En esto me ayudó mucho la bitácora para disminuir los costos de pre-organización (o dónde debería ir este liga/información). Siguiendo el método de LogSeq, si no sé donde poner algo, empiezo colocando un enlace o info en una bitácora del día en que organizo dicho info (incluso días después de haberla encontrado). De este modo, el proceso se vuelve fluído:

• Si sé donde debería ir un enlace, edito el tiddler donde debería ir.
• Si no lo sé, la coloco en la bitácora del día en que organizo dicha info.

El etiquetado y cierto orden emergente de cosas que encuentro regularmente harán el resto.

Mis blikis me permiten reencontrar la información fácilmente y los consulto/referencio con frecuencia. En particular para ofrecer información que solicitan una y otra vez, como hojas de vida y portafolios de proyectos.

Si bien a veces creo índices con otros tiddlers, en general confío y uso los sistemas de etiquetados y de búsqueda que ya vienen incorporados en la herramienta para reencontrar la información y el sistema de filtros y operadores para ofrecer vistas específicas de la misma a terceros, como mi portafolio o mi perfil.

No reorganizo la información en publicaciones más grandes y lineales como artículos o libros, como lo haría Luhmann, básicamente por dos cosas:

1. me gusta estar lejos de las mafias de publicación académica clásica y sus circuitos y
2. quisiera que las publicaciones vivas, tipo wiki, blikis, datasets y repositorios de código tuvieran un estatus epistémico en sí mismo, sin tener que tomar la forma de las publicaciones académicas clásicas que han sido tan gentrificadas.

Lo anterior requerirá otras métricas, en las cuales vamos trabajando lento.

De esta manera venía usando mi vieja instancia de TiddlyWiki (desde 2010) y también la nueva, (retomada en 2020) incluso antes de descubrir el Zettelkasten entre estas dos décadas.

El autor luego refiere dos herramientas para recordoar, una lista grande, inspirada en GTD y la bitácora diaria inspirada en el bullet journal. En mi caso, yo también usé TW para agendas y proyectos (es decir combinando GTD y actividades diarias), pero con el tiempo me decanté por Taskwarrior, porque estaba siempre convenientemente a la mano en mi ubicua y privada consola de comandos, en lugar de estar perdido en alguna de las desbordadas solapas del navegador, requiriendo autenticación y permisos y con el temor de que el estado de una nueva solapa sobreescribiera cosas importantes que tenía en la solapa ya abierta (en algún momento coloqué en MiniDocs una utilidad de agenda que se conectaba con Taskwarrior, llamada Acrobatask y le proveía una interfaz web, pero no avanzó mucho y quizás se integre mejor con Cardumem).

Ahora Taskwarrior en la consola se ocupa de los proyectos y los listados de tareas personales, mientras que para lo colectivo estoy probando NocoDB

Me enfrenté a la duda sobre los identificadores únicos para documentos en Grafoscopio y finalmente opté por NanoID, debido a la baja posibilidad de colisión con tan sólo 11 caracteres, lo que es más corto que una fecha única (pues hay que capturar los milisegundos para hacer un indicador único), funciona incluso cuando se producen muchos trozos de información rápidamente (por ejemplo en una importación de información donde hasta los milisegundos podrían coincidir) y, de todos modos, la fecha es un metadato que se puede capturar en otro lado y por alguna razón para mí no es tan memorable. Si bien el NanoID es un indicador no memorable, los videos en YouTube se hacen virales por otros datos (como su título/autor o los algoritmos de recomendación) y no por la memorabilidad de su enlace (todos acá son no memorables).

Para lidiar con la memorabilidad y organización para humanos, el identificador único es un sufijo en lugar de un prefijo, que va al final del nombre del archivo, separado por doble guiones y antes de la extensión, por ejemplo, cartofonias--e45j7.md.html). Dado que los navegadores web recuerdan partes del enlace, incluso si el nombre se cambiara, pero recordáramos (en parte) el identificador único (en e ejemplo e45j7), nos sugeriría diferentes títulos que incluyen esa secuencia de caracteres.

Los fármacos se eliminan sin cambios o transformados en metabolitos. Los fármacos liposolubles primero se vuelven más polares (hidrosolubles) para poder ser eliminados. NOTA: El riñón es el principal órgano de excreción.

Es el proceso que convierte al fármaco en formas más fáciles de eliminar. Como aplastar la basura para que quepa en un bote, en nuestro caso, el cuerpo transforma el fármaco para que pueda ser excretado (ej, por los riñones).

Un fármaco se reparte entre líquidos y tejidos del cuerpo según:

*Sus propiedades fisicoquímicas

*Cuánta sangre llega a cada órgano

*Y cómo cada tejido interactúa con él

Es como si quisiéramos repartir comida en un campamento, depende de qué tan fácil sea transportarla, a dónde la lleves y si la gente del lugar la acepta o la usa.

O sea, que tanto fármaco útil llega hasta el sitio de acción y es capaz de actuar. Como si mandas 4 soldados a una misión y solo llegan 2 al objetivo.

"Entonces, la farmacocinética es lo que hace el cuerpo con el fármaco.

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Los blikis (blogs + wikis) son formas de pensar en público y tener modos de lectura y escritura está bien, pues no todos los que se aproximan al wiki lo van a editar y la mayoría de internautas sólo lo va leer.

Además sistemas tipo TiddlyWiki (y en el futuro Cardumem) alientan en buena medida la reedición de unidades de contenido (wiki refactoring). Lo clave es que las unidades sean pequeñas y se puedan usar para organizar otras unidades (es decir usarlos como etiquetas).

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public review):

A) The presentation of the paper must be strengthened. Inconsistencies, mislabelling, duplicated text, typos, and inappropriate colour code should be changed.

We spotted and corrected several inconsistencies and mislabelling issues throughout the text and figures. Thanks!  

B) Some claims are not supported by the data. For example, the sentence that says that "adolescent mice showed lower discrimination performance than adults (l.22) should be rewritten, as the data does not show that for the easy task (Figure 1F and Figure 1H).

We carefully reviewed the specific claims and fixed some of the wording so it adheres to the data shown.

C) In Figure 7 for example, are the quantified properties not distinct across primary and secondary areas?

We now carried out additional analysis to test this. We found that while AUDp and AUDv exhibit distinct tuning properties, they show similar differences between adolescent and adult neurons (see Supplementary Table 6, Fig. S7-1a-h). Note that TEa and AUDd could not be evaluated due to low numbers of modulated neurons in this protocol.

D) Some analysis interpretations should be more cautious. (..) A lower lick rate in general could reflect a weaker ability to withhold licking- as indicated on l.164, but also so many other things, like a lower frustration threshold, lower satiation, more energy, etc).

That is a fair comment, and we refined our interpretations. Moreover, we also addressed whether impulsiveness impacted lick rates. In the Educage, we found that adolescent mice had shorter ITIs only after FAs (Fig. S2-1). In the head-fixed setup, we examined (1) the proportion of ITIs where licks occurred (Fig. S3-1c) and (2) the number of licks in these ITIs (Fig. S3-1d). We found no differences between adolescents and adults, indicating that the differences observed in the main task are not due to general differences in impulsiveness (Fig. S2-1, Fig. S3-1c, d). Finally, we note that potential differences in satiation were already addressed in the original manuscript by carefully examining the number of trials completed across the session. See also Review 3, comment #1 below.

Reviewer #2 (Public review):

A) For some of the analyses that the authors conducted it is unclear what the rationale behind them is and, consequently, what conclusion we can draw from them.

We reviewed the manuscript carefully and revised the relevant sections to clarify the rationale behind the analyses. See detailed responses to all the reviewer’s specific comments.

B) The results of optogenetic manipulation, while very interesting, warrant a more in-depth discussion.

We expanded our discussion on these experiments (L495-511) and also added an additional analysis to strengthen our findings (Fig. S3-2e).

Reviewer #3 (Public review):

(1) The authors report that "adolescent mice showed lower auditory discrimination performance compared to adults" and that this performance deficit was due to (among other things) "weaker cognitive control". I'm not fully convinced of this interpretation, for a few reasons. First, the adolescents may simply have been thirstier, and therefore more willing to lick indiscriminately. The high false alarm rates in that case would not reflect a "weaker cognitive control" but rather, an elevated homeostatic drive to obtain water. Second, even the adult animals had relatively high (~40%) false alarm rates on the freely moving version of the task, suggesting that their behavior was not particularly well controlled either. One fact that could help shed light on this would be to know how often the animals licked the spout in between trials. Finally, for the head-fixed version of the task, only d' values are reported. Without the corresponding hit and false alarm rates (and frequency of licking in the intertrial interval), it's hard to know what exactly the animals were doing.

irst, as requested, we added the Hit rates and FA rates for the head-fixed task (Fig. S3-1a). Second, as requested by the reviewr, we performed additional analyses in both the Educage and head-fixed versions of the task. Specifically, we analyzed the ITI duration following each trial outcome. We found that adolescent mice had shorter ITIs only after Fas (Fig. S2-1). In the head-fixed setup, we examined (1) the proportion of ITIs during which licks occurred (Fig. S3-1c) and (2) the number of licks in these ITIs (Fig. S3-1d). We found no differences between adolescents and adults, indicating that the differences observed in the main task are not due to general differences in impulsiveness (Fig. S2-1, Fig. S3-1c, d). See also comment #D of reviewer #1 above.

B) There are some instances where the citations provided do not support the preceding claim. For example, in lines 64-66, the authors highlight the fact that the critical period for pure tone processing in the auditory cortex closes relatively early (by ~P15). However, one of the references cited (ref 14) used FM sweeps, not pure tones, and even provided evidence that the critical period for this more complex stimulus occurred later in development (P31-38). Similarly, on lines 72-74, the authors state that "ACx neurons in adolescents exhibit high neuronal variability and lower tone sensitivity as compared to adults." The reference cited here (ref 4) used AM noise with a broadband carrier, not tones.

We carefully checked the text to ensure that each claim is accurately supported by the corresponding reference.

C) Given that the authors report that neuronal firing properties differ across auditory cortical subregions (as many others have previously reported), why did the authors choose to pool neurons indiscriminately across so many different brain regions?

We appreciate the reviewer’s concern. While we acknowledge that pooling neurons across auditory cortical subregions may obscure region-specific effects, our primary focus in this study is on developmental differences between adolescents and adults, which were far more pronounced than subregional differences.

To address this potential limitation: (1) We analyzed firing differences across subregions during task engagement (see Fig. S4-1, S4-2, S4-3; Supplementary Tables 2 and 3). (2) We have now added new analyses for the passive listening condition in AUDp and AUDv (Fig. S7-1; Supplementary Table 6).

These analyses support our conclusion that developmental stage has a greater impact on auditory cortical activity than subregional location in the contexts examined. For clarity and cohesion, the main text emphasizes developmental differences, while subregional analyses are presented in the Supplement.

D) And why did they focus on layers 5/6? (Is there some reason to think that age-related differences would be more pronounced in the output layers of the auditory cortex than in other layers?)

We agree that other cortical layers, particularly supragranular layers, are important for auditory processing and plasticity. Our focus on layers 5/6 was driven by both methodological and biological considerations. Methodologically, our electrode penetrations were optimized to span multiple auditory cortical areas, and deeper layers provided greater mechanical stability for chronic recordings. Biologically, layers 5/6 contain the principal output neurons of the auditory cortex and are well-positioned to influence downstream decision-making circuits. We acknowledge the limitation of our recordings to these layers in the manuscript (L268; L464-8).

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

(1) The presentation of the paper must be strengthened. As it is now, it makes it difficult to appreciate the strengths of the results. Here are some points that should be addressed:

a) The manuscript is full of inconsistencies that should be fixed to improve the reader's understanding. For example, the description on l.217 and the Figure. S3-1b, the D' value of 0 rounded to 0.01 on l. 735 (isn't it rather the z-scored value that is rounded? A D' of 0 is not a problem), the definition of lick bias on l. 750 and the values in Fig.2, the legend of Figure 7F and what is displayed on the graph (is it population sparseness or responsiveness?), etc.

We adjusted the legend and description of former Fig. S3-1b (now Fig. S3-2b).

We now clarify that the rounded values refer to z-scored hit and false alarm rates that we used in the d’ calculation. We adjusted the definition of the lick bias in Fig. 2 and Fig. S3-1b (L804).

We replaced ‘population responsiveness’ with ‘population sparseness’ throughout the figures, legend and the text.

b) References to figures are sometimes wrong (for example on l. 737,739).

c) Some text is duplicated (for example l. 814 and l. 837).

d) Typos should be corrected (for example l. 127, 'the', l. 787, 'upto').

We deleted the incorrect references of this section, removed the duplicated text, and corrected the typos.

e) Color code should be changed (for example the shades of blue for easy and hard tasks - they are extremely difficult to differentiate).

After consideration, we decided to retain the blue color code (i.e., Fig. 1d, Fig. 3d, Fig. 4e-g, Fig. 5c, Fig. 6d–g), where the distinction between the shades of blue appears sufficiently clear and maintains visual consistency and aesthetic appeal. We did however, made changes in the other color codes (Fig. 4, Fig. 5, Fig. 6, Fig. 7).

f) Figure design should be improved. For example, why is a different logic used for displaying Figure 5A or B and Figure 1E?

We adjusted the color scheme in Fig. 5. We chose to represent the data in Fig. 5 according to task difficulty, as this arrangement best illustrates the more pronounced deficits in population decoding in adolescents during the hard task.

f) Why use a 3D representation in Figure 4G? (2)

The 3D representation in Fig. 4g was chosen to illustrate the 3-way interactions between onset-latency, maximal discriminability, and duration of discrimination.

g) Figure 1A, lower right panel- should "response" not be completed by "lick", "no lick"?

We changed the labels to “Lick” and “No Lick” in Fig. 1a.

h) l.18 the age mentioned is misleading, because the learning itself actually started 20 days earlier than what is cited here.

Corrected.

i) Explain what AAV5-... is on l.212.

We added an explanation of virus components (see L216-220).

(2) The comparison of CV in Figure 2 H-J is interesting. I am curious to know whether the differences in the easy and hard tasks could be due to a decrease in CV in adults, rather than an increase in CV in adolescents? Also, could the difference in J be due to 3 outliers?

We agree that the observed CV differences may reflect a reduction in variability in adults rather than an increase in adolescents. We have revised the Results section accordingly to acknowledge this interpretation.

Regarding the concern about potential outliers in Fig. 2J, we tested the data for outliers using the isoutlier function in MATLAB (defining outliers as values exceeding three standard deviations from the mean) and found no such cases.

(3) Figure 2c shows that there is no difference in perceptual sensitivity between adolescents and adults, whereas the conclusion from Figure 4 is that adolescents exhibit lower discriminability in stimulus-related activity. Aren't these results contradictory?

This is a nuanced point. The similar slopes of the psychometric functions (Fig. 2c) indicating comparable perceptual sensitivity and the lower AUC observed in the ACx of adolescents (Fig. 4) do not necessarily contradict each other. These two measures capture related but distinct issues: psychometric slopes reflect behavioral output, which integrates both sensory encoding and processing downstream to ACx, while the AUC analysis reflects stimulus-related neural activity in ACx, which may still include decision-related components.<br /> Note that stimulus-related neural discriminability outside the context of the task is not different between adolescent and adult experts (Fig. 7h; p = 0.9374, Kruskal Willis Test after Tukey-Kramer correction for multiple comparisons; not discussed in the manuscript). This suggests that there are differences that emerge when we measure during behavior. Also note that behavior may rely on processing beyond ACx, and it is possible that downstream areas compensate for weaker cortical discriminability in adolescents — but this issue merits further investigation.

(4) Why do you think that the discrimination in hard tasks decreases with learning (Figure 6D vs Figure 6F)?

This is another nuanced point, and we can only speculate at this stage. While it may appear counterintuitive that single-neuron discriminability (AUC) for the hard task is reduced after learning (Fig. 6D vs. 6F), we believe this may reflect a shift in sensory coding in expert animals. In a recent study (Haimson et al., 2024; Science Advances), we found that learning alters single-neuron responses in the easy versus hard task in complex and distinct ways, which may account for this result. It is also possible that, in expert mice, top-down mechanisms such as feedback from higher-order areas act to suppress or stabilize sensory responses in auditory cortex, reducing the apparent stimulus selectivity of single neurons (e.g., AUC), even as behaviorally relevant information is preserved or enhanced at the population level.

Reviewer #2 (Recommendations for the authors):

This is very interesting work and I enjoyed reading the manuscript. See below for my comments, queries and suggestions, which I hope will help you improve an already very good paper.

We thank the reviewer for the meticulous and thoughtful review.

(1) Line 107: x-axis of panel 1e says 'pre-adolescent'.

(2) Line 130: replace 'less' with 'fewer'.

(3) Line 153: 'both learned and catch trials': I find the terminology here a bit confusing. I would typically understand a catch trial to be a trial without a stimulus but these 'catch' trials here have a stimulus. It's just that they are not rewarded/punished. What about calling them probe trials instead?

We corrected the labelling (1), reworded to ‘fewer’ and ‘probe trials’ (2,3).

(4) Line 210: The results of the optogenetics experiments are very interesting. In particular, because the effect is so dramatic and much bigger than what has been reported in the literature previously, I believe. Lick rates are dramatically reduced suggesting that the mice have pretty much stopped engaging in the task and the authors very rightly state that the 'execution' of the behavior is affected. I think it would be worth discussing the implications of these results more thoroughly, perhaps also with respect to some of the lesion work. Useful discussions on the topic can be found, for instance, in Otchy et al., 2015; Hong et al., 2018; O'Sullivan et al., 2019; Ceballo et al., 2019 and Lee et al., 2024. Are the mice unable to hear anything in laser trials and that is why they stopped licking? If they merely had trouble distinguishing them then we would perhaps expect the psychometric curves to approach chance level, i.e. to be flat near the line indicating a lick rate of 0.5. Could the dramatic decrease in lick rate be a motor issue? Can we rule out spillover of the virus to relevant motor areas? (I understand all of the 200nL of the virus were injected at a single location) Or are the effects much more dramatic than what has been reported previously simply because the GtACR2 is much more effective at silencing the auditory cortex? Could the effect be down to off-target effects, e.g. by removing excitation from a target area of the auditory cortex, rather than the disruption of cortical processing?

We have now expanded the discussion in the manuscript to more thoroughly consider alternative interpretations of the strong behavioral effect observed during ACx silencing (L495–511). In particular, we acknowledge that the suppression of licking may reflect not only impaired sensory discrimination but also broader disruptions to arousal, motivation, or motor readiness. We also discuss the potential impact of viral spread, circuit-level off-target effects, and the potency of GtACR2 as possible contributors. We highlight the need for future work using more graded or temporally precise manipulations to resolve these issues.

(5) Line 226: Reference 19 (Talwar and Gerstein 2001) is not particularly relevant as it is mostly concerned with microstimulation-induced A1 plasticity. There are, however, several other papers that should be cited (and potentially discussed) in this context. In particular, O'Sullivan et al., 2019 and Ceballo et al., 2019 as these papers investigate the effects of optogenetic silencing on frequency discrimination in head-fixed mice and find relatively modest impairments. Also relevant may be Kato et al., 2015 and Lee et al., 2024, although they look at sound detection rather than discrimination.

We changed the references and pointed the reader to the (new section) Discussion.

(6) Line 253: 'engaged [in] the task.

(7) Figure 4: It appears that panel S4-1d is not referred to anywhere in the main text.

Fixed.

