DE JURISDIÇÃO. DESCONSIDERAÇÃO DA PERSONALIDADE JURÍDICA, AUSÊNCIA

Outras ocorrências Partes (1)

• ARE 1306779 AgR
• Órgão julgador: Segunda Turma
• Relator(a): Min. EDSON FACHIN
• Julgamento: 03/05/2023
• Publicação: 09/05/2023
• 2. Ademais, este Supremo Tribunal Federal já assentou a <u>plena possibilidade</u> de a Corte de Contas, no cumprimento de seu mister constitucional, decretar a indisponibilidade de bens e de outras medidas assecuratórias do interesse público, diante de circunstâncias graves que justifiquem a necessidade de proteção efetiva do patrimônio público.
• 1. O Plenário também já afirmou a plena possibilidade de que o TCU, orientação que também se aplica às Cortes de Contas Estaduais, determine a aplicação de medidas cautelares, como verdadeira competência constitucional implícita para cumprimento de suas atribuições, nos termos do artigo 71 da Carta Magna.

Synthèse des Outils Numériques et des Procédures Associées au Lycée

Résumé

Ce document synthétise l'écosystème des outils numériques utilisés au sein de l'établissement, en se basant sur un tutoriel détaillé.

L'objectif principal est de clarifier le rôle de chaque plateforme et de fournir un guide pratique pour les parents et les élèves. Le point d'entrée fondamental de cet écosystème est le compte EduConnect, qui sert de clé d'accès unique à la quasi-totalité des services.

Sans un compte EduConnect fonctionnel, l'accès aux autres outils est impossible.

Les plateformes principales sont :

1. EduConnect et les Téléservices : Le portail national pour l'identité numérique, indispensable pour les démarches administratives telles que les demandes de bourses, le paiement de la cantine et la mise à jour des informations personnelles.

2. Mon Bureau Numérique (MBN) : L'environnement de travail fourni par la Région Grand Est, servant de portail central pour la vie scolaire quotidienne, incluant le cahier de textes, la messagerie, l'agenda et l'accès aux ressources de la classe.

3. Pronote : Accessible via MBN, cet outil est spécialisé dans le suivi pédagogique : consultation des notes, gestion des absences, emploi du temps mis à jour et prise de rendez-vous pour les rencontres parents-professeurs.

4. Parcoursup : La plateforme nationale pour l'orientation post-bac, essentielle pour les élèves de Terminale et une ressource d'information cruciale pour les élèves de Seconde et Première.

5. Outils Complémentaires : Un système de prise de rendez-vous en ligne pour les psychologues de l'Éducation Nationale (PsyEN) et la fourniture de licences Microsoft Office gratuites pour les élèves.

Un support informatique est disponible en cas de difficulté via l'adresse e-mail X.

Il est souligné qu'une part significative de parents (600) n'a pas encore activé son compte EduConnect, ce qui les prive d'accès à des services essentiels, notamment le vote pour les représentants des parents d'élèves.

1. Le Portail EduConnect : La Clé d'Accès Universelle

EduConnect est le portail d'authentification de l'Éducation Nationale. Il ne s'agit pas d'un outil utilisé au quotidien, mais de la porte d'entrée indispensable à tous les autres services.

Rôle et Importance

• Identifiant Unique : Fournit un identifiant et un mot de passe uniques pour accéder à la majorité des outils numériques de l'établissement.

• Point de Départ Obligatoire : Si le compte EduConnect ne fonctionne pas, aucun autre service n'est accessible.

• Comptes Multiples : Chaque responsable légal (père, mère, responsable 1 et 2) dispose de son propre compte individuel.

Activation et Dépannage

• Problème d'Activation : Un constat a été fait que 600 parents n'ont pas activé leur compte, souvent car un seul des deux parents gère le suivi scolaire.

Cela pose problème, par exemple, pour le vote aux élections des parents d'élèves, où chaque parent peut voter.

• Procédure en cas de problème :

1. Contacter le support informatique du lycée à l'adresse : X.   

2. Vérifier le dossier "spam" de sa messagerie, car les e-mails de réinitialisation peuvent y atterrir.   

3. En dernier recours, l'établissement peut imprimer un document papier avec des identifiants temporaires.

• Complexités Possibles : Des difficultés peuvent survenir en cas de :

◦ Plusieurs enfants scolarisés dans différents établissements.    ◦ Homonymes entre parents d'élèves.    ◦ Changement d'académie.

• Mise à jour des e-mails : Le bon fonctionnement du compte dépend de l'exactitude de l'adresse e-mail enregistrée dans la base de données de l'établissement (base Siècle), car c'est par ce biais que sont envoyés les liens de réinitialisation. Actuellement, une quarantaine d'adresses e-mail sont encore erronées sur environ 2800.

2. Téléservices (Scolarité Services) : La Gestion Administrative

Accessibles via le compte EduConnect, les Téléservices sont la plateforme pour toutes les démarches administratives numérisées.

Fonctionnalités Principales

• Demandes de Bourse : Permet de soumettre une demande de bourse en ligne, sans document papier.

• Fiche de Renseignements : Consultation et demande de modification des coordonnées (adresse postale, e-mail, téléphone). Toute modification doit être validée par le secrétariat de l'établissement.

• Paiement de la Demi-Pension :

◦ Les factures sont publiées sur cette plateforme (la première vers mi-novembre).   

◦ Il est possible de payer chaque facture trimestrielle en deux fois, permettant ainsi un étalement sur six paiements dans l'année.

La mensualisation complète est envisagée pour l'année suivante.

• Documents Officiels : Téléchargement du certificat de scolarité. Ce document est également disponible sur Pronote.

• Orientation : La plateforme sera utilisée pour les phases d'orientation, notamment pour les élèves de Seconde.

3. Mon Bureau Numérique (MBN) : L'Espace de Travail Quotidien

MBN est l'environnement numérique de travail (ENT) fourni par la Région Grand Est. Il s'agit du portail central pour la communication et les informations pédagogiques.

Accès

• La connexion s'effectue via les identifiants EduConnect.

• Après connexion au portail académique, il est nécessaire de sélectionner "Lycée Louis-Vincent" pour accéder à l'espace de l'établissement.

• Il est conseillé d'ajouter le site MBN aux favoris de son navigateur pour un accès plus rapide et écologique.

Fonctionnalités Clés pour les Parents

• Cahier de Textes : Permet de consulter le travail à faire donné aux élèves jour par jour, ainsi que le contenu des séances passées.

• Messagerie (Zimbra) :

◦ Permet de contacter les enseignants de la classe de son enfant, la direction ou d'autres personnels via un assistant destinataire.   

◦ Note importante : Le Proviseur (Olivier Palaise) préfère être contacté sur son adresse académique (olivier.palaise@ac-nancy-metz.fr) plutôt que via la messagerie MBN pour des raisons de gestion de flux (recevant entre 100 et 200 e-mails par jour).

• Agenda de l'Établissement : Un calendrier complet des événements du lycée (Fête de la Science, vacances, sessions d'évaluation Pix, etc.).

• Espace Classe : Section où les enseignants peuvent partager des ressources spécifiques à une classe ou une spécialité (ex: règles pour le travail en mathématiques, liens vers des manuels).

• Accès à Pronote : MBN est le portail d'entrée pour se connecter à Pronote.

Vue Élève : Le Médiacentre

• Les élèves disposent d'un onglet supplémentaire, "Médiacentre", qui donne accès à l'ensemble des manuels scolaires numériques fournis par la Région Grand Est.

• Avantages : Gratuité pour les familles et allègement significatif du poids des cartables.

• Inconvénients : L'utilisation d'un manuel sur écran peut être moins pratique qu'un format papier.

• Autres ressources : Le Médiacentre donne également accès à des outils comme "Do you speak grand test", une plateforme gratuite d'apprentissage des langues.

4. Pronote : Le Suivi Pédagogique Détaillé

Pronote est le logiciel de vie scolaire utilisé pour le suivi précis des résultats et de l'assiduité des élèves.

Accès et Fonctionnalités

• Accès : Se fait en cliquant sur l'icône Pronote depuis Mon Bureau Numérique (MBN).

• Suivi des Notes : Consultation de l'ensemble des notes obtenues. Les parents y ont accès quelques jours après les élèves, pour permettre aux professeurs d'expliquer d'abord les résultats en classe.

• Emploi du Temps : C'est la source la plus fiable et la plus à jour pour l'emploi du temps de l'élève, incluant les absences de professeurs et les modifications de salles.

• Élections des Parents d'Élèves : C'est via Pronote que les parents accèdent à la plateforme de vote électronique. Il est rappelé que les deux parents peuvent voter, et que le vote nécessite un compte EduConnect activé.

https://youtu.be/b7gst-DjVd8?t=1979

• Réunions Parents-Professeurs : La prise de rendez-vous pour les rencontres se fera via cet espace (ex: à partir du 17-18 novembre pour la réunion des classes de Seconde du 29 novembre).

5. Parcoursup : La Plateforme d'Orientation Post-Bac

Parcoursup est l'outil national incontournable pour la gestion des vœux d'affectation dans l'enseignement supérieur.

Utilisation et Calendrier

• Public concerné : Indispensable pour les élèves de Terminale, il est fortement conseillé aux élèves de Première et de Seconde de s'y familiariser pour préparer leurs choix de spécialités et de filières.

• Disponibilité : Même lorsque la plateforme est "fermée" (avant la mise à jour des formations de l'année en cours), elle reste une mine d'informations (vidéos, tutoriels, carte des formations de l'année précédente).

Exploration des Formations

• La recherche d'une formation donne accès à une fiche détaillée contenant des informations capitales.

• Exemple concret - PASS (Parcours d'Accès Spécifique Santé) à Nancy :

Critères de Sélection (2025)

Poids

Détails

Résultats Scolaires

45%

Notes de Première et de Terminale.

Méthodes de Travail

Évaluées via les remarques des enseignants.

Savoir-être

Motivation (lettre), comportement.

Engagement

Activités extra-scolaires valorisées (délégué, SNU, etc.).

• Chiffres Clés (2025) :

◦ Candidats : 2 990    ◦ Propositions d'admission reçues : 922   

◦ Admis finaux : 78    ◦ Profil des admis : 99% issus d'un bac général.

Des émissions dédiées à Parcoursup seront proposées plus tard dans l'année pour approfondir son fonctionnement.

6. Outils et Support Complémentaires

Prise de Rendez-vous avec les Psychologues de l'Éducation Nationale (PsyEN)

• Un site dédié, fonctionnant sur le modèle de Doctolib, a été mis en place pour prendre rendez-vous avec les deux PsyEN de l'établissement (Mme Morau et Mme Berdou).

• Adresse du site : orientation.lcer-luis-vincent.fr

• Fonctionnement : Les parents ou élèves choisissent un créneau disponible, remplissent leurs informations (nom, prénom, e-mail, classe) et reçoivent une confirmation et un rappel par e-mail.

• Recommandation : Il est conseillé de réserver deux créneaux successifs pour disposer d'une heure complète d'entretien.

Licences Logicielles

• Les élèves bénéficient de licences Microsoft Office (Word, Excel, Publisher) gratuites, fournies par le lycée et la Région Grand Est.

• Chaque licence peut être installée sur 10 appareils (ordinateurs, tablettes, téléphones).

• L'identifiant est de la forme `. Le mot de passe a été distribué en début d'année.

Support Technique

• Pour toute difficulté technique avec l'un de ces outils, l'adresse de contact unique .

Reviewer #3 (Public review):

Summary:

IRG1 is highly expressed in activated human and mouse myeloid cells. It encodes the mitochondrial enzyme cis-aconitate decarboxylase 1 (ACOD1) that generates itaconate. Itaconate has anti-microbial activity and acts immunoregulatory by interfering with cellular metabolism, signaling to cytokine production, and multiple other processes.

The authors perform a phylogenetic analysis of IRG1 to obtain insight into the evolution of itaconate biosynthesis. Combining BLAST with human IRG1 and a MmgE/Ptrp domain search, they find CAD in all domains of life, but the presence of IRG1 homologs is patchy in eukaryotes, indicating that itaconate biosynthesis is not essential. The phylogenetic analysis showed a more distant relationship of fungal and metazoan CAD/IRG1 to many prokaryotic sequences, suggesting independent acquisition of these metazoan and fungal CAD genes. In metazoans, three subbranches of paleo-IRG1 (in mollusks/early chordates) and two paralogous vertebrate forms (IRG1 and IRG1-like) were identified, with the latter derived from paleo-IRG1, and by genome duplication. While most jawed vertebrates have both IRG1 and IRG1L, metatherian and eutherian mammals have lost IRG1L and contain only IRG1.

Interestingly, sequence analysis of both paralogues showed that many IRG1L genes contain an N-terminal mitochondrial targeting sequence (MTS) that is absent from most IRG1 sequences. Limited proteolysis of submitochondrial localization confirmed that zebrafish IRG1L is only sensitive to proteases in the presence of high Triton X-100, indicative of association with mitochondrial matrix. In contrast, a recent paper from the Galan lab (Lian 2003 Nature Microbiology) reported that human IRG1 is not localized to the mitochondrial matrix, although enriched in mitochondria. Here, the authors generated a matrix-targeted human IRG1 by adding the N-terminal MTS and found that it localizes to the matrix based on a limited proteolysis assay. The loss of MTS-containing IRG1L from most mammals appears, therefore, to indicate that itaconate generation is directed to the cytoplasm, potentially reducing inhibition of TCA cycle activity in the mitochondria.

Next, the authors confirmed that the recombinant IRG1L protein has CAD activity in vitro. The last part of the manuscript addresses the expression of paleo-IRG1 in oysters and amphioxus, where they found high mRNA levels in oyster hemocytes which was further increased by poly(I:C), which was also the case in amphioxus tissues after feeding of LPS or poly(I:C), indicating a role for paleo-IRG1/itaconate in early metazoan innate immunity.

Strengths

(1) Phylogenetic perspective largely lacking so far in the IRG1/itaconate field.

(2) Manuscript clearly written and understandable across disciplines.

(3) Phylogenetic analyses complemented by biochemical and gene expression analyses to link to function.

(4) Lack of MTS in IRG1 and change in localization from mitochondria, highly relevant antimicrobial and cellular effects of itaconate.

Weaknesses:

(1) Biochemical and functional analysis of different CAD mRNA and proteins lacks depth.

(2) The submitochondrial localization assay lacks a native human IRG1 control.

(3) CAD activity shown for IRG1L but not paleo-IRG1.

(4) Itaconate production by early metazoans after PAMP stimulation?

(5) No measurement of energy metabolism (trade-offs?).

I acknowledge that some of these limitations are inevitable because the range of detailed experimental analysis is necessarily limited. However, some of these data would be important to support central claims of the manuscript (further discussed below).

Author response:

Reviewer #1 (Public review): 

Summary: 

The taxonomic analysis of IRG1 evolution is compelling and fills an important gap in the literature. However, the experimental evidence for IRG1 localization requires greater detail and confirmation. 

Strengths: 

The phylogenetic analysis of IRG1 evolution fills an important gap in the literature. The identification of independent acquisition of metazoan and fungal IRG1 from prokaryotic sources is novel, and the observation that human IRG1 lost mitochondrial matrix localization is particularly interesting, with potentially significant implications for the study of itaconate biology. 

We thank the reviewer for appreciating the novelty of our study in exploring IRG1 evolution.  

Weaknesses: 

The protease protection assay was conducted with MTS-IRG1 but not with wild-type IRG1, which should also be tested. Moreover, no complementary methods, such as microscopy, were employed to validate localization. Beyond humans, the structure and localization of mouse IRG1, highly relevant given the widespread use of the mouse as a model for IRG1 functional studies, are not addressed. 

Regarding submitochondrial localization of IRG1, we want to draw attention to the published data that a protease protection assay for wild-type mammalian IRG1 has been performed by Lian et al. 2023 (Extended Data Fig. 4), which convincingly demonstrated an outer-mitochondrial membrane localization of endogenous mouse IRG1 in mouse DC2.4 cells upon LPS stimulation that induces IRG1 expression. 

Regarding complementary microscopy evidence, the same paper performed two-color,  DNA-paint super-resolution imaging to demonstrate an enrichment of IRG1 to mitochondria with a lack of co-localization of the inner membrane/matrix marker Cox IV. 

Given the direct visualization of sub-mitochondrial localization, we consider applying super-resolution microscopy to revisit the sub-mitochondrial localization of di[erent IRG1 constructs in the study.   

Reference:

Lian H, Park D, Chen M, Schueder F, Lara-Tejero M, Liu J, Galán JE. Parkinson's disease kinase LRRK2 coordinates a cell-intrinsic itaconate-dependent defence pathway against intracellular Salmonella. Nat Microbiol. 2023 Oct;8(10):1880-1895. doi: 10.1038/s41564-023-01459-y. Epub 2023 Aug 28. PMID: 37640963; PMCID: PMC10962312.

Finally, if itaconate is indeed synthesized outside the mitochondrial matrix to safeguard metabolic activity, it is not discussed how this reconciles with its reported inhibitory e[ect on SDH. 

We thank the excellent point raised by the reviewer. Indeed, itaconate has been proposed to inhibit matrix SDH exhibiting anti-inflammation function (Lampropoulou, Cell Metab 2016). While the mitochondrial transport of itaconate has not been fully characterized in vivo or in cells, a specific itaconate transport activity has been shown for the mitochondrial 2-oxoglutarate transporter OGC using in vitro proteoliposome system (Mills et al. Nature 2018). 

We plan to discuss this important point on mitochondrial itaconate transport in the revision. 

Reference: 

Lampropoulou V, Sergushichev A, Bambouskova M, Nair S, Vincent EE, Loginicheva E, Cervantes-Barragan L, Ma X, Huang SC, Griss T, Weinheimer CJ, Khader S, Randolph GJ, Pearce EJ, Jones RG, Diwan A, Diamond MS, Artyomov MN. Itaconate Links Inhibition of Succinate Dehydrogenase with Macrophage Metabolic Remodeling and Regulation of Inflammation. Cell Metab. 2016 Jul 12;24(1):158-66. doi: 10.1016/j.cmet.2016.06.004. Epub 2016 Jun 30. PMID: 27374498; PMCID: PMC5108454.  

Mills EL, Ryan DG, Prag HA, Dikovskaya D, Menon D, Zaslona Z, Jedrychowski MP, Costa ASH, Higgins M, Hams E, Szpyt J, Runtsch MC, King MS, McGouran JF, Fischer R, Kessler BM, McGettrick AF, Hughes MM, Carroll RG, Booty LM, Knatko EV, Meakin PJ, Ashford MLJ, Modis LK, Brunori G, Sévin DC, Fallon PG, Caldwell ST, Kunji ERS, Chouchani ET, Frezza C, Dinkova-Kostova AT, Hartley RC, Murphy MP, O'Neill LA. Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1. Nature. 2018 Apr 5;556(7699):113117. doi: 10.1038/nature25986. Epub 2018 Mar 28. PMID: 29590092; PMCID: PMC6047741.

Reviewer #2 (Public review): 

Summary: 

The authors are trying to explain how the metabolite itaconate evolved, since although it's involved in host defense, it can also limit mitochondrial function. They are trying to probe the trade-o[ between these two functions. 

Strengths: 

The evolutionary aspect is novel; this is the first time to my knowledge that the evolution of IRG1 has been analysed, and there are interesting findings here. The key finding appears to be that subcellular localisation is an important aspect, allowing host defense in some organisms without compromising bioenergetics. This is an interesting finding in the context of immunomebolism, although it needs extra analysis. 

Weaknesses: 

The work concerning sub-mitochondrial localisation is confusing and needs better analysis. 

We thank the reviewer for the constructive feedback. As in our response to reviewer 1, we want to draw attention to the published data in which the outer mitochondrial membrane localization of IRG1 has been demonstrated by protease protection assay and explored using super-resolution imaging by Lian et al. 2023 (Extended Data Fig. 4). Given the direct visualization of sub-mitochondrial localization by super-resolution imaging, we plan to revisit and to apply the method to di[erent IRG1 constructs used in the paper.

Reviewer #3 (Public review): 

Summary: 

IRG1 is highly expressed in activated human and mouse myeloid cells. It encodes the mitochondrial enzyme cis-aconitate decarboxylase 1 (ACOD1) that generates itaconate. Itaconate has anti-microbial activity and acts immunoregulatory by interfering with cellular metabolism, signaling to cytokine production, and multiple other processes. 

The authors perform a phylogenetic analysis of IRG1 to obtain insight into the evolution of itaconate biosynthesis. Combining BLAST with human IRG1 and a MmgE/Ptrp domain search, they find CAD in all domains of life, but the presence of IRG1 homologs is patchy in eukaryotes, indicating that itaconate biosynthesis is not essential. The phylogenetic analysis showed a more distant relationship of fungal and metazoan CAD/IRG1 to many prokaryotic sequences, suggesting independent acquisition of these metazoan and fungal CAD genes. In metazoans, three subbranches of paleo-IRG1 (in mollusks/early chordates) and two paralogous vertebrate forms (IRG1 and IRG1-like) were identified, with the latter derived from paleo-IRG1, and by genome duplication. While most jawed vertebrates have both IRG1 and IRG1L, metatherian and eutherian mammals have lost IRG1L and contain only IRG1. 

Interestingly, sequence analysis of both paralogues showed that many IRG1L genes contain an N-terminal mitochondrial targeting sequence (MTS) that is absent from most IRG1 sequences. Limited proteolysis of submitochondrial localization confirmed that zebrafish IRG1L is only sensitive to proteases in the presence of high Triton X-100, indicative of association with mitochondrial matrix. In contrast, a recent paper from the Galan lab (Lian 2003 Nature Microbiology) reported that human IRG1 is not localized to the mitochondrial matrix, although enriched in mitochondria. Here, the authors generated a matrix-targeted human IRG1 by adding the N-terminal MTS and found that it localizes to the matrix based on a limited proteolysis assay. The loss of MTS-containing IRG1L from most mammals appears, therefore, to indicate that itaconate generation is directed to the cytoplasm, potentially reducing inhibition of TCA cycle activity in the mitochondria. 

Next, the authors confirmed that the recombinant IRG1L protein has CAD activity in vitro. The last part of the manuscript addresses the expression of paleo-IRG1 in oysters and amphioxus, where they found high mRNA levels in oyster hemocytes which was further increased by poly(I:C), which was also the case in amphioxus tissues after feeding of LPS or poly(I:C), indicating a role for paleo-IRG1/itaconate in early metazoan innate immunity. 

Strengths 

(1) Phylogenetic perspective largely lacking so far in the IRG1/itaconate field. 

(2) Manuscript clearly written and understandable across disciplines. 

(3) Phylogenetic analyses complemented by biochemical and gene expression analyses to link to function. 

(4) Lack of MTS in IRG1 and change in localization from mitochondria, highly relevant antimicrobial and cellular e[ects of itaconate. 

We thank the reviewer for the positive comments with the strengths.  

Weaknesses: 

(1) Biochemical and functional analysis of di[erent CAD mRNA and proteins lacks depth. 

We plan to explore two types of experiments: 

First, we plan to purify di[erent CAD recombinant proteins; and if successful, we will test their in vitro enzymatic activity in synthesize itaconate. The positive data will also answer question (3) below.

Second, we plan to measure itaconate level in oyster hemocytes after PAMP stimulation, to demonstrate an in vivo itaconate production activity by paleo-IRG1. The data will also address question (4) below. 

(2) The submitochondrial localization assay lacks a native human IRG1 control. 

As in our response to reviewer 1, we believe Lian et al. 2023. provided strong evidence supporting an outer mitochondrial membrane localization of wild-type endogenous, mouse IRG1. Given the direct visualization using suer-resolution imaging, we plan to revisit submitochondrial localization of di[erent IRG1 constructs using super-resolution imaging. 

(3) CAD activity shown for IRG1L but not paleo-IRG1. 

We plan to purify di[erent CAD recombinant proteins; and if successful, we will test their in vitro enzymatic activity in producing itaconate.

(4) Itaconate production by early metazoans after PAMP stimulation? 

We plan to measure itaconate level in oyster hemocytes after PAMP stimulation, to demonstrate an in vivo itaconate production activity by paleo-IRG1.

(5) No measurement of energy metabolism (trade-o[s?). 

Because PAMP signaling might trigger other downstream e[ects that also impair mitochondrial function, for instance nitric oxide that inhibits complex IV, we plan to avoid PAMP condition and direct test the e[ect of itaconate production. We plan to compare the impact on mitochondrial bioenergetics, if the same CAD enzymes (thus with the same activity) can be expressed at the same level intra-mitochondrially and extramitochondrially, for instance in the case of MTS-hACOD1 and hACOD1.