(8) Line 260: Might be useful to explain a bit more about the motivation behind focusing on L5/L6. Are there mostly theoretical considerations, i.e. would we expect the infragranular layers to be more relevant for understanding the difference in task performance? Or were there also practical considerations, e. g. did the data set contain mostly L5/L6 neurons because those were easier to record from given the angle at which the probe was inserted? If those kinds of practical considerations played a role, then there is nothing wrong with that but it would be helpful to explain them for the benefit of others who might try a similar recording approach.

There were no deep theoretical considerations for targeting L5/6.  Our focus on layers 5/6 was driven by both methodological and biological considerations. Methodologically, our electrode penetrations were optimized to span multiple auditory cortical areas, and deeper layers provided greater mechanical stability for chronic recordings. Biologically, layers 5/6 contain the principal output neurons of the auditory cortex and are well-positioned to influence downstream decision-making circuits. We acknowledge the limitation of our recordings to these layers in the manuscript (L268; L463–467). See also comment D of reviewer 3.

(9) Supplementary Table 2: The numbers in brackets indicate fractions rather than percentages.

Fixed.

(10) Figure S4-3: The figure legend implies that the number of neurons with significant discriminability for the hard stimulus and significant discriminability for choice was identical. (adolescent neurons = 368, mice = 5, recordings = 10; adult n = 544, mice = 6, recordings = 12 in both cases). Presumably, that is not actually the case and rather the result of a copy/paste operation gone wrong. Furthermore, I think it would be helpful to state the fractions of neurons that can discriminate between the stimuli and between the choices that the animal made in the main text.

Thank you for spotting the mistake. We corrected the n’s and added the percentage of neurons that discriminate stimulus and choice in the main text and the figure legend.

(11) Line 301: 'We used a ... decoder to quantify hit versus correct reject trial outcomes': I'm not sure I understand the rationale here. For the single unit analysis hit and false alarm trials were compared to assess their ability to discriminate the stimuli. FA and CR trials were compared to assess whether neurons can encode the choice of the mice. But the hit and CR trials which are contrasted here differ in terms of both stimulus and behavior/choice so what is supposed to be decoded here, what is supposed to be achieved with this analysis?

Thank you for this important point. You're correct that comparing hit and CR trials captures differences in both stimulus and choice, or task-related differences. We chose this contrast for the population decoding analysis to achieve higher trial counts per session and similar number of trials which are necessary for the reliability of the analysis. While this approach does not isolate stimulus from choice encoding, it provides an overall measure of how well population activity distinguishes task-relevant outcomes. We explicitly acknowledge this issue in L313-314.

(12) Line 332: What do you mean when you say the novice mice were 'otherwise fully engaged' in the task when they were not trained to do the task and are not doing the task?

By "otherwise fully engaged," we mean that novice mice were actively participating in the task environment, similar to expert mice — they were motivated by thirst and licked the spout to obtain water. The key distinction is that novice mice had not yet learned the task rules and likely relied on trial-and-error strategies, rather than performing the task proficiently.

(13) Line 334: 'regardless of trial outcome': Why is the trial outcome not taken into account? What is the rationale for this analysis? Furthermore, in novice mice a substantial proportion of the 'go' trials are misses. In expert mice, however, the proportion of 'miss trials' (and presumably false alarms) will by definition be much smaller. Given this, I find it difficult to interpret the results of this section.

This approach was chosen to reliably decode a sufficient number of trials for each task difficulty (i.e. expert mice predominantly performed CRs on No-Go trials and novice mice often showed FAs). Utilizing all trial outcomes ensured that we had enough trials for each stimulus type to accurately estimate the AUCs. This approach avoids introducing biases due to uneven trial numbers across learning stages.

(14) Line 378: 'differences between adolescents and adults arise primarily from age': Are there differences in any of the metrics shown in 7e-h between adolescents and adults?

We confirm that differences between adolescents and adults are indeed present in some metrics but not others in Figure 7e–h. Specifically, while tuning bandwidth was similar in novice animals, it was significantly lower in adult experts (Fig. 7e; novice: p = 0.0882; expert: p = 0.0001 Kruskal Willis Test after Tukey-Kramer correction for multiple comparisons; not discussed in the manuscript). The population sparseness was similar in both novice and expert adolescent and adult neurons (Fig. 7f; novice: p = 0.2873; expert: p = 0.1017, Kruskal Willis Test after Tukey-Kramer correction for multiple comparisons; not discussed in the manuscript). The distance to the easy go stimulus was similar in novice animals, but lower in adult experts (Fig. 7g; novice: p = 0.7727; expert: p = 0.0001, Kruskal Willis Test after Tukey-Kramer correction for multiple comparisons; not discussed in the manuscript). The neuronal d-prime was similar in both novice and expert adolescent and adult neurons (Fig. 7h; novice: p = 0.7727; expert: p = 0.0001, Kruskal Willis Test after Tukey-Kramer correction for multiple comparisons; not discussed in the manuscript).

(15) Line 475: '...well and beyond...': something seems to be missing in this statement.

(16) Line 487: 'onto' should be 'into', I think.

(17) Line 610 and 613: '3 seconds' ... '2.5 seconds': Was the response window 3s or 2.5s?

(18) Line 638: 'set' should be 'setup', I believe.

All the mistakes mentioned above, were fixed. Thanks.

(19) Line 643: 'Reward-reinforcement was delayed to 0.5 seconds after the tone offset': Presumably, if they completed their fifth lick later than 0.5 seconds after the tone, the reward delivery was also delayed?

Apologies for the lack of clarity. In the head-fixed version, there was no lick threshold. Mice were reinforced after a single lick. If that lick occurred after the 0.5-second reinforcement delay following tone offset, the reward or punishment was delivered immediately upon licking.

(20) Line 661: 'effect [of] ACx'.

(21) Line 680: 'a base-station connected to chassis'. The sentence sounds incomplete.

(22) Line 746: 'infliction', I believe, should say 'inflection'.

(23) Line 769: 'non-auditory responsive units': Shouldn't that simply say 'non-responsive units'? The way it is currently written I understand it to mean that these units were responsive (to some other modality perhaps) but not to auditory stimulation.

(24) Line 791: 'bins [of] 50ms'.

(25) Line 811: 'all of' > 'of all'.

(26) Line 814: Looks like the previous paragraph on single unit analysis was accidentally repeated under the wrong heading.

(27) Line 817: 'encoded' should say 'calculated', I believe.

All the mistakes mentioned above were fixed. Thanks.

(28) Line 869: 'bandwidth of excited units': Not sure I understand how exactly the bandwidth, i.e. tuning width was measured.

We acknowledge that our previous answer was unclear and expanded the Methods section. To calculate bandwidth, we identified significant tone-evoked responses by comparing activity during the tone window to baseline firing rates at 62 dB SPL (p < 0.05). For each neuron, we counted the number of contiguous frequencies with significant excitatory responses, subtracting isolated false positives to correct for chance. We then converted this count into an octave-based bandwidth by multiplying the number of frequency bins by the octave spacing between them (0.1661 octaves per step).

(29) Line 871: 'population sparseness': Is that the fraction of tone frequencies that produced a significant response? I would have thought that this measure is very highly correlated to your measure of bandwidth, to the point of being redundant, but I may have misunderstood how one or the other is calculated. Furthermore, the Y label of Figure 7f says 'responsiveness' rather than sparseness and that would seem to be the more appropriate term because, unless I am misunderstanding this, a larger value here implies that the neuron responded to more frequencies, i.e. in a less sparse manner.

We have clarified the use of the term "population sparseness" and updated the Y-axis label in Figure 7f to better reflect this measure. This metric reflects the fraction of tone–attenuation combinations that elicited a significant excitatory response across the entire population of neurons, not within individual units.

While this measure is related to bandwidth, it captures a distinct property of the data. Bandwidth quantifies how broadly or narrowly a single neuron responds across frequencies at a fixed intensity, whereas population sparseness reflects how distributed responsiveness is across the population as a whole. Although the two measures are related, since broadly tuned neurons often contribute to lower population sparseness, they capture distinct aspects of neural coding and are not redundant.

(30) Line 881: I think this line should refer to Figure 7h rather than 7g.

Fixed.

Reviewer #3 (Recommendations for the authors):

(1) In the Educage, water was only available when animals engaged in the task; however, there is no mention of whether/how animal weight was monitored.

In the Educage, mice had continuous access to water by voluntarily engaging in the task, which they could perform at any time. Although body weight was not directly monitored, water access was essentially ad libitum, and mice performed hundreds of trials per day, thereby ensuring sufficient daily intake. This approach allowed us to monitor hydration (ad libitum food is supplied in the home cage). The 24/7 setup, including automated monitoring of trial counts and water consumption, was reviewed and approved by our institutional animal care and use committee (IACUC).

(2) In Figure 2B-C and Figure 2E, the y-axis reads "lick rate". At first glance, I took this to mean "the frequency of licking" (i.e. an animal typically licks at a rate of 5 Hz). However, what the authors actually are plotting here is the proportion of trials on which an animal elicited >= 5 licks during the response window (i.e. the proportion of "yes" responses). I recommend editing the y-axis and the text for clarity.

We replaced the y-label and adjusted the figure legend (Fig. 2).

(3) I didn't see any examples of raw (filtered) voltage traces. It would be worth including some to demonstrate the quality of the data.

We have added an example of a filtered voltage trace aligned to tone onset in Fig. S4-1a to illustrate data quality. In addition, all raw and processed voltage traces, along with relevant analysis code, are available through our GitHub repository and the corresponding dataset on Zenodo.

(4) The description of the calculation of bias (C) in the methods section (lines 749-750) is incorrect. The correct formula is C = -0.5 * [z(hit rate) + z(fa rate)]. I believe this is the formula that the authors used, as they report negative C values. Please clarify or correct.

Thanks for spotting this. It is now corrected.

(5) The authors use the terms 'naïve' and 'novice' interchangeably. I suggest sticking with one term to avoid potential confusion.

(6) Multiple instances: "less trials/day" should be "fewer trials/day"

(7) Supplementary Table 2: The values reported are proportions, not percentages. Please correct.

(8) Line 270: Table 2 does not show the number of neurons in the dataset categorized by region. Perhaps the authors meant Supplementary Table 2?

Fixed. Thank you for pointing these mistakes out.

(9) Figure 5C: the data from the hard task are entirely obscured by the data from the easy task. I recommend splitting it into two different plots.

We agree and split the decoding of the easy and the hard task into two graphs (left: easy task; right: hard task). Thank you!

(10) How many mice contributed to each analyzed data set? Could the authors provide a breakdown in a table somewhere of how many neurons were recorded in each mouse and which ones were included in which analyses?

We added an overview of the analyzed datasets in supplementary Table 7. Please note that the number of mice and neurons used in each analysis is also reported in the main text and legends. Importantly, all primary analyses were conducted using LME models, which explicitly account for hierarchical data structure and inter-mouse variability, thereby addressing potential concerns about data imbalance or bias.

Author Response:

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

Reviewer #1 (Recommendations for the authors): 

Overall, the manuscript could be clearer and more beneficial to the readers with the following suggested revisions:  

(1) The abstract should include information on the comparative performance of 89Zr 64Cu and 18F labeled nanobodies, especially noting the challenges with DFO-89Zr and NOTA-64Cu. 

(2) The abstract should explicitly note the types of transplants assessed and the specific PET findings.

(3) The abstract should note the negative results in terms of brain PET findings. 

We thank reviewer 1 for these three suggestions. We have now included this information in the abstract.

(4)  Based on the data shown in Fig. 1 and Table 1, it seems that the nanobodies bind to quite a few proteins other than TfR. This should be discussed frankly as a limitation. 

The presence of multiple other bands and proteins identified by LC/MS in Figure 1 is typical for immunoprecipitation experiments, as performed under the conditions used: all proteins other than TfR that are identified in Table 1 are abundant cytoplasmic (cytoskeletal) and/or nuclear proteins.  More rigorous washing would perhaps have removed some of these contaminants at the risk of losing some of the specific signal as well. We have added a comment to this effect.  In an in vivo setting, this would be of minor concern, as these proteins would be inaccessible to our nanobodies. In fact, when VHH123 radioconjugates are injected in huTfr+/+ mice (or VHH188 in C57BL/6), we observe no specific signal – which supports this conclusion. 

We therefore state: “We show that both V_HHs bind only to the appropriate TfR, with no obvious cross-reactivity to other surface-expressed proteins by immunoblot, LC/MSMS analysis of immunoprecipitates, SDS-PAGE of ³⁵S-labelled proteins and flow cytometry (Fig 1;Table 1).”. We have added some clarification to make this clearer, and we also include the full LC/MSMS data tables are also added in supplemental materials, as supplementary Table 1. We have included subcellular localization information for each protein identified through LC/MSMS in Table 1 as well.

(5)  Why did the authors use DFO, which is well known to leak Zr, rather than the current standard for 89Zr PET, DFO* (DFO-star)? 

We used DFO rather than DFO-star for several reasons: 1) because we had already conducted and published numerous other studies using DFO-conjugated nanobodies and not observed any release of ⁸⁹Zr, 2) commercially sourced clickchemistry enabled DFO-star (such as DFO*-DBCO) was not available at the time of the study. 

(6) Figure 2B appears to show complex structures, more complex than just GGG-DFOazide, and GGG-NOTA-azide. This should be explained in detail. 

We have added two supplemental figures and methods that recapitulate how we generated what we have termed as GGG-DFO-Azide and GGG-NOTA-Azide. We have updated the legend of Figure 2B. 

(7) Why is there a double band in Suppl. Fig 9 for VHH123-NOTA-Azide? 

Under optimal conditions, sortase A-mediated transpeptidation is efficient,  resulting in the formation of a peptide bond between the C-terminally LPETG-tagged protein and the GGG-probe. However, extended reaction times or suboptimal concentrations of modified GGG-probes (which are often in limited supply) in the reaction mixture, allow hydrolysis of the sortase A-LPET-protein intermediate. The hydrolysis product can no longer participate in a sortase A reaction. This is what explains the doublet in the reaction used to generate VHH123-NOTA-N₃ – the upper band is VHH123-NOTA-N₃ and the lower band is the hydrolysis product.  VHH123-LPET, is unable to react with PEG_20kDa-DBCO (the lower band that appears at the same position of migration in the next lane on the gel). We noticed that an adjacent lane was mislabelled as ‘VHH188-NOTA-PEG_20kDa’ when in fact it was ‘VHH123-NOTA-PEG_20kDa’. This has been corrected.

The hydrolysis product, VHH123-LPET, has a short circulatory half-life and obviously lacks the PEG moiety as well as the chelator. It therefore cannot chelate ⁶⁴Cu. Its presence should not interfere with PET imaging.  Since all animals were injected with the same measured dose of ⁶⁴Cu labeled-conjugate, the presence of an unlabeled TfRbinding competitor in the form of VHH123-LPET - at a << 1:1 molar ratio to the labelled nanobody – would be of no consequence.

(8) More details should be provided about the tetrazine-TCO click chemistry for 18F labeling. 

We have added supplementary methods and figures that detail how ¹⁸F-TCO was generated. For the principle of TCO-tetrazine click-chemistry, a brief description was added in the text, as well as a reference to a review on the subject.

(9) For the data shown in Figure 3H, the authors should state whether the brain tissues were capillary depleted, and if so, how this was performed and how complete the procedure was. 

No capillary depletion of the brain tissues was performed, as this was challenging to perform in compliance with the radiosafety protocols in place at our institution. We have updated the legend of figure 3H and methods to include this important detail. Whole blood gamma-counting did not show any obvious di  erence of activity across the 4 groups in figure 3G (same mice as in figure 3H), which would go against the interpretation that activity di  erences in the brain (figure 3H) are solely attributable to residual activity from blood in the capillaries. 

(10) The authors should experimentally test the hypotheses that the PEG adduct reduced BBB transcytosis. 

Reviewer 1 is correct to point out that we have not tested un-PEGylated conjugates of ⁶⁴Cu and ⁸⁹Zr with the anti-TfR nanobodies and we currently do not have the means to perform additional experiments. However, the ¹⁸F conjugates were not PEGylated, and these also fail to show any detectable signal in the CNS by PET/CT (see figure 4A). PEGylation alone cannot be the sole factor that limits transcytosis across the BBB.

(11) It was interesting to note that the Cu appears to dissociate from the NOTA chelator. The authors should provide more information about the kinetics of this process.  

We have not tested the kinetics of dissociation between ⁶⁴Cu and the NOTA conjugates in vitro, like we have done for ⁸⁹Zr and DFO (supplemental figure 2), because previous work (see references 35 and 36 by Dearling JL and Mirick GR and colleagues) has shown that NOTA and other copper chelators tend to release free copper radioisotopes in the liver, a commonly reported artifact. We have also included a new set of images that show the biodistribution of VHH123-NOTA-⁶⁴Cu in huTfR+/+ mice, where we still observe a substantial signal in the liver, indicating release of ⁶⁴Cu from NOTA, in the absence of the anti-TfR VHH binding to its target. This was clearly not seen using the DFO-⁸⁹Zr conjugates.  Binding of the VHH to TfR, followed by internalization, appears to be required for the release of ⁸⁹Zr from DFO, prompting us to investigate this phenomenon further.

(12) The authors should increase the sample size, and test two different radiolabels for the transplant imaging results (Figs. 5 and 6), since these seem to be the ones they feel are the most important, based on the title and abstract. 

We agree with reviewer 1 that more repeats would increase the significance of our findings, but we unfortunately do not have the means of performing additional experiments at this time (the lab at Boston Children’s Hospital has closed as Dr. Ploegh has retired). We believe that the results are compelling and will be of use to the in vivo imaging community.

(13) Fig. 6G appears to show a false positive result for the kidney imaging. Is this real, or an artifact of small sample size?

We agree with reviewer 1 that the kidney signals in figure 6 are somewhat puzzling. The difference between the tumor-bearing mice that received VHH123 and VHHEnh conjugates is not significant – with the obvious caveat that the VHHEnh group is comprised of only 2 mice, so sample size may well be a factor here. If we compare the signals of the VHH123 conjugate in tumor-bearing mice vs. tumor-free mice, the VHH123 conjugates would have cleared much faster in the tumor-free mice over 24 hours (since no epitope is present for VHH123 to bind to), thus weakening the kidney signal observed after 24 hours. The same would be true for all the other tissues – except for the liver (where free ⁶⁴Cu that leaks from NOTA accumulates). VHHEnh conjugates in tumor-bearing mice show a significant kidney signal – although no VHH123 target epitope is present in these mice. B16.F10 tumors at 4 weeks of growth tend to be necrotic and can passively retain any radiotracer – this generates the weak lung signal visible in Fig 6D – thus the radiotracer would clear at a slower rate than VHH123 conjugates in tumor-free mice giving a higher kidney signal at 24 hours. 

No tumors were found in the kidneys post-necropsy. We attribute the differences in kidney signals to di erent kinetics of clearance of the radioconjugates. We have added this explanation to the results and discussion.

(14) Are the results shown in Fig. 7 generalizable? The authors should the constructs with 18F labeling and without the PEG adduct. 

We agree with reviewer 1 that it would be very interesting to confirm these observations using 18F radioconjugates. The results should be generalizable, as the difference between signals can only be attributed to the presence of the recognized epitope in the placenta– which is in fact the only variable that differs between the two groups. At the time of conducting the study, we had not planned to perform the same experiments with 18F radioconjugates – partly because synthesis of 18F radioconjugates is more challenging (and costly) than the production of 89Zr-labeled nanobodies.  

(15) The authors should discuss the relative safety of 89Zr and 64Cu. It is likely to be quite a bit worse than for 18F, since the 89Zr and 64Cu have longer half-lives, dissociate from their chelators, and lodge in off-target tissues. An alternative interpretation of the authors' data could be that 89Zr and 64Cu labeling in this context are unsuitable for the stated purposes of PET imaging. In this case, the key experiments shown in Figs. 5-7 should be repeated with the 18F labeled nanobody constructs. 

Our vision was to o er a tool to the scientific community interested in in vivo tracking of cells in di erent preclinical disease models. The question of safety regarding 89Zr and 64Cu for clinical use was therefore not a factor we then considered. However, we have now included a section in the discussion about the potential safety issue of ⁸⁹Zr release and bone accumulation in clinical settings, especially for radioconjugates that target an internalizing surface protein. 

(16) The authors should remark on the somewhat surprisingly modest amount of BBB transcytosis in the discussion. What were the a inities of the nanobodies? 

The a inities and binding kinetics of both nanobodies was described in a separate work that is referenced in the introduction (references 21 and 22 by Wouters Y and colleagues). Through other methods that rely on a highly sensitive bio-assay, it was shown that both VHH123 and VHH188 are capable of transcytosis: both nanobodies coupled to a neurotensin peptide induced a drop of temperature after i.v. injection in matching mouse strains (VHH123 in C57BL/6 and VHH188 in huTfr +/+). The lack of any compelling CNS signal by PET/CT is discussed in the manuscript.

(17) More details of the methods should be provided in the supplement. 

a.  What was the source of the penta-mutant Sortase A-His6? 

Sortase A pentamutant is produced in-house, by cytoplasmic expression in E.coli (BL21 strain), using a plasmid vector encoding a truncated and mutated version of Sortase A. References were added, as well as the Addgene repository number (51140).

b.  What was the yield of the sortase reactions? 

For small proteins, such as nanobodies/ V_HHs, we find that the yield of a sortase A reaction typically is > 75%. This is what we observed for all our conjugations. The methods section was updated to include this information.

c.  What was the source of the GGG-Azide-DFO and GGG-Azide NOTA? Based on the structures shown in Fig. 2, these appear to be more complex that was noted in the text. 

We have now detailed the synthesis of GGG-DFO-Azide and GGG-NOTA-Azide in the supplementary methods.

d.  More details about the source and purity of the tetrazine and TCO labeling reagents should be provided. 

We have included information on the synthesis of GGG-tetrazine in the supplementary methods. Concerning the synthesis of ¹⁸F-TCO, we have also included a detailed description of the compound in supplementary methods. The reaction between GGG-tetrazine and ¹⁸F-TCO is now further detailed in the manuscript. 

e.  The TCO-agarose slurry purification should be explained in more detail, and the results should be shown. 

We have included a detailed procedure of how the TCO-agarose slurry purification was performed in the methods sections. We had already included the Radio-Thin Layer Chromatography QC data of the final VHH123-18F and VHH188-18F purifications in the supplementary figures – which are obtained immediately after TCOagarose slurry purification. The detailed yields of the TCO-agarose slurry purification in terms of activity of each collected fraction is now detailed in the methods section.

f.   The CT parameters should be provided.  

We have now added more information about the PET/CT imaging procedure in the methods section of the manuscript.

Reviewer #2 (Recommendations for the authors): 

Authors should discuss the possibility of the TfR as a rejection antigen. Murine TfR is foreign for hTfR+/+ mice and vice versa. 

We have not discussed this possibility, as we believe the risk of rejection of huTfR+ cells in moTfR+ mice (or vice versa) is negligible. The cells and mice are of the same genetic background – save for the coding region of ectodomain of the TfR (spanning amino acids ~194 to 390 of the full length TfR, which is 763 AA). The pairwise identity of both human and mouse TfR ectodomains is of 73% after alignment of both AA sequences using Clustal Omega. We agree that we cannot formally exclude the possibility of an immune rejection, and have now mentioned this possibility in the discussion.

Is there any clinical use of the anti-human TfR receptor PET tracer? 

We do not currently envision an application for the anti-human TfR VHH in PET/CT in a clinical setting.  

Why is the in vivo anti-mouse TfR uptake level in C57BL/6 mice consistently higher than the anti-human TfR receptor PET tracer in hTfR+/+ mice? Is this due to differences in characteristics of the VHH's (e.g. a inity, internalization properties), or rather due to a different biological behavior of the hTfR-transgene (e.g. reduced internalization properties)? 

We indeed observed that VHH123 uptake and binding appears to be more robust than that of VHH188 to their respective targets. Moreover, after later times post-injection (> 48h), VHH188 appears to display a very low reactivity to C57BL/6 (moTfR+) cells (see Figure 3B). We attribute this to the respective affinities and specificities of both VHHs. We have not investigated the VHH binding kinetics of the mouse versus humanectodomain TfR proteins in vitro. Internalization should be mildly different at best, as ⁸⁹Zr release from DFO occurs with both VHHs in both C57BL/6 and huTfR +/+ mouse models (when injected in a matched configuration). The huTfR +/+ mice rely exclusively on the huTfr for their iron supply. They are healthy with no obvious pathological features. The behavior of the huTfr is therefore presumably similar, if not identical to that of the mouse Tfr, bearing in mind that the huTfr and the mouse Tfr are both reliant on mouse Tf as their ligand

The anti-TfR VHHs were initially developed as a carrier for BBB-transport of VHH-based drug conjugates (previous publications). The data shown here reduces enthusiasm towards this application. Uptake in the brain is several log-factors lower than physiological uptake elsewhere. Potential consequences of off-brain uptake on potential toxicity of VHH-based drug-conjugates could be better emphasized in the discussion. 

We did not observe a significant presence of the anti-TfR VHHs in the CNS by PET/CT. We have addressed several possibilities: longer circulation times post-injection may favor transcytosis of the VHHs through the BBB. However, because transcytosis requires endocytosis –⁸⁹Zr may be released by their chelating moiety at this step. The only radiotracers with a covalent bond between the radio-isotope and the VHHs in our work are the ¹⁸F VHHs, but the signal acquisition window may have been too short to observe transcytosis and accumulation in the CNS. Another possible caveat is that PEGylation of the radiotracers may be an obstacle to transcytosis. The circulatory halflife of unpegylated VHHs is too low to allow adequate visualization after 24 hours postinjection, as the conjugates rapidly clear from the circulation (t ½ = 30 minutes or less). We have updated the discussion to address these points.

In several locations (I have counted 5) a space is missing between words, please double-check. 

We carefully checked the manuscript to remove any remaining typos.

It is unclear to me why for the melanoma-tracking experiment the tracer is switched from the 89Zr-labeled variant to the 64Cu-labeled variant. 

The decision to switch to the ⁶⁴Cu labeled VHHs for the melanoma experiment stemmed from a wish to 1) evaluate the performance of the ⁶⁴Cu-radioconjugates in detecting transplanted cells as we had done with the ⁸⁹Zr conjugates and 2) assess how the (non-specific) liver signal seen with ⁶⁴Cu contrasts with a specific signal.  

typo in discussion: C57BL/6 instead of C57B/6         

We have corrected the typo.

It is unclear to me why in FIG1B cells are labeled with 35S. Is it correct that the signals seen are due to staining membranes with anti-TfR mAbs? Or is this an autoradiography of the gel? 

In Figure 1B cells were labeled with 35S-Met/Cys, while the images shown are indeed those of Western Blots, using an anti-TfR monoclonal antibody as the primary antibody to detect human and mouse TfR retrieved by the anti Tfr VHHs. Autoradiography using the same lysates showed the presence of contaminants in the VHH eluates, as commonly seen in immunoprecipitates from metabolically labeled cells (as distinct from IP/Westerns). For this reason, we performed a Western Blot on the same samples to confirm TfR pull-down. As written in the results section, we also performed LCMS analysis of the immunoprecipitated proteins to better characterize contaminating proteins (Table 1). To clarify this, we have now added the autoradiographs in supplementary data (supplementary figure 15) and added a reference to these observation in the results. 

ROI quantifications in all figures: these should be expressed as %ID/cc instead of %ID/g. Ex vivo tissue counts should be in %ID/g instead of cpm. 

We have converted all ROI quantification figures as %ID/cc based on the assumption that 1mL (1cc) = 1g. For ex vivo tissue counts, %ID/g has been calculated based on injected dose (except for figure 3G, where the comparisons in %ID/G are not possible due to the uncertain nature of bone marrow and whole blood). All figures have now been updated.

Fig4: it would be good to also see respective mouse controls (C57BL6 vs hTfR+/+) for the 64Cu- and 18F-labeled VHH123 tracers. Each radiolabeling methodology changes in vivo biodistribution and specificity, which can be better assessed by using appropriate controls. 

We had performed these controls but they were not included in the manuscript as deemed redundant with the results of Figure 3. We have now separated Figure 4 in two panels (Figure 4A and 4B) with figure 4A showing the 1h timepoint post-injection of VHH123 radiotracers in C57BL/6 vs huTfr^+/+ and Figure 4B showing the 24h timepoint in the same configuration. ROI analyses were also done on the huTfR^+/+ controls and were included in Figure 4C as well.

Fig7: is it correct that mouse imaging is performed at 24h p.i. and dissected embryo's at 72h p.i.? Why are there 2 days between each procedure of the same animals? 

We acquired images at di erent timepoints, specifically at 1h, 24h, 48h and 72 hours after radio-tracer injection. As 72 h was the last timepoint, the mice were sacrificed the same day and embryo dissection performed thereafter, at 72 hours post radiotracer injection. We decided to show the 24h timepoint images as they were the most representative of the series, o ering the best signal-to-noise ratio. The signal pattern did not change over the course from 24h to 72h. We have now added those timepoints in the supplementary data.

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What is this?

I get an error message here. I have followed all explanation of R code mentioned here step by step. I've checked the spelling it's O.K. I don't know what the problem is. The error is: Error in eval(family$initialize) : y values must be 0 <= y <= 1

Author response:

The following is the authors’ response to the original reviews

Public Reviews: 

Reviewer #1 (Public review): 

Summary: 

This work integrates two timepoints from the Adolescent Brain Cognitive Development (ABCD) Study to understand how neuroimaging, genetic, and environmental data contribute to the predictive power of mental health variables in predicting cognition in a large early adolescent sample. Their multimodal and multivariate prediction framework involves a novel opportunistic stacking model to handle complex types of information to predict variables that are important in understanding mental health-cognitive performance associations. 

Strengths: 

The authors are commended for incorporating and directly comparing the contribution of multiple imaging modalities (task fMRI, resting state fMRI, diffusion MRI, structural MRI), neurodevelopmental markers, environmental factors, and polygenic risk scores in a novel multivariate framework (via opportunistic stacking), as well as interpreting mental health-cognition associations with latent factors derived from partial least squares. The authors also use a large well-characterized and diverse cohort of adolescents from the ABCD Study. The paper is also strengthened by commonality analyses to understand the shared and unique contribution of different categories of factors (e.g., neuroimaging vs mental health vs polygenic scores vs sociodemographic and adverse developmental events) in explaining variance in cognitive performance 

Weaknesses: 

The paper is framed with an over-reliance on the RDoC framework in the introduction, despite deviations from the RDoC framework in the methods. The field is also learning more about RDoC's limitations when mapping cognitive performance to biology. The authors also focus on a single general factor of cognition as the core outcome of interest as opposed to different domains of cognition. The authors could consider predicting mental health rather than cognition. Using mental health as a predictor could be limited by the included 9-11 year age range at baseline (where many mental health concerns are likely to be low or not well captured), as well as the nature of how the data was collected, i.e., either by self-report or from parent/caregiver report. 

Thank you so much for your encouragement.

We appreciate your comments on the strengths of our manuscript.

Regarding the weaknesses, the reliance on the RDoC framework is by design. Even with its limitations, following RDoC allows us to investigate mental health holistically. In our case, RDoC enabled us to focus on a) a functional domain (i.e., cognitive ability), b) the biological units of analysis of this functional domain (i.e., neuroimaging and polygenic scores), c) potential contribution of environments, and d) the continuous individual deviation in this domain (as opposed to distinct categories). We are unaware of any framework with all these four features.

Focusing on modelling biological units of analysis of a functional domain, as opposed to mental health per se, has some empirical support from the literature. For instance, in Marek and colleagues’ (2022) study, as mentioned by a previous reviewer, fMRI is shown to have a more robust prediction for cognitive ability than mental health. Accordingly, our reasons for predicting cognitive ability instead of mental health in this study are motivated theoretically (i.e., through RDoC) and empirically (i.e., through fMRI findings). We have clarified this reason in the introduction of the manuscript.

We are aware of the debates surrounding the actual structure of functional domains where the originally proposed RDoC’s specific constructs might not fit the data as well as the data-driven approach (Beam et al., 2021; Quah et al., 2025). However, we consider this debate as an attempt to improve the characterisation of functional domains of RDoC, not an effort to invalidate its holistic, neurobiological and basicfunctioning approach. Our use of a latent-variable modelling approach through factor analyses moves towards a data-driven direction. We made the changes to the second-to-last paragraph in the introduction to make this point clear:

“In this study, inspired by RDoC, we a) focused on cognitive abilities as a functional domain, b) created predictive models to capture the continuous individual variation (as opposed to distinct categories) in cognitive abilities, c) computed two neurobiological units of analysis of cognitive abilities: multimodal neuroimaging and PGS, and d) investigated the potential contributions of environmental factors. To operationalise cognitive abilities, we estimated a latent variable representing behavioural performance across various cognitive tasks, commonly referred to as general cognitive ability or the gfactor (Deary, 2012). The g-factor was computed from various cognitive tasks pertinent to RDoC constructs, including attention, working memory, declarative memory, language, and cognitive control. However, using the g-factor to operationalise cognitive abilities caused this study to diverge from the original conceptualisation of RDoC, which emphasises studying separate constructs within cognitive abilities (Morris et al., 2022; Morris & Cuthbert, 2012). Recent studies suggest an improvement to the structure of functional domains by including a general factor, such as the g-factor, in the model, rather than treating each construct separately (Beam et al., 2021; Quah et al., 2025). The g-factor in children is also longitudinally stable and can forecast future health outcomes (Calvin et al., 2017; Deary et al., 2013). Notably, our previous research found that neuroimaging predicts the g-factor more accurately than predicting performance from separate individual cognitive tasks (Pat et al., 2023). Accordingly, we decided to conduct predictive models on the g-factor while keeping the RDoC’s holistic, neurobiological, and basic-functioning characteristics.”

Reviewer #2 (Public review):

Summary: 

This paper by Wang et al. uses rich brain, behaviour, and genetics data from the ABCD cohort to ask how well cognitive abilities can be predicted from mental-health-related measures, and how brain and genetics influence that prediction. They obtain an out-ofsample correlation of 0.4, with neuroimaging (in particular task fMRI) proving the key mediator. Polygenic scores contributed less. 

Strengths: 

This paper is characterized by the intelligent use of a superb sample (ABCD) alongside strong statistical learning methods and a clear set of questions. The outcome - the moderate level of prediction between the brain, cognition, genetics, and mental health - is interesting. Particularly important is the dissection of which features best mediate that prediction and how developmental and lifestyle factors play a role. 

Thank you so much for the encouragement. 

Weaknesses: 

There are relatively few weaknesses to this paper. It has already undergone review at a different journal, and the authors clearly took the original set of comments into account in revising their paper. Overall, while the ABCD sample is superb for the questions asked, it would have been highly informative to extend the analyses to datasets containing more participants with neurological/psychiatric diagnoses (e.g. HBN, POND) or extend it into adolescent/early adult onset psychopathology cohorts. But it is fair enough that the authors want to leave that for future work. 

Thank you very much for providing this valuable comment and for your flexibility.

For the current manuscript, we have drawn inspiration from the RDoC framework, which emphasises the variation from normal to abnormal in normative samples (Morris et al., 2022). The ABCD samples align well with this framework.

We hope to extend this framework to include participants with neurological and psychiatric diagnoses in the future. We have begun applying neurobiological units of analysis for cognitive abilities, assessed through multimodal neuroimaging and polygenic scores (PGS), to other datasets containing more participants with neurological and psychiatric diagnoses. However, this is beyond the scope of the current manuscript. We have listed this as one of the limitations in the discussion section:

“Similarly, our ABCD samples were young and community-based, likely limiting the severity of their psychopathological issues (Kessler et al., 2007). Future work needs to test if the results found here are generalisable to adults and participants with stronger severity.”

In terms of more practical concerns, much of the paper relies on comparing r or R2 measures between different tests. These are always presented as point estimates without uncertainty. There would be some value, I think, in incorporating uncertainty from repeated sampling to better understand the improvements/differences between the reported correlations. 

This is a good suggestion. We have now included bootstrapped 95% confidence intervals in all of our scatter plots, showing the uncertainty of predictive performance.

The focus on mental health in a largely normative sample leads to the predictions being largely based on the normal range. It would be interesting to subsample the data and ask how well the extremes are predicted. 

We appreciate this comment. Similar to our response to Reviewer 2’s Weakness #1, our approach has drawn inspiration from the RDoC framework, which emphasises the variation from normal to abnormal in normative samples (Morris et al., 2022). Subsampling the data would make us deviate from our original motivation. 

Moreover, we used 17 mental healh variables in our predictive models: 8 CBCL subscales, 4 BIS/BAS subscales and 5 UPSS subscales. It is difficult to subsample them. Perhaps a better approach is to test the applicability of our neurobiological units of analysis for cognitive abilities (multimodal neuroimaging and PGS) in other datasets that include more extreme samples. We are working on this line of studies at the moment, and hope to show that in our future work. 

Reviewer 2’s Weakness #4

A minor query - why are only cortical features shown in Figure 3? 

We presented both cortical and subcortical features in Figure 3. The cortical features are shown on the surface space, while the subcortical features are displayed on the coronal plane. Below is an example of these cortical and subcortical features from the ENBack contrast. The subcortical features are presented in the far-right coronal image.

We separated the presentation of cortical and subcortical features because the ABCD uses the CIFTI format (https://www.humanconnectome.org/software/workbenchcommand/-cifti-help). CIFTI-format images combine cortical surface (in vertices) with subcortical volume (in voxels). For task fMRI, the ABCD parcellated cortical vertices using Freesurfer’s Destrieux atlas and subcortical voxels using Freesurfer’s automatically segmented brain volume (ASEG).

Due to the size of the images in Figure 3, it may have been difficult for Reviewer 2 to see the subcortical features clearly. We have now added zoomed-in versions of this figure as Supplementary Figures 4–13.

Recommendations for the authors:

Reviewer #1 (Recommendations for the autors):

(1) In the abstract, could the authors mention which imaging modalities contribute most to the prediction of cognitive abilities (e.g., working memory-related task fMRI)? 

Thank you for the suggestion. Following this advice, we now mention which imaging modalities led to the highest predictive performance. Please see the abstract below.

“Cognitive abilities are often linked to mental health across various disorders, a pattern observed even in childhood. However, the extent to which this relationship is represented by different neurobiological units of analysis, such as multimodal neuroimaging and polygenic scores (PGS), remains unclear. 