Reviewer #1 (Public review):

Summary:

This paper investigates whether transformer-based models can represent sentence-level semantics in a human-like way. The authors designed a set of 108 sentences specifically to dissociate lexical semantics from sentence-level information and collected 7T fMRI data from 30 participants reading these sentences. They conducted representational similarity analysis (RSA) comparing brain data and model representations, as well as the human behavioral ratings. It is found that transformer-based models match brain representation better than a static word embedding baseline, which ignores word order, but fall short of models that encode the structural relations between words. The main contributions of this paper are:

(1) The construction of a sentence set that disentangles sentence structure from word meaning.

(2) A comprehensive comparison of neural sentence representations (via fMRI), human behavior, and multiple computational models at the sentence level.

Strengths:

(1) The paper evaluates a wide variety of models, including layer-wise analysis for transformers and region-wise analysis in the human brain.

(2) The stimulus design allows precise dissociation between lexical and sentence-level semantics. The RSA-based approach is empirically sound and intuitive.

(3) The constructed sentences, along with the fMRI and behavioral data, represent a valuable resource for studying sentence representation.

Weaknesses:

(1) The rationale behind averaging sentence embeddings across multiple transformer models (with different architectures and training objectives) is unclear. These transformer-based models have different training paradigms and model architectures, which may result in misaligned semantic spaces. The averaging operation may dilute the distinct sentence representations learned by each model, potentially weakening the overall semantic encoding for sentences. Please clarify this choice or cite supporting methodology.

(2) All structure-sensitive models discussed incorporate semantics to some extent. Including a purely syntactic baseline, such as a model based on context-free grammar, would help confirm the importance of syntactic structures.

(3) In Figure 2, human behavioral judgments show weak correlations with neural data, and even fall below those of computational models, suggesting the behavioral judgments may not reflect the sentence structures in a brain-like way. This discrepancy between behavioral and neural data should be clarified, as it affects the interpretation of the results.

(4) To better contextualize model and neural performance, sentence similarity should be anchored to a notion of semantic "ground truth", such as the matrix shown in Figure 1a. Comparing this reference with human judgments, brain responses, and model similarities would help establish an upper bound.

(5) The structure of this paper is confusing. For instance, Figure 5 is cited early but appears much later. Reordering sections and figures would enhance readability.

(6) While the analysis is broad and comprehensive, it lacks depth in some respects. For instance, it remains unclear what specific insights are gained from comparing across brain regions (e.g., whole brain, language network, and other subregions). Similarly, the results of simple-average and group-average RSA appear quite similar and may not advance the interpretation.

(7) While explaining the grid-like pattern due to sentence length is important, this part feels somewhat disconnected from the central question of this paper (word order). It might be better placed in supplementary material.

Reviewer #2 (Public review):

Summary:

The paper used fMRI data while reading a set of sentences. The sentences are designed to disentangle syntax from meaning. RSA was performed using voxel activations and a variety of language models. The results show that transformers are inferior to models with explicit syntactic representation in terms of matching brain representations.

Strengths:

(1) The study controls for some variables that allow for an investigation of sentence structure in the brain. This controlled setting has an advantage over naturalistic stimuli in targeting more specific linguistic phenomena.

(2) The study combines fMRI data with behavioral similarity ratings and a variety of language models (static, transformers, graph-based models).

Weaknesses:

(1) The stimuli are not fully controlled for lexical content across conditions. Residual lexical differences between sentences could still influence both brain and model similarity patterns. To more cleanly isolate syntactic effects, it would be useful to systematically vary only a single structural element while keeping all other lexical content constant (e.g., the boy kicked the ball / the ball kicked the boy). It would be better to engage more with the minimal pair paradigm, which is widely used in large language model probing research.

(2) The comparisons are done across fundamentally different model types, including static embeddings, graph-based parsers, and transformers. The inherent differences in dimensionality and training objectives might make the conclusion drawn from RSA inconclusive. Transformer embeddings typically occupy much higher-dimensional, anisotropic representational spaces, and their similarity structure may reflect richer, more heterogeneous information than models explicitly encoding semantic roles. A lower RSA correlation in this study does not necessarily imply that transformers fail to encode syntactic information; rather, they may represent additional aspects of meaning or context that diverge from the narrow structural contrasts probed here.

(3) The interpretation of the RSA correlation largely depends on the understanding of models. The authors suggest that because hybrid models correlate better than transformers, this implies that transformers are inferior at representing syntax. However, this is not a direct test of syntactic ability. Transformers may encode syntactic information, but it may not be expressed in a way that aligns with the RSA paradigm or the chosen stimuli. RSA does not reveal what the model encodes, and the models might achieve a good correlation for non-syntactic reasons (e.g., length of sentence, orthographic similarity, lexical features).

Reviewer #3 (Public review):

Summary:

Large Language Models have revolutionized Artificial Intelligence and can now match or surpass human language abilities on many tasks. This has fueled interest in cognitive neuroscience in exposing representational similarities between Language Models and brain recordings of language comprehension. The current study breaks from this mold by: (1) Systematically identifying sentence structures for which brain and Large Language Model representations diverge. (2) Demonstrating that brain representations for these sentences can be better accounted for by a model structured by the semantic roles of words in the sentence. As such, the study may now fuel interest in characterizing how Large Language Models and brain representations differ, which may prompt new, more brain-like language models.

Strengths:

(1) This study presents a bold and solid challenge to a literature trend that has touted similarities between Transformer models and human cognition based on representational correlations with brain activity. This challenge is substantiated by identifying sentences for which brain and model representations of sentences diverge and explaining those divergences using models structured by semantic roles/syntax.

(2) This study conducts a rigorous pre-registered analysis of a comprehensive selection of the state-of-the-art Large Language Models, on a controlled sentence comprehension fMRI dataset. The analysis is conducted within a Representation Similarity framework to support similarity comparisons between graph structures and brain activity without needing to vectorize graphs. Transformer models are predicted and shown to diverge from brain representations on subsets of sentences with similar word-level content but different sentence structures.

(3) The study introduces a 7T fMRI sentence comprehension dataset and accompanying human sentence similarity ratings, which may be a fruitful resource for developing more human-like language models. Unlike other model-based sentence datasets, the relation between grammatical structure and word-level content is controlled, and subsets of sentences for which models and brains diverge are identified.

Weaknesses:

(1) The interpretation of findings is nuanced. Although Transformers underperform as brain models on the critical subsets of controlled sentences, a Transformer outperforms all other models when evaluated on the union of all sentences when both word-level content and structure vary. Transformers also yield equivalent or better models of human behavioral data. Thus, although Transformers have demonstrable flaws as human models, which are pinpointed here, in the general case, (some) Transformers are more human-like than the other models considered.

(2) There may be confounds between the critical sentence structure manipulations and visual representations of sentence stimuli. This is inconvenient because activation in brain regions that process semantics tends to partially correlate with visual cortex representations, and computational models tend to reflect the number of words/tokens/elements in sentences. Although the study commendably controls for confounds associated with sentence length, there could still be residual effects that remain. For instance, the Graph model correlates most strongly with the visual cortex despite these sentence length controls.

(3) Sentence similarity computations are emphasized as the basis for unifying comparative analyses of graph structures and vector data. A strength of this approach is that correlation is not always the ideal similarity metric. However, a weakness is that similarity computations are not unified across models. This has practical consequences here because different similarity metrics applied to the same model produce positive or negative correlations with brain data.

Author response:

We thank the reviewers for their insightful comments on our manuscript. Here we briefly highlight our responses to several issues raised by reviewers, and also provide a summary of planned changes to be made with the next draft.

Reviewer 1:

(1) The reviewer questions the rationale for averaging sentence embeddings across different models. However, our method involves computing correlations separately for each model, then averaging the correlations. We also report model correlations for each model separately in Fig S2. We will clarify this in our revised manuscript.

(2) We agree with the reviewer that including a context-free grammar model as a comparison would be informative. We will incorporate this in the revised manuscript.

(3) The reviewer raises questions about the low correlation between behavioural and brain similarities. While the behavioural judgements are made by different participants and involve a different task than the neuroimaging results, nonetheless we agree the difference is surprising and warrants more detailed consideration. We will provide additional discussion of the relationship between behavioural judgements and brain data in the revised manuscript.

(4) The reviewer suggests contrasting our models with a ‘semantic ground truth’, as in our design matrix shown in Fig 1. While our design matrix served as the basis for constructing a set of stimuli with systematic modifications, we respectfully suggest that it should not be regarded as a ‘semantic ground truth’. In particular, sentence pairs within each category will not have the same degrees of semantic similarity since the words and context differ across sentences in a graded manner. Furthermore, while we anticipated ‘different’ sentence pairs would be less similar than ‘swapped’ sentence pairs, and that within each of the six block diagonals the ‘modified’ or ‘substituted’ sentence pairs would be the most similar, we did not have any prediction about the magnitude of these differences. Our goal was to construct a set of sentence pairs which spanned a range of semantic similarities, and allowed for dissociation between lexical similarity and overall similarity in meaning. The design matrix is not intended to represent a ‘ground truth’ that human judgements or brain representations would be expected to conform with.

(5) In the revised draft we will modify the location of Fig. 5 so that it flows better with the text.

(6) We agree that the discussion of the differences between brain regions could be expanded. We will include this in the revised version of our manuscript. The reviewer questions our inclusion of the simple-average and group-average RSA analysis as they show similar results. We included both analyses in line with our preregistration, and also because we believe the fact that two distinct approaches to analyzing the data yield similar results strengthens our conclusions.

(7) We believe that the grid-like pattern in the RSA results is an important unexpected finding that warrants discussion in the main manuscript.

Reviewer 2:

(1) The reviewer argues that our stimuli do not fully control for lexical content across conditions, and that a more appropriate paradigm may be to utilise minimal pairs in which only a single variable of interest (such as sentence structure) is modified. We agree that most of our sentence pairs do not constitute minimal pairs, however this was not our objective. Our study design aimed to synthesise traditional minimal pair approaches with more recent research paradigms using naturalistic stimuli. As such, we selected stimuli which are more complex and contain more variable features than traditional minimal pair studies, but which also are tailored to highlight differences which are of particular theoretical interest. Because we are interested in comparing the effects of multiple sentence elements and semantic roles, a systematic pairwise comparison of minimal pairs is not necessarily optimal. Instead, we designed our stimuli to leverage the advantage of fMRI in that we can measure the brain representations corresponding to each sentence, and hence can conduct a full series of pairwise comparisons of sentence representations. Most of these comparisons will not be between minimal pairs, but we selected sentences so as to provide a range of semantic similarities (low to high), while also providing for semantic contrasts of theoretical interest (such as the ‘swapped’ and ‘substituted’ sentence pairs). We do not claim this approach to be universally superior to a minimal pair approach, but we do believe our novel approach provides additional insights and a new perspective on semantic representation relative to minimal pair studies. We will add additional detail in the revised manuscript providing additional explanation for how stimuli were chosen, and contrasting this with minimal pair approaches.

(2) The reviewer notes that low RSA correlations do not imply that transformers fail to encode syntactic information. We acknowledge this in our discussion (page 10), where we also highlight that our focus is not on whether transformers encode such information, but rather what transformer representations can tell us about how sentence structure is represented in the brain. Our results indicate that transformer embeddings do not have the same geometric properties as brain representations of sentence meaning, at least for certain types of sentences where lexical information is insufficient to determine overall meaning. The reviewer also notes that transformer embeddings are highly anisotropic, however we adjust for this by normalising each feature as discussed on page 14. Finally, the reviewer notes that the transformers we examine differ in architecture and training objectives. This is not critical for our study because we are not seeking to determine which architecture or training objectives are best. Our goal is simply to compare a range of approaches and see which, if any, have similar sentence representations to those formed by the brain. In fact, our results indicate that architecture and training regime make relatively little difference for our stimuli.

(3) The reviewer argues that RSA correlations do not measure the extent to which a model encodes syntactic information. This is very similar to the previous point. We do not claim that our results show that transformers do not encode syntactic information. Rather, our claim is that sentence embeddings derived from transformers have different geometric properties to brain representations, and that brain representations are better described by models explicitly representing key semantic roles. From this we conclude that, at least for the sentences we present, the brain is highly sensitive to semantic roles in a way that transformer representations are not (at least to the same extent). We also respectfully disagree with the reviewer’s suggestions that sentence length and orthographic or lexical similarities may drive model correlations with brain activity. As we discuss on page 19, we explicitly control for differences in sentence length when computing correlations. Our process for constructing our sentence set also controls for lexical similarity by generating pairs of sentences with all or mostly the same words but different orderings. We did not explicitly address orthographic similarity, but this will be strongly correlated with lexical similarity.

Reviewer 3:

(1) The reviewer emphasises the need for nuance in our conclusions, given that some of the transformers achieve higher correlations when assessed over the full set of sentences. We agree with this comment, and will modify the discussion section in the revised manuscript to address this point. Having said that, we would like to note one of the disadvantages of transformers as a model of mind or brain representations is that they are largely a ‘black box’ whose workings are poorly understood. One advantage of hybrid models like our simple semantic role model is that they can be much easier to interpret, thereby enabling them to be used to determine which features are most important for brain representations of sentence meaning, and what mechanisms are used to combine individual words into a full sentence. Given their relative simplicity and interpretability, we believe hybrid models have considerable value as scientific tools, even in cases where they achieve comparable correlations to transformers. We will highlight this issue more clearly in our revised manuscript.

(2) The reviewer notes that despite our existing controls, residual confounds of sentence length may remain. We agree that this is a potential issue, and will add discussion to the revised manuscript. We also will present further supplementary analyses which we believe indicate that sentence length effects do not drive our main results. At the same time, we believe the fact that our results are robust to simultaneously controlling for sentence length and the ‘minimum length effect’ (Fig. S5) indicates they are not primarily driven by sentence length effects.

(3) The reviewer notes that the method for computing similarities differs between the vector-based (mean and transformer) models, and the hybrid and syntax-based models, thereby potentially adding an additional confound to our results. We agree that this is a potential limitation, and our correlations should always be understood as applying to a model paired with a similarity metric. However, we believe that this is mostly unavoidable when comparing different formalisms. An alterative approach of first embedding a graph into a vector and then training an encoding model on the graph embeddings has a similar limitation of being dependent not just on the graph representation, but also on the way it was embedded into a vector and the way the encoding model was trained. Arguably this process is more opaque than similarity methods, since it is unclear to what extent the graph embeddings preserve the logic and properties of a graph-based representation. Further, it not clear whether there is any single method which can overcome the difficulty of comparing distinct formalisms for representing semantics. The reviewer also highlights how the correlations measured for the syntax model differ greatly depending on whether the Smatch or WWLK similarity metrics are used. We believe this highlights the need for careful examination of commonly used graph similarity metrics, as has been noted in previous research. We will include additional discussion of this issue in our revised manuscript.

Reviewer #1 (Public review):

The authors describe a new computational pipeline designed to identify smFISH probes with improved RNA detection compared to preexisting approaches. smFISH is a powerful and relatively straightforward technique to detect single RNAs in cells at subcellular resolution, which is critical for understanding gene expression regulation at the RNA level. However, existing methods for designing smFISH oligos suffer from several limitations, including off-target binding that produces high background signals, as well as a restricted number of probes that are sufficiently specific to target shorter-than-average mRNAs. To address these challenges, the authors developed TrueProbes, a computational method that aims to minimize off-target-mediated background fluorescence.

Overall, the study addresses a technically relevant problem. If improved, this would allow researchers to study gene expression regulation more effectively using single-molecule FISH. However, based on the current presentation of data, it is not yet clear that TrueProbes offers significant advantages over preexisting pipelines. In the following section, I describe some concerns, which should be adequately addressed.

Major Comments:

(1) The manuscript currently presents only one example in which different pipelines were tested to generate probes (targeting ARF4). While the images suggest that both TrueProbes and Stellaris outperform the other pipelines, the comparison is potentially misleading because the number of probes used differs substantially. I recommend that the authors include at least three independent examples in which an equal number of probes are designed across pipelines, so that signal-to-noise can be assessed in a controlled and comparable way. This would allow the probe number to be held constant while directly evaluating performance.

(2) It is also unclear how many biological replicates were performed for the ARF4 experiments. If only a single replicate was included, it is difficult to conclude that TrueProbes consistently outperforms other pipelines in a robust and reproducible manner. I suggest the authors include data from at least three biological replicates with appropriate statistical analysis, and ideally extend this to additional smFISH targets as outlined in Comment 1.

(3) No controls are presented to demonstrate that the TrueProbes-designed smFISH spots are specifically detecting ARF4. The current experiment primarily measures signal-to-noise, but it remains possible that some detected spots do not correspond to ARF4 mRNAs. Since one of the major criteria used by TrueProbes is to limit cross-hybridization, the authors should perform ARF4 knockdown experiments and demonstrate that nearly all ARF4 smFISH signal is lost. A similar approach should be applied to the additional examples recommended in Comment 1.

(4) In the limitations of the study, the authors note that "RNA secondary and tertiary structures are not included, which may lead to inaccuracies if binding sites are structurally occluded." However, I am not convinced that this is a true limitation, since formamide in the smFISH protocol should denature secondary structures and allow oligo access to the RNA. I recommend that the authors comment on this point and clarify whether secondary structure poses a practical limitation in smFISH probe design.

(5) The authors also correctly acknowledge in their limitations that "RNA-protein interactions, which can modulate accessibility of the transcript, are not modeled." I suggest referencing relevant studies on this issue, particularly Buxbaum et al. (2014, Science), which would provide important context.

Reviewer #2 (Public review):

Summary:

Hughes et al present a new single-molecule RNA fluorescence in situ hybridization (smFISH) probe design software, termed "TrueProbes" in this manuscript. They claim that all existing smFISH (and variants) probe design software packages have limitations that ultimately impact experimental performance. The author's claim to address the majority of these limitations in TrueProbes by introducing multiple computational steps to ensure high-quality probe design. The manuscript's goal is clear, and the authors provide some evidence by designing and targeting one gene. Overall, the manuscript lacks rigorous evidence to support the claims, does not demonstrate its suitability for a variety of smFISH-type experiments, and some of the provided quantification data are unclear. While TrueProbes clearly has potential, more data is required, or the authors should tone down the claims.

Strengths:

(1) The problem is well-articulated in the abstract and the introduction.

(2) Figures 3 and 4 follow a consistent color scheme where each probe design method has its own color, which helps the reader visually compare methods.

(3) The authors compared multiple probe design software packages both computationally and experimentally.

(4) TrueProbes does produce visually and quantitatively better results when compared to 2 of the 4 existing smFISH probe design packages (Paintshop and MERFISH panel designer).

(5) The authors introduce a comprehensive steady-state thermodynamic model to help optimally guide probe design.

Weaknesses:

(1) The abstract describes the problem well and introduces the solution (the TrueProbes software), but fails to provide specific ways in which the TrueProbes software performs better. The authors state that "...[TrueProbes] consistently outperformed alternatives across multiple computational metrics and experimental validation assays", but specific, quantitative evidence of improved performance would strengthen the statement.

(2) The text claims that TrueProbes outperforms all other probe design software, but Figure 3 indicates that TrueProbes has neither the greatest number of on-target binding nor the lowest number of off-target binding. The data in Figure 3 does not support the claims made in the text. Specifically, the authors claim that "RNA FISH Experimental Results Demonstrate that Off Target and Binding Affinity Inclusive Probe Design Improve RNA FISH Signal Discrimination" (lines 217-218). However, despite their claim that Stellaris and Oligostan-HT produce more off-target probes when evaluated with the TrueProbes framework, the experiment results are nearly identical. The authors should consider modifying their claims or performing new experiments that more clearly demonstrate their claims.

(3) The bar graphs in Figure 3 do not seem to agree with the probability graphs in Figure 4. For example, Figure 3 indicates that Stellaris probes have higher off-target binding than TrueProbes; however, in Figure 4, their probability graphs lie almost on top of each other.

(4) The authors performed validation for only one gene (ARF4), because "...it had the highest gene expression (in TPM units) and the fewest isoforms among all candidate genes for the Jurkat cell line" (lines 176-177). While the results do look good, this is a minimal use case and does not really showcase the power of their method. One experiment that could be helpful would be two-color (or more) smFISH in tissue, where the chances for off-target binding contributing to higher errors are much greater than in an adherent cell line.

(5) A common strategy for both smFISH and highly multiplexed methods is to use secondary DNA oligos with dye molecules instead of direct conjugation. Given that this is a primary design goal of PaintSHOP and the Zhuang lab's MERFISH probe design code, it would be helpful to demonstrate that TrueProbes can design a two-layer probe strategy for high-quality RNA-FISH labeling.

(6) The authors claim, "For every probe set, TrueProbes can simulate expected smRNA FISH outcomes including optimal probe, RNA, and salt concentrations and optionally account for probe secondary structure, hybridization temperature, multiple targets, fluorophore choice, DNA, nascent RNA, and photon count statistics (Figures S2A, S2B). The model can be used to generate predictions for temperature and cell line sensitivity, multi-target discrimination, multiple fluorophore colocalization; when provided transcript expression levels and probe/background intensity, it can start to generate predictions for spot intensity, background, signal to noise ratio, and false negative rates (Figure S2C)." (lines 156-163). Figure S2 is a flow chart and does not provide evidence for any of these items. The authors should provide evidence for these claims, either as a figure or an example script in their software repository. If that is not possible, then it should be removed.

(7) All thermodynamic equations are performed at steady state. The authors do not justify this assumption, and there is no discussion of the potential impacts of either low molecule numbers or violations of the well-mixed assumption. Can the authors please include a discussion on the potential impacts non non-steady state dynamics?

Reviewer #3 (Public review):

Summary:

This manuscript introduces a new platform termed "TrueProbes" for designing mRNA FISH probes. In comparison to existing design strategies, the authors incorporate a comprehensive thermodynamic and kinetic model to account for probe states that may contribute to nonspecific background. The authors validate their design pipeline using Jurkat cells and provide evidence of improved probe performance.

Strengths:

A notable strength of TrueProbes is the consideration of genome-wide binding affinities, which aims to minimize off-target signals. The work will be of interest to researchers employing mRNA FISH in certain human cell lines.

Weaknesses:

However, in my view, the experimental validation is not sufficient to justify the broad claims of the platform. Given the number of assumptions in the model, additional experimental comparisons across probe design methods, ideally targeting transcripts with different expression levels, would be necessary to establish the general superiority of this approach.

Author response:

Reviewer #1 (Public Review):

The authors describe a new computational pipeline designed to identify smFISH probes with improved RNA detection compared to preexisting approaches. smFISH is a powerful and relatively straightforward technique to detect single RNAs in cells at subcellular resolution, which is critical for understanding gene expression regulation at the RNA level. However, existing methods for designing smFISH oligos suffer from several limitations, including off-target binding that produces high background signals, as well as a restricted number of probes that are sufficiently specific to target shorter-than-average mRNAs. To address these challenges, the authors developed TrueProbes, a computational method that aims to minimize off-target-mediated background fluorescence.

Overall, the study addresses a technically relevant problem. If improved, this would allow researchers to study gene expression regulation more effectively using single-molecule FISH. However, based on the current presentation of data, it is not yet clear that TrueProbes offers significant advantages over preexisting pipelines. In the following section, I describe some concerns, which should be adequately addressed.

Major Comments:

(1) The manuscript currently presents only one example in which different pipelines were tested to generate probes (targeting ARF4). While the images suggest that both TrueProbes and Stellaris outperform the other pipelines, the comparison is potentially misleading because the number of probes used differs substantially. I recommend that the authors include at least three independent examples in which an equal number of probes are designed across pipelines, so that signal-to-noise can be assessed in a controlled and comparable way. This would allow the probe number to be held constant while directly evaluating performance.

This is an important observation. We have already addressed this issue in Figures 3E-G and Supplementary Figure 4E-G, where we plotted the number of OFF-targets for each ON-target probe. If we select longer genes to ensure an equal number of designed probes with strong signals, we will still end up with the same number of ON-target probes. Consequently, Figures 3B-D and 3E-G would show similar trends, albeit with different values on the y-axis. Additionally, we will conduct an analysis using Stellaris at its highest probe design stringency setting to compare the software under its strictest design conditions. Additional experiments are outside the scope of the current manuscript.

(2) It is also unclear how many biological replicates were performed for the ARF4 experiments. If only a single replicate was included, it is difficult to conclude that TrueProbes consistently outperforms other pipelines in a robust and reproducible manner. I suggest the authors include data from at least three biological replicates with appropriate statistical analysis, and ideally extend this to additional smFISH targets as outlined in Comment 1.

Three biological replicates were utilized for the ARF4 experiments. As stated in the original submission, the average data from all three replicates is presented in Figure 4, while the data for each individual replicate can be found in Figure S5. Statistical analyses were conducted for both the pooled data in Figure 4 and the individual data in Figure S5. The results of all statistical calculations are detailed in Supplemental Table 1. We will update the text to clearly indicate the number of biological replicates and the outcomes of the statistical analysis.

(3) No controls are presented to demonstrate that the TrueProbes-designed smFISH spots are specifically detecting ARF4. The current experiment primarily measures signal-to-noise, but it remains possible that some detected spots do not correspond to ARF4 mRNAs. Since one of the major criteria used by TrueProbes is to limit cross-hybridization, the authors should perform ARF4 knockdown experiments and demonstrate that nearly all ARF4 smFISH signal is lost. A similar approach should be applied to the additional examples recommended in Comment 1.