Using large-scale data from the Adolescent Brain Cognitive Development (ABCD) Study, we first quantified the relationship between cognitive abilities and mental health by applying multivariate models to predict cognitive abilities from mental health in children aged 9-10, finding an out-of-sample r\=.36 . We then applied similar multivariate models to predict cognitive abilities from multimodal neuroimaging, polygenic scores (PGS) and environmental factors. Multimodal neuroimaging was based on 45 types of brain MRI (e.g., task fMRI contrasts, resting-state fMRI, structural MRI, and diffusion tensor imaging). Among these MRI types, the fMRI contrast, 2-Back vs. 0-Back, from the ENBack task provided the highest predictive performance (r\=.4). Combining information across all 45 types of brain MRI led to the predictive performance of r\=.54. The PGS, based on previous genome-wide association studies on cognitive abilities, achieved a predictive performance of r\=.25. Environmental factors, including socio-demographics (e.g., parent’s income and education), lifestyles (e.g., extracurricular activities, sleep) and developmental adverse events (e.g., parental use of alcohol/tobacco, pregnancy complications), led to a predictive performance of r\=.49. 

In a series of separate commonality analyses, we found that the relationship between cognitive abilities and mental health was primarily represented by multimodal neuroimaging (66%) and, to a lesser extent, by PGS (21%). Additionally, environmental factors accounted for 63% of the variance in the relationship between cognitive abilities and mental health. The multimodal neuroimaging and PGS then explained 58% and 21% of the variance due to environmental factors, respectively. Notably, these patterns remained stable over two years. 

Our findings underscore the significance of neurobiological units of analysis for cognitive abilities, as measured by multimodal neuroimaging and PGS, in understanding both a) the relationship between cognitive abilities and mental health and b) the variance in this relationship shared with environmental factors.”

(2) Could the authors clarify what they mean by "completing the transdiagnostic aetiology of mental health" in the introduction? (Second paragraph). 

Thank you. 

We intended to convey that understanding the transdiagnostic aetiology of mental health would be enhanced by knowing how neurobiological units of cognitive abilities, from the brain to genes, capture variations due to environmental factors. We realise this sentence might be confusing. Removing it does not alter the intended meaning of the paragraph, as we clarified this point later. The paragraph now reads:

“According to the National Institute of Mental Health’s Research Domain Criteria (RDoC) framework (Insel et al., 2010), cognitive abilities should be investigated not only behaviourally but also neurobiologically, from the brain to genes. It remains unclear to what extent the relationship between cognitive abilities and mental health is represented in part by different neurobiological units of analysis -- such as neural and genetic levels measured by multimodal neuroimaging and polygenic scores (PGS). To fully comprehend the role of neurobiology in the relationship between cognitive abilities and mental health, we must also consider how these neurobiological units capture variations due to environmental factors, such as sociodemographics, lifestyles, and childhood developmental adverse events (Morris et al., 2022). Our study investigated the extent to which a) environmental factors explain the relationship between cognitive abilities and mental health, and b) cognitive abilities at the neural and genetic levels capture these associations due to environmental factors. Specifically, we conducted these investigations in a large normative group of children from the ABCD study (Casey et al., 2018). We chose to examine children because, while their emotional and behavioural problems might not meet full diagnostic criteria (Kessler et al., 2007), issues at a young age often forecast adult psychopathology (Reef et al., 2010; Roza et al., 2003). Moreover, the associations among different emotional and behavioural problems in children reflect transdiagnostic dimensions of psychopathology (Michelini et al., 2019; Pat et al., 2022), making children an appropriate population to study the transdiagnostic aetiology of mental health, especially within a framework that emphasises normative variation from normal to abnormal, such as the RDoC (Morris et al., 2022).“

(3) It is unclear to me what the authors mean by this statement in the introduction: "Note that using the word 'proxy measure' does not necessarily mean that the predictive model for a particular measure has a high predictive performance - some proxy measures have better predictive performance than others". 

We added this sentence to address a previous reviewer’s comment: “The authors use the phrasing throughout 'proxy measures of cognitive abilities' when they discuss PRS, neuroimaging, sociodemographics/lifestyle, and developmental factors. Indeed, the authors are able to explain a large proportion of variance with different combinations of these measures, but I think it may be a leap to call all of these proxy measures of cognition. I would suggest keeping the language more objective and stating these measures are associated with cognition.” 

Because of this comment, we assumed that the reviewers wanted us to avoid the misinterpretation that a proxy measure implies high predictive performance. This term is used in machine learning literature (for instance, Dadi et al., 2021). We added the aforementioned sentence to ensure readers that using the term 'proxy measure' does not necessarily mean that the predictive model for a particular measure has high predictive performance. However, it seems that our intention led to an even more confusing message. Therefore, we decided to delete that sentence but keep an earlier sentence that explains the meaning of a proxy measure (see below).

“With opportunistic stacking, we created a ‘proxy’ measure of cognitive abilities (i.e., predicted value from the model) at the neural unit of analysis using multimodal neuroimaging.”

(4) Overall, despite comments from reviewers at another journal, I think the authors still refer to RDoC more than needed in the intro given the restructuring of the manuscript. For instance, at the end of page 4 and top of page 5, it becomes a bit confusing when the authors mention how they deviated from the RDoC framework, but their choice of cognitive domains is still motivated by RDoC. I think the chosen cognitive constructs are consistent with what is in ABCD and what other studies have incorporated into the g factor and do not require the authors to further justify their choice through RDoC. Also, there is emerging work showing that RDoC is limited in its ability to parse apart meaningful neuroimaging-based patterns; see for instance, Quah et al., Nature 2025 (https://doi.org/10.1038/s41467-025-55831-z). 

Thank you very much for your comment. We have addressed it in our Response to Reviewer 1’s summary, strengths, and weaknesses above. We have rewritten the paragraph to clarify the relevance of our work to the RDoC framework and to recent studies aiming to improve RDoC constructs (including that from Quah and colleagues).

(5) I am still on the fence about the use of 'proxy measures of cognitive abilities' given that it is defined as the predictive performance of mental health measures in predicting cognition - what about just calling these mental health predictors? Also, it would be easier to follow this train of thought throughout the manuscript. But I leave it to the authors if they decide to keep their current language of 'proxy measure of cognition'. 

Thank you so much for your flexibility. As we explained previously, this ‘proxy measures’ term is used in machine learning literature (for instance, Dadi et al., 2021). We thought about other terms, such as “score”, which is used in genetics, i.e., polygenic scores (Choi et al., 2020). and has recently been used in neuroimaging, i.e., neuroscore (Rodrigue et al., 2024). However, using a ‘score’ is a bit awkward for mental health and socio-demographics, lifestyle and developmental adverse events. Accordingly, we decided to keep the term ‘proxy measures’.

(6) It is unclear which cognitive abilities are being predicted in Figure 1, given the various domains that authors describe in their intro. Is it the g-factor from CFA? This should be clarified in all figure captions. 

Yes, cognitive abilities are operationalised using a second-order latent variable, the g-factor from a CFA. We now added the following sentence to Figure 1, 2, 4 to make this point clearer. Thank you for the suggestion:

“Cognitive abilities are based on the second-order latent variable, the g-factor, based on a confirmatory factor analysis of six cognitive tasks.”

(7) I think it may also be worthwhile to showcase the explanatory power cognitive abilities have in predicting mental health or at least comment on this in the discussion. Certainly, there may be a bidirectional relationship here. The prediction direction from cognition to mental health may be an altogether different objective than what the paper currently presents, but many researchers working in psychiatry may take the stance (with support from the literature) that cognitive performance may serve as premorbid markers for later mental health concerns, particularly given the age range that the authors are working with in ABCD. 

Thank you for this comment. 

It is important to note that we do not make a directional claim in these cross-sectional analyses. The term "prediction" is used in a machine learning sense, implying only that we made an out-of-sample prediction (Yarkoni & Westfall, 2017). Specifically, we built predictive models on some samples (i.e., training participants) and applied our models to test participants who were not part of the model-building process. Accordingly, our predictive models cannot determine whether mental health “causes” cognitive abilities or vice versa, regardless of whether we treat mental health or cognitive abilities as feature/explanatory/independent variables or as target/response/outcome variables in the models. To demonstrate directionality, we would need to conduct a longitudinal analysis with many more repeated samples and use appropriate techniques, such as a cross-lagged panel model. It is beyond the scope of this manuscript and will need future releases of the ABCD data.

We decided to use cognitive abilities as a target variable here, rather than a feature variable, mainly for theoretical reasons. This work was inspired by the RDoC framework, which emphasises functional domains. Cognitive abilities is the functional domain in the current study. We created predictive models to predict cognitive abilities based on a) mental health, b) multimodal neuroimaging, c) polygenic scores, and d) environmental factors. We could not treat cognitive abilities as a functional domain if we used them as a feature variable. For instance, if we predicted mental health (instead of cognitive abilities) from multimodal neuroimaging and polygenic scores, we would no longer capture the neurobiological units of analysis for cognitive abilities.

We now made it clearer in the discussion that our use of predictive models cannot provide the directional of the effects

“Our predictive modelling revealed a medium-sized predictive relationship between cognitive abilities and mental health. This finding aligns with recent meta-analyses of case-control studies that link cognitive abilities and mental disorders across various psychiatric conditions (Abramovitch et al., 2021; East-Richard et al., 2020). Unlike previous studies, we estimated the predictive, out-of-sample relationship between cognitive abilities and mental disorders in a large normative sample of children. Although our predictive models, like other cross-sectional models, cannot determine the directionality of the effects, the strength of the relationship between cognitive abilities and mental health estimated here should be more robust than when calculated using the same sample as the model itself, known as in-sample prediction/association (Marek et al., 2022; Yarkoni & Westfall, 2017). Examining the PLS loadings of our predictive models revealed that the relationship was driven by various aspects of mental health, including thought and externalising symptoms, as well as motivation. This suggests that there are multiple pathways—encompassing a broad range of emotional and behavioural problems and temperaments—through which cognitive abilities and mental health are linked.”

(8) There is a lot of information packed into Figure 3 in the brain maps; I understand the authors wanted to fit this onto one page, and perhaps a higher resolution figure would resolve this, but the brain maps are very hard to read and/or compare, particularly the coronal sections. 

Thank you for this suggestion. We agree with Reviewer 1 that we need to have a better visualisation of the feature-importance brain maps. To ensure that readers can clearly see the feature importance, we added a Zoom-in version of the feature-importance brain maps as Supplementary Figures 4 – 13.

(9) It would be helpful for authors to cluster features in the resting state functional connectivity correlation matrices, and perhaps use shorter names/acronyms for the labels. 

Thank you for this suggestion. 

We have now added a zoomed-in version of the feature importance for rs-fmri as Supplementary Figure 7 (for baseline) and 12 (for follow-up).

(10) Figures 4a) and 4b): please elaborate on "developmental adverse" in the title. I am assuming this is referring to childhood adverse events, or "developmental adversities". 

Thank you so much for pointing this out. We meant ‘developmental adverse events’. We have made changes to this figure in the current manuscript.

(11) For the "follow-up" analyses, I would recommend the authors present this using only the features that are indeed available at follow-up, even if the list of features is lower, otherwise it becomes a bit confusing with the mix of baseline and follow-up features. Or perhaps the authors could make this more clear in the figures by perhaps having a different color for baseline vs follow-up features along the y-axis labels. 

Thank you for this advice. We have now added an indicator in the plot to show whether the features were collected in the baseline or follow-up. We also added colours to indicate which type of environmental factors they were. It is now clear that the majority of the features that were collected at baseline, but were used for the followup predictive model, were developmental adverse events.

(12) Minor: Makowski et al 2023 reference can be updated to Makowski et al 2024, published in Cerebral Cortex. 

Thank you for pointing this out. We have updated the citation accordingly. 

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Reply to the reviewers

Note : The original preprint version of our manuscript has been reviewed by 3 subject experts for Review Commons. All the three reviewers’ comments on the original version of our manuscript have been fully addressed. Their input was extremely valuable in helping us clarify and refine the presentation of our results and conclusions. Their feedback contributed to making the study both more thoroughly developed and more accessible to a broad readership, while preserving its mechanistic depth. We believe that this revised version more effectively highlights the conceptual advances brought by our findings.

Reviewer #1

Evidence, reproducibility and clarity

The manuscript "Key roles of the zona pellucida and perivitelline space in promoting gamete fusion and fast block to polyspermy inferred from the choreography of spermatozoa in mice oocytes" by Dr. Gourier and colleagues explores the poorly understood process of gamete fusion and the subsequent block to polyspermy by live-cell imaging of mouse oocytes with intact zona pellucida in vitro. The new component in this study is the presence of the ZP, which in prior studies of live-cell imaging had been removed before. This allowed the authos to examine contributions of the ZP to the block in polyspermy in relation to the timing of sperm penetrating the ZP and sperm fusing with the oocyte. By carefully analysing the timing of the cascade of events, the authors find that the first sperm that reaches the membrane of the mouse oocyte is not necessarily the one that fertilizes the oocytes, revealing that other mechanisms post-ZP-penetration influence the success of individual sperm. While the rate of ZP penetration remains constant in unfertilized oocytes, it decreases upon fertilization for subsequent sperm, providing direct evidence for the known 'slow block to polyspermy' provided by changes to the ZP adhesion/ability to be penetrated. Careful statistical analyses allow the authors to revisit the role of the ZP in preventing polyspermy: They show that the ZP block resulting from the cortical reaction is too slow (in the range of an hour) to contribute to the immediate prevention of polyspermy in mice. The presented analyses reveal that the ZP does contribute to the block to polyspermy in two other ways, namely by effectively limiting the number of sperm that reach the oocyte surface in a fertilization-independent manner, and by retaining components like JUNO and CD9, that are shed from the oocyte plasma membrane after fertilization, in the perivitelline space, which may help neutralize surplus spermatozoa that are already present in the PVS. Lastly, the authors report that the ZP may also contribute to channeling the flagellar oscillations of spermatozoa in the PVS to promote their fusion competence.

Major comments:

• Are the key conclusions convincing?

The authors provide a careful analysis of the dynamics of events, though the analyses are correlative, and can only be suggestive of causation. While this is a limitation of the study, it provides important analysis for future research. Moreover, by analysing also control oocytes without fertilization and the timing of events, the authors have in some instances clear 'negative controls' for comparison.

Some claims would benefit from rewording or rephrasing to put the findings better in the context of what is already known and what is novel:

• the phrasing 'challenging prior dogma' might be too strong since it had been observed before that it is not necessarily the first sperm that gets through the ZP that fertilizes the egg (though I am afraid that I do not have any citations or references for this). However, given that in the field people generally think it is not necessarily and always the first sperm, the authors may want to consider weakening this claim.

Only real-time imaging of in vitro fertilization of zona pellucida-intact oocytes, as performed in our study, is capable of determining which spermatozoon crossing the zona pellucida fuses with the oocyte. However, such studies are rare, and most do not specifically address this question. As Reviewers 1 & 3, we have not found any citation or reference telling or showing that it is not necessarily the first spermatozoon to penetrate the zona pellucida that fertilizes the egg. In contrast, at least one reference (Sato et al., 1979) explicitly reports the opposite. If, as suggested by Reviewer 1 and 3, it has indeed been observed before that the first sperm to pass the ZP is not always the one that fertilizes, and if this idea is generally accepted in the field, then it is all the more important that a study demonstrates and publishes this point. This is precisely what our study makes possible. However, in case we may have overlooked a previous reference making the same observation as ours, we have removed the phrasing ‘challenging prior dogma’. That being said, the key issue is not so much that it is not necessarily the first spermatozoon penetrating the perivitelline space that fertilizes, but rather why spermatozoa that successfully reach the PVS of an unfertilized oocyte may fail to achieve fertilization. This is one of the central questions our study sought to address.

• I do think the cortical granule release could still contribute to the block to polyspermy though - as the authors here nicely show - at a later time-point only, and thus not the major and not the immediate block as previously thought. The wording in the abstract should therefore be adjusted (since it could still contribute...)

We are concerned that we may disagree on this point. The penetration block resulting from cortical granule release progressively reduces the permeability of the zona pellucida to spermatozoa, relative to its baseline permeability prior to sperm–oocyte fusion. Any decrease in this baseline permeability occurring before the fusion block becomes fully effective can contribute to the prevention of polyspermy by limiting the number of sperm that can access the oolemma at a time when fusion is still possible. In contrast, once the fusion block is fully established, limiting the number of spermatozoa traversing the ZP becomes irrelevant regarding the block to polyspermy, as the fusion block alone is sufficient to prevent additional fertilizations, rendering the penetration block obsolete. The only scenario that could challenge this obsolescence is if the fusion block were transient. In that case, as Reviewer 1 suggests, the penetration block could indeed play a role at a later time-point. However, taken together, our study and that of Nozawa et al. (2018) support the conclusion that this is not the case in mice:

• Our in vitro study using kinetic tracking shows that the time constant for completion of the fusion block is typically 6.2 ± 1.3 minutes. During this time window, we observe that the permeability of the zona pellucida to spermatozoa does not yet decrease significantly from the baseline level it exhibited prior to sperm–oocyte fusion (see Figures 5B and S1B in the revised manuscript, and Figures 5A and 5B in the initial version). Consequently, before the fusion block is fully established, the penetration block can contribute only marginally—if at all—to the prevention of polyspermy. In contrast, the naturally low baseline permeability of the ZP—independent of any fertilization-triggered penetration block—as well as the relatively long timing of fusion ( minutes on average) after sperm penetration in the perivitelline space, are factors that contribute to the preservation of monospermic while the fusion block is still being established.
• Our in vitro study using kinetic tracking shows that once the fusion block is completed following the first fusion event, no additional spermatozoa are able to fuse with the oocyte until the end of the experiment, 4 hours post-insemination (see blue points and fitting curve in Figure 5C). Meanwhile, one or more additional spermatozoa—most of them motile and therefore viable—are present in the perivitelline space in 50% of the oocytes analyzed (purple point in Figure 5C). This demonstrates that, once established, the fusion block remains effective for at least the entire duration of the experiment, supporting the idea of a fully functional and long-lasting fusion block.
• Nozawa et al. (2018) found that female mice lacking ovastacin—the protease released during the cortical reaction that renders the zona pellucida impenetrable—are normally fertile. They additionally reported that the oocytes recovered from these females after mating are monospermic despite the systematic presence of additional spermatozoa in the perivitelline space. These findings further support the conclusion that in mice the fusion block is both permanent and sufficient to prevent polyspermy. For all these reasons, we believe that even at a later time-point, the penetration block does not contribute to the prevention of polyspermy in mice.

To clarify the fact that the penetration block does not necessarily contribute to prevent polyspermy, which indeed challenges the commonly accepted view, we have substantially revised the discussion. Furthermore, Figure 9 from the initial version of the manuscript has been replaced by Figure 8 in the revised version. This new figure provides a more didactic illustration of the inefficacy of the penetration block in preventing polyspermy in mice, by showing the respective impact of the fusion block, the penetration block, as well as fusion timing and the natural baseline permeability of the zona pellucida, on the occurrence of polyspermy.

As for the abstract, it has also been thoroughly revised. The content related to this section is now expressed in a way that emphasizes the factors that actively contribute to the prevention of polyspermy in mice, rather than those with no or marginal contribution (such as the penetration block in this case).

• release of OPM components - in the abstract it's unclear what the authors mean by this - in the results part it becomes clear. Please already make it clear in the abstract that it is the fertility factors JUNO/CD9 that could bind to sperm heads upon their release and thus 'neutralize' them? I would also recommend not referring to it as 'outer' plasma membrane (there is no 'inner plasma membrane'). Moreover, in the abstract please clarify that this release is happening only after fusion of the first sperm and not all the time. In the abstract it sounds as if this was a completely new idea, but there is good prior evidence that this is in fact happening (as also then cited in the results part) - maybe frame it more as the retention inside the PVS as new finding.