Thank you for your suggestion. Currently, we lack the expertise in our lab to conduct such experiments, so they are beyond the scope of this manuscript. However, we will create additional supplementary figures to demonstrate that the likelihood of false positives is low, based on the assumption that current publicly available BLAST algorithms, genome annotations, and reference transcription expression data are accurate.

We will include a comparison in our supplementary materials showing the off-target RNA that can bind the highest number of probes simultaneously for each software. Additionally, we will perform a correlation analysis to illustrate the relationship between spot intensity for different software and the number of probes they design. This will help us estimate how the number of probes bound to RNA correlates with expected spot intensity ranges.

Using this information, along with autofluorescence background intensity measurements from no-probe controls, we will estimate the minimum number of probes that need to bind to targets to be detected as single spots. If this minimum is higher than the maximum number of simultaneous off-target probe bindings, we anticipate that the detected spot signal will primarily reflect ARF4 rather than other transcripts.

(4) In the limitations of the study, the authors note that "RNA secondary and tertiary structures are not included, which may lead to inaccuracies if binding sites are structurally occluded." However, I am not convinced that this is a true limitation, since formamide in the smFISH protocol should denature secondary structures and allow oligo access to the RNA. I recommend that the authors comment on this point and clarify whether secondary structure poses a practical limitation in smFISH probe design.

Thank you for pointing this out. We will revise the manuscript to clarify: "We did not include RNA secondary and tertiary structures in the model because the use of formamide in RNA-FISH experiments denatures these structures, allowing oligonucleotides to access the RNA."

(5) The authors also correctly acknowledge in their limitations that "RNA-protein interactions, which can modulate accessibility of the transcript, are not modeled." I suggest referencing relevant studies on this issue, particularly Buxbaum et al. (2014, Science), which would provide important context.

Thank you for highlighting the literature that supports this limitation. We will include Buxbaum et al. (2014, Science) and additional studies that discuss how RNA-protein interactions can affect RNA-FISH experiments.

Reviewer #2 (Public review):

Summary:

Hughes et al present a new single-molecule RNA fluorescence in situ hybridization (smFISH) probe design software, termed "TrueProbes" in this manuscript. They claim that all existing smFISH (and variants) probe design software packages have limitations that ultimately impact experimental performance. The author's claim to address the majority of these limitations in TrueProbes by introducing multiple computational steps to ensure high-quality probe design. The manuscript's goal is clear, and the authors provide some evidence by designing and targeting one gene. Overall, the manuscript lacks rigorous evidence to support the claims, does not demonstrate its suitability for a variety of smFISH-type experiments, and some of the provided quantification data are unclear. While TrueProbes clearly has potential, more data is required, or the authors should tone down the claims.

We appreciate the reviewer’s thoughtful feedback. We will revise the text to ensure that all claims are backed by computational or experimental evidence. For claims that do not have supporting results, we will relocate them to the discussion section as potential future extensions. Since our probe design is open access, both we and the community can further develop our codes as needed.

Strengths:

(1) The problem is well-articulated in the abstract and the introduction.

(2) Figures 3 and 4 follow a consistent color scheme where each probe design method has its own color, which helps the reader visually compare methods.

(3) The authors compared multiple probe design software packages both computationally and experimentally.

(4) TrueProbes does produce visually and quantitatively better results when compared to 2 of the 4 existing smFISH probe design packages (Paintshop and MERFISH panel designer).

(5) The authors introduce a comprehensive steady-state thermodynamic model to help optimally guide probe design.

We like to thank the reviewer for pointing out the strength of the manuscript.

Weaknesses:

(1) The abstract describes the problem well and introduces the solution (the TrueProbes software), but fails to provide specific ways in which the TrueProbes software performs better. The authors state that "...[TrueProbes] consistently outperformed alternatives across multiple computational metrics and experimental validation assays", but specific, quantitative evidence of improved performance would strengthen the statement.

Thank you for acknowledging the clarity of the abstract and introduction. We will revise the abstract to provide more specific details on how TrueProbes outperforms other software. Additionally, we will include specific computational and experimental metrics that demonstrate TrueProbes' improved performance compared to other software.

(2) The text claims that TrueProbes outperforms all other probe design software, but Figure 3 indicates that TrueProbes has neither the greatest number of on-target binding nor the lowest number of off-target binding. The data in Figure 3 does not support the claims made in the text. Specifically, the authors claim that "RNA FISH Experimental Results Demonstrate that Off Target and Binding Affinity Inclusive Probe Design Improve RNA FISH Signal Discrimination" (lines 217-218). However, despite their claim that Stellaris and Oligostan-HT produce more off-target probes when evaluated with the TrueProbes framework, the experiment results are nearly identical. The authors should consider modifying their claims or performing new experiments that more clearly demonstrate their claims.

In Figure 3, we aim to convey two main points. 

The first point is to compare the number of ON-target probes designed by each software using their most stringent design criteria (Figure 3A). Currently, we are using a medium strict design criterion for Stellaris (level 3). As shown in the new supplementary figure XX, when we apply the most stringent design criteria for Stellaris (level 5), the number of ON-target probes decreases to XX probes. This clearly indicates that, based on theoretical calculations, TrueProbes can design more probes than any of its competitors.

The second point is to compare the number of OFF-targets produced by each probe design. To illustrate this, we used two different metrics. In Figures 3B-D, we compare the total number of probes bound to OFF-target RNA. However, since each software generates a different number of ON-target probes, the number of OFF-targets may vary simply due to the differences in ON-target probe counts. Therefore, we introduced a second metric to compare OFF-targets. In Figures 3E-G, we present the number of OFF-targets normalized by the number of ON-targets. Using this metric, TrueProbes shows the lowest number of OFF-targets. We will updat the manuscript to clarify this point.

Regarding the experiments and their comparison to theoretical calculations: The theoretical calculations consider only the reference DNA and RNA genomes along with the oligonucleotide sequences for the probes. We then use a thermodynamic model to identify ON- and OFF-targets. Thus, these theoretical calculations represent an upper bound on the maximum possible number of ON-targets and the minimum number of OFF-targets. All other design software evaluated in this manuscript relies on the same or less reference data and makes certain assumptions. None of these methods quantitatively compare their computational designs with experimental results; they simply design probes based on unverified assumptions, conduct experiments, and present spot data to conclude that their probe designs are effective.

We will update the manuscript to clarify the goals of the theoretical model and its relationship to the experiments. Future work will be necessary to enhance our theoretical model to fully account for additional aspects of RNA-FISH experiments (e.g., formaldehyde crosslinking, hybridization conditions, washing steps) to better predict the experimental data shown in Figure 4. We will also adjuste our claims to accurately reflect the current capabilities of our theoretical framework and its relation to experimental outcomes.

(3) The bar graphs in Figure 3 do not seem to agree with the probability graphs in Figure 4. For example, Figure 3 indicates that Stellaris probes have higher off-target binding than TrueProbes; however, in Figure 4, their probability graphs lie almost on top of each other.

The predictions in Figure 3 regarding the number of probe off-target binding events, based on reference gene expression data, do not necessarily encompass all the information required to predict RNA-FISH signal intensity. Therefore, these predictions should not be expected to translate directly into the experimental results shown in Figure 4, particularly concerning the background signal.

While our software aims to minimize off-target probe binding, this does not automatically lead to a reduction in off-target background signal. Numerous other factors influence the spot background and overall signal-to-noise ratio (SNR) performance, beyond just probe-target binding interactions. Although we strive to minimize off-target background through probe binding, this approach is not designed to directly predict the SNR. Extending the computational analysis of probe binding dynamics to RNA-FISH signal intensity dynamics is beyond the scope of this study.

We have revised our text to clearly separate computational results from experimental results into two distinct sections. We will use different terminology to describe the outcomes of computational performance versus experimental performance, reducing potential confusion between these two aspects. Additionally, we will clarify our conceptual overview in Figure 1 regarding traditional probe design limitations related to sensitivity and specificity. We will specify how the signal from the number of probes bound to ON-target RNA, relative to those bound to OFF-targets and cellular autofluorescence, translates—either linearly or non-linearly—into the signal-to-noise ratio.

(4) The authors performed validation for only one gene (ARF4), because "...it had the highest gene expression (in TPM units) and the fewest isoforms among all candidate genes for the Jurkat cell line" (lines 176-177). While the results do look good, this is a minimal use case and does not really showcase the power of their method. One experiment that could be helpful would be two-color (or more) smFISH in tissue, where the chances for off-target binding contributing to higher errors are much greater than in an adherent cell line.

Thank you for highlighting these valuable experiments. Currently, our lab lacks the expertise to generate tissue samples beyond culturing cells. Additionally, implementing a two-color probe design in tissues containing different cell types with unknown expression levels presents further challenges. Due to these limitations, designing and conducting two-color experiments in tissue samples is beyond the scope of the current manuscript, but we plan to pursue this in the future.

(5) A common strategy for both smFISH and highly multiplexed methods is to use secondary DNA oligos with dye molecules instead of direct conjugation. Given that this is a primary design goal of PaintSHOP and the Zhuang lab's MERFISH probe design code, it would be helpful to demonstrate that TrueProbes can design a two-layer probe strategy for high-quality RNA-FISH labeling.

Thank you for bringing this to our attention. TrueProbes is currently designed and tested specifically for primary smRNA-FISH probes. Our focus is on demonstrating a new approach to designing these probes without the added complexities of secondary probes and multiplexing. Future work will expand on this foundation to incorporate secondary probe detection and transcript multiplexing.

(6) The authors claim, "For every probe set, TrueProbes can simulate expected smRNA FISH outcomes including optimal probe, RNA, and salt concentrations and optionally account for probe secondary structure, hybridization temperature, multiple targets, fluorophore choice, DNA, nascent RNA, and photon count statistics (Figures S2A, S2B). The model can be used to generate predictions for temperature and cell line sensitivity, multi-target discrimination, multiple fluorophore colocalization; when provided transcript expression levels and probe/background intensity, it can start to generate predictions for spot intensity, background, signal to noise ratio, and false negative rates (Figure S2C)." (lines 156-163). Figure S2 is a flow chart and does not provide evidence for any of these items. The authors should provide evidence for these claims, either as a figure or an example script in their software repository. If that is not possible, then it should be removed.

The supplemental information of the article will be updated to include figures that illustrate predictions for each capability currently offered by TrueProbes, along with the scripts used to generate these predictions. Any capabilities that do not have corresponding scripts will be removed from this section and instead referred to as potential improvements or future additions to the TrueProbes framework in the discussion section.

(7) All thermodynamic equations are performed at steady state. The authors do not justify this assumption, and there is no discussion of the potential impacts of either low molecule numbers or violations of the well-mixed assumption. Can the authors please include a discussion on the potential impacts non non-steady state dynamics?

Thermodynamic equations are calculated at steady state because RNA-FISH hybridization reactions typically last from eight to twenty hours. This duration allows probes adequate time to localize to their targets and reach binding equilibrium, based on current estimates of DNA oligonucleotide association and dissociation rate constants. We will address the potential violation of the well-mixed assumption in the assumptions and limitations section, specifically discussing how RNA localization can affect the spatial distribution of both on-target and off-target probes within cells, which may disrupt the well-mixed condition.

Low molecule numbers are not a significant concern, as probe DNA oligonucleotide concentrations in RNA-FISH protocols are much higher than the number of transcripts present in cells, by several orders of magnitude.

The assumptions and limitations section will be revised to clearly state: “Probe hybridization reactions were computed at steady state because most RNA-FISH protocols utilize probe hybridization incubation steps lasting over eight hours, which should provide sufficient time to reach equilibrium based on current estimates of forward and reverse reaction rate constants. Predictions from the equilibrium model may be less accurate for RNA-FISH experiments with shorter hybridization times, where non-steady state dynamics can result in different transient outcomes depending on the duration of hybridization.”

Reviewer #3 (Public review):

Summary:

This manuscript introduces a new platform termed "TrueProbes" for designing mRNA FISH probes. In comparison to existing design strategies, the authors incorporate a comprehensive thermodynamic and kinetic model to account for probe states that may contribute to nonspecific background. The authors validate their design pipeline using Jurkat cells and provide evidence of improved probe performance.

Strengths:

A notable strength of TrueProbes is the consideration of genome-wide binding affinities, which aims to minimize off-target signals. The work will be of interest to researchers employing mRNA FISH in certain human cell lines.

Weaknesses:

However, in my view, the experimental validation is not sufficient to justify the broad claims of the platform. Given the number of assumptions in the model, additional experimental comparisons across probe design methods, ideally targeting transcripts with different expression levels, would be necessary to establish the general superiority of this approach.

We will revise our text to make our claims more specific and clearer, avoiding overgeneralizations and ensuring that all claims are adequately supported by the data we present.

Reviewer #1 (Public review):

Summary:

The manuscript reports a series of experiments designed to test whether optogenetic activation of infralimbic (IL) neurons facilitates extinction retrieval and whether this depends on animals' prior experience. In Experiment 1, rats underwent fear conditioning followed by either one or two extinction sessions, with IL stimulation given during the second extinction; stimulation facilitated extinction retrieval only in rats with prior extinction experience. Experiments 2 and 3 examined whether backward conditioning (CS presented after the US) could establish inhibitory properties that allowed IL stimulation to enhance extinction, and whether this effect was specific to the same stimulus or generalized to different stimuli. Experiments 5 - 7 extended this approach to appetitive learning: rats received backward or forward appetitive conditioning followed by extinction, and then fear conditioning, to determine whether IL stimulation could enhance extinction in contexts beyond aversive learning and across conditioning sequences. Across studies, the key claim is that IL activation facilitates extinction retrieval only when animals possess a prior inhibitory memory, and that this effect generalizes across aversive and appetitive paradigms.

Strengths:

(1) The design attempts to dissect the role of IL activity as a function of prior learning, which is conceptually valuable.

(2) The experimental design of probing different inhibitory learning approaches to probe how IL activation facilitates extinction learning was creative and innovative.

Weaknesses:

(1) Non-specific manipulation.

ChR2 was expressed in IL without distinction between glutamatergic and GABAergic populations. Without knowing the relative contribution of these cell types or the percentage of neurons affected, the circuit-level interpretation of the results is unclear.

(2) Extinction retrieval test conflates processes

The retrieval test included 8 tones. Averaging across this many tone presentations conflate extinction retrieval/expression (early tones) with further extinction learning (later tones). A more appropriate analysis would focus on the first 2-4 tones to capture retrieval only. As currently presented, the data do not isolate extinction retrieval.

(3) Under-sampling and poor group matching.

Sample sizes appear small, which may explain why groups are not well matched in several figures (e.g., 2b, 3b, 6b, 6c) and why there are several instances of unexpected interactions (protocol, virus, and period). This baseline mismatch raises concerns about the reliability of group differences.

(4) Incomplete presentation of conditioning data.

Figure 3 only shows a single conditioning session despite five days of training. Without the full dataset, it is difficult to evaluate learning dynamics or whether groups were equivalent before testing.

(5) Interpretation stronger than evidence.

The authors conclude that IL activation facilitates extinction retrieval only when an inhibitory memory has been formed. However, given the caveats above, the data are insufficient to support such a strong mechanistic claim. The results could reflect non-specific facilitation or disruption of behavior by broad prefrontal activation. Moreover, there is compelling evidence that optogenetic activation of IL during fear extinction does facilitate subsequent extinction retrieval without prior extinction training (Do-Monte et al 2015, Chen et al 2021), which the authors do not directly test in this study.

Impact:

The role of IL in extinction retrieval remains a central question in the fear learning literature. However, because the test used conflates extinction retrieval with new learning and the manipulations lack cell-type specificity, the evidence presented here does not convincingly support the main claims. The study highlights the need for more precise manipulations and more rigorous behavioral testing to resolve this issue.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors examine the mechanisms by which stimulation of the infralimbic cortex (IL) facilitates the retention and retrieval of inhibitory memories. Previous work has shown that optogenetic stimulation of the IL suppresses freezing during extinction but does not improve extinction recall when extinction memory is probed one day later. When stimulation occurs during a second extinction session (following a prior stimulation-free extinction session), freezing is suppressed during the second extinction as well as during the tone test the following day. The current study was designed to further explore the facilitatory role of the IL in inhibitory learning and memory recall. The authors conducted a series of experiments to determine whether recruitment of IL extends to other forms of inhibitory learning (e.g., backward conditioning) and to inhibitory learning involving appetitive conditioning. Further, they assessed whether their effects could be explained by stimulus familiarity. The results of their experiments show that backward conditioning, another form of inhibitory learning, also enabled IL stimulation to enhance fear extinction. This phenomenon was not specific to aversive learning, as backward appetitive conditioning similarly allowed IL stimulation to facilitate extinction of aversive memories. Finally, the authors ruled out the possibility that IL facilitated extinction merely because of prior experience with the stimulus (e.g., reducing the novelty of the stimulus). These findings significantly advance our understanding of the contribution of IL to inhibitory learning. Namely, they show that the IL is recruited during various forms of inhibitory learning, and its involvement is independent of the motivational value associated with the unconditioned stimulus.

Strengths:

(1) Transparency about the inclusion of both sexes and the representation of data from both sexes in figures.

(2) Very clear representation of groups and experimental design for each figure.

(3) The authors were very rigorous in determining the neurobehavioral basis for the effects of IL stimulation on extinction. They considered multiple interpretations and designed experiments to address these possible accounts of their data.

(4) The rationale for and the design of the experiments in this manuscript are clearly based on a wealth of knowledge about learning theory. The authors leveraged this expertise to narrow down how the IL encodes and retrieves inhibitory memories.

Weaknesses:

(1) In Experiment 1, although not statistically significant, it does appear as though the stimulation groups (OFF and ON) differ during Extinction 1. It seems like this may be due to a difference between these groups after the first forward conditioning. Could the authors have prevented this potential group difference in Extinction 1 by re-balancing group assignment after the first forward conditioning session to minimize the differences in fear acquisition (the authors do report a marginally significant effect between the groups that would undergo one vs. two extinction sessions in their freezing during the first conditioning session)?

(2) Across all experiments (except for Experiment 1), the authors state that freezing during the initial conditioning increased across "days". The figures that correspond to this text, however, show that freezing changes across trials. In the methods, the authors report that backward conditioning occurred over 5 days. It would be helpful to understand how these data were analyzed and collated to create the final figures. Was the freezing averaged across the five days for each trial for analyses and figures?

(3) In Experiment 3, the authors report a significant Protocol X Virus interaction. It would be useful if the authors could conduct post-hoc analyses to determine the source of this interaction. Inspection of Figure 4B suggests that freezing during the two different variants of backward conditioning differs between the virus groups. Did the authors expect to see a difference in backward conditioning depending on the stimulus used in the conditioning procedure (light vs. tone)? The authors don't really address this confounding interaction, but I do think a discussion is warranted.

(4) In this same experiment, the authors state that freezing decreased during extinction; however, freezing in the Diff-EYFP group at the start of extinction (first bin of trials) doesn't look appreciably different than their freezing at the end of the session. Did this group actually extinguish their fear? Freezing on the tone test day also does not look too different from freezing during the last block of extinction trials.

(5) The Discussion explored the outcomes of the experiments in detail, but it would be useful for the authors to discuss the implications of their findings for our understanding of circuits in which the IL is embedded that are involved in inhibitory learning and memory. It would also be useful for the authors to acknowledge in the Discussion that although they did not have the statistical power to detect sex differences, future work is needed to explore whether IL functions similarly in both sexes.

Reviewer #3 (Public review):

Summary:

This is a really nice manuscript with different lines of evidence to show that the IL encodes inhibitory memories that can then be manipulated by optogenetic stimulation of these neurons during extinction. The behavioral designs are excellent, with converging evidence using extinction/re-extinction, backwards/forwards aversive conditioning, and backwards appetitive/forwards aversive conditioning. Additional factors, such as nonassociative effects of the CS or US, are also considered, and the authors evaluate the inhibitory properties of the CS with tests of conditioned inhibition.

Strengths:

The experimental designs are very rigorous with an unusual level of behavioral sophistication.

Weaknesses:

(1) More justification for parametric choices (number of days of backwards vs forwards conditioning) could be provided.

(2) The current discussion could be condensed and could focus on broader implications for the literature.

Author response:

Reviewer #1 (Public review):

Summary:

The manuscript reports a series of experiments designed to test whether optogenetic activation of infralimbic (IL) neurons facilitates extinction retrieval and whether this depends on animals' prior experience. In Experiment 1, rats underwent fear conditioning followed by either one or two extinction sessions, with IL stimulation given during the second extinction; stimulation facilitated extinction retrieval only in rats with prior extinction experience. Experiments 2 and 3 examined whether backward conditioning (CS presented after the US) could establish inhibitory properties that allowed IL stimulation to enhance extinction, and whether this effect was specific to the same stimulus or generalized to different stimuli. Experiments 5 - 7 extended this approach to appetitive learning: rats received backward or forward appetitive conditioning followed by extinction, and then fear conditioning, to determine whether IL stimulation could enhance extinction in contexts beyond aversive learning and across conditioning sequences. Across studies, the key claim is that IL activation facilitates extinction retrieval only when animals possess a prior inhibitory memory, and that this effect generalizes across aversive and appetitive paradigms.

Strengths:

(1) The design attempts to dissect the role of IL activity as a function of prior learning, which is conceptually valuable.

We thank the Reviewer for their positive assessment.

(2) The experimental design of probing different inhibitory learning approaches to probe how IL activation facilitates extinction learning was creative and innovative.

We thank the Reviewer for their positive assessment.

Weaknesses:

(1) Non-specific manipulation.

ChR2 was expressed in IL without distinction between glutamatergic and GABAergic populations. Without knowing the relative contribution of these cell types or the percentage of neurons affected, the circuit-level interpretation of the results is unclear.

ChR2 was intentionally expressed in the infralimbic cortex (IL) without distinction between local neuronal populations for two reasons. First, this manuscript aimed to uncover some of the features characterizing the encoding of inhibitory memories in the IL, and this encoding likely engages interactions among various neuronal populations within the IL. Second, the hypotheses tested in the manuscript derived from findings that indiscriminately stimulated the IL using the GABA_A receptor antagonist picrotoxin, which is best mimicked by the approach taken. We agree that it is also important to determine the respective contributions of distinct IL neuronal populations to inhibitory encoding; however, the global approach implemented in the present experiments represents a necessary initial step. This rationale will be incorporated into the revised manuscript, which will also make reference to the need to identify the relative contributions of the various neuronal populations within the IL. 

(2) Extinction retrieval test conflates processes

The retrieval test included 8 tones. Averaging across this many tone presentations conflate extinction retrieval/expression (early tones) with further extinction learning (later tones). A more appropriate analysis would focus on the first 2-4 tones to capture retrieval only. As currently presented, the data do not isolate extinction retrieval.

It is unclear when retrieval of what has been learned across extinction ceases and additional extinction learning occurs. In fact, it is only the first stimulus presentation that unequivocally permits a distinction between retrieval and additional extinction learning, as the conditions for this additional learning have not been fulfilled at that presentation. However, confining evidence for retrieval to the first stimulus presentation introduces concerns that other factors could influence performance. For instance, processing of the stimulus present at the start of the session may differ from that present at the end of the previous session, thereby affecting what is retrieved. Such differences between the stimuli present at the start and end of an extinction session have been long recognized as a potential explanation for spontaneous recovery (Estes, 1955). More importantly, whether the test data presented confound retrieval and additional extinction learning or not, the interpretation remains the same with respect to the effects of a prior history of inhibitory learning on enabling the facilitative effects of IL stimulation. Finally, it is unclear how these facilitative effects could occur in the absence of the subjects retrieving the extinction memory formed under the stimulation. Nevertheless, the revised manuscript will provide the trial-by-trial performance during the post-extinction retrieval tests and discuss this issue.

(3) Under-sampling and poor group matching.

Sample sizes appear small, which may explain why groups are not well matched in several figures (e.g., 2b, 3b, 6b, 6c) and why there are several instances of unexpected interactions (protocol, virus, and period). This baseline mismatch raises concerns about the reliability of group differences.

Efforts were made to match group performance upon completion of each training stage and before IL stimulation. Unfortunately, these efforts were not completely successful due to exclusions following post-mortem analyses. However, we acknowledge that the unexpected interactions deserve further discussion, and this will be incorporated into the revised manuscript (see also comment from Reviewer 2). Although we cannot exclude that sample sizes may have contributed to some of these interactions, we remain confident about the reliability of the main findings reported, especially given their replication across the various protocols. Overall, the manuscript provides evidence that IL stimulation does not facilitate brief extinction in the absence of prior inhibitory experience in five different experiments, replicating previous findings (Lingawi et al., 2018; Lingawi et al., 2017). It also replicates these previous findings by showing that prior experience with either fear or appetitive extinction enables IL stimulation to facilitate subsequent fear extinction. Furthermore, the facilitative effects of such stimulation following fear or appetitive backward conditioning are replicated in the present manuscript.  