We thank reviewer 1 for pointing out the lack of precision in the abstract regarding the “components” released from the oolemma, and the fact that our phrasing may have given the impression that the post-fertilization release of CD9 and JUNO is a novel observation. The new observation is that CD9 and JUNO, which are known to be massively released from the oolemma after fertilization, bind to spermatozoa in the perivitelline space. However, we cannot rule out the possibility that other oocyte-derived molecules not investigated here may undergo a similar process. This is why we employed the broader term “components”, which encompasses both CD9 and JUNO as well as potential additional molecules. That said, we acknowledge the lack of precision introduced by this terminology. To address this, we have revised the corresponding sentence in the abstract to better reflect our new findings relative to previous ones, and to eliminate the ambiguity introduced by the word “component”.

The revised sentence of the abstract reads as follows:

“Our observation that non-fertilizing spermatozoa in the perivitelline space are coated with CD9 and JUNO oocyte’s proteins, which are known to be massively released from the oolemma after gamete fusion, supports the hypothesis that the fusion block involves an effective perivitelline space-block contribution consisting in the neutralization of supernumerary spermatozoa in the perivitelline space by these and potentially other oocyte-derived factors.”

Moreover, we cannot state in the abstract that the release of CD9 and JUNO occurs only after the fusion of the first spermatozoon and not before, since some CD9 and JUNO are already detectable in the perivitelline space (PVS) prior to fusion. What our study shows is that, before fertilization, CD9 and JUNO are predominantly localized at the oocyte membrane. In contrast, after fusion (four hours post-insemination), oocyte CD9 is distributed between the membrane and the PVS, and the only JUNO signal detectable in the oocyte is found in the PVS. This is what we describe in the Results section on page 15.

Regarding the acronym “OPM” in the initial version of the manuscript, although it was defined in the introduction as referring to the oocyte plasma membrane and not the outer plasma membrane (which, indeed, would not be meaningful), we acknowledge that it may have caused confusion to people in the field due to its resemblance to the commonly used meaningful acronym “OAM” for outer acrosomal membrane. To avoid any ambiguity, we have replaced the acronym “OPM” throughout the revised manuscript with the term “oolemma”, which unambiguously refers to the plasma membrane of the oocyte.

It is unclear to me what the relevance of dividing the post-fusion/post-engulfment into different phases as done in Fig 2 (phase 1, and phase 2) - also for the conclusions of this paper this seems rather irrelevant and overly complicated, since the authors never get back to it and don't need it (it's not related to the polyspermy block analyses). I would remove it from the main figures and not divide into those phases since it is distracting from the main focus.

Sperm engulfment and PB2 extrusion are two processes that follow sperm–oocyte fusion. As such, they are clear indicators that fusion has occurred and that meiosis has resumed. Their progression over time is readily identifiable in bright-field imaging: sperm engulfment is characterized by the gradual disappearance of the spermatozoon head from the oolemma, whereas PB2 extrusion is observed as the progressive emergence of a rounded protrusion from the oocyte membrane (Figure 2 in the initial manuscript and Figure S2 A&B in the revised version). The kinetics of these events, measured from the arrest of “push-up–like” movement of the sperm head against the oolemma —assumed to coincide with sperm-oocyte fusion, as further justified in a later response to Reviewer 1—provide reliable temporal landmarks for estimating the timing of fusion when the fusion event itself is not directly observed in real time (Figure S2 C&D).

The four landmarks used in this estimation are:

(i) the disappearance of the sperm head from the oolemma due to internalization (28 ± 2 minutes post-arrest, mean ± SD);

(ii) the onset of PB2 protrusion from the oolemma (28 ± 2 minutes post-arrest);

(iii) the moment when the contact angle between the PB2 protrusion and the oolemma shifts from greater than to less than 90° (49 ± 6 minutes post-arrest);

(iv) the completion of PB2 extrusion (73 ± 10 minutes post-arrest).

The approach used to determine the fusion time window of a fertilizing spermatozoon from these landmarks is detailed in the “Determination of the Fertilization Time Windows” section of the Materials and Methods. Compared to the initial version of the manuscript, we have added a paragraph explaining the rationale for using the arrest of the push-up–like movement as a reliable indicator for sperm–oocyte fusion and have clarified the description of the approach used to determine fertilization timing.

The timed characterization of sperm engulfment and PB2 extrusion kinetics is highly relevant to the analysis of the penetration and fusion blocks, however we agree that its place is more appropriate in the Supplementary Information than in the main text. In accordance with the reviewer’s recommendation, this section has therefore been moved to the Supplementary Information SI2.

For the statistical analysis, I am not sure whether the assumption "assumption that the probability distribution of penetration or fertilization is uniform within a given time window" is in fact true since the probability of fertilizing decreases after the first fertilization event.... Maybe I misunderstood this, but this needs to be explained (or clarified) better, or the limitation of this assumption needs to be highlighted.

During in vitro fertilization experiments with kinetic tracking, each oocyte is observed sequentially in turn. As a result, sperm penetration into the perivitelline space or fusion with the oolemma may occur either during an observation round or in the interval between two rounds. In the former case, penetration or fusion is directly observed in real time, allowing for high temporal precision in determining the moment of the event. In contrast, when penetration or fusion occurs between two observation rounds, the precise timing cannot be directly determined. We can only ascertain that the event took place within the time window we have determined. Because, within a given penetration or fusion time window, we do not know the exact moment at which the event occurred, there is no reason to favor one time over another. This justifies the assumption that all time points within the window are equally probable. This explanation has been added in the section Statistical treatment of penetration and fertilization chronograms to study the kinetics of fertilization, penetration block and fusion block of the main text and in the section Statistical treatment of penetrations and fertilizations chronograms to study penetration and fusion blocks of the material and methods.

-Suggestion for additional experiments:

If I understood correctly, the onset of fusion in Fig 2C is defined by stopping of sperm beating? If it is by the sudden stop of the beating flagellum, this should be confirmed in this situation (with the ZP intact) that it correctly defines the time-point of fusion since this has not been measured in this set-up before as far as I understand. In order to measure this accurately, the authors will need to measure this accurate to be able to acquire those numbers (of time from fusion to end of engulfment), e.g. by pre-loading the oocyte with Hoechst to transfer Hoechst to the fusing sperm upon membrane fusion.

The nuclear dye Hoechst is widely used as a marker of gamete fusion, as it transfers from the ooplasm—when preloaded with the dye—into the sperm nucleus upon membrane fusion, thereby signaling the happening of the fusion event. This technique is applicable in the context of in vitro fertilization using ZP-free oocytes. However, it is not suitable when cumulus–oocyte complexes are inseminated, as is the case in both in vitro experimental conditions of the present study (standard IVF and IVF with kinetic tracking). Indeed, when cumulus–oocyte complexes are incubated with Hoechst to preload the oocytes, the numerous surrounding cumulus cells also take up the dye. Consequently, upon insemination, spermatozoa acquire fluorescence while traversing and dispersing the cumulus mass—before reaching the ZP—thus rendering Hoechst labeling ineffective as a specific marker of membrane fusion. This remains true even under optimized conditions involving brief Hoechst incubation of cumulus–oocyte complexes ( Nonetheless, we have strong evidence supporting the use of the arrest of sperm movement as a surrogate marker for the moment of fusion. In our previous study (Ravaux et al., 2016; ref. 4 in the revised manuscript), we investigated the temporal relationship between the abrupt cessation of sperm head movement on the oolemma—resulting from strong flagellar beating arrest—and the fusion event, using ZP-free oocytes preloaded with Hoechst. That study revealed a temporal delay of less than one minute between the cessation of sperm oscillations and the actual membrane fusion, thereby supporting the conclusion that in ZP-free oocytes, the arrest of vigorous sperm movement at the oolemma is a reliable indicator of the moment at which fusion occurs. In the same study, the kinetics of sperm head internalization into the ooplasm were also characterized, typically concluding within 20–30 minutes after movement cessation. These findings are fully consistent with our current observations in ZP-intact oocytes, where sperm head engulfment was completed approximately 24 ± 3 minutes after the arrest of sperm oscillations. Taken together, these results strongly support the conclusion that, in both ZP-free and ZP-intact oocytes, the arrest of sperm movement is a reliable indicator of the fusion event. This assumption formed the basis for our determination of fertilization time points in the present study.

These justifications were not fully detailed in the original version of the manuscript. We have addressed this in the revised version by explicitly presenting this rationale in the Materials and Methods section under Determination of the Fertilization Time Windows.

Fig 8: 2 comments

• To better show JUNO/CD9 pre-fusion attachment to the oocyte surface and post-fusion loss from the oocyte surface (but persistence in the PVS), an image after removal of the ZP (both for pre-fertilization and post-fertilization) would be helpful - the combination of those images with the ones you have (ZP intact) would make your point more visible.

We have followed this recommendation. Figure 8 of the initial manuscript has been replaced by Figure 6 in the revised manuscript, which illustrates the four situations encountered in this study: fertilized and unfertilized oocytes, each with and without unfused spermatozoa in their PVS. To better show JUNO/CD9 pre-fusion presence to the oocyte plasma membrane, as well as their post-fusion partial (for CD9) and near-complete (for JUNO) loss from the oocyte membrane (but persistence in the PVS), paired images of the same oocyte before and after of ZP removal are now provided, both for unfertilized (Figure 6A) and fertilized oocytes (Figure 6C).

• You show that the heads of spermatozoa post fusion are covered in CD9 and JUNO, yet I was missing an image of sperm in the PVS pre-fertilization (which should then not yet be covered).

As staining and confocal imaging of the oocytes were performed 4 hours after insemination, images of sperm in the PVS of an oocyte “pre-fertilization” cannot be strictly obtained. However, we can have images of spermatozoa present in the PVS of oocytes that remained unfertilized. This situation, now illustrated in Figure 6B of the revised manuscript, shows that these spermatozoa are also covered in JUNO and CD9, which they may have progressively acquired over time from the baseline presence of these proteins in the PVS of unfertilized oocytes. This also may provide a mechanistic explanation for their inability to fuse with the oolemma, and, consequently, for the failure of fertilization in these oocytes.

Minor comments:

• The videos were remarkable to look at, and great to view in full. However, for the sake of time, the authors might want to consider cropping them for the individual phases to have a shorter video (with clear crop indicators) with the most important different stages visible in a for example 1 min video (e.g. video.

We have followed this recommendation. The videos have been cropped and annotated in order to highlight the key events that support the points made in the result section from page 9 to 11 in the revised manuscript.

• In general, given that the ZP, PVS and oocyte membrane are important components, a general scheme at the very beginning outlining the relative positioning of each before and during fertilization (and then possibly also including the second polar body release) would be extremely helpful for the reader to orient themselves.

A general scheme addressing Reviewer 1 request, summarizing the key components and concepts discussed in the article and intended to help guide the reader, has been added to the introduction of the revised manuscript as Figure 1.

• first header results "Multi-penetration and polyspermy under in vivo conditions and standard and kinetics in vitro fertilization conditions" is hard to understand - simplify/make clearer (comparison of in vivo and in vitro conditions? Establishing the in vitro condition as assay?)

The title of the first Results section has been revised in accordance with Reviewer 1 suggestion. It now reads: Comparative study of penetration and fertilization rates under in vivo and two distinct in vitro fertilization conditions.

• Large parts of the statistical analysis (the more technical parts) could be moved to the methods part since it disrupts the flow of the text.

In the revised version of our manuscript, we have restructured this part of the analysis to ensure that more technical or secondary elements do not disrupt the flow of the main text. Accordingly, the equations have been reduced to only what is strictly necessary to understand our approach, their notation has been greatly simplified, and the statistical analysis of unfertilized oocytes whose zona pellucida was traversed by one or more spermatozoa has been moved to the Supplementary Information (SI1).

• To me, one of the main conclusions was given in the text of the results part, namely that "This suggests that first fertilization contributes effectively to the fertilization-block, but less so to the penetration block". I would suggest that the authors use this conclusion to strengthen their rationale and storyline in the abstract.

We agree with Reviewer 1 suggestion. Accordingly, we have not only thoroughly revised our abstract, but also the introduction and discussion, in order to better highlight the rationale of our study, its storyline, and the new findings which not only challenge certain established views but also open new research directions in the mechanisms of gamete fusion and polyspermy prevention.

• Wording: To characterize the kinetics with which penetration of spermatozoa in the PVS falls down after a first fertilization," falls down should be replaced with decreases (page 10 and page 12)

Falls down has been removed from the new version and replaced with decreases

Significance

Overall, this manuscript provides very interesting and carefully obtained data which provides important new insights particularly for reproductive biology. I applaud the authors on first establishing the in vivo conditions (how often do multiple sperm even penetrate the ZP in vivo) since studies have usually just started with in vitro condition where sperm at much higher concentration is added to isolated oocyte complexes. Thank you for providing an in vivo benchmark for the frequency of multiple sperm being in the PVS. While this frequency is rather low (somewhat expectedly, with 16% showing 2-3 sperm in the PVS), this condition clearly exists, providing a clear rationale for the investigation of mechanisms that can prevent additional sperm from entering.

My own expertise is experimentally - thus I don't have sufficient expertise to evaluate the statistical methods employed here.

__ __

Reviewer #2

Evidence, reproducibility and clarity

Overall, this is a very interesting and relevant work for the field of fertilization. In general, the experimental strategies are adequate and well carried out. I have some questions and suggestions that should be considered before the work is published.

1) Why are the cumulus cells not mentioned when the AR is triggered before or while the sperms cross it? It seems the paper assumes from previous work that all sperm that reach ZP and the OPM have carried out the acrosome reaction. This, though probably correct, is still a matter of controversy and should be discussed. It is in a way strange that the authors do not make some controls using sperm from mice expressing GFP in the acrosome, as they have used in their previous work.

We do not mention the cumulus cells or whether the acrosome reaction is triggered before, during, or after their traversal (i.e., upon sperm binding to the ZP), as this question, while scientifically relevant, pertains to a distinct line of investigation that lies beyond the scope of the present study. Even with the use of spermatozoa expressing GFP in the acrosome, addressing this question would require a complete redesign of our kinetic tracking protocol, which was specifically conceived to monitor in bright field the dynamic behavior of spermatozoa from the moment they begin to penetrate the perivitelline space of an oocyte. Accordingly, we imaged oocytes that were isolated 15 minutes after insemination of the cumulus–oocyte complexes, by which time most (if not all) cumulus cells had detached from the oocytes, as explained in the fourth paragraph of the material and methods of both the initial and revised versions of the manuscript. The spermatozoa we had access to were therefore already bound to the zona pellucida at the time of removal from the insemination medium, and had thus necessarily passed through the cumulus layer. It is unclear for us why Reviewer 2 believes that we “assume from previous work that all sperm that reach ZP has carried out the acrosome reaction”. We could not find any statement in our manuscript suggesting, let alone asserting, such an assumption, which we know to be incorrect. Based on both published work from Hirohashi’s group in 2011 (Jin et al., 2011, DOI: 10.1073/pnas.1018202108) and our own unpublished observation (both involving cumulus-oocyte masses inseminated with spermatozoa expressing GFP in the acrosome), it is established that only a subset of spermatozoa reaching the ZP after crossing the cumulus layer has undergone acrosome reaction. Moreover, from the same sources—as well as from a recent publication by Buffone’s group (Jabloñsky et al., 2023 DOI: 10.7554/eLife.93792 ) which is the one to which reviewer 2 refers in her/his 3rd comment, it is also well established that spermatozoa have all undergone acrosome reaction when they enter the PVS. To the best of our knowledge, this latter point has long been widely accepted and is not questioned. Therefore, stating this in the first paragraph of the Discussion in the revised manuscript, while referencing the two aforementioned published studies, should be appropriate. What remains a matter of ongoing debate, however, is the timing and the physiological trigger(s) of the acrosome reaction in fertilizing spermatozoa. The 2011 study by Hirohashi’s group challenged the previously accepted view that ZP binding induces the acrosome reaction, showing instead that most spermatozoa capable of crossing the ZP and fertilizing the oocyte had already undergone the acrosome reaction prior to ZP binding. However, as this issue lies beyond the scope of our study, we do not consider it appropriate to include a discussion of it in the manuscript.

2) In the penetration block equations, it is not clear to me why (𝑡𝑃𝐹1) refers to both PIPF1 and 𝜎𝜎𝑃I𝑃𝐹1. Is it as function off?

That is correct: (tPF1) means function of the time post-first fertilization. Both the post-first fertilization penetration index (i.e. PIPF1) and its incertainty (i.e. 𝜎𝑃I𝑃𝐹1 ) vary as a function of this time. However, as mentioned in a previous response to Reviewer 1, this section has been rewritten to improve clarity and readability. The equations have been limited to those strictly necessary for understanding our approach, and their notation has been significantly simplified.

3) Why do the authors think that the flagella stops. The submission date was 2024-10-01 07:27:26 and there has been a paper in biorxiv for a while that merits mention and discussion in this work (bioRxiv [Preprint]. 2024 Jul 2:2023.06.22.546073. doi: 10.1101/2023.06.22.546073.PMID: 37904966).

Our experimental approach allows us to determine when the spermatozoon stops moving, but not why it stops. We thank Reviewer 3 for pointing out this very relevant paper from Buffone’s group (doi: 10.7554/eLife.93792) which shows the existence of two distinct populations of live, acrosome-reacted spermatozoa. These correspond to two successive stages, which occur either immediately upon acrosome reaction in a subset of spermatozoa, or after a variable delay in others, during which the sperm transitions from a motile to an immotile state. The transition from the first to the second stage was shown to follow a defined sequence: an increase in the sperm calcium concentration, followed by midpiece contraction associated with a local reorganization of the helical actin cortex, and ultimately the arrest of sperm motility. For fertilizing spermatozoa in the PVS, this transition was shown to occur upon fusion. However, it was also reported in some non-fertilizing spermatozoa that this transition took place within the PVS. These findings are consistent with the requirement for sperm motility in order to achieve fusion with the oolemma. Moreover, the fact that some spermatozoa may prematurely transition to the immotile state within the PVS can therefore be added to the list of possible reasons why a spermatozoon that penetrates the PVS of an oocyte might fail to fuse.

This discussion has been added to the first paragraph of the Discussion section of our revised manuscript.

4) Please correct at the beginning of Materials and Methos: Sperm was obtained from WT male mice, it should say were.

Thank you, the correction has been done.

5) This is also the case in the fourth paragraph of this section: oocyte were not was.

The sentence in question has been modified as followed: “In the in vitro fertilization experiments with kinetic tracking, a subset of oocytes—together with their associated ZP-bound spermatozoa—was isolated 15 minutes post-insemination and transferred individually into microdrops of fertilization medium to enable identification.”

Significance

Understanding mammalian gamete fusion and polyspermy inhibition has not been fully achieved. The authors examined real time brightfield and confocal images of inseminated ZP-intact mouse oocytes and used statistical analyses to accurately determine the dynamics of the events that lead to fusion and involve polyspermy prevention under conditions as physiological as possible. Their kinetic observations in mice gamete interactions challenge present paradigms, as they document that the first sperm is not necessarily the one that fertilizes, suggesting the existence of other post-penetration fertilization factors. The authors find that the zona pellucida (ZP) block triggered by the cortical reaction is too slow to prevent polyspermy in this species. In contrast, their findings indicate that ZP directly contributes to the polyspermy block operating as a naturally effective entry barrier inhibiting the exit from the perivitelline space (PVS) of components released from the oocyte plasma membrane (OPM), neutralizing unwanted sperm fusion, aside from any block caused by fertilization. Furthermore, the authors unveil a new important ZP role regulating flagellar beat in fertilization by promoting sperm fusion in the PVS.