(4) Incomplete presentation of conditioning data.

Figure 3 only shows a single conditioning session despite five days of training. Without the full dataset, it is difficult to evaluate learning dynamics or whether groups were equivalent before testing.

We apologize, as we incorrectly labeled the X axis for the backward conditioning data set in Figures 3B, 4B, 4D and 5B. It should have indicated “Days” instead of “Trials”. This error will be corrected in the revised manuscript.

(5) Interpretation stronger than evidence.

The authors conclude that IL activation facilitates extinction retrieval only when an inhibitory memory has been formed. However, given the caveats above, the data are insufficient to support such a strong mechanistic claim. The results could reflect non-specific facilitation or disruption of behavior by broad prefrontal activation. Moreover, there is compelling evidence that optogenetic activation of IL during fear extinction does facilitate subsequent extinction retrieval without prior extinction training (Do-Monte et al 2015, Chen et al 2021), which the authors do not directly test in this study.

As noted above, the revised manuscript will show that the interpretations of the main findings stand whether ore the test data confounds retrieval with additional extinction learning. The revised manuscript will also clarify the plotting of the data for the backward conditioning stages. We do agree that further discussion of the unexpected interactions is necessary, and this will also be incorporated into the revised manuscript. However, the various replications of the core findings provide strong evidence for their reliability and the interpretations advanced in the original manuscript. The proposal that the results reflect non-specific facilitation or disruption of behavior seems highly unlikely. Indeed, the present experiments and previous findings (Lingawi et al., 2018; Lingawi et al., 2017) provide multiple demonstrations that IL stimulation fails to produce any facilitation in the absence of prior inhibitory experience with the target stimulus. Although these demonstrations appear inconsistent with previous studies (Do-Monte et al., 2015; Chen et al., 2021), this inconsistency is likely explained by the fact that these studies manipulated activity in specific IL neuronal populations. Previous work has already revealed differences between manipulations targeting discrete IL neuronal populations as opposed to general IL activity (Kim et al., 2016). Importantly, as previously noted, the present manuscript aimed to generally explore inhibitory encoding in the IL that, as we will acknowledge, is likely to engage several neuronal populations within the IL. Adequate statements on these matters will be included in the revised manuscript.

Impact:

The role of IL in extinction retrieval remains a central question in the fear learning literature. However, because the test used conflates extinction retrieval with new learning and the manipulations lack cell-type specificity, the evidence presented here does not convincingly support the main claims. The study highlights the need for more precise manipulations and more rigorous behavioral testing to resolve this issue.

As noted in our responses, the interpretations of the data presented remain identical whether the test data conflate extinction retrieval with additional extinction learning or not. Although we agree that it is important to establish the role of specific IL neuronal populations in extinction learning, this was beyond the scope of the manuscript and the findings reported remain valuable to our understanding of inhibitory encoding within the IL.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors examine the mechanisms by which stimulation of the infralimbic cortex (IL) facilitates the retention and retrieval of inhibitory memories. Previous work has shown that optogenetic stimulation of the IL suppresses freezing during extinction but does not improve extinction recall when extinction memory is probed one day later. When stimulation occurs during a second extinction session (following a prior stimulation-free extinction session), freezing is suppressed during the second extinction as well as during the tone test the following day. The current study was designed to further explore the facilitatory role of the IL in inhibitory learning and memory recall. The authors conducted a series of experiments to determine whether recruitment of IL extends to other forms of inhibitory learning (e.g., backward conditioning) and to inhibitory learning involving appetitive conditioning. Further, they assessed whether their effects could be explained by stimulus familiarity. The results of their experiments show that backward conditioning, another form of inhibitory learning, also enabled IL stimulation to enhance fear extinction. This phenomenon was not specific to aversive learning, as backward appetitive conditioning similarly allowed IL stimulation to facilitate extinction of aversive memories. Finally, the authors ruled out the possibility that IL facilitated extinction merely because of prior experience with the stimulus (e.g., reducing the novelty of the stimulus). These findings significantly advance our understanding of the contribution of IL to inhibitory learning. Namely, they show that the IL is recruited during various forms of inhibitory learning, and its involvement is independent of the motivational value associated with the unconditioned stimulus.

Strengths:

(1) Transparency about the inclusion of both sexes and the representation of data from both sexes in figures.

We thank the Reviewer for their positive assessment.

(2) Very clear representation of groups and experimental design for each figure.

We thank the Reviewer for their positive assessment.

(3) The authors were very rigorous in determining the neurobehavioral basis for the effects of IL stimulation on extinction. They considered multiple interpretations and designed experiments to address these possible accounts of their data.

We thank the Reviewer for their positive assessment.

(4) The rationale for and the design of the experiments in this manuscript are clearly based on a wealth of knowledge about learning theory. The authors leveraged this expertise to narrow down how the IL encodes and retrieves inhibitory memories.

We thank the Reviewer for their positive assessment.

Weaknesses:

(1) In Experiment 1, although not statistically significant, it does appear as though the stimulation groups (OFF and ON) differ during Extinction 1. It seems like this may be due to a difference between these groups after the first forward conditioning. Could the authors have prevented this potential group difference in Extinction 1 by re-balancing group assignment after the first forward conditioning session to minimize the differences in fear acquisition (the authors do report a marginally significant effect between the groups that would undergo one vs. two extinction sessions in their freezing during the first conditioning session)?

As noted (see response to Reviewer 1), efforts were made daily to match group performance across the training stages, but these efforts were ultimately hampered by the necessary exclusions following post-mortem analyses. This will be made explicit in the revised manuscript. Regarding freezing during Extinction 1, as noted by the Reviewer, the difference, which was not statistically significant, was absent across trials during the subsequent forward fear conditioning stage. Likewise, the protocol difference observed during the initial forward fear conditioning was absent in subsequent stages. We are therefore confident that these initial differences (significant or not) did not impact the main findings at test. Importantly, these findings replicate previous work using identical protocols in which no differences were present during the training stages. These considerations will be addressed in the revised manuscript.

(2) Across all experiments (except for Experiment 1), the authors state that freezing during the initial conditioning increased across "days". The figures that correspond to this text, however, show that freezing changes across trials. In the methods, the authors report that backward conditioning occurred over 5 days. It would be helpful to understand how these data were analyzed and collated to create the final figures. Was the freezing averaged across the five days for each trial for analyses and figures?

We apologize, as noted above, we incorrectly labeled the X axis for the backward conditioning data sets in Figures 3B, 4B, 4D and 5B. It should have indicated “Days” instead of “Trials”. The data shown in these Figures use the average of all trials on a given day. This will be clarified in the methods section of the revised manuscript. The labeling errors on the Figures will be corrected.

(3) In Experiment 3, the authors report a significant Protocol X Virus interaction. It would be useful if the authors could conduct post-hoc analyses to determine the source of this interaction. Inspection of Figure 4B suggests that freezing during the two different variants of backward conditioning differs between the virus groups. Did the authors expect to see a difference in backward conditioning depending on the stimulus used in the conditioning procedure (light vs. tone)? The authors don't really address this confounding interaction, but I do think a discussion is warranted.

We agree with the Reviewer that further discussion of the Protocol x Virus interaction that emerged during the backward conditioning and forward conditioning stages of Experiment 3 is warranted. This will be provided in the revised manuscript. Briefly, during both stages, follow-up analyses did not reveal any differences (main effects or interactions) between the two groups trained with the light stimulus (Diff-EYFP and Diff-ChR2). By contrast, the ChR2 group trained with the tone (Back-ChR2) froze more overall than the EYFP group (Back-EYFP), but there were no other significant differences between the two groups. Based on these analyses, the Protocol x Virus interaction appears to be driven by greater freezing in the ChR2 group trained with the tone rather than a difference in the backward conditioning performance based on stimulus identity. Consistent with this, the statistical analyses did not reveal a main effect of Protocol during either the backward conditioning stage or the stimulus trials during the forward conditioning stage. Nevertheless, during this latter stage, a main effect of Protocol emerged during baseline performance, but once again, this seems to be driven by the Back-ChR2 group. Critically, it is unclear how greater stimulus freezing in the Back-ChR2 group during forward conditioning would lead to lower freezing during the post-extinction retrieval test.  

(4) In this same experiment, the authors state that freezing decreased during extinction; however, freezing in the Diff-EYFP group at the start of extinction (first bin of trials) doesn't look appreciably different than their freezing at the end of the session. Did this group actually extinguish their fear? Freezing on the tone test day also does not look too different from freezing during the last block of extinction trials.

We confirm that overall, there was a significant decline in freezing across the extinction session shown in Figure 4B. The Reviewer is correct to point out that this decline was modest (if not negligible) in the Diff-EYFP group, which was receiving its first inhibitory training with the target tone stimulus. It is worth noting that across all experiments, most groups that did not receive infralimbic stimulation displayed a modest decline in freezing during the extinction session since it was relatively brief, involving only 6 or 8 tone alone presentations. This was intentional, as we aimed for the brief extinction session to generate minimal inhibitory learning and thereby to detect any facilitatory effect of infralimbic stimulation. This issue will be clarified and explained in the revised version of the manuscript.

(5) The Discussion explored the outcomes of the experiments in detail, but it would be useful for the authors to discuss the implications of their findings for our understanding of circuits in which the IL is embedded that are involved in inhibitory learning and memory. It would also be useful for the authors to acknowledge in the Discussion that although they did not have the statistical power to detect sex differences, future work is needed to explore whether IL functions similarly in both sexes.

In line with the Reviewer’s suggestion (see also Reviewer 3), the revised manuscript will include a discussion of the broader implications of the findings regarding inhibitory brain circuitry and will acknowledge the need to further explore sex differences and IL functions.

Reviewer #3 (Public review):

Summary:

This is a really nice manuscript with different lines of evidence to show that the IL encodes inhibitory memories that can then be manipulated by optogenetic stimulation of these neurons during extinction. The behavioral designs are excellent, with converging evidence using extinction/re-extinction, backwards/forwards aversive conditioning, and backwards appetitive/forwards aversive conditioning. Additional factors, such as nonassociative effects of the CS or US, are also considered, and the authors evaluate the inhibitory properties of the CS with tests of conditioned inhibition.

Strengths:

The experimental designs are very rigorous with an unusual level of behavioral sophistication.

We thank the Reviewer for their positive assessment.

Weaknesses:

(1) More justification for parametric choices (number of days of backwards vs forwards conditioning) could be provided.

All experimental parameters were based on previously published experiments showing the capacity of the backward conditioning protocols to generate inhibitory learning and the forward conditioning protocols to produce excitatory learning. Although this was mentioned in the methods section, we acknowledge that further explanation is required to justify the need for multiple days of backward training. This will be provided in the revised manuscript.

(2) The current discussion could be condensed and could focus on broader implications for the literature.

The revised manuscript will make an effort to condense the discussion and focus on broader implications for the literature.

References

Chen, Y.-H., Wu, J.-L., Hu, N.-Y., Zhuang, J.-P., Li, W.-P., Zhang, S.-R., Li, X.-W., Yang, J.-M., & Gao, T.-M. (2021). Distinct projections from the infralimbic cortex exert opposing effects in modulating anxiety and fear. J Clin Invest, 131(14), e145692. https://doi.org/10.1172/JCI145692

Do-Monte, F. H., Manzano-Nieves, G., Quiñones-Laracuente, K., Ramos-Medina, L., & Quirk, G. J. (2015). Revisiting the role of infralimbic cortex in fear extinction with optogenetics. J Neurosci, 35(8), 3607-3615. https://doi.org/10.1523/JNEUROSCI.3137-14.2015

Estes, W. K. (1955). Statistical theory of spontaneous recovery and regression. Psychol Rev, 62(3), 145-154. https://doi.org/10.1037/h0048509

Kim, H.-S., Cho, H.-Y., Augustine, G. J., & Han, J.-H. (2016). Selective Control of Fear Expression by Optogenetic Manipulation of Infralimbic Cortex after Extinction. Neuropsychopharmacology, 41(5), 1261-1273. https://doi.org/10.1038/npp.2015.276

Lingawi, N. W., Holmes, N. M., Westbrook, R. F., & Laurent, V. (2018). The infralimbic cortex encodes inhibition irrespective of motivational significance. Neurobiol Learn Mem, 150, 64-74. https://doi.org/10.1016/j.nlm.2018.03.001

Lingawi, N. W., Westbrook, R. F., & Laurent, V. (2017). Extinction and Latent Inhibition Involve a Similar Form of Inhibitory Learning that is Stored in and Retrieved from the Infralimbic Cortex. Cereb Cortex, 27(12), 5547-5556. https://doi.org/10.1093/cercor/bhw322

HEAT BITES 1. Itchy private area from the heat 2. Coffee - news reporter 3. Isolating due to hot weather 4. Mailman Moe 5. Ally Gator 6. Libby & Nibbles 7. Daisy 8. Jeff Tracy 9. Walking With Dinosaurs toys 10. Officers Brogan and Haldane

Reviewer #1 (Public review):

Summary:

In this paper, the authors analyze connectome data from Drosophila and compare the physical wiring with functional connectivity estimated from calcium imaging data. They quantify structure-function relationships as a correlation of the two connectivity modalities. They report correlations roughly comparable to what has been described in the literature on sc/fc relationships in mammalian connectome data at the meso-scale. They then repeat their analysis, focusing on segregated versus unsegregated synapses. They derive separate connectomes using one or the other class of synapse. They show differential contributions to the sc/fc relationships by segregated versus unsegregated synapses.

Strengths:

There is nice synthesis of multimodal imaging data (Ca and EM data from flies and meso-scale data from human and marmoset).

Weaknesses:

(1) The paper is written in an unusual way. The introduction intermingles results with background, making it hard to figure out what precisely is being tested.

(2) There are also major methodological gaps. Though the mammalian connectomes are used as a point of reference, no descriptions of their origins or processing are included.

(3) A major weakness stems from the actual calculation of the sc/fc correlation. In general, SC is sparse. In the case of the EM connectomes, it is *exceptionally* sparse (most neural elements are not connected to one another). The authors calculated sc/fc coupling by correlating the off-diagonal elements of sc (the logarithm of its edge weights) and fc matrices with one another. The logarithmic transformation yields a value of infinity for all zero entries. The authors simply impute these elements with 0. This makes no sense and, depending on whether these zero elements are distributed systematically versus uniformly random, could either inflate or deflate the sc/fc correlations. Care must be taken here.

(4) Further, in constructing the segregated versus unsegregated connectomes, they use absolute thresholds for collecting synapses. It is unclear, however, whether similar numbers of synapses were included in both matrices. If the number is different, that might explain the differential relationship with fc; one matrix has more non-zero entries (and as noted earlier, those zero entries are problematic).

(5) There was also considerable text (in the results) describing the processing of the Ca data. In this section, the authors frequently refer to some pipelines as "better" or "worse" (more or less effective). But it is not clear what measures they adopted to assess the effectiveness of a pipeline.

Reviewer #2 (Public review):

Summary:

Okuno et al. investigate the structure-function relationship in the fruit fly Drosophila melanogaster. To do so, they combine published data from two recent synapse-level connectomes ("hemibrain" and "FlyWire") with a dataset comprising functional whole-brain calcium imaging and behavioural data. First, they investigate the applicability of fMRI pre-processing techniques on data from calcium imaging. They then cross-correlate this pre-processed functional data with structural data extracted from the connectomes, including a comparison to humans. The authors proceed to compare the two connectomes and find significant differences, which they attribute to differences in the accuracy of the synapse detections. Next, they present a novel algorithm to quantify whether neurons are segregated (pre- and postsynapses are spatially separate) or unsegregated (pre- and postsynapses are mixed). Using this approach, they find that unsegregated neurons may contribute more to function than segregated neurons. Applying a general linear model to the functional dataset suggests that activity in two brain areas (Wedge and AVLP) is suppressed during walking. The authors identify a GABAergic neuron in the connectome that could be responsible for this effect and suggest it may provide feedback to the fly's "compass" in the central complex.

Strengths:

The study tackles a relevant question in connectomics by exploring the relationship between structural and functional connectivity in the Drosophila brain. The authors apply a range of established and adapted analytical methods, including fMRI-style preprocessing and a novel synaptic segregation index. The effort to integrate multiple datasets and to compare across species reflects a broad and methodical approach.

Weaknesses:

The manuscript would benefit from a clearer overarching narrative to unify the various analyses, which currently appear somewhat disjointed. While the technical methods are extensive, the writing is often convoluted and lacks crucial details, making it difficult to follow the logic and interpret key findings. Additionally, the conclusions are relatively incremental and lack a compelling conceptual advance, limiting the overall impact of the work.

(1) The introduction currently contains a number of findings and conclusions that would be better placed in the results and discussion to clearly delineate past findings from new results and speculations.

(2) The narrative would benefit greatly from some clear statements along the lines of "we wanted to find out X, therefore we did Y".

(3) More concise terminology would be helpful. For example, the connectomes are currently referred to as either "hemibrain", "FlyEM", "whole-brain", or "FlyWire".

(4) The abstract claims "a new, more robust method to quantify the degree of pre- and post-synaptic segregation". However, the study fails to provide evidence that this method is indeed more robust than existing methods.

(5) The authors define unsegregated neurons as having mixed pre- and postsynapses in the same space. However, this ignores the neurons' topology: a neuron can exhibit a clearly defined dendrite with (mostly) postsynapses and a clearly defined axon with (mostly) presynapses, which then occupy the same space. This is different from genuinely unsegregated neurons with no distinct dendritic and axonal compartments, such as CT1.

(6) It is not entirely clear where the marmoset dataset originates from. Was it generated for this study? If not, why is there a note in the Ethics Declaration?

(7) On the differences between hemibrain and FlyWire: What is the "18.8 million post-synapses" for FlyWire referring to? The (thresholded) FlyWire synapse table has 130M connections (=postsynapses). Subsetting that synapse cloud to the hemibrain volume still gives ~47M synapses. Further subsetting to only connections between proofread neurons inside the hemibrain volume gives 19.4M - perhaps the authors did something like that? Similarly, the hemibrain synapse table contains 64M postsynapses. Do the 21M "FlyEM" post-synapses refer to proofread neurons only? If the authors indeed used only (post-)synapses from proofread neurons, they need to make that explicit in results and methods, and account for differences in reconstruction status when making any comparisons. For example, the mushroom body in the hemibrain got a lot more attention than in FlyWire, which would explain the differences reported here. For that reason, connection weights are often expressed as, e.g., a fraction of the target's inputs instead of the total number of synapses when comparing connectivity across connectomic datasets. Furthermore, in Figure 3b, it looks like the FlyWire synapse cloud was not trimmed to the exact hemibrain boundaries: for example, the trimmed FlyWire synapse cloud seems to extend further into the optic lobes than the hemibrain volume does.

Reviewer #3 (Public review):

Summary:

In this manuscript, Okuno et al. re-analyze whole-brain imaging data collected in another paper (Brezovec et al., 2024) in the context of the two currently available Drosophila connectome datasets: the partial "FlyEM" (hemibrain) dataset (Scheffer et al., 2020) and the whole-brain "FlyWire" dataset (Dorkenwald et al., 2024). They apply existing fMRI signal processing algorithms to the fly imaging data and compute function-structure correlations across a variety of post-processing parameters (noise reduction methods, ROI size), demonstrating an inverse relationship between ROI size and FC-SC correlation. The authors go on to look at structural connectivity amongst more polarized or less polarized neurons, and suggest that stronger FC-SC correlations are driven by more polarized neurons.

Strengths:

(1) The result that larger mesoscale ROIs have a higher correlation with structural data is interesting. This has been previously discussed in Drosophila in Turner et al., 2021, but here it is quantified more extensively.

(2) The quantification of neuron polarization (PPSSI) as applied to these structural data is a promising approach for quantifying differences in spatial synapse distribution.

Weaknesses:

One should not score noise/nuisance removal methods solely by their impact on FC-SC correlation values, because we do not know a priori that direct structural connections correspond with strong functional correlations. In fact, work in C. elegans, where we have access to both a connectome and neuron-resolution functional data, suggests that this relationship is weak (Yemini et al., 2021; Randi et al., 2023). Similarly, I don't think it's appropriate to tune the confidence scores on the EM datasets using FC-SC correlations as an output metric.

Any discussion of FC-SC comparisons should include an analysis of excitatory/inhibitory neurotransmitters, which are available in the fly connectome dataset. However, here the authors do not perform any analyses with neurotransmitter information.<br /> Comparisons between fly and human MRI data are also premature here. Firstly, the fly connectomes, which are derived from neuron-scale EM reconstructions, are a qualitatively different kind of data from human connectomes, which are derived from DSI imaging of large-scale tracts. Likewise, calcium data and fMRI data are very different functional data acquisition methods-the fact that similar processing steps can be used on time-series data does not make them surprisingly similar, and does not in my view, constitute evidence of "similar design concepts."

The comparison of FlyEM/FlyWire connectomes concludes that differences are more likely a result of data processing than of inter-individual variability. If this is the case, the title should not claim that the manuscript covers individual variability.<br /> The analysis of the wedge-AVLP neuron strikes me as highly speculative, given that the alignment precision between the connectome and the functional data is around 5 microns (Brezovec* et al, PNAS 2024).

Reviewer #3 (Public review):

In the manuscript by Lapao et al., the authors uncover a role for the RAB27A effector protein SYTL5 in regulating mitochondrial function and apparent selective turnover of mitochondrial components. The authors find that SYTL5 localizes to mitochondria in a RAB27A dependent way and that loss of SYTL5 (or RAB27A) impairs lysosomal turnover of MTCO1 (but not a matrix-based reporter/other mitochondrial proteins). The authors go on to show that loss of SYTL5 impacts mitochondrial respiration and ECAR and as such may influence the Warburg effect and tumorigenesis. Of relevance here, the authors go on to show that SYTL5 expression is reduced in adrenocortical carcinomas and this correlates with reduced survival rates.

As previously reviewed, this is a very intriguing body of work and reveals a new role for SYTL5/RAB27A at the mitochondria. Unfortunately, it appears that SYTL5 is challenging protein to detect endogenously and the authors' cell lines "comprise a heterogenous pool with high variability", which means that a lot of my original concerns remain. It is still also not clear if the conventional autophagy machinery is required for this pathway, especially if SYTL5/RAB27A mitochondrial recruitment is upstream of this. Hopefully, in future work, the authors (and/or others) will be able to address this and build on the mechanisms of this interesting and potentially important pathway.

DOI: 10.1038/s41598-020-63169-3

Resource: RRID:BDSC_68283

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_68283

What is this?

DOI: 10.1038/s41598-020-63169-3

Resource: RRID:BDSC_50373

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_50373

What is this?

DOI: 10.1038/s41598-020-63169-3

Resource: RRID:BDSC_51953

Curator: @scibot

SciCrunch record: RRID:BDSC_51953

What is this?

DOI: 10.1038/s41598-020-63169-3

Resource: RRID:BDSC_68294

Curator: @scibot

SciCrunch record: RRID:BDSC_68294

What is this?

DOI: 10.1038/s41598-020-63169-3

Resource: RRID:BDSC_49539

Curator: @scibot

SciCrunch record: RRID:BDSC_49539

What is this?

This passage shows the deep economic and racial divide within Orange County. The contrast between cities like Laguna Beach and Santa Ana highlights how inequality exists even within close distances. It’s striking to see how income and race shape opportunities so differently, emphasizing the need to address these structural disparities.

There have been big changes in the lives of many people who used to live in Los Angeles and San Diego. Many of them have moved to Orange County.

Large-scale changes in population changed the picture of a lot of people living in Los Angeles and San Diego, with many moving to Orange County.

There is a significant disparity within Orange County, with a clear social and economic divide.

This statement highlights that Orange County has undergone significant changes over the past 40 years due to a dramatic increase in population, and its original image no longer aligns with the current reality.

This section highlights the need to value the strengths of low-income students and build a mentoring culture that promotes activism and confidence. I agree that teachers should work with parents and communities to support students’ growth. It’s a reminder that educators must stay true to their purpose—challenging inequality and helping all students find hope and opportunity.

Élections au Conseil de la Vie Lycéenne 2025 : Analyse des Programmes

Résumé

Le présent document synthétise les professions de foi des candidats aux élections du Conseil de la Vie Lycéenne (CVL) du lycée Louis Vincent pour le mandat 2025.

Les élections se dérouleront du 30 septembre au 2 octobre via la plateforme Pronote, où cinq binômes seront élus pour un mandat de deux ans.

L'objectif commun déclaré par l'ensemble des candidats est d'améliorer les conditions et la qualité de vie au sein de l'établissement.

L'analyse des programmes révèle plusieurs thèmes transversaux et récurrents :

1. Convivialité et Événements : La quasi-totalité des candidats propose l'organisation de journées à thème (pyjama, sans sac, carnaval, maillot de foot), de ventes de nourriture et de boissons chaudes (notamment en hiver), et de grands événements fédérateurs comme un festival de fin d'année ouvert à tous les niveaux, des tournois sportifs interclasses ou un bal des terminales.

2. Solidarité et Actions Caritatives : Un fort accent est mis sur la solidarité, avec des propositions de collectes (alimentaires, produits d'hygiène, fournitures scolaires) et de levées de fonds dont les bénéfices seraient reversés à des associations caritatives (Restos du cœur, un cahier un crayon) ou à des lycéens dans le besoin de manière anonyme.

3. Culture et Pédagogie : De nombreux programmes ambitionnent de dynamiser la vie culturelle du lycée.

Les idées incluent une meilleure utilisation de la salle de cinéma pour des projections pédagogiques ou des séances gratuites, l'organisation de sorties culturelles, la création de clubs (lecture, débat), et la mise en place de systèmes de tutorat et de parrainage entre élèves.

4. Bien-être et Cadre de Vie : L'amélioration du bien-être des élèves est une priorité centrale.

Les propositions visent à créer des espaces de détente et de travail plus calmes et accueillants, à aménager des espaces verts (potager, jardin partagé), à gérer le stress via des ateliers, et à améliorer les infrastructures pratiques comme l'ajout de casiers.

5. Communication et Représentation : Plusieurs binômes souhaitent renforcer le lien entre le CVL et les élèves par la gestion active d'un compte Instagram, la mise en place d'une boîte à idées, la publication de comptes-rendus mensuels et une promesse générale d'écoute.

Un candidat, Baptiste, fonde même l'intégralité de sa démarche sur l'absence de programme prédéfini afin de représenter directement les demandes des élèves.

En somme, les candidatures de 2025 témoignent d'une volonté partagée de rendre le lycée Louis Vincent plus dynamique, solidaire, culturellement riche et attentif au bien-être de chaque élève.

Présentation des Candidats et de Leurs Plateformes

Le tableau suivant récapitule les différents binômes candidats, leur niveau de classe et les points saillants de leur programme.

Candidats (Titulaires et Suppléants)

Classe(s)

Qualités Revendiquées

Points Clés du Programme

VM & CR

Terminale 6 & Première 2

Créatives, motivées, sérieuses, expérience et énergie

Projet "H24" (camping au lycée), journées à thème (maillot de foot, sans sac, pyjama).

SP et OP

Seconde 12 & Seconde 13

Bienveillants, honnêtes, altruistes, intègres, ouverts au dialogue

Ventes de snacks/boissons chaudes, 2ème service à la cantine, création d'un lieu de travail/repos, tournois sportifs interclasses.

MV et KM

Non spécifié

-

4 axes : écologie (coin nature, potager), vie scolaire (journées à thème, menus spéciaux), culture (concours de talents, tutorat, sorties), bien-être (coin calme).

MO et LO

Première STL & Première 11

Engagée, sympathique, responsable, réfléchi, à l'écoute, sérieux, ambitieux

Récolte caritative, grande journée conviviale de fin d'année, vente de gourdes écoresponsables, communication améliorée (compte-rendu, boîte à idées).

AI et IP

Terminale 2 & Première 7

Inspirés par les anciens CVL

Vente de fournitures scolaires, vente de thé/chocolat chaud pour des associations, festival de fin d'année pour tous les niveaux.

AP et GL

Première 2 & Première 4

Expérience (délégué, ancien CVC)

Ventes de pâtisseries/boissons, gestion active du compte Instagram du CVL (sondages, menus), ajout de casiers pour tous, décorations du lycée.

RS et LH

Seconde 4 & Seconde 1

À l'écoute

Utilisation de la salle de cinéma à but pédagogique, organisation de goûters thématiques pour financer des associations ou des sorties, décorations festives.

HS et JPM

Terminale & Première 6

-

Optimisation des TV de l'accueil (horaires de bus, menus), dynamisation de la web TV, séances de cinéma gratuites, collecte de fournitures, vente de pulls du lycée, changement de la sonnerie via vote.

RB et NL

Seconde 4 & Seconde 6

-

Grand repas mensuel à la cantine, collecte de produits d'hygiène/vêtements pour les SDF, semaines à thème (cinéma), tutorat entre 15h et 18h.

B

Seconde

-

Absence de programme défini. Volonté de représenter directement les idées et besoins des 1600 élèves. Souhaite renouveler les initiatives type "Journées du Patrimoine".

SV et LL

Terminale 4 & Première 9

Souriantes, à l'écoute, motivées

Ateliers de gestion du stress, espaces de détente, tutorat Terminales/Secondes, création d'espaces verts (potager), sorties culturelles, amélioration des salles de travail.

Analyse Détaillée des Thèmes de Campagne

1. Convivialité, Événements et Vie Sociale

Ce thème est le plus largement partagé par les candidats, qui souhaitent briser la routine et renforcer les liens entre les élèves.

• Journées à Thème : Une proposition quasi unanime.

◦ Journée pyjama : Proposée par Victorine & Capucine, et Mabine & Victoria.   

◦ Journée sans sac : Proposée par Victorine & Capucine, Mabine & Victoria, Henry & Maxime, et Sacha & Lili.  

◦ Journée en maillot de foot : Proposée par Victorine & Capucine. 

◦ Journée carnaval : Proposée par Mabine & Victoria, et Sacha & Lili.  

◦ Journée de l'élégance : Proposée par Sacha & Lili.

• Événements de Fin d'Année :

◦ Festival pour tous les niveaux : Avazov & Isaac proposent un événement avec stands de nourriture, concours de talents et concerts pour inclure les Secondes et Premières, souvent exclus du bal des terminales.  

◦ Grande journée conviviale : Méina & Lilian suggèrent une journée avec jeux, sport et musique pour élèves, professeurs et personnel.  

◦ Bal des terminales et "Perçant" : Arnaud & Grégoire s'engagent à organiser ces événements traditionnels.

• Ventes de Nourriture et Boissons :

◦ Ventes de snacks et boissons chaudes (hiver) : Proposées par Sacha & Oscar, Avazov & Isaac.    ◦ Ventes de pâtisseries : Proposées par Arnaud & Grégoire.  

◦ Goûters thématiques (Noël, Halloween) : Proposés par Roman & Louison.

• Événements Sportifs et Sociaux :

◦ Tournois sportifs interclasses : Proposés par Sacha & Oscar.  

◦ Grand repas mensuel à la cantine : Suggéré par Romain & Noa pour favoriser les rencontres.

2. Solidarité, Écologie et Actions Caritatives

De nombreux candidats placent l'engagement solidaire et écologique au cœur de leur projet.

• Collectes et Dons :

◦ Récoltes caritatives générales : Proposées par Méina & Lilian, avec une redistribution anonyme possible aux lycéens dans le besoin.  

◦ Collectes pour des associations (SDF, etc.) : Romain & Noa proposent de récolter des produits d'hygiène, de la nourriture et des vêtements chauds. Mabine & Victoria souhaitent organiser des collectes solidaires régulières.  

◦ Collecte de fournitures scolaires : Henry & Maxime suggèrent que les terminales fassent don de leur matériel (ex: calculatrices) aux nouveaux élèves.

• Financement d'Associations :

◦ Plusieurs binômes (Avazov & Isaac, Roman & Louison) proposent que les bénéfices des ventes de nourriture soient reversés à des associations comme les Restos du cœur ou Un cahier, un crayon.

• Écologie et Cadre de Vie :

◦ Espaces verts : Mabine & Victoria et Sacha & Lili veulent créer un "coin nature", un jardin partagé ou un potager pédagogique.  

◦ Gourdes écoresponsables : Méina & Lilian proposent de créer et vendre une gourde en matériaux recyclés.

3. Culture, Éducation et Entraide

L'accès à la culture et le soutien scolaire sont des axes de développement majeurs.

• Utilisation de la Salle de Cinéma : Un projet récurrent.

◦ Usage pédagogique : Roman & Louison souhaitent encourager les professeurs à utiliser la salle pour des projections en lien avec les cours.  

◦ Accès gratuit à la culture : Henry & Maxime et Mabine & Victoria veulent organiser des séances de cinéma gratuites et régulières pour tous.

• Tutorat et Parrainage :

◦ Aide aux devoirs : Romain & Noa suggèrent un système de tutorat entre 15h et 18h pour les élèves en difficulté.   

◦ Parrainage Secondes/Terminales : Sacha & Lili proposent un parrainage pour faciliter l'intégration des Secondes, une idée également mentionnée par Mabine & Victoria.

• Enrichissement Culturel :

◦ Sorties culturelles et sportives : Mabine & Victoria et Sacha & Lili souhaitent en organiser davantage.  

◦ Rencontres avec d'anciens élèves : Mabine & Victoria proposent d'inviter des anciens pour partager leur parcours.   

◦ Concours de talents : Mabine & Victoria veulent lancer un concours annuel (photo, écriture, musique, sport).  

◦ Clubs : L'idée de clubs (lecture, débat) est avancée par Mabine & Victoria.

4. Améliorations Pratiques et Bien-être

Les candidats sont attentifs aux aspects concrets de la vie lycéenne et à la santé mentale des élèves.

• Espaces de Vie et de Travail :

◦ Création d'un lieu de repos/travail : Sacha & Oscar constatent que la Maison des Lycéens est souvent encombrée et bruyante et proposent un nouvel espace.  

◦ Aménagement d'un coin calme et détente : Une priorité pour Mabine & Victoria et Sacha & Lili, qui souhaitent aussi mettre en place des ateliers de gestion du stress.

• Infrastructures et Services :

◦ Ajout de casiers : Arnaud & Grégoire insistent sur le manque de casiers, notamment pour les demi-pensionnaires aux journées longues.  

◦ Deuxième service à la cantine : Proposé par Sacha & Oscar.    ◦ Vente de fournitures scolaires : Avazov & Isaac suggèrent une vente au foyer pour les oublis avant une évaluation.

• Ambiance Sonore et Visuelle :

◦ Changement de la sonnerie : Henry & Maxime proposent un vote via Instagram pour changer la sonnerie plusieurs fois par an.  

◦ Décorations du lycée : Arnaud & Grégoire et Roman & Louison veulent décorer l'établissement pour les fêtes (Noël, Halloween).

5. Communication et Démocratie Lycéenne

Améliorer la transmission de l'information et l'écoute des élèves est un enjeu clé.

• Outils de Communication Numérique :

◦ Gestion du compte Instagram du CVL : Arnaud & Grégoire veulent le rendre plus actif avec des sondages et des informations pratiques (menus de la cantine).   

◦ Utilisation des télévisions de l'accueil : Henry & Maxime veulent y diffuser des informations utiles (horaires de bus, actualités du CVL).

• Mécanismes de Participation :

◦ Boîte à idées : Proposée par Méina & Lilian pour que chaque élève puisse soumettre des propositions.  

◦ Comptes-rendus mensuels : Également une idée de Méina & Lilian pour plus de transparence sur les actions du CVL.

• Philosophie de Représentation :

◦ La quasi-totalité des candidats se disent "à l'écoute".  

◦ Baptiste se distingue par une approche radicale, refusant un programme pour se faire le porte-parole direct et exclusif des demandes formulées par les élèves.

Synthèse des Métiers : Perspectives et Exigences

Résumé

Ce document propose une synthèse exhaustive des informations présentées sur un large éventail de professions, allant de l'ingénierie à la santé, en passant par les arts et le droit.

L'analyse des données révèle plusieurs thèmes transversaux. Premièrement, l'accès à de nombreux métiers spécialisés, notamment dans les secteurs de la santé et de la technologie, exige un parcours académique long et rigoureux, souvent au niveau Bac+5 et pouvant dépasser Bac+10. Deuxièmement, la réussite professionnelle repose systématiquement sur une double compétence : une expertise technique pointue ("hard skills") et des qualités interpersonnelles solides ("soft skills") comme la communication, la gestion du stress et le travail d'équipe.

Troisièmement, la notion de "vocation" ou de "passion" est un moteur essentiel, particulièrement dans les domaines exigeants qui demandent des sacrifices personnels importants.

Enfin, le marché du travail est caractérisé par une forte variabilité des rémunérations, non seulement entre les secteurs mais aussi en fonction de l'expérience, du statut (salarié, libéral, public, privé) et de la nécessité omniprésente d'une formation continue pour s'adapter aux évolutions technologiques et réglementaires.

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Ingénierie et Technologie

Cette section regroupe les métiers au cœur de l'innovation, de la conception et du développement technologique. Ces professions exigent une forte expertise scientifique et une capacité à résoudre des problèmes complexes.

Ingénieur Ferroviaire

• Rôle et Missions : Gérer la sécurité des circulations ferroviaires en concevant et entretenant des systèmes robustes. Collabore avec des services variés comme le BTP, l'architecture et l'environnement.

• Formation et Diplômes : Niveau Bac+5 minimum, via une école d'ingénieurs (ex: École des Ponts ParisTech, Conservatoire national des arts et métiers).

• Compétences et Qualités Requises : Inventif, stratégique, organisé, curieux, capable de diriger une équipe.

• Rémunération : Salaire net mensuel débutant à 2 250 €, pouvant atteindre 5 000 € en fin de carrière.

• Avantages et Inconvénients :

◦ Avantages : Secteur en plein essor qui recrute, polyvalence, belles évolutions de carrière, nombreuses primes.    ◦ Inconvénients : Études longues et exigeantes, nécessité de se mettre à jour constamment, amplitudes horaires parfois excessives.

Ingénieur en Construction Automobile

• Rôle et Missions : Créer, développer et construire les pièces des véhicules pour optimiser les modèles actuels et futurs. Travaille à partir d'un cahier des charges, réalise des calculs, des essais sur ordinateur et des tests sur prototypes.

• Formation et Diplômes : Bac+5 en école d'ingénieur ou master en mécanique/électronique.

• Compétences et Qualités Requises : Passionné, imaginatif, rigoureux, persévérant. Nécessite également une bonne vue, une bonne condition physique et de la dextérité.

• Conditions de Travail : Principalement en bureau mais peut nécessiter des déplacements. Semaines de 35 à 40 heures, voire plus selon les projets.

• Rémunération : Début de carrière autour de 2 000 € net/mois. Le salaire annuel brut peut passer de 37 300 € à 98 000 € en fin de carrière.

• Perspectives d'Évolution : Insertion professionnelle facile malgré des débuts parfois difficiles en sortie d'école.

Ingénieur en Intelligence Artificielle (IA)

• Rôle et Missions : Concevoir et développer des algorithmes capables d'apprendre et de prendre des décisions de manière autonome. Programme des modèles d'IA, analyse de grandes quantités de données.

• Formation et Diplômes : Bac+5 (diplôme d'ingénieur ou master spécialisé en IA). Spécialités NSI, Maths et Physique recommandées au lycée. Formation continue essentielle.

• Compétences et Qualités Requises : Programmation (Python, R), maîtrise des mathématiques appliquées, esprit d'analyse, rigueur, créativité, curiosité.

• Conditions de Travail : Travail en équipe avec des data scientists et développeurs, horaires flexibles, télétravail courant.

• Rémunération :

◦ Source 1 : Salaire annuel de 45 000 € à 55 000 € en début de carrière, pouvant dépasser 100 000 € avec l'expérience.    ◦ Source 2 : Salaire brut mensuel de 3 500 € à 4 500 € pour un débutant, pouvant atteindre 7 000 € ou plus.

• Perspectives d'Évolution : Secteur en pleine explosion avec une très forte demande et des opportunités dans de nombreux domaines (santé, finance, automobile).

Ingénieur Aéronautique et Aérospatial

• Rôle et Missions : Concevoir, développer, tester et améliorer les aéronefs (avions, hélicoptères, drones) et les engins spatiaux (fusées, satellites). Travaille sur les composants, les moteurs, les systèmes de navigation.

• Formation et Diplômes : Bac+5 minimum, via une école d'ingénieur spécialisée (ENAC, ESTACA, IPSA) ou généraliste.

• Compétences et Qualités Requises : Solides connaissances en mécanique des fluides, aérodynamique, thermodynamique, matériaux. Maîtrise de l'anglais, esprit d'équipe, capacité à travailler sous pression.

• Conditions de Travail : S'exerce dans des usines, des bureaux d'études ou des agences (NASA, ESA). Déplacements fréquents sur les chantiers.

• Rémunération :

◦ Ingénieur aéronautique : Commence à 3 400 € brut/mois, peut atteindre 123 000 €/an avec l'expérience.    ◦ Ingénieur aérospatial : Salaire annuel de 40 000 € à 50 000 € en sortie d'école, jusqu'à 80 000 € après 10 ans, et peut dépasser 100 000 €.

• Perspectives d'Évolution : Secteur très demandé. Évolution vers des postes de chef de projet, directeur technique ou expert.

Ingénieur Chimiste

• Rôle et Missions : Concevoir de nouveaux produits, mettre en œuvre des démarches scientifiques, réaliser des contrôles qualité et rédiger des fiches de données de sécurité (FDS).

• Formation et Diplômes : Bac+5 obtenu en école d'ingénieur.

• Compétences et Qualités Requises : Patience, rigueur, sociabilité, bonnes capacités rédactionnelles, maîtrise de l'anglais et excellentes compétences en physique-chimie et mathématiques.

• Conditions de Travail : Travail en petits groupes (laboratoire, usine), mobile pour répondre aux besoins des entreprises. Journées de 8 heures maximum.

• Rémunération : Environ 2 000 € dans le public et 3 000 € dans le privé. Des pays comme l'Allemagne ou le Luxembourg offrent des salaires plus élevés.

Ingénieur en Conception Mécanique

• Rôle et Missions : Développement d'objets techniques de demain (recherche et développement). Conçoit le produit, son mécanisme, réalise des modélisations et des essais.

• Formation et Diplômes : Bac+5 (diplôme d'ingénieur ou master) avec une mention en mécanique ou génie mécanique.

• Compétences et Qualités Requises : Curiosité, persévérance, goût pour l'innovation, maîtrise des logiciels de conception, solides connaissances théoriques (aérodynamique, résistance des matériaux).

• Conditions de Travail : Travail en bureau ou en laboratoire, en équipe pluridisciplinaire. Temps de travail moyen de 40 heures/semaine.

• Rémunération : Salaire brut mensuel de 2 800 € en sortie d'école, évoluant vers 3 500 € et pouvant atteindre 5 000 €.

Concepteur Développeur / Ingénieur Logiciel

• Rôle et Missions : Créer, développer et mettre en place des applications, logiciels ou sites web selon un cahier des charges. Analyse les besoins, écrit le code, effectue des tests et peut former les utilisateurs.

• Formation et Diplômes : Niveau Bac+2 (DUT/BTS) à Bac+5 (diplôme d'ingénieur, Master MIAGE).

• Compétences et Qualités Requises : Maîtrise technique des langages de programmation (HTML, CSS, PHP, etc.), rigueur, capacité d'adaptation, sens de l'organisation, écoute du client, travail en équipe.

• Conditions de Travail : Travail sédentaire mais collaboratif. Délais parfois courts, environ 9 heures de travail par jour.

• Rémunération :

◦ Concepteur Développeur : Jusqu'à 2 000 € brut/mois en début de carrière, environ 5 000 € pour un profil expérimenté.    ◦ Ingénieur Logiciel : 2 830 € brut/mois en début de carrière (2 200 € net), jusqu'à 4 500 € brut (3 600 € net) avec l'expérience.

Développeur de Jeux Vidéo

• Rôle et Missions : Écrire et modifier le code source pour assurer le bon fonctionnement d'un jeu. Optimise les graphismes, l'IA et la fluidité. Utilise des moteurs de jeu (Unity, Unreal Engine) et des langages (C++, Python).

• Formation et Diplômes : Bac+3 à Bac+5 en informatique, développement logiciel ou jeux vidéo. Des écoles spécialisées (Isart, Supinfogame) sont une voie possible. Formation continue indispensable.

• Compétences et Qualités Requises : Logique, rigueur, patience, esprit d'analyse, capacité à résoudre des problèmes techniques.

• Conditions de Travail : Travail en studio ou en freelance, principalement sédentaire. Collaboration étroite avec les graphistes et game designers. Horaires classiques mais "périodes de crunch time" intenses en fin de projet.

• Rémunération : Salaire annuel brut de 30 000 € à 40 000 € pour un débutant, pouvant atteindre 60 000 € et plus avec l'expérience.

Domoticien

• Rôle et Missions : Installer et programmer des systèmes automatisés dans les habitations (volets, alarmes, thermostats) pour améliorer le confort, la sécurité et l'efficacité énergétique.

• Formation et Diplômes : Bac Pro Systèmes Numériques, BTS Domotique, ou BUT Génie Électrique et Informatique Industrielle. Formation régulière nécessaire.

• Compétences et Qualités Requises : Connaissances en électronique et informatique, logique, précision, esprit d'analyse, patience.

• Conditions de Travail : Métier dynamique, partagé entre chantiers et bureaux d'études. Déplacements fréquents, collaboration avec d'autres corps de métier. Semaines de 35 à 40 heures, avec possibles heures supplémentaires.

• Rémunération : Salaire brut mensuel de 1 800 € à 2 200 € en début de carrière, pouvant atteindre 3 000 € à 4 000 € avec l'expérience.

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Santé et Sciences du Vivant

Ce domaine regroupe des professions dédiées au soin, au diagnostic et à l'amélioration de la santé humaine. Elles se caractérisent par un parcours d'études long, un fort sens des responsabilités et un contact humain central.

Médecin (Chirurgien, Pédiatre, Urgentiste, Médecin Légiste)

Spécialité

Formation

Rôle et Missions

Conditions de Travail

Rémunération (Début de carrière)

Chirurgien

11-12 ans post-bac

Opérer des patients pour soigner ou réparer.

Très intense, longues heures, forte pression, gardes. Travail d'équipe.

4 000 - 5 000 € / mois

Pédiatre

10-11 ans post-bac

Soigner les enfants de la naissance à 18 ans, suivi médical, diagnostic.

En cabinet, hôpital, PMI. Contact humain et psychologique essentiel.

3 000 - 3 500 € net / mois

Médecin Urgentiste

10 ans post-bac min.

Prise en charge de patients en situation d'urgence.

Forte pression, décisions rapides, travail d'équipe pluridisciplinaire.

4 500 - 5 000 € brut / mois

Médecin Légiste

9-11 ans post-bac + DES

Analyser corps et blessures dans un cadre judiciaire (autopsies, examens).

Stress élevé, résistance émotionnelle requise, horaires variables et urgences.

3 000 - 3 500 € net / mois

Professionnels Paramédicaux et de la Santé

Profession

Formation

Rôle et Missions

Conditions de Travail

Rémunération (Début de carrière)

Infirmière

Bac + IFSI (3 ans)

Prodiguer des soins, surveiller l'état de santé, accompagner les patients.

Très variées (hôpital, libéral, école, armée). Horaires décalés, stress.

Varie fortement : 1 860 € à 3 075 € brut / mois

Kinésithérapeute

5 ans post-bac

Soigner et rééduquer les personnes ayant des troubles du mouvement.

En libéral, hôpital, centre de rééducation. Métier mobile, horaires flexibles mais longs.

Non spécifié

Pharmacien

6-9 ans post-bac

Gérer et distribuer les médicaments, conseiller les patients.

En officine, hôpital, industrie. Horaires variables, gardes. Forte responsabilité.

3 000 - 3 500 € brut / mois

Diététicienne

Bac+2 (BTS/BUT)

Conseiller et accompagner les personnes dans la gestion de leur alimentation.

En cabinet, hôpital, scolaire. Métier plutôt sédentaire.

1 800 - 2 200 € brut / mois

Chirurgiens-Dentistes et Spécialistes

• Rôle et Missions : Prévention, soins conservateurs (caries, détartrage), pose de prothèses et actes chirurgicaux (extractions, implants).

• Formation :

◦ Chirurgien-dentiste : 6 ans post-bac (PASS/LAS + 5 ans d'études).    ◦ Dentiste pédiatrique : Spécialisation après le cursus de chirurgie dentaire.

• Compétences et Qualités Requises : Minutie, méthode, empathie, écoute, dextérité manuelle. Le dentiste pédiatrique doit savoir rassurer les enfants.

• Conditions de Travail : Métier sédentaire, en cabinet libéral ou hôpital. Travail en équipe (assistant, secrétaire).

• Rémunération :

◦ Chirurgien-dentiste : De 2 500 € à 7 500 € / mois selon l'expérience.    ◦ Dentiste pédiatrique : 2 500 € à 6 000 € / mois en libéral ; 2 000 € à 3 500 € en salariat.

Biologiste Médical

• Rôle et Missions : Analyser des prélèvements biologiques (sang, urine) pour aider au diagnostic. Valide les prescriptions, interprète les résultats, participe à la recherche.

• Formation et Diplômes : Bac+9 minimum (Doctorat en Pharmacie ou Médecine + DES de biologie médicale).