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

SUMMARY: This study by Dubois et al. utilizes live-cell imaging studies of mouse oocytes undergoing fertilization. A strength of this study is their use of three different conditions for analyses of events of fertilization: (1) eggs undergoing fertilization retrieved from females at 15 hr after mating (n = 211 oocytes); (2) cumulus-oocyte complexes inseminated in vitro (n = 220 oocytes), and (3) zona pellucida (ZP)-intact eggs inseminated in vitro, transferred from insemination culture once sperm were observed bound to the ZP for subsequent live-cell imaging (93 oocytes). This dataset and these analyses are valuable for the field of fertilization biology. Limitations of this manuscript are challenges arise with some conclusions, and the presentation of the manuscript. There are some factual errors, and also some places where clearer explanations should to be provided, in the text and potentially augmented with illustrations to provide more clarity on the models that the authors interpret from their data.

MAJOR COMMENTS:

The authors are congratulated on their impressive collection of data from live-cell imaging. However, the writing in several sections is challenging to understand or seems to be of questionable accuracy. The lack of accuracy is suspected to be more an effect of overly ambitious attempts with writing style, rather than to mislead readers. Nevertheless, these aspects of the writing should be corrected. There also are multiple places where the manuscript contradicts itself. These contradictions should be corrected. Finally, there are factual points from previous studies that need correction.

Second, certain claims and the conclusions as presented are not always clearly supported by the data. This may be connected to the issues with writing style, word and phrasing choices, etc. The conclusions could be expressed more clearly, and thus may not require additional experiments or analyses to support them. The authors might also consider illustrations as ways to highlight the points they wish to make. (Figure 7 is a strong example of how they use illustrations to complement the text).

In response to Reviewer 3's concern about the writing style, which made several sections difficult to understand, we have thoroughly revised the entire manuscript to improve clarity, and precision. To further enhance comprehension, we have added illustrations in the revised version of the manuscript:

• Figure 1A presents the gamete components; Figure 1B depicts the main steps of fertilization considered in the present study; and Figure 1C illustrates the penetration and fusion blocks, along with the respective contributing mechanisms: the ZP-block for the penetration block, and the membrane-block and PVS-block for the fusion block

• Figure 2A provides a description of the three experimental protocols used in this study: Condition 1, in vivo fertilization after mating; Condition 2, standard in vitro fertilization following insemination of cumulus-oocyte complexes; and Condition 3, in vitro fertilization with kinetic tracking of oocytes isolated from the insemination medium 15 min after insemination of the cumulus-oocyte complexes.

• Figure 4 (formerly Figure 7 in the initial version) now highlights all fusing and non-fusing situations documented in videos 1-6 and associated paragraphs of the Results section.

• In the Discussion, Figure 9 from the original version has been replaced by Figure 8, which now provides a more pedagogical illustration of the inefficacy of the penetration block in preventing polyspermy in mice. This figure illustrates the respective contributions of the fusion block, the penetration block, fusion timing, and the intrinsic permeability of the zona pellucida to the occurrence of polyspermy.

We hope that this revised version of the article will guide the reader smoothly throughout, without causing confusion.

Regarding the various points that Reviewer 3 perceives as contradictions or factual errors, or the claims and the conclusions which, as presented, should not always supported by the data, we will provide our perspective on each of them as they are raised in the review.

SPECIFIC COMMENTS:

(1) The authors should use greater care in describing the blocks to polyspermy, particularly because they appear to be wishing to reframe views about prevention of polyspermic fertilization. The title mentions of "the fast block to polyspermy;" this problematic for a couple of different reasons. There is no strong evidence for block to polyspermy in mammals that occurs quickly, particularly not in the same time scale as the first-characterized fast block to polyspermy. To many biologists, the term "fast block to polyspermy" refers to the block that has been described in species like sea urchins and frogs, meaning a rapid depolarization of the egg plasma membrane. However, such depolarization events of the egg membrane have not been detected in multiple mammalian species. Moreover, the change in the egg membrane after fertilization does not occur in as fast a time scale as the membrane block in sea urchins and frogs (i.e., is not "fast" per se), and instead occurs in a comparable time frame as the conversation of the ZP associated with the cleavage of ZP2. Thus, it is misleading to use the terms "fast block" and "slow block" when talking about mammalian fertilization. This also is an instance of where the authors contradict themselves in the manuscript, stating, "the membrane block and the ZP block are established in approximatively the same time frame" (third paragraph of Introduction). This statement is indeed accurate, unlike the reference to a fast block to polyspermy in mammals.

We fully agree with Reviewer 3 on the importance of clearly defining the two blocks examined in the present study—the penetration block and the fusion block (as referred to in the revised version) —and of situating them in relation to the three blocks described in the literature: the ZP-block, membrane-block, and PVS-block. We acknowledge that this distinction was not sufficiently clear in the original version of the manuscript. In the revised version, these two blocks and their relationship to the ZP-, membrane-, and PVS-blocks are now clearly introduced in the second paragraph of the Introduction section and illustrated in the first figure of the manuscript (Fig. 1C). They are then discussed in detail in two dedicated paragraphs of the Discussion, entitled Relation between the penetration block and the ZP-block and Relation between the fusion block and the membrane- and PVS-blocks.

The penetration block refers to the time-dependent decrease in the number of spermatozoa penetrating the perivitelline space (PVS) following fertilization, whereas the fusion block refers to the time-dependent decrease in sperm-oolemma fusion events after fertilization. It is precisely to the characterization of these two blocks that our in vitro fertilization experiments with kinetic tracking allow us to access.

In this study, as in the literature, fusion-triggered modifications of the ZP that hinder sperm traversal of the ZP are referred to as the ZP-block (also known as ZP hardening). The ZP-block thus contributes to the post-fertilization reduction in sperm penetration into the PVS and thereby underlies the penetration block. Similarly, fusion-triggered alterations of the PVS and the oolemma that reduce the likelihood of spermatozoa that have reached the PVS successfully to fuse with the oolemma are referred to as the PVS-block and membrane-block, respectively. These two blocks act together to reduce the probability of sperm-oolemma fusion after fertilization, and thus contribute to the fusion block.

The time constant of the penetration block was found to be 48.3 ± 9.7 minutes, which is consistent with the typical timeframe of ZP-block completion—approximately one hour post-fertilization in mice—as reported in the literature. By contrast, the time constant of the fusion block was determined to be 6.2 ± 1.3 minutes, which is markedly faster than the time typically reported in the literature for the completion of the fusion-block (more than one hour in mice). This strongly suggests that the kinetics of the fusion block are not primarily governed by its membrane-block component, but rather by its PVS-block component—about which little to nothing was previously known.

Contrary to what Reviewer 3 appears to have understood from our initial formulation, there is therefore no contradiction or error in stating that "the membrane block and the ZP block are established within approximately the same timeframe", while the fusion block, which proceeds much more rapidly, is likely to rely predominantly on the PVS-block. We have thoroughly revised the manuscript to clarify this key message of the study.

However, we understand Reviewer 3’s objection to referring to the fusion block (or the PVS-block) as a fast block, given that this term is conventionally reserved for the immediate fertilization-triggered membrane depolarization occurring in sea urchins and frogs. Although the kinetics we report for the fusion block are considerably faster than those of the penetration block, they occur on the scale of minutes, and not seconds. In line with the reviewer's recommendation, we have therefore modified both the title and the relevant passages in the text to remove all references to the term fast block in the revised version.

(2) The authors aim to make the case that events occurring in the perivitelline space (PVS) prevent polyspermic fertilization, but the data that they present is not strong enough to make this conclusion. Additional experiments would optional for this study, but data from such additional experiments are needed to support the authors' claims regarding these functions in fertilization. Without additional data, the authors need to be much more conservative in interpretations of their data. The authors have indeed observed phenomena (the presence of CD9 and JUNO in the PVS) that could be consistent with a molecular basis of a means to prevent fertilization by a second sperm. However, the authors would need additional data from additional experimental studies, such as interfering with the release of CD9 and JUNO and showing that this experimental manipulation leads to increased polyspermy, or creating an experimental situation that mimics the presence of CD9 and JUNO (in essence, what the authors call "sperm inhibiting medium" on page 20) and showing that this prevents fertilization.

A major section of the Results section here (starting with "The consequence is that ... ") is speculation. Rather than be in the Results section, this should be in the Discussion. The language should be also softened regarding the roles of these proteins in the perivitelline space in other portions of the manuscript, such as the abstract and the introduction.

Finally, the authors should do more to discuss their results with the results of Miyado et al. (2008), which interestingly, posited that CD9 is released from the oocytes and that this facilitates fertilization by rendering sperm more fusion-competent. There admittedly are two reports that present data that suggest lack of detection of CD9-containing exosomes from eggs (as proposed by Miyado et al.), but nevertheless, the authors should put their results in context with previous findings.

We generally agree with all the remarks and suggestions made here. In the revised version of the manuscript, we have retained in the Results section (pp. 14–15) only the factual data concerning the localization of CD9 and JUNO in unfertilized and fertilized oocytes, as well as in the spermatozoa present in the PVS of these oocytes. We have taken care not to include any interpretive elements in this section, which are now presented exclusively in a dedicated paragraph of the Discussion, entitled “Possible molecular bases of the membrane-block and ZP-block contributing to the fusion block” (p. 21). There, we develop our hypothesis and discuss it in light of both the findings from the present study and previous work by other groups. In doing so, we also address the data reported by Miyado et al. (2008, https://doi.org/10.1073/pnas.0710608105), as well as subsequent studies by two other groups—Gupta et al. (2009, https://doi.org/10.1002/mrd.21040) and Barraud-Lange et al. (2012, https://doi.org/10.1530/REP-12-0040)—that have challenged Miyado’s findings.

We are fully aware that our interpretation of the coverage of unfused sperm heads in the perivitelline space (PVS) by CD9 and JUNO, released from the oolemma—as a potential mechanism of sperm neutralization contributing to the PVS block—remains, at this stage, a plausible hypothesis or working model that, as such, warrants further experimental investigation. It is precisely in this spirit that we present it—first in the abstract (p.1), then in the Discussion section (p. 21), and subsequently in the perspective part of the Conclusion section (p. 22).

(3) Many of the authors' conclusions focus on their prior analyses of sperm interaction - beautifully illustrated in Figure 7. However, the authors need to be cautious in their interpretations of these data and generalizing them to mammalian fertilization as a whole, because mouse and other rodent sperm have sperm head morphology that is quite different from most other mammalian species.

In a similar vein, the authors should be cautious in their interpretations regarding the extension of these results to mammalian species other than mouse, given data on numbers of perivitelline sperm (ranging from 100s in some species to virtually none in other species), suggesting that different species rely on different egg-based blocks to polyspermy to varying extents. While these observations of embryos from natural matings are subject to numerous nuances, they nevertheless suggest that conclusions from mouse might not be able to be extended to all mammalian species.

It is not clear to us whether Reviewer 3’s comment implies that we have, at some point in the manuscript, generalized conclusions obtained in mice to other mammalian species—which we have not—or whether it is simply a general, common-sense remark with which we fully agree: that findings established in one species cannot, by default, be assumed to apply to another.

We would like to emphasize that throughout the manuscript, we have taken care to restrict our interpretations and conclusions to the mouse model, and we have avoided any unwarranted extrapolation to other species.

To definitively close this matter—if there is indeed a matter—we have added the following clarifying statements in the revised version of the manuscript:

In the introduction, second paragraph (pp. 2–3):"The variability across mammalian species in both the rate of fertilized oocytes with additional spermatozoa in their PVS (from 0 to more than 80%) after natural mating and the number of spermatozoa present in the PVS of these oocytes (from 0 to more than a hundred) suggests that the time for completion of the penetration block and thus its efficiency to prevent polyspermy can vary significantly between species."

At the end of the preamble to the Results section (p. 4):"This experimental study was conducted in mice, which are the most widely used model for studying fertilization and polyspermy blocks in mammals. While there are many interspecies similarities, the findings presented here should not be directly extrapolated to humans or other mammalian species without species-specific validation."

In the Conclusion, the first sentence is (p.22) : “This study sheds new light on the complex mechanisms that enable fertilization and ensure monospermy in mouse model.”

Within the Conclusion section, among the perspectives of this work (p. 22):"In parallel, comparative studies in other mammalian species will be needed to assess the generality of the PVS-block and its contribution relative to the membrane-block and ZP-blocks, as well as the generality of the mechanical role played by flagellar beating and ZP mechanical constraint in membrane fusion."

(4) Results, page 4 - It is very valuable that the authors clearly define what they mean by a penetrating spermatozoon and a fertilizing spermatozoon. However, they sometimes appear not to adhere to these definitions in other parts of the manuscript. An example of this is on page 10; the description of penetration of spermatozoon seems to be referring to membrane fusion with the oocyte plasma membrane, which the authors have alternatively called "fertilizing" or fertilization - although this is not entirely clear. The authors should go through all parts of the manuscript very carefully and ensure consistent use of their intended terminology.

Overall, while these definitions on page 4 are valuable, it is still recommended that the authors explicitly state when they are addressing penetration of the ZP and fertilization via fusion of the sperm with the oocyte plasma membrane. This help significantly in comprehension by readers. An example is the section header in the middle of page 9 - this could be "Spermatozoa can penetrate the ZP after the fertilization, but have very low chances to fertilize."

We chose to define our use of the term penetration at the beginning of the Results section because, as readers of fertilization studies, we have encountered on multiple occasions ambiguity as to whether this term was referring to sperm entry into the perivitelline space following zona pellucida traversal, or to the fusion of the sperm with the oolemma. To avoid such ambiguity, we were particularly careful throughout the writing of our original manuscript to use the term penetration exclusively to describe sperm entry into the PVS. The terms fertilizing and fusion were reserved specifically for membrane fusion between the gametes. However, as occasional lapses are always possible, we followed Reviewer 3’s recommendation and carefully re-examined the entire manuscript to ensure consistent use of our intended terminology. We did not identify any inconsistencies, including on page 10, which was cited as an example by Reviewer 3. We therefore confirm that, in accordance with our predefined terminology, all uses of the term penetration, on that page and anywhere else in our original manuscript, refer exclusively to sperm entry into the PVS and do not pertain to fusion with the oolemma.

That said, it is important that all readers— including those who may only consult selected parts of the article—are able to understand it clearly. Therefore, despite the potential risk of slightly overloading the text, Reviewer 3’s suggestion to systematically associate the term penetration with ZP seems to us a sound one. However, we have opted instead to associate penetration with PVS, as our study focuses on the timing of sperm penetration into the perivitelline space, rather than on the traversal of the zona pellucida itself. Accordingly, except in a few rare instances where ambiguity seemed impossible, we have systematically used the phrasing “penetration into the PVS” throughout the revised version of the manuscript.

Another variation of this is in the middle of page 9, where the authors use the terms "fertilization block" and "penetration block." These are not conventional terms, and venture into being jargon, which could leave some readers confused. The authors could clearly define what they mean, particularly with respect to "penetration block,"

This point has already been addressed in our response to Comment 1 from Reviewer 3. We invite Reviewer 3 to refer to that response.

This extends to other portions of the manuscript as well, such as Figure 2C, with the label on the y-axis being "Time after fertilization." It seems that what the authors actually observed here was the cessation of sperm tail motility. (It is not evident they they did an assessment of sperm-oocyte fusion here.)

Regarding Figure 2C (original version), it has been merged with Figure 2B (original version) to form a single figure (Figure S2D), now included in Supplementary Information SI2. This new figure retains all the information originally presented in Figure 2C and indicates the time axis origin as the time when oscillatory movements of the sperm cease.

That said, for the reasons detailed in our response to Reviewer 1 and in the Materials and Methods, we explain why it is legitimate to use the cessation of sperm head oscillations on the oolemma as a marker for the timing of the fusion event. We invite the reviewers to refer to that response for a full explanation of our rationale.

(5) Several points that the authors try to make with several pieces of data do not come across clearly in the text, including Figure 2 on page 6, Figure 4 on page 9, and the various states utilized for the statistical treatment, "post-first penetration, post-first fertilization, no fertilization, penetration block and polyspermy block" on page 10. Either re-writing and clearer definitions'explanations are needed, and/or schematic illustrations could be considered to augment re-written text. Illustrations could be a valuable way present the intended concepts to readers more clearly and accurately. For example, Figure 4 and the associated text on page 9 get particularly confusing - although this sounds like a quite impressive dataset with observations of 138 sperm. Illustrations could be helpful, in the spirit of "a picture is worth 1000 words," to show what seem to be three different situations of sequences of events with the sperm they observed. Finally, the text in the Results about the 138 sperm is quite difficult to follow. It also might help comprehension to augment the percentages with the actual numbers of sperm - e.g., is 48.6% referring 67 of the total 138 sperm analyzed? Does the 85.1% refer to 57 of these 67 sperm?

Figure 2 in the original version of our manuscript concerns sperm engulfment and PB2 extrusion. As already mentioned in our response to Reviewer 1, the characterization of sperm engulfment and PB2 extrusion kinetics is highly relevant to the analysis of the penetration and fusion blocks. However, we agree that its presence in the main text may distract the reader from the main focus of the study. Therefore, this figure and the associated text have been moved to the Supplementary Information in the revised manuscript (SI 2, pages 26–27).

Regarding Figure 4 (original version), in response to Reviewer 3’s concern about the difficulty in grasping the message conveyed in its three graphs and associated text we have completely rethought the way these data are presented. Since the three graphs of Figure 4 were directly derived from the experimental timing data of sperm entry in the PVS and fusion with the oolemma in fertilized oocytes (originally shown in Figure 3A), we have combined them into a single figure in the revised manuscript: Figure 3 (page 8). This new Figure 3 now comprises three components:

• Figure 3A remains unchanged from the original version and shows the timing of sperm penetration and fusion in fertilized oocytes. Each sperm category (fused or non-fused , penetrated in the PVS before fusion or after fusion) is represented using a color code clearly explained in the main text (last paragraph of page 7).
• Figure 3B focuses specifically on the first spermatozoon to penetrate the PVS of each oocyte. It reports how many of these first-penetrating spermatozoa succeeded in fusing versus how many failed to do so, highlighting that being the first to arrive is not sufficient for fusion—other factors are involved. This is explained simply in the first paragraph of page 9.
• Figure 3C considers all spermatozoa that entered the PVS of fertilized oocytes, classifying them into three categories: those that penetrated the PVS before fertilization, those that did so after fertilization, and those for which the timing could not be precisely determined. Such classification makes it apparent that the number of spermatozoa penetrating before and after fertilization is of the same order of magnitude, indicating that fertilization is not very effective at preventing further sperm entry into the PVS for the duration of our observations (~4 hours). To facilitate the identification of these three categories, the same color code used in Figure 3A is applied. In addition, within each category, the number of spermatozoa that successfully fused are indicated in black. This allows the reader to quickly assess the fertilization probability for each category—high for sperm entering before fertilization, very low or null for those entering after fertilization. This analysis shows that fertilization is far more effective at blocking sperm fusion than at blocking sperm penetration. This is clearly explained in the second paragraph of page 9. Regarding__ statistical analysis__, as already mentioned in our responses to Reviewers 1 and 2, this section has been rewritten to improve clarity and readability. The notation has also been significantly simplified. To improve the overall fluidity of the text related to the statistical analysis, Figure 3B (original version), which presented the timing of penetration into the perivitelline space of oocytes that remained unfertilized, along with its associated statistical analysis previously in Figure 5B), have been revised and transferred together in a single Figure S1 of the Supplementary Information (SI1, pages 26; now Figures S1A and S1B).