• Compétences et Qualités Requises : Solides connaissances scientifiques, compétences en gestion, esprit d'initiative, sens du dialogue.

• Conditions de Travail : Travail sédentaire en laboratoire (privé ou hospitalier), en équipe. Semaines de 30 à 40 heures avec gardes possibles.

• Rémunération : À partir de 4 500 € brut/mois (public) ou 5 500 € brut/mois (privé).

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Art, Création et Communication

Ce secteur rassemble des métiers où la créativité, le sens esthétique et la capacité à transmettre un message sont primordiaux. Ils sont souvent passionnants mais peuvent être exigeants et concurrentiels.

Directeur Artistique

• Rôle et Missions : Donner une identité visuelle forte à un projet (campagne publicitaire, site web, magazine). Élabore des concepts, choisit couleurs et typographies, supervise la production graphique.

• Formation et Diplômes : Formation en art graphique, design ou communication visuelle (Beaux-Arts, Gobelins, etc.). Licence (Bac+3) minimum, Master (Bac+5) recommandé.

• Compétences et Qualités Requises : Créatif, curieux, à l'affût des tendances, compétences techniques (Photoshop, Illustrator), rigueur, gestion du stress.

• Conditions de Travail : Exigeant, pression des délais, horaires parfois irréguliers en agence.

• Rémunération : Salaire annuel brut de 30 000 € à 40 000 € pour un débutant, 50 000 € à 70 000 € (voire plus) pour un profil confirmé.

Journaliste

• Rôle et Missions : Rechercher, vérifier et transmettre des informations au public. Peut se spécialiser (grand reporter, journaliste politique, etc.).

• Formation et Diplômes : Formation post-bac dans l'une des 14 écoles de journalisme reconnues en France.

• Compétences et Qualités Requises : Rigueur, curiosité, disponibilité, maîtrise des outils multimédias.

• Conditions de Travail : Peut s'exercer sur le terrain ou en bureau. Horaires très variables, soumis à l'actualité (weekends, nuits). Débuts souvent précaires (pigiste).

• Rémunération : Très grandes variations de salaire, de 1 140 € à 45 000 € par mois.

Metteur en Scène et Chorégraphe

• Rôle et Missions :

◦ Metteur en scène : Crée des spectacles, dirige une équipe artistique et gère des aspects variés (rédaction de dossiers, budgets).    ◦ Chorégraphe : Crée les mouvements pour des danseurs ou des comédiens. Souvent danseur à l'origine.

• Formation et Diplômes : Parcours universitaires (études théâtrales) ou expérience directe en tant qu'artiste (danseur).

• Compétences et Qualités Requises : Curiosité, travailleur, savoir-faire variés, capacité à diriger une équipe. La passion est décrite comme une "nécessité".

• Conditions de Travail : Métier difficile, conditions financières souvent précaires.

• Rémunération : Non spécifiée, mais la précarité est soulignée.

Joaillier

• Rôle et Missions : Concevoir, fabriquer, réparer et restaurer des bijoux en métaux précieux. Combine savoir-faire artisanal, précision technique et créativité.

• Formation et Diplômes : Filières artisanales (CAP, BMA) ou artistiques (DN MADE, École Boulle, Haute École de Joaillerie).

• Conditions de Travail : Travail sédentaire en atelier, seul ou en équipe. Horaires de 35-39h/semaine pour les salariés, jusqu'à 50-60h pour les indépendants.

• Rémunération : Non spécifiée.

Architecte d'Intérieur

• Rôle et Missions : Concevoir et réaliser l'aménagement d'espaces intérieurs. Visite les sites, dessine les plans, suit les chantiers.

• Formation et Diplômes : BTS Étude et réalisation d'agencement (Bac+2), DN MADE (Bac+3), Master (Bac+5).

• Compétences et Qualités Requises : Créativité, innovation, savoir dessiner.

• Conditions de Travail : Jongle entre le bureau (sédentaire) et le chantier (mobile). Le temps de travail est très variable, grande disponibilité requise.

• Rémunération : Débute à environ 2 300 €/mois, peut atteindre 3 800 €/mois en entreprise.

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Droit, Sécurité et Service Public

Ces professions sont au service de la justice, de la protection des citoyens et de l'ordre public. Elles exigent un grand sens de l'éthique, de la rigueur et une forte résistance au stress.

Avocat

• Rôle et Missions : Conseiller, défendre et représenter les intérêts de ses clients (particuliers, entreprises) devant les juridictions. Peut être généraliste ou spécialisé.

• Formation et Diplômes : Master en droit (Bac+4/5) + examen d'entrée à l'École d'avocats (EDA) + 18 mois de formation pour obtenir le CAPA.

• Compétences et Qualités Requises : Patience, écoute, organisation, persévérance, connaissance approfondie du droit.

• Conditions de Travail : Travail mobile (tribunal) et sédentaire (cabinet). Profession majoritairement libérale, avec un temps de travail variable.

• Rémunération :

◦ Source 1 : 1 800 € à 2 700 € / mois en début de carrière.    ◦ Source 2 : 30 000 € à 40 000 € / an en début de carrière, peut dépasser 100 000 € / an avec l'expérience.

Sapeur-Pompier Professionnel

• Rôle et Missions : Secourir les personnes et protéger les biens lors d'incendies, d'accidents et autres sinistres.

• Formation et Diplômes : Diplôme national du brevet minimum, suivi d'un concours.

• Compétences et Qualités Requises : Excellente condition physique et mentale, capacité à travailler en équipe, respect des ordres, gestion des émotions face à des situations choquantes.

• Conditions de Travail : Métier difficile, physiquement et mentalement. Forte hiérarchie et responsabilité croissante avec le grade.

• Rémunération : Varie selon le grade, complétée par des primes.

Officier de Police Judiciaire (OPJ)

• Rôle et Missions : Constater les infractions, recevoir les plaintes, mener des enquêtes et placer des suspects en garde à vue sous l'autorité du procureur.

• Formation et Diplômes : Licence (Bac+3) + concours de l'École Nationale Supérieure de la Police (ENSP) (formation de 18 mois).

• Compétences et Qualités Requises : Rigueur, courage, droiture, sens du collectif.

• Conditions de Travail : Travail en commissariat, souvent en horaires décalés (nuit, weekends). Métier stressant, exigeant des décisions rapides et le respect strict des procédures.

• Rémunération : Un lieutenant débute à 2 420 € brut/mois, peut atteindre 4 000 € brut/mois en tant que commissaire.

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Commerce, Gestion et Construction

Ce secteur couvre la conception et la réalisation de bâtiments ainsi que la gestion des activités commerciales. Il requiert des compétences en organisation, en management et en communication.

Architecte

• Rôle et Missions : Concevoir un projet architectural (plans) et suivre sa réalisation sur le chantier. Peut concerner des constructions neuves ou des rénovations.

• Formation et Diplômes : Diplôme d'architecte DPLG, obtenu après des études en école d'architecture. Formation continue exigée.

• Conditions de Travail : Moitié sédentaire (dessin, administratif), moitié mobile (relevés, réunions de chantier). Travail d'équipe indispensable. Temps de travail élevé (environ 50h/semaine pour un gérant).

• Rémunération : Non spécifiée.

Conducteur de Travaux

• Rôle et Missions : Responsable de la gestion et de la coordination d'un chantier de construction. Veille au respect des délais, du budget et des normes de sécurité.

• Formation et Diplômes : DUT Génie Civil ou diplôme d'école d'ingénieur en construction.

• Compétences et Qualités Requises : Organisé, sens de la gestion, rigoureux, capable de résoudre des problèmes rapidement, bonne endurance physique.

• Conditions de Travail : Combine travail sur le chantier (extérieur) et au bureau (administratif). Travail en équipe. Semaines de 35 à 40 heures avec possibles heures supplémentaires.

• Rémunération : Salaire brut mensuel de 2 000 € à 2 500 € en début de carrière, pouvant atteindre 4 000 € ou plus.

Manager Commercial

• Rôle et Missions : Gérer un ou plusieurs rayons d'un magasin, ce qui inclut le rangement, la mise en avant des produits et la gestion des achats.

• Formation et Diplômes : DUT Management ou Master Commercial.

• Compétences et Qualités Requises : Organisé, sociable, capable de calculer et de gérer des stocks.

• Conditions de Travail : Travail mobile au sein du magasin, en équipe. Semaines de 35 heures, mais peut inclure le travail le week-end et des horaires matinaux ou tardifs.

• Rémunération : Environ 1 100 € net/mois pour un alternant, jusqu'à 2 800 € net/mois pour un manager confirmé.

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Science et Recherche

Ces métiers sont dédiés à l'avancement des connaissances. Ils demandent un très haut niveau d'études, de la rigueur intellectuelle et une grande persévérance.

Paléontologue

• Rôle et Missions : Étudier les restes fossiles des êtres vivants du passé. Extrait, préserve, étudie et reconstitue des squelettes.

• Formation et Diplômes : Longues études jusqu'au Doctorat (Bac+8).

• Compétences et Qualités Requises : Connaissances en biologie et géologie, maîtrise des technologies de fouille, patience et persévérance.

• Rémunération : Débute à 1 900 € brut/mois.

Astrophysicien

• Rôle et Missions : Étudier le ciel, les objets célestes (étoiles, planètes, galaxies) et leurs caractéristiques physiques. Collecte et analyse des données de télescopes et satellites.

• Formation et Diplômes : Doctorat (thèse) incontournable. Voies possibles via l'université ou une école d'ingénieur.

• Compétences et Qualités Requises : Grande rigueur, capacité à se représenter des concepts abstraits, savoir travailler en équipe.

• Conditions de Travail : Principalement un métier de bureau, mais avec des déplacements pour les conférences. Horaires souples mais pouvant atteindre 40h/semaine.

• Rémunération : Varie selon les agences, de 3 000 € à 5 000 € net/mois.

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

Autres Métiers Spécialisés

Libraire

• Rôle et Missions : Sélectionner, acheter et vendre des ouvrages. Conseille les clients, gère les stocks et organise des événements culturels.

• Formation et Diplômes : Formations possibles du CAP au DUT et Licence Professionnelle "Métiers du livre".

• Compétences et Qualités Requises : Bonne culture générale, goût pour la lecture, excellente mémoire, capable de rester debout longtemps.

• Conditions de Travail : Métier sédentaire en librairie, horaires de commerce (35-39h/semaine, incluant le samedi).

• Rémunération : Salaire brut mensuel de 1 500 € à 1 800 € pour un débutant.

Accompagnateur en Moyenne Montagne

• Rôle et Missions : Accompagner des groupes de personnes en moyenne montagne (randonnée, raquettes). Ne peut pas marcher sur des glaciers ou utiliser des techniques d'alpinisme.

• Formation et Diplômes : Diplôme d'État, préparé au Centre National de Ski Nordique et de Moyenne Montagne.

• Compétences et Qualités Requises : Excellente condition physique, amour de la nature, savoir travailler en groupe.

• Conditions de Travail : Travail en extérieur par tous les temps. Statut souvent indépendant, nécessitant de se faire connaître.

• Rémunération : Gagne entre 170 € et 270 € par sortie.

I experienced something very similar in my high school Spanish classes. It was common knowledge amongst students at the school that the teaching in the Spanish classes were mediocre, less than satisfactory, and undesirable. Additionally the expectations were very low and I learned very little despite taking 3 years of courses.

Stoneditch pointed out an unusual period of media concentration in the United States in the 1900s. This centralization creates a more unified national narrative that feels very different from today’s fragmented social media environment. This “solidarity” can silence minority voices because, as the passage points out, those mainstream newsrooms are dominated by white people, resulting in limited diversity in the news media, further increasing the promotion of a single group narrative.

I find the simplification of data very interesting, because that is an aspect of coding that I have never really considered before. Even in our everyday lives, information is being simplified in a way that is easy for people to process and digest, so why should coding be any different? In particular, the examples of the complexity of even simple questions like "what country are you from" was interesting because I had never really considered the complexities behind questions like that.

While reading §4.2 and the debate over Twitter bots, I was struck by how definitions can quietly gain influence. For a class project, I modified a spam heuristic (number of URLs + account age), and my bot estimate went from 3% to 14%—the same data, just simplified. This convinced me that the "<5%" number wasn't a fact, but a governance decision about what's considered criticaI. Platforms should publish ranges based on uncertain scenarios and disclose the underlying assumptions. Question: Why don't we require confidence intervals and other definitions for platform metrics, as we do in epidemioIogy?

• grand, rhetorical vision of science which Victor feeds on

• making small discoveries at a time

• fictions

https://www.youtube.com/watch?v=T-ibUVMh9XE

See this video for more information on how to do this.

The creation and research for my project will follow a similar list of components. 1) Locate and find the data: I have to select artworks that I believe are rich in narrative, to allow for an expressive animation. 2) Research and collect: Find as much information about the narrative as possible, and collect as much context as I can. The more the better, as it brings more content for the story. 3) Analyze the Data and Visuals: What parts are the best for animation? What would bring the narrative to life the most? 4) Communicating the Story in the Data: Using Krita and bringing the project to light.

My project ties into the themes of digital humanities as well. Digital humanities is how we apply digital technologies to study culture, history and the arts. Because my project requires a lot of research with these 3 categories, my work with Krita will integrate with the concept of digital humanities with archeology.

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Referee #3

Evidence, reproducibility and clarity

Summary

Marcu et al. demonstrate a gut-neuron axis that is required for the lifespan-shortening effects mediated by gut bacteria. They show that the presence of commensal bacteria-particularly Acetobacter pomorum-promotes Tk expression in the gut, which then binds to neuronal tachykinin receptors to shorten lifespan. Tk has also recently been reported to extend lifespan via adipokinetic hormone (Akh) signaling (Ahrentløv et al., Nat Metab 7, 2025), but the mechanism here appears distinct. The lifespan shortening by Ap via Tk seems to be partially dependent on foxo and independent of both insulin signaling and Akh-mediated lipid mobilization. Although the detailed mechanistic link to lifespan is not fully resolved, the experiment and its results clearly shows the involvement of the molecules tested. This work adds a valuable dimension to our growing understanding of how gut bacteria influence host longevity. However, there are some points that should be addressed.

1. Tk+ EEC activity should be assessed directly, rather than relying solely on transcript levels. Approaches such as CaLexA or GCaMP could be used.
2. In Line243, the manuscript states that the reporter activity was not increased in the posterior midgut. However, based on the presented results in Fig4E, there is seemingly not apparent regional specificity. A more detailed explanation is necessary.
3. If feasible, assessing foxo activation would add mechanistic depth. This could be done by monitoring foxo nuclear localization or measuring the expression levels of downstream target genes.
4. Fig1C uses Adh for normalization. Given the high variability of the result, the authors should (1) check whether Adh expression levels changed via bacterial association and/or (2) compare the results using different genes as internal standard.
5. While the difficulty of maintaining lifelong axenic conditions is understandable, it may still be feasible to assess the induction of Tk (ie. Tk transcription or EE activity upregulation) by the microbiome on males.
6. We also had some concerns regarding the wording of the title. Fig6B and C suggests that TkR86C, in addition to TkR99D, may be involved in the A. pomorum-lifespan interaction. Consider revising the title to refer more generally to the "tachykinin receptor" rather than only TkR99D. The difference between "aging" and "lifespan" should also be addressed. While the study shows a role for Tk in lifespan, assessment of aging phenotypes (eg. Climbing assay, ISC proliferation) beyond the smurf assay is required to make conclusions about aging.
7. The statement in Line 82 that EEs express 14 peptide hormones should be supported with an appropriate reference, if available.

Referees cross-commenting

I agree with the other reviewers that the study has been done very well and hence additional experiments are not mandatory to be published such as calcium imaging. However, I still believe that testing Tk's elevation by the Ap in males should greatly increase the generality of the finding, no matter what the outcome would be. Too many studies use only females.

Significance

General assessment

The main strength of this study is the careful and extensive lifespan analyses, which convincingly demonstrate the role of gut microbiota in regulating longevity. The authors clarify an important aspect of how microbial factors contribute to lifespan control. The main limitation is that the study primarily confirms the involvement of previously reported signaling pathways, without identifying novel molecular players or previously unrecognized mechanisms of lifespan regulation.

Advance

The lifespan-shortening effect of Acetobacter pomorum (Ap) has been reported previously, as has the lifespan-shortening effect of Tachykinin (Tk). However, this study is the first to link these two factors mechanistically, which represents a significant and original contribution to the field. The advance is primarily mechanistic, providing new insight into how microbial cues converge on host signaling pathways to influence ageing.

Audience

This work will be of particular interest to a specialized audience of basic researchers in ageing biology. It will also attract interest from microbiome researchers who are investigating host-microbe interactions and their physiological consequences. The findings will be useful as a conceptual framework for future mechanistic studies in this area.

Field of expertise

Drosophila ageing, lifespan, microbiome, metabolism

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Referee #2

Evidence, reproducibility and clarity

The main finding of this work is that microbiota impacts lifespan though regulating the expression of a gut hormone (Tk) which in turn acts on its receptor expressed on neurons. This conclusion is robust and based on a number of experimental observation, carefully using techniques in fly genetics and physiology: 1) microbiota regulates Tk expression, 2) lifespan reduction by microbiota is absent when Tk is knocked down in gut (specifically in the EEs), 3) Tk knockdown extends lifespan and this is recapitulated by knockdown of a Tk receptor in neurons. These key conclusions are very convincing. Additional data are presented detailing the relationship between Tk and insulin/IGF signalling and Akh in this context. These are two other important endocrine signalling pathways in flies. The presentation and analysis of the data are excellent.

There are only a few experiments or edits that I would suggest as important to confirm or refine the conclusions of this manuscript. These are:

1. When comparing the effects of microbiota (or single bacterial species) in different genetic backgrounds or experimental conditions, I think it would be good to show that the bacterial levels are not impacted by the other intervention(s). For example, the lifespan results observed in Figure 2A are consistent with Tk acting downstream of the microbes but also with Tk RNAi having an impact on the microbiota itself. I think this simple, additional control could be done for a few key experiments. Similarly, the authors could compare the two bacterial species to see if the differences in their effects come from different ability to colonise the flies.
2. The effect of Tk RNAi on TAG is opposite in CR and Ax or CR and Ap flies, and the knockdown shows an effect in either case (Figure 2E, Figure 3D). Why is this? Better clarification is required.
3. With respect to insulin signalling, all the experiments bar one indicate that insulin is mediating the effects of Tk. The one experiment that does not is using dilpGS to knock down TkR99D. Is it possible that this driver is simply not resulting in an efficient KD of the receptor? I would be inclined to check this, but as a minimum I would be a bit more cautious with the interpretation of these data.
4. Is it possible to perform at least one lifespan repeat with the other Tk RNAi line mentioned? This would further clarify that there are no off-target effects that can account for the phenotypes.

There are a few other experiments that I could suggest as I think they could enrich the current manuscript, but I do not believe they are essential for publication: 5. The manuscript could be extended with a little more biochemical/cell biology analysis. For example, is it possible to look at Tk protein levels, Tk levels in circulation, or even TkR receptor activation or activation of its downstream signalling pathways? Comparing Ax and CR or Ap and CR one would expect to find differences consistent with the model proposed. This would add depth to the genetic analysis already conducted. Similarly, for insulin signalling - would it be possible to use some readout of the pathway activity and compare between Ax and CR or Ap and CR? 6. The authors use a pan-acetyl-K antibody but are specifically interested in acetylated histones. Would it be possible to use antibodies for acetylated histones? This would have the added benefit that one can confirm the changes are not in the levels of histones themselves. 7. I think the presentation of the results could be tightened a bit, with fewer sections and one figure per section.

Referees cross-commenting

Reviewer 1

I generally agree with this reviewer but for

"I'm convinced by the data showing that FOXO is required for TkRNAi to prevent lifespan shortening by Ap, but FOXO doesn't only respond to insulin signalling and can't be taken by itself to indicate a role for insulin signalling which the authors appear to do here."

To the best of my knowledge, Foxo has only been shown to be required for lifespan extension/modulation by a reduction in insulin-like signalling. I.e. it does respond to other pathways but this is the only one where Foxo activity is known to modulate lifespan.

Reviewer 3

I agree with reviewer 1 that point raised under (1) does not appear strictly required for the conclusions of the manuscript.

Both reviewers 1 and 3:

I have a different take on the results of experiments where IPCs are manipulated. To me, Figure 7D and E show that ablating the IPCs removes the difference between Ax and Ap i.e. the IPCs are involved and insulin-like signalling is likely involved. The fact that RNAi against the TKR99D receptor does not have the same effect, does not matter (the sensing could happen in different neurons). Similarly, dilp expression is only a minor readout of what is happening with insulin-like signalling - dilps are controlled at the level of secretion.

However, I would be happy for the authors to present different arguments and make a reasonable conclusion, which may differ from mine. But I think the arguments I present above should be taken into account.

Significance

The main contribution of this manuscript is the identification of a mechanism that links the microbiota to lifespan. This is very exciting and topical for several reasons:

1) The microbiota is very important for overall health but it is still unclear how. Studying the interaction between microbiota and health is an emerging, growing field, and one that has attracted a lot of interest, but one that is often lacking in mechanistic insight. Identifying mechanisms provides opportunities for therapies. The main impact of this study comes from using the fruit fly to identify a mechanism.

2) It is very interesting that the authors focus on an endocrine mechanism, especially with the clear clinical relevance of gut hormones to human health recently demonstrated with new, effective therapies (e.g. Wegovy).

3) Tk is emerging as an important fly hormone and this study adds a new and interesting dimension by placing TK between microbiota and lifespan.

I think the manuscript will be of great interest to researchers in ageing, human and animal physiology and in gut endocrinology and gut function.

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Referee #1

Evidence, reproducibility and clarity

Summary:

In this study the authors use a Drosophila model to demonstrate that Tachykinin (Tk) expression is regulated by the microbiota. In Drosophila conventionally reared (CR) flies are typically shorter lived than those raised without a microbiota (axenic). Here, knockdown of Tk expression is found to prevent lifespan shortening by the microbiota and the reduction of lipid stores typically seen in CR flies when compared to axenic counterparts. It does so without reducing food intake or fecundity which are often seen as necessary trade-offs for lifespan extension. Further, the strength of the interaction between Tk and the microbiota is found to be bacteria specific and is stronger in Acetobacter pomorum (Ap) monoassociated flies compared to Levilactobacillus brevis (Lb) monoassociation. The impact on lipid storage was also only apparent in Ap-flies. Building on these findings the authors show that gut specific knockdown is largely sufficient to explain these phenotypes. Knockdown of the Tk receptor, TkR99D, in neurons recapitulates the lifespan phenotype of intestinal Tk knockdown supporting a model whereby Tk from the gut signals to TkR99D expressing neurons to regulate lifespan. In addition, the authors show that FOXO may have a role in lifespan regulation by the Tk-microbiota interaction. However, they rule out a role for insulin producing cells or Akh-producing cells suggesting the microbiota-Tk interaction regulates lifespan through other, yet unidentified, mechanisms.

Major comments:

Overall, I find the key conclusions of the paper convincing. The authors present an extensive amount of experimental work, and their conclusions are well founded in the data. In particular, the impact of TkRNAi on lifespan and lipid levels, the central finding in this study, has been demonstrated multiple times in different experiments and using different genetic tools. As a result, I don't feel that additional experimental work is necessary to support the current conclusions. However, I find it hard to assess the robustness of the lifespan data from the other manipulations used (TkR99DRNAi, TkRNAi in dFoxo mutants etc.) because information on the population size and whether these experiments have been replicated is lacking. Can the authors state in the figure legends the numbers of flies used for each lifespan and whether replicates have been done? For all other data it is clear how many replicates have been done, and the methods give enough detail for all experiments to be reproduced.

Minor comments:

While I feel the conclusions of this study are well supported by the data I found this to be a complex read and in places hard to follow. I feel some work is necessary in the writing to help the reader follow the authors logic. Below I describe some of the issues that confused me and provide some suggestions that I hope the authors will find helpful.

Survival curves The authors state that the lifespan difference between CR and axenic flies disappears with TkRNAi because TkRNAi CR flies are longer lived, rather than because TkRNAi axenic flies are shorter lived. Is this consistent in every TkRNAi experiment? It's hard for the reader to assess this because the relevant lifespan curves are presented on separate plots. I'd encourage the authors to provide lifespan plots that enable comparison between all conditions. For example, in figures 2 and 6 the reader wants to directly compare between RU- and RU+ but can't easily do so. Additional plots could be made available in the supplementary figures showing the comparisons that are not easy to make on the main figures.

Consistent framing of the data Do the interventions shorten lifespan relative to the axenic cohort? Or do they prevent lifespan extension by axenic conditions? Both statements are valid, and the authors need to be consistent in which one they use to avoid confusing the reader. For example, line 325 says TkR86CRNAi prevents lifespan extension in axenic flies. Given the framing in the previous sections, it might be clearer to say that TkR86CRNAi shortens the lifespan of axenic flies to that of CR flies in contrast to TkRNAi and TkR99DRNAi which don't.

The impact of TkRNAi on lipid levels in axenic flies TkRNAi consistently reduces lipid levels in axenic flies (Figs 2E, 3D), essentially phenocopying the loss of lipid stores seen in control conventionally reared (CR) flies relative to control axenic. This suggests that the previously reported role of Tk in lipid storage - demonstrated through increased lipid levels in TkRNAi flies (Song et al (2014) Cell Rep 9(1): 40) - is dependent on the microbiota. In the absence of the microbiota TkRNAi reduces lipid levels. The lack of acknowledgement of this in the text is confusing for the reader because it is inconsistent with the microbiota driving both higher Tk expression and higher lipid storage. If the microbiota increases Tk expression and this results in reduced lipid storage, why does reduced Tk expression also result in reduced lipid storage in axenic flies? This could further highlight the unique impact that the interaction between TkRNAi and the microbiota has on lipid storage, given it reverses both the impact of the microbiota alone and TkRNAi alone. I feel this aspect of the data should be given more attention in the text both for clarity and because it may be telling us something important about the function of Tk. The current framing around pleiotropic effects is valid, and the impact of Tk on lipid storage is clearly independent of its impact on lifespan and so is not central to this study. However, I feel a short additional paragraph to acknowledge this nuance of the data is needed. It can be made clear in the text that further exploration is beyond the scope of the current study.

Role of insulin signalling and insulin producing cells I'm convinced by the data showing that FOXO is required for TkRNAi to prevent lifespan shortening by Ap, but FOXO doesn't only respond to insulin signalling and can't be taken by itself to indicate a role for insulin signalling which the authors appear to do here.

I would expect ablation of IPCs to have the opposite effect to foxo mutation and to increase FOXO activity throughout the organism due to a reduction in Dilp levels and so reduced insulin signalling. So, I have struggled to follow the authors logic in ablating the IPCs and feel a clear statement on what they expected the outcome to be would help the reader. They find that TkRNAi still prevents lifespan shortening by Ap when IPCs are ablated and that TkR99DRNAi in IPCs also doesn't block lifespan shortening by Ap despite reducing the expression of dilp3 and dilp5. To me these data rule out a role for insulin signalling despite the requirement for FOXO and yet the authors conclude that insulin signalling is involved in the response to Ap and TkRNAi, although not obligately (lines 420 - 422 and 511 - 512). Can the authors clarify their logic in concluding a role for insulin signalling, and qualify this conclusion with appropriate consideration of alternative hypotheses? The potential involvement of other signalling inputs to FOXO activity, e.g. immune signalling and JNK, should be acknowledged and warrants some discussion.

Typographical errors:

Incomplete sentence line 121 to 122 - starting "Cox proportional hazards.... and posthoc tests (Fig 2b).

Line 123 "EMMs" - define abbreviation on first use

References to Fig 2b (first given on line 122), should be capitalised to Fig 2B for consistency.

Lines 231 and 317 - the phrase "steady state (microbiota independent) expression" in reference to flyATLAS 2 data could be misleading. The term "microbiota independent" could suggest that expression levels have been shown not to be regulated by the microbiota and this is not the case. The authors should change this to simply state they are referring to steady state expression in conventionally reared flies.

Referees cross-commenting

Below are brief comments on the revision suggestions that reviewers 2 and 3 have requested.

Reviewer 2

1. I agree that confirmation that TkRNAi doesn't impact microbial levels could be helpful and would be straightforward for the authors to do. However, I don't feel it's essential to support the central claims of the paper.
2. I agree.
3. I don't feel that any of these experiments supports a role for insulin signalling, so I don't feel that this additional control is needed.
4. It would be a good addition to have lifespan data from a separate knockdown line for corroboration. However, this has already been done in several different genetic backgrounds through crosses with different driver lines in multiple tissues, so I feel it's unnecessary given the time and resources that lifespan experiments take. There's also the caveat that different RNAi lines can knockdown to different extents so that would have to be assessed as well and if there's a difference it may mean that ultimately not much can be concluded from this additional experiment.
5. A good suggestion, but not straightforward and depends on the availability of the necessary tools, or possibly the generation of new tools. One for a follow up study.
6. I feel this is not important enough to the central findings of the study to warrant the extra work.
7. I agree.

Reviewer 3 1. Imaging calcium signalling is not straightforward unless a lab already has the tools available and optimised. If Tk+ EEs show changes in calcium signalling I'm not convinced that this tells us anything specific to the Tk-microbiota interaction. The point is the role of Tk itself, not the broader activity of the cells that express it. 2. I agree this needs clarification. 3. I agree that this would add depth, if feasible, but feel it's not essential to support the current conclusions. 4. This is a minor point and given the RT-qPCR data and the RNAseq data corroborate each other I'm convinced that Tk levels are elevated. 5. I feel exploring this in males is opening an additional line of enquiry beyond the scope of the current study. Either the phenotypes are the same - in which case what is added? - or they are different but there's no scope to assess why. A good suggestion for a follow up study. 6. No comment. 7. Agreed.

One final comment. It's true that FOXO has only been shown to regulate lifespan in the context of insulin signalling. However, as far as I'm aware it hasn't been shown not to regulate lifespan downstream of it's other activators, this simply hasn't been explored due to the historical focus on insulin signalling in this field. In the context of host-microbiota interactions considering other pathways the activate FOXO, such as immune and JNK signals, would make sense.

Reviewed by Dr Rebecca Clark, Department of Biosciences, Durham University

Significance

Overall, I find the key conclusions of the paper convincing. The authors present an extensive amount of experimental work, and their conclusions are well founded in the data. We have known that the microbiota influence lifespan for some time but the mechanisms by which they do so have remained elusive. This study identifies one such mechanism and as a result opens several avenues for further research. The Tk-microbiota interaction is shown to be important for both lifespan and lipid homeostasis, although it's clear these are independent phenotypes. The fact that the outcome of the Tk-microbiota interaction depends on the bacterial species is of particular interest because it supports the idea that manipulation of the microbiota, or specific aspects of the host-microbiota interaction, may have therapeutic potential.<br /> These findings will be of interest to a broad readership spanning host-microbiota interactions and their influence on host health. They move forward the study of microbial regulation of host longevity and have relevance to our understanding of microbial regulation of host lipid homeostasis. They will also be of significant interest to those studying the mechanisms of action and physiological roles of Tachykinins.

Field of expertise: Drosophila, gut, ageing, microbiota, innate immunity

taxnegative = distortionary taxes and unproductive spending, or nondistortionary taxes, lowers growth -0.18

taxambigous = lower -0.11

taxpositive = 0.02 growtyh

taxpositive = positive economic growth. taxnegative = negative growth. this is the theory on what forms of taxation create this

past methods

Document d'information : La Santé Mentale des Mineurs en France

video Présentation du rapport à l'Assemblée nationale

Résumé

Ce document synthétise les conclusions du rapport d'information de l'Assemblée Nationale sur la santé mentale des mineurs.

Le constat principal est un écart grandissant et critique entre une demande de soins en forte augmentation et une offre de soins publique saturée, sous-financée et géographiquement inégale.

Cette crise systémique entraîne des prises en charge tardives, un recours accru aux psychotropes et une saturation des services d'urgence.

La hausse de la demande est un phénomène multifactoriel, résultant d'une meilleure détection des troubles et d'une moindre stigmatisation, mais aussi de l'impact croissant de déterminants sociaux, environnementaux et numériques.

Les violences intrafamiliales, la précarité, la pression scolaire (notamment via Parcoursup), l'exposition aux écrans et un contexte général anxiogène (géopolitique, environnemental) sont identifiés comme des facteurs majeurs.

Les populations les plus vulnérables, notamment les enfants suivis par l'Aide Sociale à l'Enfance (ASE) et les mineurs non accompagnés (MNA), sont particulièrement touchées et leur prise en charge est souvent défaillante.

Face à cette situation, le rapport préconise une double stratégie ambitieuse.

D'une part, il appelle à une consolidation de l'offre de soins existante, en réaffirmant les principes fondateurs de la psychiatrie de secteur.

Cela implique de renforcer le maillage territorial, de rendre effective une gradation des soins (médecins généralistes, Maisons des Adolescents en première ligne ; Centres Médico-Psychologiques comme pivot ; centres de crise pour les urgences) et d'améliorer la coordination entre les secteurs sanitaire, social et éducatif.

D'autre part, le rapport insiste sur la nécessité de déployer des politiques de prévention robustes, en agissant sur trois environnements clés :

1. L'environnement familial : via un soutien renforcé à la parentalité, notamment durant la période périnatale (les 1000 premiers jours).

2. L'environnement scolaire : en restaurant la capacité de détection précoce des services de santé scolaire et en apaisant un climat jugé trop compétitif et anxiogène.

3. L'environnement numérique : par une régulation de l'usage des écrans et des campagnes de prévention sur les dangers de l'addiction.

La mise en œuvre de ces recommandations nécessite un investissement financier pérenne, une revalorisation des professions de la pédopsychiatrie pour renforcer leur attractivité, et une meilleure formation de l'ensemble des professionnels au contact des enfants.

Perspectives des Rapporteures

Les deux co-rapporteures, bien que partageant de nombreuses préconisations, soulignent des priorités et des analyses distinctes dans leurs avant-propos.

Mme Nathalie Colin-Oesterlé met l'accent sur :

• La prévention comme axe clé, en particulier durant la période de la conception aux deux ans de l'enfant.

• Le rôle décisif des parents, insistant sur les conséquences de leur absence psychique ou physique et le manque de cadre.

• Les dangers de l'addiction aux écrans, soutenant la majorité numérique et une "pause numérique" dans les établissements scolaires.

• La nécessité d'une meilleure gradation des soins et d'une coopération renforcée entre les secteurs public et privé pour désengorger les Centres Médico-Psychologiques (CMP).

Mme Anne Stambach-Terrenoir insiste sur :

• Le manque structurel de moyens de la pédopsychiatrie publique, critiquant une "réponse néolibérale" qui privilégie la réorganisation à l'investissement.

• Les déterminants sociaux des troubles mentaux, liant la dégradation de la santé mentale à l'augmentation des inégalités sociales, à la précarité et aux évolutions du monde du travail (horaires atypiques).

• Les effets anxiogènes du système scolaire, citant Parcoursup et les réformes Blanquer comme des facteurs aggravants.

• L'urgence de redonner des moyens financiers et humains aux services publics existants (santé, Éducation nationale, ASE) plutôt que de financer des "projets innovants" non pérennes.

I. Un Écart Croissant entre la Demande et l'Offre de Soins

Le rapport dresse le constat d'une crise profonde, caractérisée par une demande de soins psychiques exponentielle face à une offre contrainte, fragmentée et souvent inaccessible.

A. Un Enjeu de Santé Publique Majeur

1. Augmentation et Nature de la Demande de Soins

• Prévalence : Selon la DGOS, 1,6 million d'enfants et d'adolescents en France souffriraient d'un trouble psychique.

• Données Épidémiologiques :

◦ L'enquête Enabee (2022) révèle que 13 % des enfants de 6 à 11 ans et 8,3 % des 3-6 ans présentent un trouble probable de santé mentale.   

◦ L'enquête Enclass (2022) sur les collégiens et lycéens montre que 14 % des collégiens et 15 % des lycéens présentent un risque important de dépression. 24 % des lycéens déclarent des pensées suicidaires au cours des 12 derniers mois.

• Différences de Genre : Les enquêtes confirment une prévalence plus forte des troubles internalisés (anxio-dépressifs) chez les filles, et des troubles externalisés (comportement, hyperactivité) chez les garçons.

• Interprétation des Données : Plusieurs experts auditionnés appellent à la prudence, soulignant que la notion de "trouble probable" ne constitue pas un diagnostic clinique et qu'il existe un risque de médicalisation excessive de phénomènes développementaux normaux ou de difficultés sociales.

• Une Parole Libérée : L'augmentation des demandes s'explique aussi par une meilleure connaissance des troubles, une déstigmatisation progressive et une plus grande capacité des jeunes et de leurs parents à exprimer une souffrance psychique.

2. Un Phénomène Multifactoriel

La dégradation de la santé mentale des mineurs est le produit de multiples facteurs :

• Violences : 30 % des patients en pédopsychiatrie sont victimes de maltraitance parentale. L'exposition aux violences (physiques, sexuelles, psychologiques, intrafamiliales) est un déterminant majeur.

• Déterminants Sociaux : La précarité, les conditions de logement et le niveau de revenu sont corrélés à un risque plus élevé de développer des troubles mentaux.

• Environnement Numérique : L'exposition excessive aux écrans est unanimement identifiée comme un facteur aggravant les troubles anxieux dépressifs. Elle perturbe le sommeil, isole et expose à des contenus préjudiciables (cyberharcèlement, contenus violents, pornographiques).

• Environnement Social Anxiogène :

◦ Milieu scolaire : Le harcèlement (5% des élèves du CE2 au CM2, 6% des collégiens), la pression liée à la performance, l'inadaptation de certains programmes et un système d'orientation jugé anxiogène (Parcoursup) contribuent au stress et au "refus scolaire anxieux".  

◦ Contexte global : L'éco-anxiété et les peurs liées au contexte géopolitique (guerres, attentats) participent également à l'angoisse des jeunes.

3. Prise en Charge Défaillante des Populations les plus Vulnérables

• Aide Sociale à l'Enfance (ASE) : Les enfants suivis par l'ASE sont surreprésentés dans les services de psychiatrie (occupant jusqu'à 40-50 % des lits d'hospitalisation à temps plein). Leurs parcours chaotiques et les carences affectives engendrent des troubles sévères, mais leur suivi médical est souvent défaillant et discontinu.

L'hôpital se substitue parfois à un lieu d'hébergement.

• Mineurs Non Accompagnés (MNA) : Ces jeunes souffrent massivement de stress post-traumatique lié à des parcours migratoires extrêmement violents (guerres, torture, naufrages).

Leurs conditions d'accueil et les procédures d'évaluation de leur minorité aggravent leur vulnérabilité et complexifient leur accès aux soins.

B. Une Offre de Soins Insuffisante et Illisible

Le système de la "sectorisation", conçu pour garantir un accès équitable aux soins, est aujourd'hui saturé.

1. Une Offre Contrainte face à une Demande Croissante

• Moyens Inadaptés : 58 % des lits d'hospitalisation en pédopsychiatrie ont été supprimés entre 1986 et 2013. De nombreux jeunes sont hospitalisés en services pour adultes, avec les risques que cela comporte.

• Pénurie de Soignants : La situation est critique, notamment pour les pédopsychiatres, dont le nombre a diminué de 34 % entre 2010 et 2022.

La profession est vieillissante (moyenne d'âge de 60 ans) et peu attractive pour les jeunes médecins.

La pénurie concerne également les infirmiers, les médecins scolaires et les psychologues dans le secteur public.

• Disparités Territoriales : L'accès aux soins est très inégal. La densité de pédopsychiatres varie fortement d'une région à l'autre (ex: 9 pour 100 000 jeunes en PACA, 3 en Hauts-de-France).

Les zones rurales et les départements d'outre-mer sont particulièrement sous-dotés.

2. Une Multiplicité d'Acteurs Rend l'Offre Illisible

Bien que des structures comme les Maisons des Adolescents (MDA) et les Équipes Mobiles (EMPEA) jouent un rôle crucial, la multiplication des dispositifs et des centres experts (qui posent des diagnostics sans assurer de suivi) rend le parcours de soins complexe et illisible pour les familles.

3. Des Conséquences Déléteres

• Prise en Charge Tardive : Les délais d'attente pour un premier rendez-vous en Centre Médico-Psychologique (CMP), pivot du système, peuvent atteindre 12 à 18 mois, conduisant à une aggravation des troubles.

• Recours Accru aux Psychotropes : Face à la saturation des dispositifs de suivi thérapeutique, la prescription de psychotropes aux mineurs a augmenté de 18 % entre 2019 et 2023.

Cette médicalisation est souvent perçue comme une solution par défaut.

• Saturation des Urgences : Faute de prise en charge en amont, les services d'urgence deviennent le lieu du premier diagnostic lors de crises aiguës, ce qui contribue à leur engorgement.

II. Stratégies de Consolidation de l'Offre et de Prévention

Le rapport formule 53 recommandations visant à la fois à renforcer le système de soins existant et à mettre en œuvre une politique de prévention ambitieuse.

A. Améliorer la Prise en Charge

1. Rendre l'Offre de Soins Graduée et Effective

• Réaffirmer le Rôle du Secteur : La priorité est de consolider le maillage existant plutôt que de créer de nouveaux dispositifs. Le CMP doit rester le pivot du parcours, avec un accueil inconditionnel.

• Mettre en Place une Gradation Claire :

◦ Premier niveau : Médecins généralistes, pédiatres, psychologues de ville et Maisons des Adolescents (MDA), qui doivent être adossées à des centres de soins.  

◦ Deuxième niveau : Les CMP, dont les moyens et le maillage doivent être renforcés (objectif : un accès en moins de 30 minutes pour chaque enfant).   

◦ Gestion des crises : Création de centres de crise adossés aux urgences pédiatriques pour les épisodes aigus, limitant ainsi le recours aux urgences générales.

2. Renforcer la Formation et l'Attractivité des Professions

• Formation : Il est crucial de mieux former tous les professionnels : médecins généralistes (détection précoce), infirmiers (stage obligatoire en psychiatrie, développement de la pratique avancée), professionnels de la petite enfance (théorie de l'attachement).

• Attractivité : Pour lutter contre la pénurie, il est préconisé de revaloriser les salaires et les tarifs des consultations, d'améliorer les conditions de travail et de créer une chaire de psychiatrie de l'enfant et de l'adolescent dans chaque CHU pour revaloriser la discipline.

3. Améliorer la Coordination et le Financement

• Coordination Intersectorielle : Une meilleure articulation est nécessaire avec l'ASE (création de structures mixtes soin-hébergement), l'Éducation nationale (redynamisation des RASED, intégration de la santé scolaire dans les projets de santé mentale) et le secteur médico-social (adossement des CMPP au secteur sanitaire).

• Financement : Le rapport appelle à pérenniser et flécher les budgets de la pédopsychiatrie, à privilégier les dotations pluriannuelles aux appels à projets, et à réformer le codage des actes pour valoriser le temps de coordination et d'échange avec les familles, qui est au cœur du soin pédopsychiatrique.

B. Privilégier les Politiques Préventives

1. Agir sur l'Environnement Familial

• Périnatalité et 1000 Premiers Jours : C'est une période cruciale. Il faut développer les équipes dédiées à la périnatalité dans chaque CMP, étoffer les unités d'hospitalisation conjointes parents-bébé et généraliser les "maisons des 1000 premiers jours" pour accompagner tous les parents.

• Soutien à la Parentalité : Les parents sont des acteurs essentiels de la prise en charge.

Il faut renforcer les dispositifs qui les accompagnent et les forment, notamment via des associations et des groupes de parole.

2. Agir sur l'Environnement Scolaire

• Renforcer la Santé Scolaire : Il est urgent d'engager un plan de recrutement massif de médecins scolaires, d'infirmiers et de psychologues de l'Éducation nationale pour atteindre des ratios acceptables.

• Rendre la Détection Précoce Effective : Les bilans de santé à 6 ans et 12 ans doivent être systématiquement réalisés et inclure un volet de dépistage des troubles de santé mentale.

• Apaiser le Climat Scolaire : L'école doit devenir un lieu moins anxiogène, en favorisant les compétences psychosociales (cours d'empathie) et en réformant un système d'orientation jugé trop précoce et compétitif.

3. Agir sur l'Environnement Numérique

• Responsabiliser les Parents : Il est essentiel de sensibiliser les parents à leur propre usage des écrans et à l'"absence psychique" qui en découle, ainsi qu'à la nécessité de passer du temps qualitatif sans écrans avec leurs enfants.

• Déployer des Campagnes de Prévention : Des campagnes massives sur les dangers de l'addiction aux écrans sont nécessaires.

• Réguler à l'École : La "pause numérique" (interdiction des téléphones portables) doit être généralisée dans tous les établissements scolaires.

La majorité numérique doit également être rendue effective pour protéger les plus jeunes.

3. É possível indeferir pedido de desistência recursal, veiculado anteriormente ao julgamento, desde que a negativa de desistência seja avaliada pelo colegiado em questão de ordem, levando-se em consideração:

• (i) se tratar de tema nunca enfrentado no STJ ("leading case"),

• (ii) haver indícios de estratagema a evitar jurisprudência em pedidos de desistência homologados anteriormente envolvendo o mesmo desistente,

• (iii) o sorteio de relatoria preceder a apresentação do pedido de desistência e

• (iv) haver forte interesse público no enfrentamento do objeto recursal a recomendar orientação jurisprudencial nacional.

(REsp n. 2.172.296/RJ, relatora Ministra Nancy Andrighi, Terceira Turma, julgado em 4/2/2025, DJEN de 7/2/2025.)

• Informativo nº 844
• 25 de março de 2025.
• RECURSOS REPETITIVOS
• Processo: REsp 2.021.665-MS, Rel. Ministro Moura Ribeiro, Corte Especial, por maioria, julgado em 13/3/2025. (Tema 1198).

Ramo do Direito DIREITO CIVIL, DIREITO PROCESSUAL CIVIL

TemaPaz, Justiça e Instituições Eficazes <br /> Demandas abusivas. Documentos capazes de comprovar a seriedade da demanda. Exigência. Finalidade. Coibição de fraude processual. Tema 1198.

Destaque - Constatados indícios de litigância abusiva, o juiz pode exigir, de modo fundamentado e com observância à razoabilidade do caso concreto, a emenda da petição inicial a fim de demonstrar o interesse de agir e a autenticidade da postulação, respeitadas as regras de distribuição do ônus da prova.

Informações do Inteiro Teor - Cinge-se a controvérsia em verificar a possibilidade de o juiz, em um estágio inicial do processo, exigir que a parte apresente documentos capazes de evidenciar a verossimilhança do direito alegado, pavimentando, dessa forma, o caminho para a entrega de uma tutela jurisdicional efetiva e coibindo, a um só tempo, a prática de fraudes processuais. Ou seja, saber até que ponto ou em qual medida o juiz, antevendo a natureza temerária da lide, pode exigir da parte autora que apresente documentos capazes de confirmar a seriedade da pretensão deduzida em juízo.

• Nas sociedades de massa, em que a grande maioria da população integra processos de produção, distribuição e consumo de larga escala, é esperado o surgimento de demandas e lides também massificadas. Essa litigância de massa, conquanto apresente novos desafios ao Poder Judiciário, constitui manifestação legítima do direito de ação.

• Observa-se, no entanto, em várias regiões do país, verdadeira avalanche de processos infundados, marcados pelo exercício de advocacia abusiva, predatória, que não encontra respaldo no legítimo direito de ação. Tais feitos não apenas embaraçam o exercício de uma jurisdição efetiva, mas verdadeiramente criam sérios problemas de política pública, conforme identificado por órgãos de inteligência de vários tribunais do país.

• A possibilidade de o juiz exigir a apresentação de documentos para comprovar o interesse de agir ou a verossimilhança do direito alegado tem sido admitida por esta Corte e também pelo Supremo Tribunal Federal (STF) em diversas situações.

• Por isso, poderá o juiz, a fim de coibir o uso fraudulento do processo, exigir que o autor apresente extratos bancários, cópias de contratos, comprovante de residência, procuração atualizada e com poderes específicos, dentre outros documentos, a depender de cada caso concreto.

• A procuração outorgada para determinada causa em regra não subsiste para outras ações distintas e desvinculadas, porque uma vez executado o negócio cessa o mandato para o qual outorgado (art. 682, IV, do Código Civil - CC). Assim, caso o advogado apresente instrumento muito antigo, dando margem a descrença de que não existe mais relação atual com o cliente, é lícito ao juiz determinar que a situação seja esclarecida, com juntada de um eventual novo instrumento.

• A cautela indicada tem respaldo em princípios constitucionais de acesso à justiça, de proteção ao consumidor e de duração razoável do processo, harmonizando-se, ainda, com os postulados legais que privilegiam o julgamento de mérito e impõem o dever de cooperação entre os sujeitos do processo que, afinal, precisa ter desenvolvimento válido e regular.

• O risco de exigências judiciais excessivas, como de resto o de qualquer decisão judicial equivocada, constitui realidade inexpugnável, ínsita ao sistema de Justiça, mas que deve ser controlado pontualmente em cada processo, não podendo ser invocado como obstáculo à adoção de boas práticas na condução judicial do feito.

1. Nos termos do entendimento do Superior Tribunal de Justiça, a "ausência de legitimidade ativa, por se tratar de uma das condições da ação, é matéria de ordem pública cognoscível a qualquer tempo e grau, sendo insuscetível de preclusão nas instâncias ordinárias" (REsp 1.731.214/AL, relator Ministro Herman Benjamin, Segunda Turma, julgado em 8/5/2018, DJe de 19/11/2018). (AgInt no AgInt no AREsp n. 2.399.315/MA, relator Ministro Paulo Sérgio Domingues, Primeira Turma, julgado em 30/9/2024, DJe de 7/10/2024.)