(6) Introduction, page 2 - it is inaccurate to state that only diploid zygotes can develop into a "new being." Triploid zygotes typically fail early in develop, but can survive and, for example, contribute to molar pregnancies. Additionally, it would be beneficial to be more scientifically precise term than saying "development into a new being." This is recommended not only for scientific accuracy, but also due to current debates, including in lay public circles, about what defines "life" or human life.

In response to Reviewer 3’s comment, we no longer state in the revised version of the manuscript that only diploid zygotes can develop into a new being. We have modified our wording as follows, on page 2, second paragraph: “In mammals, oocytes fertilized by more than one spermatozoon cannot develop into viable offspring.”

(7) Introduction, page 2 - The mammalian sperm must pass through three layers, not just two as stated in the first paragraph of the Introduction. The authors should include the cumulus layer in this list of events of fertilization.

The sentence from the introduction from the original manuscript mentioned by Reviewer 3 was: “To fertilize, a spermatozoon must successively pass two oocyte’s barriers.” This statement is accurate in the sense that the cumulus cell layer is not part of the oocyte itself, unlike the two oocyte’s barriers: the zona pellucida and the oolemma. Moreover, the traversal of the cumulus layer is not within the scope of our study, unlike the traversal of the zona pellucida and fusion with the oolemma. However, it is also correct that in our study the spermatozoa have passed through the cumulus layer before reaching the oocyte. Therefore, in response to Reviewer 3’s comment, we have revised the sentence to clarify this point as follows:

“Once a spermatozoon has passed through the cumulus cell layer surrounding the oocyte, it still must overcome two oocyte’s barriers to complete fertilization.”

(8) Introduction, page 2 - While there is evidence that zinc is released from mouse egg upon fertilization, the evidence is not convincing or conclusive that zinc is released from cortical granules or via cortical granule exocytosis.

To better highlight the rationale, storyline, and scope of our study, the introduction has been thoroughly streamlined. In this context, the section discussing the cortical reaction and zinc release seemed more appropriate in the Discussion, specifically within the paragraph titled “Relationship between the penetration block and the ZP-block.”

To address the uncertainty raised by Reviewer 3 regarding the origin of the zinc spark release, we have rephrased this part as follows:

“The fertilization-triggered processes responsible for the changes in ZP properties are generally attributed to the cortical reaction—a calcium-induced exocytosis of secretory granules (cortical granules) present in the cortex of unfertilized mammalian oocytes—and to zinc sparks. As a result, proteases, glycosidases, lectins, and zinc are released into the perivitelline space (PVS), where they act on the components of the zona pellucida. This leads to a series of modifications collectively referred to as ZP hardening or the ZP-block”.

(9) The authors inaccurately state, "only if monospermic multi-penetrated oocytes are able to develop normally, which to our knowledge has never been proven in mice" (page 4) - This was demonstrated with the Astl knockout, assuming that the authors use of "multi-penetrated oocytes" here refers to the definition of penetration that they use, namely penetrating the ZP. This also is one of the instances where the authors contradict themselves, as they note the results with this knockout on page 18.

Thank you for bringing this point to our attention. Nozawa et al. (2018) found that female mice lacking ovastacin (Astl)—the protease released during the cortical reaction that plays a key role in rendering the zona pellucida impenetrable—are normally fertile. They also reported that oocytes recovered from these females after mating were monospermic, despite the consistent presence of additional spermatozoa in the perivitelline space. We can indeed consider that taken together these findings demonstrate that the presence of multiple spermatozoa in the PVS does not impair normal development, as long as the oocyte remains monospermic. In our study, we re-demonstrated this in a different way (by reimplantation of monospermic oocytes with additional spermatozoa in their PVS) in a more physiological context of WT oocytes, but we agree that we cannot state: “which to our knowledge has never been proven in mice.” This part of the sentence has therefore been removed. In the revised version of the manuscript, the sentence is now formulated in the first paragraph of page 5 as follows: “However, the contribution of the fusion block to prevent polyspermy has physiological significance only if monospermic oocytes with additional spermatozoa in their PVS can develop into viable pups.”

Minor comments:

There are numerous places where this reader marked places of confusion in the text. A sample of some of these:

We will indicate hereinafter how we have modified the text in the specific examples provided by Reviewer 3. Beyond these, however, we would like to emphasize that we have thoroughly revised the entire manuscript to improve clarity and precision.

Page 4 - "continuously relayed by other if they detach" - don't know what this means

Replaced now p 5 by “can be replaced by others if they detach”

Page 6 - "hernia" - do the authors mean "protrusion" on the oocyte surface?

The paragraph from the Results section in question has now been moved to the Supplementary Information, on pages 26 and 27. The term hernia has been systematically replaced with protrusion, including in the Materials and Methods section on page 24.

Page 10 - "penetration of spermatozoa in the PVS falls down" - don't know what this means

Falls down has been removed from the new version and replaced with decreases

Page 12 - "spermatozoa linked to the oocyte ZP" - not clear what "linked" means here

Replaced now page 16 by “spermatozoa bound to the oocyte ZP”

Page 14 - "by dint of oscillations" - don't know what this means

Replaced now page 10 by “the persistent flagellum movements”

Specifics for Materials and Methods:

Exact timing of females receiving hCG and then being put with males for mating - assume this was immediate but this is an important detail regarding the timing for the creation of embryos in vivo.

That is correct: females were placed with males for mating immediately after receiving hCG. This clarification has been added in the revised version of the manuscript.

Please provide the volumes in which inseminations occurred, and how many eggs were placed in this volume with the 10^6 sperm/ml.

The number of eggs may vary from one cumulus–oocyte complex to another. It is therefore not possible to specify exactly how many eggs were inseminated. However, we now indicate on page 23 the number of cumulus–oocyte complexes inseminated (4 per experiment), the volume in which insemination was performed (200 mL), and the sperm concentration used 106 sperm/mL.

**Referees cross-commenting**

I concur with Reviewer 1's comment, that the 'challenging prior dogma' about the first sperm not always being the one to fertilize the egg is too strong. As Reviewer 1 notes, "it had been observed before that it is not necessarily the first sperm that gets through the ZP that fertilizes the egg." I even thought about adding this comment to my review, although held off (I was hoping to find references, but that was taking too long).

Please refer to our response to Reviewer 1 regarding this point.

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

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Referee #3

Evidence, reproducibility and clarity

Summary:

This study by Dubois et al. utilizes live-cell imaging studies of mouse oocytes undergoing fertilization. A strength of this study is their use of three different conditions for analyses of events of fertilization: (1) eggs undergoing fertilization retrieved from females at 15 hr after mating (n = 211 oocytes); (2) cumulus-oocyte complexes inseminated in vitro (n = 220 oocytes), and (3) zona pellucida (ZP)-intact eggs inseminated in vitro, transferred from insemination culture once sperm were observed bound to the ZP for subsequent live-cell imaging (93 oocytes). This dataset and these analyses are valuable for the field of fertilization biology. Limitations of this manuscript are challenges arise with some conclusions, and the presentation of the manuscript. There are some factual errors, and also some places where clearer explanations should to be provided, in the text and potentially augmented with illustrations to provide more clarity on the models that the authors interpret from their data.

Major comments:

The authors are congratulated on their impressive collection of data from live-cell imaging. However, the writing in several sections is challenging to understand or seems to be of questionable accuracy. The lack of accuracy is suspected to be more an effect of overly ambitious attempts with writing style, rather than to mislead readers. Nevertheless, these aspects of the writing should be corrected. There also are multiple places where the manuscript contradicts itself. These contradictions should be corrected. Finally, there are factual points from previous studies that need correction.

Second, certain claims and the conclusions as presented are not always clearly supported by the data. This may be connected to the issues with writing style, word and phrasing choices, etc. The conclusions could be expressed more clearly, and thus may not require additional experiments or analyses to support them. The authors might also consider illustrations as ways to highlight the points they wish to make. (Figure 7 is a strong example of how they use illustrations to complement the text).

Specific comments:

1. The authors should use greater care in describing the blocks to polyspermy, particularly because they appear to be wishing to reframe views about prevention of polyspermic fertilization. The title mentions of "the fast block to polyspermy;" this problematic for a couple of different reasons. There is no strong evidence for block to polyspermy in mammals that occurs quickly, particularly not in the same time scale as the first-characterized fast block to polyspermy. To many biologists, the term "fast block to polyspermy" refers to the block that has been described in species like sea urchins and frogs, meaning a rapid depolarization of the egg plasma membrane. However, such depolarization events of the egg membrane have not been detected in multiple mammalian species. Moreover, the change in the egg membrane after fertilization does not occur in as fast a time scale as the membrane block in sea urchins and frogs (i.e., is not "fast" per se), and instead occurs in a comparable time frame as the conversation of the ZP associated with the cleavage of ZP2. Thus, it is misleading to use the terms "fast block" and "slow block" when talking about mammalian fertilization.

This also is an instance of where the authors contradict themselves in the manuscript, stating, "the membrane block and the ZP block are established in approximatively the same time frame" (third paragraph of Introduction). This statement is indeed accurate, unlike the reference to a fast block to polyspermy in mammals.<br /> 2. The authors aim to make the case that events occurring in the perivitelline space (PVS) prevent polyspermic fertilization, but the data that they present is not strong enough to make this conclusion. Additional experiments would optional for this study, but data from such additional experiments are needed to support the authors' claims regarding these functions in fertilization. Without additional data, the authors need to be much more conservative in interpretations of their data. The authors have indeed observed phenomena (the presence of CD9 and JUNO in the PVS) that could be consistent with a molecular basis of a means to prevent fertilization by a second sperm. However, the authors would need additional data from additional experimental studies, such as interfering with the release of CD9 and JUNO and showing that this experimental manipulation leads to increased polyspermy, or creating an experimental situation that mimics the presence of CD9 and JUNO (in essence, what the authors call "sperm inhibiting medium" on page 20) and showing that this prevents fertilization.

A major section of the Results section here (starting with "The consequence is that ... ") is speculation. Rather than be in the Results section, this should be in the Discussion. The language should be also softened regarding the roles of these proteins in the perivitelline space in other portions of the manuscript, such as the abstract and the introduction.

Finally, the authors should do more to discuss their results with the results of Miyado et al. (2008), which interestingly, posited that CD9 is released from the oocytes and that this facilitates fertilization by rendering sperm more fusion-competent. There admittedly are two reports that present data that suggest lack of detection of CD9-containing exosomes from eggs (as proposed by Miyado et al.), but nevertheless, the authors should put their results in context with previous findings. 3. Many of the authors' conclusions focus on their prior analyses of sperm interaction - beautifully illustrated in Figure 7. However, the authors need to be cautious in their interpretations of these data and generalizing them to mammalian fertilization as a whole, because mouse and other rodent sperm have sperm head morphology that is quite different from most other mammalian species.

In a similar vein, the authors should be cautious in their interpretations regarding the extension of these results to mammalian species other than mouse, given data on numbers of perivitelline sperm (ranging from 100s in some species to virtually none in other species), suggesting that different species rely on different egg-based blocks to polyspermy to varying extents. While these observations of embryos from natural matings are subject to numerous nuances, they nevertheless suggest that conclusions from mouse might not be able to be extended to all mammalian species.<br /> 4. Results, page 4 - It is very valuable that the authors clearly define what they mean by a penetrating spermatozoon and a fertilizing spermatozoon. However, they sometimes appear not to adhere to these definitions in other parts of the manuscript. An example of this is on page 10; the description of penetration of spermatozoon seems to be referring to membrane fusion with the oocyte plasma membrane, which the authors have alternatively called "fertilizing" or fertilization - although this is not entirely clear. The authors should go through all parts of the manuscript very carefully and ensure consistent use of their intended terminology.

Overall, while these definitions on page 4 are valuable, it is still recommended that the authors explicitly state when they are addressing penetration of the ZP and fertilization via fusion of the sperm with the oocyte plasma membrane. This help significantly in comprehension by readers. An example is the section header in the middle of page 9 - this could be "Spermatozoa can penetrate the ZP after the fertilization, but have very low chances to fertilize."

Another variation of this is in the middle of page 9, where the authors use the terms "fertilization block" and "penetration block." These are not conventional terms, and venture into being jargon, which could leave some readers confused. The authors could clearly define what they mean, particularly with respect to "penetration block,"

This extends to other portions of the manuscript as well, such as Figure 2C, with the label on the y-axis being "Time after fertilization." It seems that what the authors actually observed here was the cessation of sperm tail motility. (It is not evident they they did an assessment of sperm-oocyte fusion here.) 5. Several points that the authors try to make with several pieces of data do not come across clearly in the text, including Figure 2 on page 6, Figure 4 on page 9, and the various states utilized for the statistical treatment, "post-first penetration, post-first fertilization, no fertilization, penetration block and polyspermy block" on page 10 . Either re-writing and clearer definitions'explanations are needed, and/or schematic illustrations could be considered to augment re-written text. Illustrations could be a valuable way present the intended concepts to readers more clearly and accurately. For example, Figure 4 and the associated text on page 9 get particularly confusing - although this sounds like a quite impressive dataset with observations of 138 sperm. Illustrations could be helpful, in the spirit of "a picture is worth 1000 words," to show what seem to be three different situations of sequences of events with the sperm they observed. Finally, the text in the Results about the 138 sperm is quite difficult to follow. It also might help comprehension to augment the percentages with the actual numbers of sperm - e.g., is 48.6% referring 67 of the total 138 sperm analyzed? Does the 85.1% refer to 57 of these 67 sperm?<br /> 6. Introduction, page 2 - it is inaccurate to state that only diploid zygotes can develop into a "new being." Triploid zygotes typically fail early in develop, but can survive and, for example, contribute to molar pregnancies. Additionally, it would be beneficial to be more scientifically precise term than saying "development into a new being." This is recommended not only for scientific accuracy, but also due to current debates, including in lay public circles, about what defines "life" or human life. <br /> 7. Introduction, page 2 - The mammalian sperm must pass through three layers, not just two as stated in the first paragraph of the Introduction. The authors should include the cumulus layer in this list of events of fertilization. 8. Introduction, page 2 - While there is evidence that zinc is released from mouse egg upon fertilization, the evidence is not convincing or conclusive that zinc is released from cortical granules or via cortical granule exocytosis.<br /> 9. The authors inaccurately state, "only if monospermic multi-penetrated oocytes are able to develop normally, which to our knowledge has never been proven in mice" (page 4) - This was demonstrated with the Astl knockout, assuming that the authors use of "multi-penetrated oocytes" here refers to the definition of penetration that they use, namely penetrating the ZP. This also is one of the instances where the authors contradict themselves, as they note the results with this knockout on page 18.

Minor comments:

There are numerous places where this reader marked places of confusion in the text. A sample of some of these:

Page 4 - "continuously relayed by other if they detach" - don't know what this means

Page 6 - "hernia" - do the authors mean "protrusion" on the oocyte surface?

Page 10 - "penetration of spermatozoa in the PVS falls down" - don't know what this means

Page 12 - "spermatozoa linked to the oocyte ZP" - not clear what "linked" means here

Page 14 - "by dint of oscillations" - don't know what this means

Specifics for Materials and Methods:

Exact timing of females receiving hCG and then being put with males for mating - assume this was immediate but this is an important detail regarding the timing for the creation of embryos in vivo.

Please provide the volumes in which inseminations occurred, and how many eggs were placed in this volume with the 10^6 sperm/ml.

Referees cross-commenting

I concur with Reviewer 1's comment, that the 'challenging prior dogma' about the first sperm not always being the one to fertilize the egg is too strong. As Reviewer 1 notes, "it had been observed before that it is not necessarily the first sperm that gets through the ZP that fertilizes the egg." I even thought about adding this comment to my review, although held off (I was hoping to find references, but that was taking too long).

Significance

This manuscript brings interesting new observations for the field of gamete and fertilization biology. For very obvious reasons, the understanding of mammalian fertilization has lagged behind the understanding of fertilization of species with external fertilization. Decades ago, developmental biologists first focused on studies of fertilization on gametes from species that release sperm and egg into water, either spontaneously or with relatively easy stimulation, and gametes that could be easily cultured and enabled to create embryos as researchers watched. Studies of mammalian fertilization have since caught up, with the elucidation of conditions that support in vitro fertilization in various mammalian species, most notably mouse as an experimental model.

Author response:

The following is the authors’ response to the original reviews

Reviewing Editor Comments:

Focus and Scope:

The paper attempts to address too many topics simultaneously, resulting in a lack of focus and insufficient depth in the treatment of individual components.

We have moved this selective clinical review section that was previously Part I in the paper now to Part II, given the importance of leading off with the meta-analysis and resource before doing a selective review, which are now Part I. In the lead in to Part II, we now indicate that the review is not intended to be comprehensive, because there are other recent comprehensive reviews, which we cite. This part of the paper merely aims to generate hypotheses on the directionality of effects ripe for testing on how TUS could be used to excite or suppress function, illustrated with specific clinical examples. The importance of this section, even though not comprehensive, is that it should provide the reader with examples on how the directionality of TUS could be used specifically in a range of clinical applications. The reader will find that the same hypotheses do not apply to different clinical disorder. Therefore, patient specific hypotheses need to be motivated and then subsequently tested with empirical application of TUS, which Part II provides.

Part II. Selective TUS clinical applications review and TUS directionality hypotheses starts at line 458. Part I, the meta-analysis and resource section starts at line 199, after the Introduction on TUS and the importance on understanding how the directionality of TUS effects could be better understood.

Strengthening the Meta-Analysis:

The meta-analysis is the strongest aspect of the paper and should be expanded to include the relevant statistics. However, it currently omits several key concepts, studies, and discussion points, particularly related to replication and the dominance of results from specific groups. These omissions should be addressed even with a focus on meta-analysis.

We thank the reviewer for their enthusiasm about the meta-analysis, which we have now promoted to Part I in the revised paper. We have substantially updated the latest database (inTUS_DATABASE_1-2025.csv) and ensured that the R markdown script can re-generate all of the results and statistical values. We have inserted additional statistical values in the main manuscript, as requested. The inTUS Resource is located here (https://osf.io/arqp8/ under Cafferatti_et_al_inTUS_Resource), and we have aimed to make it as user friendly to use and contribute to as possible. For instance, the reader can find them all in the HTML link summarizing the R markdown output with all statistical values here: https://rpubs.com/BenSlaterNeuro/1268823, a part of the inTUS resource.

Since the last submission, there has been a tremendous increase in the number of TUS studies in healthy participants. We have curated and included all of the relevant studies we could find in the 1-2025 database, as the next large expansion of the database (now including 52 experiments in healthy participants). We then reran and report the results of the statistical tests via the R markdown script (starting at line 336). Finally, the online database (inTUS_DATABASE_1-2025.csv) has additional columns, suggested by the reviewers, including one to identify the same groups that conducted the TUS study, based on a social network analysis. The manuscript figures (Table 1 and Table 2) did not have the space to expand the data tables, but these additional columns are available in the database online. Finally, we have ensured that the resource is as easy to use as possible (line 862 has the Introduction to the inTUS Resource – which is also the online READ ME file), and we have been in contact with the iTRUSST consortium leads who are interested in discussing hosting the resource and helping it to become self-sustaining.