2. VI. Na forma da jurisprudência dominante desta Corte, as questões de ordem pública, apreciáveis, de ofício, em qualquer tempo e grau de jurisdição, nas instâncias ordinárias (e.g., pressupostos processuais, condições da ação, decadência, prescrição, etc.), não se sujeitam à preclusão, podendo ser suscitadas, ainda que em sede de Embargos de Declaração. Precedentes [...] (EAREsp n. 146.473/ES, relatora Ministra Assusete Magalhães, Primeira Seção, julgado em 11/10/2023, DJe de 19/10/2023.)

Lei 9.507/97 (Lei do Habeas-data)

• Art. 8° A petição inicial, que deverá preencher os requisitos dos arts. 282 a 285 do Código de Processo Civil, será apresentada em duas vias, e os documentos que instruírem a primeira serão reproduzidos por cópia na segunda.

Parágrafo único. A petição inicial deverá ser instruída com prova:

I - da recusa ao acesso às informações ou do decurso de mais de dez dias sem decisão; (Recusa administrativa)

II - da recusa em fazer-se a retificação ou do decurso de mais de quinze dias, sem decisão; ou

III - da recusa em fazer-se a anotação a que se refere o § 2° do art. 4° ou do decurso de mais de quinze dias sem decisão.

• RE 631240 (Repercussão Geral - Tema 350)
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. ROBERTO BARROSO
• Julgamento: 03/09/2014
• Publicação: 10/11/2014

Ementa Ementa: RECURSO EXTRAORDINÁRIO. REPERCUSSÃO GERAL. PRÉVIO REQUERIMENTO ADMINISTRATIVO E INTERESSE EM AGIR. 1. A instituição de condições para o regular exercício do direito de ação é compatível com o art. 5º, XXXV, da Constituição. Para se caracterizar a presença de interesse em agir, é preciso haver necessidade de ir a juízo. 2. A concessão de benefícios previdenciários depende de requerimento do interessado, não se caracterizando ameaça ou lesão a direito antes de sua apreciação e indeferimento pelo INSS, ou se excedido o prazo legal para sua análise. É bem de ver, no entanto, que a exigência de prévio requerimento não se confunde com o exaurimento das vias administrativas. 3. A exigência de prévio requerimento administrativo não deve prevalecer quando o entendimento da Administração for notória e reiteradamente contrário à postulação do segurado. 4. Na hipótese de pretensão de revisão, restabelecimento ou manutenção de benefício anteriormente concedido, considerando que o INSS tem o dever legal de conceder a prestação mais vantajosa possível, o pedido poderá ser formulado diretamente em juízo – salvo se depender da análise de matéria de fato ainda não levada ao conhecimento da Administração –, uma vez que, nesses casos, a conduta do INSS já configura o não acolhimento ao menos tácito da pretensão. 5. Tendo em vista a prolongada oscilação jurisprudencial na matéria, inclusive no Supremo Tribunal Federal, deve-se estabelecer uma fórmula de transição para lidar com as ações em curso, nos termos a seguir expostos. 6. Quanto às ações ajuizadas até a conclusão do presente julgamento (03.09.2014), sem que tenha havido prévio requerimento administrativo nas hipóteses em que exigível, será observado o seguinte: (i) caso a ação tenha sido ajuizada no âmbito de Juizado Itinerante, a ausência de anterior pedido administrativo não deverá implicar a extinção do feito; (ii) caso o INSS já tenha apresentado contestação de mérito, está caracterizado o interesse em agir pela resistência à pretensão; (iii) as demais ações que não se enquadrem nos itens (i) e (ii) ficarão sobrestadas, observando-se a sistemática a seguir. 7. Nas ações sobrestadas, o autor será intimado a dar entrada no pedido administrativo em 30 dias, sob pena de extinção do processo. Comprovada a postulação administrativa, o INSS será intimado a se manifestar acerca do pedido em até 90 dias, prazo dentro do qual a Autarquia deverá colher todas as provas eventualmente necessárias e proferir decisão. Se o pedido for acolhido administrativamente ou não puder ter o seu mérito analisado devido a razões imputáveis ao próprio requerente, extingue-se a ação. Do contrário, estará caracterizado o interesse em agir e o feito deverá prosseguir. 8. Em todos os casos acima – itens (i), (ii) e (iii) –, tanto a análise administrativa quanto a judicial deverão levar em conta a data do início da ação como data de entrada do requerimento, para todos os efeitos legais. 9. Recurso extraordinário a que se dá parcial provimento, reformando-se o acórdão recorrido para determinar a baixa dos autos ao juiz de primeiro grau, o qual deverá intimar a autora – que alega ser trabalhadora rural informal – a dar entrada no pedido administrativo em 30 dias, sob pena de extinção. Comprovada a postulação administrativa, o INSS será intimado para que, em 90 dias, colha as provas necessárias e profira decisão administrativa, considerando como data de entrada do requerimento a data do início da ação, para todos os efeitos legais. O resultado será comunicado ao juiz, que apreciará a subsistência ou não do interesse em agir.

Art. 217, Constituição Federal:

§ 1º O Poder Judiciário só admitirá ações relativas à disciplina e às competições desportivas após esgotarem-se as instâncias da justiça desportiva, regulada em lei.

CONFLITO NEGATIVO DE COMPETÊNCIA. JUSTIÇA COMUM ESTADUAL E JUSTIÇA FEDERAL. ADOÇÃO. CRIANÇA INDÍGENA. ART. 28, §6º, III, DO ECA. ART. 109, I E XI DA CF. OBRIGATORIEDADE DE INTERVENÇÃO DA FUNAI COMO ENTIDADE CONSULTIVA. DIREITO PARTICULAR DA CRIANÇA INDÍGENA QUE NÃO SE CONFUNDE COM DIREITOS INDÍGENAS PREVISTOS NO ART. 231 DA CF. COMPETÊNCIA DA JUSTIÇA ESTADUAL QUE POSSUI MELHOR ESTRUTURA E EQUIPE ESPECIALIZADA. MELHOR INTERESSE DA CRIANÇA E DO ADOLESCENTE. - 1. Conflito negativo de competência suscitado em 22/10/2024 e concluso ao gabinete em 19/12/2024. - 2. O propósito do conflito de competência consiste em decidir se: (I) é obrigatória a intervenção da Fundação Nacional dos Povos Indígenas (FUNAI) em ação de adoção de criança indígena; e (II) se sim, qual o Juízo competente para o processamento de ação de adoção de criança indígena. - 3. A intervenção da Fundação Nacional dos Povos Indígenas (FUNAI) em ações de adoção de crianças e adolescentes de origem indígena possibilita melhor verificação das condições e particularidades da família biológica, a fim de propiciar o seu adequado acolhimento na família substituta. - 4. Nos termos do art. 28, § 6º, III, do ECA, a participação da FUNAI é obrigatória perante a equipe multiprofissional ou interdisciplinar que irá acompanhar a demanda, a fim de que possa verificar o adequado acolhimento da criança adotanda e, consequentemente, a proteção de seus melhores interesses. Precedentes. - 5. O fato de a criança ou do adolescente adotandos pertencerem à etnia indígena não atrai, por si só, a competência da Justiça Federal para o processamento da ação de adoção, pois o procedimento de adoção diz respeito a direito privado, uma vez tratar-se de interesse particular de criança ou adolescente, ainda que de origem indígena, não sendo devida a aplicação da competência prevista no art. 109, I e XI, da CF. - 6. É do melhor interesse de crianças e adolescentes indígenas a competência da Justiça Estadual para processar e julgar ações de adoção, uma vez que a Vara da Infância e Juventude <u>terá maiores e melhores condições</u> de acompanhar o procedimento, contando com equipe técnica qualificada e especializada. - 7. No conflito de competência sob julgamento, a ação de adoção na origem não envolve direitos indígenas previstos no art. 231 da CF, mas diz respeito a adoção intuitu personae de criança indígena de etnia Kayapó, promovida por pessoa também indígena, que cuida da criança desde o seu nascimento. - 8. O Juízo Federal suscitante expressamente reconhece a inexistência de interesse jurídico da FUNAI na lide, sobretudo porque não envolveria direitos indígenas coletivamente considerados, nos termos da Súmula 150/STJ. - 9. Portanto, a presença <u>obrigatória</u> da FUNAI não atrai a competência automática da Justiça Federal, devendo a demanda ser processada e acompanhada pela Justiça Estadual, uma vez que a Vara da Infância e Juventude apresenta instrumentos e equipe especializada para assegurar o atendimento ao melhor interesse da criança adotanda. - 10. Conflito de competência conhecido para declarar competente o Juízo de Direito da Vara Cível da Comarca de São Félix do Xingu - PA, ora suscitado.

(CC n. 209.192/PA, relatora Ministra Nancy Andrighi, Segunda Seção, julgado em 3/4/2025, DJEN de 9/4/2025.)

• Edição Extraordinária nº 8
• Direito Público
• 17 de janeiro de 2023
• Processo: AgInt no AREsp 2.118.653-SP, Rel. Ministro Humberto Martins, Segunda Turma, por unanimidade, julgado em 28/11/2022, DJe 30/11/2022.

Ramo do Direito DIREITO PROCESSUAL CIVIL

TemaPaz, Justiça e Instituições Eficazes <br /> Recurso especial e agravo em recurso especial. Tempestividade recursal. Aferição. Prazos em curso no Tribunal de origem.

Destaque - A tempestividade do recurso especial e do respectivo agravo em recurso especial deve ser aferida de acordo com os prazos em curso na Corte de <u>origem</u>.

Informações do Inteiro Teor - A tempestividade do recurso especial e do agravo respectivo deve ser aferida de acordo com os prazos em curso na Corte de origem, e não no STJ.

• Esta é a jurisprudência do Superior Tribunal de Justiça, no sentido de que "O prazo dos recursos interpostos perante a Corte de origem ainda que estejam endereçados a este Tribunal - obedece ao calendário de funcionamento do Tribunal de origem, sendo irrelevante, para a verificação da tempestividade do recurso, a existência de recesso forense, no STJ" (AgInt no AREsp 1.149.576/SP, rel. Ministra Assusete Magalhães, Segunda Turma, julgado em 3/4/2018, DJe 10/4/2018).

Note de Synthèse : Rapport sur la Santé Mentale des Mineurs

Le rapport: https://hyp.is/go?url=https%3A%2F%2Fwww.assemblee-nationale.fr%2Fdyn%2F17%2Frapports%2Fdde%2Fl17b1700_rapport-information.pdf&group=world

Résumé

Ce document de synthèse présente les conclusions et recommandations clés du rapport parlementaire sur la santé mentale des mineurs.

Le constat principal est une dégradation alarmante de la santé psychique des enfants et adolescents en France, avec 1,6 million de mineurs souffrant d'un trouble psychique, et une augmentation particulièrement marquée de +70% chez les 10-14 ans entre 2017 et 2023.

Le système de soins est totalement débordé, avec seulement un jeune sur deux ayant accès à un suivi et des délais d'attente atteignant 18 mois pour un premier rendez-vous en Centre Médico-Psychologique (CMP).

Le rapport identifie deux crises interdépendantes : d'une part, une offre de soins illisible, sous-financée et en manque criant de personnel, et d'autre part, une prévention quasi inexistante, notamment dans le cadre scolaire.

Une surreprésentation dramatique des enfants de l'Aide Sociale à l'Enfance (ASE) au sein des services de pédopsychiatrie (jusqu'à 40% des lits pour 2% de la population mineure) met en lumière la faillite du suivi de ces publics particulièrement vulnérables.

Les 54 recommandations formulées visent à une refonte structurelle du système. Les priorités incluent :

1. La réorganisation du parcours de soins via une "gradation" claire, renforçant le premier recours (médecins généralistes, pédiatres) pour désengorger les CMP.

2. Le renforcement des CMP comme pivots du système, avec des budgets pérennes, un maillage territorial assurant un accès en 30 minutes, et une approche pluridisciplinaire.

3. L'investissement massif dans la prévention, de la périnatalité (unités parents-bébés) à la santé scolaire (atteindre les ratios européens de médecins et psychologues).

4. Une meilleure prise en charge des enfants de l'ASE avec un repérage systématique, un suivi de santé effectif et la création de structures mixtes soin-hébergement.

5. La lutte contre la pénurie de professionnels par la revalorisation des salaires, la réforme de la tarification des actes et des mesures pour améliorer l'attractivité de la pédopsychiatrie.

Enfin, le rapport souligne l'impact majeur de facteurs environnementaux comme la pression scolaire (Parcoursup), l'addiction aux écrans et les inégalités sociales, appelant à des réponses politiques plus larges.

1. Le Constat Alarmant d'une Crise de Santé Publique

Le rapport parlementaire, fruit de six mois de travail incluant 37 auditions et quatre déplacements, dresse un tableau sans équivoque de la détérioration de la santé mentale des mineurs en France.

A. Chiffres Clés de la Dégradation

• Prévalence des troubles : Selon le rapport de la Cour des comptes de 2023, 1,6 million d'enfants et d'adolescents souffrent d'un trouble psychique.

• Augmentation significative : Entre 2017 et 2023, une forte augmentation des troubles a été observée, notamment :

◦ +70 % chez les 10-14 ans.    ◦ +46 % chez les 15-20 ans.

• Actes auto-infligés : La DREES note une augmentation des hospitalisations pour tentatives de suicide et gestes auto-infligés chez les 10-14 ans.

• Précocité des troubles : La moitié des troubles psychiatriques se déclarent avant l'âge de 15 ans, soulignant l'urgence d'une intervention précoce.

B. La Vulnérabilité Extrême des Enfants Protégés

Un des constats les plus marquants du rapport est la surreprésentation des enfants relevant de l'Aide Sociale à l'Enfance (ASE) dans les services de pédopsychiatrie.

• Occupation des lits : Jusqu'à 40-50 % des lits d'hospitalisation à temps plein sont occupés par des enfants placés, alors qu'ils ne représentent que 2 % des mineurs en France.

• Facteurs de risque : La maltraitance subie par ces enfants multiplie par deux les risques de suicide et par quatre les risques de psychotrauma.

• Cercle vicieux : Le suivi médical défaillant de ces mineurs entraîne une prise en charge trop tardive, souvent via une hospitalisation prolongée qui se substitue à un lieu de vie, aggravant leur état.

En parallèle, les foyers sont démunis face à des jeunes avec des troubles lourds non pris en charge.

2. Un Système de Soins Débordé et Illisible

Face à la hausse de la demande, l'offre de soins est criante de manque de moyens et d'organisation, laissant de nombreuses familles sans solution.

A. La Saturation des Structures

• Accès aux soins limité : Sur les 1,6 million de jeunes présentant des troubles, seuls 750 000 à 850 000 sont suivis, soit environ un sur deux.

• Délais d'attente insoutenables : L'attente pour un premier rendez-vous en Centre Médico-Psychologique (CMP) peut atteindre 12 à 18 mois, parfois plus.

Cette attente laisse le temps à la situation de s'aggraver, conduisant à une prise en charge aux urgences, elles-mêmes engorgées.

B. Recommandation : Instaurer une Gradation des Soins

Pour rendre l'offre plus lisible et efficiente, le rapport préconise une meilleure organisation du parcours de soins.

• Premier niveau : Les médecins généralistes et pédiatres devraient assurer la première consultation, la détection et l'orientation.

Cela nécessite un renforcement de leur formation en psychiatrie infanto-juvénile.

Ce niveau pourrait aussi inclure des psychologues et les Maisons des Adolescents.

• Deuxième niveau (pivot) : Les Centres Médico-Psychologiques (CMP) se concentreraient sur les cas nécessitant une expertise pluridisciplinaire, tout en conservant un accueil inconditionnel sans adressage obligatoire pour ne pas éloigner les plus fragiles.

• Troisième niveau : Des centres de gestion de crise de courte durée, adossés aux urgences pédiatriques ou hôpitaux psychiatriques, pour les situations les plus aiguës.

C. Recommandation : Renforcer le Secteur de la Pédopsychiatrie

Le rapport insiste sur la nécessité de renforcer l'existant plutôt que de disperser les moyens dans des dispositifs innovants non pérennes.

• Stabilité financière : Pérenniser et flécher les budgets de la pédopsychiatrie au sein des hôpitaux et privilégier les dotations pluriannuelles aux appels à projets.

• Priorité au soin : Réorienter les moyens financiers et humains vers le soin direct plutôt que vers des plateformes de diagnostic qui, malgré leurs avancées, peuvent favoriser une approche médicamenteuse au détriment de la relation thérapeutique.

• Maillage territorial : Revoir le maillage des CMP pour que chaque enfant puisse y accéder en moins de 30 minutes de son domicile, en adaptant le découpage des secteurs aux évolutions démographiques (un secteur couvre aujourd'hui jusqu'à 500 000 habitants contre 200 000 prévus initialement).

3. L'Urgence d'une Prévention Efficace

Le rapport souligne que la prévention est le parent pauvre de la politique de santé mentale, alors qu'elle est déterminante.

A. La Période des 1000 Premiers Jours

La période allant de la conception aux deux ans de l'enfant est fondamentale.

• Soutien à la parentalité : Les "Maisons des bébés" sont des dispositifs essentiels pour prévenir les troubles précoces du lien parent-enfant.

• Psychiatrie périnatale : Les unités d'hospitalisation conjointe mère-bébé, comme celle de Toulouse, permettent de détecter les signes de souffrance dès les premiers jours et de réparer le lien d'attachement. Il est recommandé de développer ces unités et de former les professionnels à la théorie de l'attachement.

B. Le Rôle Fondamental de la Santé Scolaire

L'Éducation Nationale est un lieu stratégique pour la détection précoce, mais ses moyens sont gravement fragilisés.

Professionnel

Ratio Actuel

Ratio Recommandé (UE)

Recommandation du Rapport

Psychologue scolaire

1 pour 1 600 élèves

1 pour 800 élèves

1 pour 800 élèves

Médecin scolaire

1 pour 13 000 élèves

-

1 pour 5 000 élèves

Infirmier scolaire

1 pour 9 établissements

-

1 par établissement

• Conséquence directe : Le bilan de santé obligatoire à 6 ans est réalisé à 12 ans dans 80 % des cas, entraînant une accumulation de difficultés non détectées à l'entrée au collège.

Le rapport préconise une trajectoire de recrutement ambitieuse pour atteindre les ratios cibles.

4. La Crise des Ressources Humaines et la Question de l'Attractivité

La pénurie de professionnels qualifiés est au cœur de la crise du système.

• Pénurie de pédopsychiatres : La densité nationale est de 6,7 pédopsychiatres pour 100 000 jeunes, et la profession est vieillissante (moyenne d'âge de 60 ans).

La discipline est peu attractive pour les internes (127 inscrits pour 157 postes ouverts en 2023) car jugée trop proche des sciences humaines et aux conditions de travail difficiles.

• Manque de valorisation : Les professions (psychologues, paramédicaux) sont peu valorisées et faiblement rémunérées.

Une consultation en pédopsychiatrie est un acte long et complexe (minimum 1h) mais rémunéré seulement 67 € en moyenne, poussant les professionnels vers le secteur non conventionné ou la patientèle adulte.

• Recommandations :

• ◦ Revaloriser la rémunération des psychologues et personnels paramédicaux en CMP.  

• ◦ Augmenter significativement le tarif des consultations en pédopsychiatrie.  

• ◦ Réformer le codage des actes pour valoriser le temps de concertation avec les familles et partenaires. 

• ◦ Instaurer un quotient départemental pour les stages en internat afin de mieux répartir les futurs médecins sur le territoire.

5. Facteurs Environnementaux et Sociétaux Aggravants

Le rapport identifie plusieurs facteurs externes qui pèsent lourdement sur la santé mentale des jeunes.

• Médicalisation excessive : La consommation de psychotropes (type Ritaline) est en forte augmentation (490 000 enfants de 3 à 17 ans concernés).

Ces médicaments sont souvent prescrits par des médecins généralistes peu formés, posant la question du bon usage.

Le rapport recommande de privilégier les soins thérapeutiques à la prescription.

• Impact des écrans : L'addiction aux écrans et l'exposition à des contenus violents, pornographiques ou au cyberharcèlement sont unanimement citées comme aggravant les troubles anxio-dépressifs.

Le rapport soutient la majorité numérique, la mise en place de "pauses numériques" dans les établissements scolaires et appelle à des campagnes de prévention massives.

• Pression scolaire : Le système d'orientation, et notamment Parcoursup, est décrit comme créant un "climat extrêmement anxiogène".

Les rapporteurs proposent, avec des nuances, de réformer en profondeur (Nathalie Colin-Osterlé) ou de supprimer (autre rapporteure) ce dispositif et toute forme de sélection à l'université.

• Déterminants sociaux : Les enfants de familles défavorisées ont un risque trois fois plus important de développer un trouble mental et ont plus de difficultés d'accès aux soins.

Le rapport appelle à investir dans les services publics et les politiques de lutte contre les inégalités sociales pour agir sur les causes profondes.

Analyse du projet académique ac Versailles 2025-2029

source https://www.ac-versailles.fr/projet-academique-2025-2029-129400

Comme les fois précédentes l'académie a produit un projet académique sans consultation spécifique de la FCPE.

Ce document a pour but d'analyser les manques d'ambition pour leur engagement parental Mindmaps

Project académique 2025-2029 Mind Map (6).png

coéducation et parentalité Mind Map (6).png

Le Projet Académique 2025-2029 de l'Académie de Versailles, tel qu'articulé autour de ses quatre axes principaux, mentionne l'ambition d'une réussite de tous les élèves grâce à la mobilisation de la communauté éducative et des partenaires.

Cependant, en se basant sur le contenu fourni, plusieurs éléments cruciaux et concrets relatifs au rôle des parents, à la parentalité et à la coéducation semblent manquer ou ne sont pas suffisamment explicités pour constituer un volet fort et structurant de l'action académique.

Voici ce qui paraît absent ou insuffisamment développé dans le projet, compte tenu des enjeux soulevés par l'ensemble des sources :

I. Manque de reconnaissance explicite et de pilotage stratégique

1. Absence des termes "Coéducation" et "Parentalité" dans les axes stratégiques :

Bien que le projet réaffirme l'importance de renforcer les liens entre la communauté éducative et la société, les termes fondamentaux de « coéducation » et de « parentalité » n'apparaissent pas dans les titres ou les objectifs précis des quatre axes du projet 2025-2029.

Cette absence est d'autant plus notable que le précédent projet académique (2021-2024) mentionnait pourtant déjà le « renforcement de la coéducation et du rôle des parents dans l’apprentissage des élèves ».

2. Absence de mention spécifique des Représentants de Parents d'Élèves (RPE) :

Le projet insiste sur la mobilisation des "partenaires" et des "associations", mais ne mentionne pas explicitement les associations et les RPE (comme la FCPE) comme des acteurs privilégiés ou siégeant aux instances de gouvernance et de pilotage stratégique.

Les documents antérieurs critiquent déjà le manque d'audition des représentants des parents d'élèves pour l'élaboration du projet académique.

3. Manque de rejet du « glissement sémantique » :

Le projet utilise le terme générique de "partenaires". Ce vocabulaire est critiqué par les associations de parents, qui rappellent que les parents sont des co-éducateurs qui ne se choisissent pas et sont constitutifs de l'enfant, contrairement à un partenaire qui peut être choisi ou dont on peut se séparer.

L'absence d'une clarification institutionnelle risque de perpétuer l'idée de parents « consommateurs » ou « partenaires » plutôt que de les reconnaître comme membres légitimes et incontournables de la communauté éducative.

II. Manque de leviers concrets pour l'implication et l'accompagnement

1. Non-prise en compte de l'épuisement parental et de la santé mentale des parents :

L'Axe 1 vise à soutenir le bien-être et la santé mentale des élèves.

Or, les sources soulignent que la capacité d'un enfant à apprendre sereinement est directement liée à la qualité de la relation parents-école et au bien-être des parents.

Le projet n'aborde pas la question du burn-out parental ou du soutien à la santé mentale des parents, malgré l'importance de ce facteur sur l'environnement éducatif et la nécessité de ne pas les sur-responsabiliser.

2. Absence de dispositifs spécifiques pour l'accueil des familles éloignées :

Bien que l'Académie reconnaisse l'hétérogénéité territoriale (QPV, zones rurales, centres-villes privilégiés), le projet n'évoque pas le déploiement ou le renforcement des dispositifs d'accueil visant à combler le fossé avec les familles les moins familières des codes scolaires (les « parents invisibles »).

Des outils tels que les « Espaces Parents » ou le dispositif OEPRE (Ouvrir l'École aux Parents pour la Réussite des Enfants) ne sont pas mentionnés dans les objectifs concrets.

3. Absence de stratégie face à la fracture numérique et au droit à la déconnexion :