Conceptual Development:

The more conceptual part of the paper is underdeveloped. It lacks sufficient supporting data, a well-articulated argument, and a clear derivation or development of a concrete model.

To ensure that the conceptual sections are well developed, we have revised the introduction, including the background on TUS and bases for the interest in the directionality of effects. We have also revised the TUS mechanisms background as suggested by the reviewers. For Part I, the meta-analysis basis and hypotheses we have ensured the rationale is clearer. The hypotheses are based on several lines of research in the animal model and human literature as cited (starting with line 211). For Part II, the selective clinical review, we have revised this section as well to have each section on lowintensity TUS and end in a hypothesis on the directionality of TUS effects. Starting at line 199 we have clarified the scope of the review and ensured that all the relevant experiments in healthy participants (n = 52 experiments) have now been included in the next key update of the resource and meta-analysis in this key paper update.

Database Curation:

The authors should provide more detailed information about how the database will be curated and made accessible. They may consider collaborating with ITRUSST.

We have expanded the information on the Resource documents (starting at line 862) to make the resource as user friendly as possible. At the beginning of the resource development stage we had contacted but not heard from the ITRUSST consortium. Encouraged by this comment we again reached out and are now in contact with the ITRUSST consortium leads who are interested in discussing sustaining the resource. It would be wonderful to have the resource linked to other ITTRUST tools, since it was inspired by the organization. Practically what this means is that the resource rather than being hosted on Open Science Framework, would potentially be hosted on the ITRUSST web site (https://itrusst.com/). These discussions are in progress, but the next key update to the database (1-2025) is already available and reported in this key update to our original paper.

Reviewer #1: (Public Review)

Summary:

This paper is a relevant overview of the currently published literature on lowintensity focussed ultrasound stimulation (TUS) in humans, with a meta-analysis of this literature that explores which stimulation parameters might predict the directionality of the physiological stimulation effects.

The pool of papers to draw from is small, which is not surprising given the nascent technology. It seems nevertheless relevant to summarize the current field in the way done here, not least to mitigate and prevent some of the mistakes that other non-invasive brain stimulation techniques have suffered from, most notably the theory- and data-free permutation of the parameter space.

The meta-analysis concludes that there are, at best, weak trends toward specific parameters predicting the direction of the stimulation effects. The data have been incorporated into an open database, that will ideally continue to be populated by the community and thereby become a helpful resource as the field moves forward.

Strengths:

The current state of human TUS is concisely and well summarized. The methods of the meta-analysis are appropriate. The database is a valuable resource.

Weaknesses:

These are not so much weaknesses but rather comments and suggestions that the authors may want to consider.

We thank the reviewer for their support of the resource and meta-analysis. We have implemented the suggestions next as follows.

I may have missed this, but how will the database be curated going forward? The resource will only be as useful as the quality of data entry, which, given the complexity of TUS can easily be done incorrectly.

We have added a paragraph on how authors could use the Qualtrics form to submit their data and the curation process involved (from line 891). Currently, this process cannot be automated because we continue to find that reported papers do not report the TUS parameters that ITRUSST has encouraged the community to report (Martin et al., 2024). We can dedicate for a TUS expert to ensure that every 6 or 12 months the data base is curated and expanded. The current version is the latest 1-2025 update to the data base. Longer term we are in discussion with ITRUSST on whether the resource could become self sustaining when TUS papers regularly reporting all the relevant parameters such that the database expansion becomes trivial, and then the Resource R markdown script and other tools can be used to re-evaluate the statistical tests and the user can conduct secondary hypothesis testing on the data.

It would be helpful to report the full statistics and effect sizes for all analyses. At times, only p-values are given. The meta-analysis only provides weak evidence (judged by the p-values) for two parameters having a predictive effect on the direction of neuromodulation. This reviewer thinks a stronger statement is warranted that there is currently no good evidence for duty cycle or sonication direction predicting outcome (though I caveat this given the full stats aren't reported). The concern here is that some readers may gallop away with the impression that the evidence is compelling because the p-value is on the correct side of 0.05.

We have ensured that the R script can generate the full statistics from the tests and the effect sizes for all the analyses, and now also report more of the key statistical values in the revised paper (starting at line 336). As suggested, we have also ensured that the interpretation is sufficiently nuanced given the small sample sizes and the p-values below 0.1 but above 0.05 are interpreted as a statistical trend.

This reviewer thinks the issue of (independent) replication should be more forcefully discussed and highlighted. The overall motivation for the present paper is clearly and thoughtfully articulated, but perhaps the authors agree that the role that replication has to play in a nascent field such as TUS is worth considering.

We completely agree and have added additional columns to the online database to identify unique groups, using a social network analysis, and independent replications. These expanded tables did not fit in the manuscript versions of Tables 1 and 2 but are fully available in the Resource data tables ready for further analysis by interested resource users.

A related point is that many of the results come from the same groups (the so-called theta-TUS protocol being a clear example). The analysis could factor this in, but it may be helpful to either signpost independent replications, which studies come from the same groups, or both.

In the expanded database tables (inTUS_DATABASE_1-2025.csv: https://osf.io/arqp8/ under Cafferatti_et_al_inTUS_Resource) we have added a column to identify independent replication.

The recent study by Bao et al 2024 J Phys might be worth including, not least because it fails to replicate the results on theta TUS that had been limited to the same group so far (by reporting, in essence, the opposite result).

Thank you. We have added this study and over a dozen recent TUS studies in healthy participants to the database and redone the analyses.

The summary of TUS effects is useful and concise. Two aspects may warrant highlighting, if anything to safeguard against overly simplistic heuristics for the application of TUS from less experienced users. First, could the effects of sonication (enhancing vs suppressing) depend on the targeted structure? Across the cortex, this may be similar, but for subcortical structures such as the basal ganglia, thalamus, etc, the idiosyncratic anatomy, connectivity, and composition of neurons may well lead to different net outcomes. Do the models mentioned in this paper account for that or allow for exploring this? And is it worth highlighting that simple heuristics that assume the effects of a given TUS protocol are uniform across the entire brain risk oversimplification or could be plain wrong? Second, and related, there seems to be the implicit assumption (not necessarily made by the authors) that the effects of a given protocol in a healthy population transfer like for like to a patient population (if TUS protocol X is enhancing in healthy subjects, I can use it for enhancement in patient group Y). This reviewer does not know to which degree this is valid or not, but it seems simplistic or risky. Many neurological and psychiatric disorders alter neurotransmission, and/or lead to morphological and structural changes that would seem capable of influencing the impact of TUS. If the authors agree, this issue might be worth highlighting.

We agree that given the divergence in circuits and cellular constituents between cortical and subcortical areas, it is important to distinguish studies that have focused on cortical or subcortical brain areas. The online data tables identify the target region. The analyses can be used to focus on the cortical or subcortical sites for analysis, although for the current version of the database there are too few subcortical sites with which to conduct analyses on subcortical sites. On the second point, that pathology may have affected the results, we completely agree and have clarified that the current database only includes healthy participant experiments for this reason. We are considering future updates to the resource may include clinical patient results (Line 247).

Reviewer #1 (Recommendations for the authors):

Minor edits (I wouldn't call them "corrections").

We sincerely appreciate the constructive comments and have aimed to address them all as suggested.

Perhaps the most relevant edit pertains to the statistics.

We now report the more complete statistical results (line 336) and the R markdown script can re-generate all the statistical values for the tests.

The issue of replication also seems relevant and ought to be raised. This reviewer does not want to prescribe what to do or impose the view the authors ought to adopt.

In the online version of the data tables for the latest dataset, we have added a column in the data table as suggested that identifies independent groups and replications.

The other points are left to the authors' discretion.

We have aimed to address all of the reviewer’s points. Thank you for the constructive input which has helped to improve the expanded database and resource.

Reviewer #2: (Public Review)

Summary:

This paper describes a number of aspects of transcranial ultrasound stimulation (TUS) including a generic review of what TUS might be used for; a meta-analysis of human studies to identify ultrasound parameters that affect directionality; a comparison between one postulated mechanistic model and results in humans; and a description of a database for collecting information on studies.

Strengths:

The main strength was a meta-analysis of human studies to identify which ultrasonic parameters might result in enhancement or suppression of modulation effects. The meta-analysis suggests that none of the US parameters correlate significantly with effects. This is a useful result for researchers in the field in trying to determine how the parameter space should be further investigated to identify whether it is possible to indeed enhance or suppress brain activity with ultrasound.

The database is a good idea in principle but would be best done in collaboration with ITRUSST, an international consortium, and perhaps should be its own paper.

Weaknesses:

The paper tries to cover too many topics and some of the technical descriptions are a bit loose. The review section does not add to the current literature. The comparison with a mechanistic model is limited to comparing data with a single model at a time when there is no general agreement in the field as to how ultrasound might produce a neuromodulation effect. The comparison is therefore of limited value.

We appreciate the reviewer’s assessment and interest in the meta-analysis and database to guide the development of TUS for more systematic control of the directionality of neuromodulation. With this next key expansion of the database (inTUS_DATABASE_1-2025.csv) we have added over a dozen new studies that have been published since our original submission (n = 52 experiments). We have also moved the ‘review’ part of the paper below the meta-analysis and resource description. We have clarified that the clinical review section (now Part II in the revised manuscript) is not intended as a comprehensive review but as a selective review showing how hypotheses on the directionality of TUS effects need to be carefully developed for specific patient groups that require different effects to be induced at specific brain areas. Finally, we have gotten in contact with the ITRUSST consortium leads, as suggested, and are in discussion on whether the inTUS resource could be hosted by ITRUSST. Since these discussions are ongoing practically what this might mean is moving the resource from the Open Science Framework to ITRUSST webpages, which would be a trivial update of the link to the resource in OSF.

We also sincerely appreciate the time and care the reviewer has given to provide us with the below guidance, all of which we have aimed to take on board in the revised paper.

Reviewer #2 (Recommendations for the authors):

Line 24/25 - I suggest avoiding using the term "deep brain stimulation" in reference to TUS as the term is normally used to describe electrically implanted electrodes.

We have removed the term “deep” brain stimulation in reference to TUS to avoid confusion with electrical DBS for patient treatment [Line 24].

Line 25 - I don't think "computational modelling" has changed how TUS can be done. There is still much to be understood about mechanisms. I think the modelling aspects of the paper should be toned down. Indeed the NICE data that is presented later appears to have a weak, if any, correlation to the outcomes.

We have revised the manuscript text throughout to ensure that the computational modeling contributions are not overstated, as noted, given the lack of strong correlation to the NICE model outcomes by the meta-analysis including in the latest results with the more extensive database (n = 52).

Line 32 - "exponentially increasing" is a well-defined technical term and the increase in studies should be quantified to ensure it is indeed exponential. I agree that TUS studies in humans are increasing but a quick tally of the data by year in the meta-analysis reported here doesn't suggest that it follows an "exponential" growth.

We have changed “exponential” to “to increase”. [Line 32]

Line 50 - I would suggest using the term sub-MHz rather than 100-1,000 kHz as it is challenging to deliver ultrasound at 1 MHz through the skull. The highest frequency in the meta-analysis is 850 kHz; but the majority are in the 200-500 kHz range.

We have made this correction to sub-MHz. [Line 54]

Line 58/59 - Is the FDA publication on diagnostic imaging relevant for saying that 50 W/cm2 is a lowintensity TUS? I think it's perhaps reasonable to say that intensities below diagnostic thresholds are "low intensities" but that is not clear in the text. I would refer to ITRUSST on what is appropriate for defining what is low, medium, or high.

We have cut the reference to the FDA here since it is, as noted, not as relevant as pointing to the ITRUSST definition.

Line 65/66 - I agree that ultrasound for neuromodulation is gaining traction and there is an increase in activity, but it also has a long history with the work of the Fry brothers published in the 1950s; and extensive work of Gavrilov in humans starting in the 1970s.

We have added citations to the Fry brothers and Gavrilov to the text in this section. [Line 69/70]

Line 75 - I think the intermembrane cavitation mechanism is unlikely to be due to "microbubbles" in a lipid membrane. The predicted displacements are on the order of nanometres, so they are unlikely to generate microbubbles. The work on comparing with NICE is limited. Note there are a number of experimental papers that have reported an absence of intra-membrane cavitation, including the Yoo et al 2022 which is referenced later in the paragraph. Also, there are other models, such as Liao et al 2021 (https://www.nature.com/articles/s41598020-78553-2).

As suggested, we have removed this phrase on microbubble formation as a likely mechanism. We have also added the Liao paper to this paragraph as it is relevant.

Line 83 - "At the lower intensities..." it is not clear whether this means all TUS intensities or the lower end of intensities used in TUS.

We now use the following wording here: “low intensities”. [Line 86]  

Line 85/86 - "more continuous stimulation" the modulation paradigms haven't been described yet and so pulse vs continuous hasn't been made clear to the reader. Also "more continuous" is very loose terminology. Something is either continuous or it isn't.

We agree and have removed “more” to be clear that the stimulation is continuous. [Line 88]

Line 87/88 - "TUS does not .. cavitation ..when ..ISPTA...<14 W/cm2". You can't use ISPTA to determine cavitation. It is the peak negative pressure which is the key driver for cavitation and the MI which is the generally accepted (although grudgingly by some) metric for assessing cavitation risk. You can link the negative pressure to ISPPA but not really to ISPTA. In histotripsy for example the ISPTA is low due to the low duty cycles to avoid heating but the cavitation is a huge effect. Technical terminology is loose.

We have corrected this to “TUS does not appear to cause significant heating or cavitation of brain tissue when the intensity remains low, based on Mechanical and Thermal Index values and recommendations of use”. [Line 90/91]

Line 89 - What is meant by "low intensity TUS"? I think all TUS used in the literature counts as low intensity - in that it is below the level allowed for diagnostic imaging.

We have ensured that the text is focused on TUS being low-intensity and only in the introduction do we distinguish low intensity TUS from moderate and high intensity TUS, such as used for thermal ablation [Lines 62-66].

Line 88/89 - Most temperature rises in brain tissue in TUS are well below 1 C - will this really change membrane capacitance significantly? If so it would have been good to consider a model for it.

We have revised this statement as “thermal effects could at least minimally alter cell membrane capacitance…”. [Line 93]

Line 111 - The text refers to "recent studies" but then the next two references are from 1990 and 2005 which I would argue don't count as "recent".

We have corrected this wording to “previous studies”. [Line 114]

Lines 122/129 - This paragraph on TMS pulsing should be linked to the TUS paragraph on pulsing (lines 109/116). The intervening paragraph on anaesthesia is relevant but breaks the flow.

We have merged the paragraph on anesthesia to the prior one on TUS so that the TMS paragraph is linked more closely to it [starting on line 112].

Line 130/131 - It is not clear to me that current studies are being guided by computational models. I think there is still no generally accepted theory for mechanisms. If the authors want to do a mechanisms paper then they should compare a few.

We have revised this as suggested to not overstate the contribution of the limited computational modeling studies throughout the manuscript.

Line 132 on - There are a number of studies that suggest that NICE is likely not the mechanism by which TUS produces neuromodulation.

We have revised this sentence as follows: “Although it remains questionable whether intramembrane cavitation is a key mechanism for TUS, the NICE model simulations explored a broad set of TUS parameters, including TUS intensity and the continuity of stimulation (duty cycle) on modelled neuronal responses.” [Lines 139/142]

Lines 137-140 - Terms are defined after their use. Things like ISPPTA, PRF, TI, and MI have been discussed already and so the terms should have been defined earlier. The authors should think carefully about how the material is presented to make it more logical for the reader.

We have ensured that the definitions precede the use of abbreviations and have added abbreviations to the tables.

Part I Line 180-437 - The review of potential applications for TUS reads like an introductory chapter of a thesis. It is entirely proper for a thesis to have a chapter like this, but it is not really relevant for a peer-reviewed research article. There are also numerous applications, e.g. mapping areas associated with decisions, or treating patients with addiction, which are not included, so it is not exhaustive. I would suggest this part be removed.

We have moved the ‘review’ part of the paper to Part II, given the metaanalysis and resource should be more prominent as Part I. In the review now Part II of the paper we also now make it clear that there are recent comprehensive reviews of the clinical literature ( line 465/467). Namely, the purpose of our selective review is to demonstrate how directionality of TUS effects need to be specific for the clinical application intended, given the great variability in clinical effects that might be desired, brain areas targeted and pathology being treated. We have also aimed to ensure that each section summary is scholarly and academically written to a high level. All the co-authors contributed to these sections so we have also edited to have some consistency across sections, with sections ending with directionality of TUS hypotheses that could be developed for empirical testing.

Line 453 - It is stated that "ISPTA, which mathematically integrates ISSPA by the sonication DC" It sounds rather grand to mathematically integrate but you can't integrate with respect to DC, you can integrate with respect to time. If you integrate intensity with respect to time over pulse and over the sonication time then one finds that ISPTA = DC x ISPPA, multiplication is also an important mathematical function and should be given its due. Lastly, I think there is a typo and ISSPA should read ISPPA

We have corrected the typo and the statement to “mathematically multiplies ISPPA by the continuity of sonication”. [Line 221/222]

Line 454 - I don't think ISPTA is a good measure of "dose." In radiation physics dose is well defined in terms of absorbed energy. The equivalent has yet to be defined for TUS so I would avoid using dose. The ISPTA does relate to TI - although it depends not just on the spatial peak but also on the spatial distribution and the frequency-dependent absorption coefficient of the tissue. I would just avoid the use of "dose" until the field has a better idea of what is going on.

We have cut this phrase on dose as suggested.

Page 16 Box 1 - TI is defined as diagnostic ultrasound imaging it is based on. Also, I think TI is dimensionless; it is referenced to a 1-degree temperature rise and so it can be interpreted in terms of celsius or kelvin; but to be technically accurate it is dimensionless.

We have made TI dimensionless in Box 1

Page 17 Box 2 - Here you have no units for TI - which is correct but inconsistent with Box 1. But the legend suggests a 2 K temperature rise where as your Box allows for 6 K. The value of 6 is consistent with FDA but my understanding of the BMUS guidelines is the TI must be less than or equal to 0.7 for unlimited time or less than 3 if the duration is less than 1 minute. I accept that the table is labelled FDA limits, but the bold table caption is "Recommendations for TUS parameters" I think you should give the ITRUSST values rather than FDA.

We have revised this Box legend to better distinguish the FDA and ITRUSST recommendation where they differ (e.g., the importance of ISPTA and the TI values). See revised legend for Box 2.

Page 18 Box 3 - Not sure what this is trying to show? Also, what is "higher intensity" and "lower intensity"?

Why not just give a range of values in each box?

We agree that the higher and lower intensities likely to lead to enhancement or suppression are poorly defined and have noted this in the legend: “Note that the threshold for ISPPA qualifying as ‘higher’ or ‘lower’ intensity is currently poorly understood, or may non-linearly interact with other factors” [Line 751/754, Box 3].

Line 444 - The hypotheses should be stated more clearly. Maybe I am just dense, but it is not obvious to me from box 3.

We provide the basis for the hypotheses in the manuscript text on the paragraph [Lines 106-179].

Line 481/482 - The intensity of a diagnostic ultrasound system is very well characterised. It just might be that the authors didn't report it. It is not clear what is meant by the "continuity." I guess it's to do with pulsing - which is also well defined but perhaps also not reported.

We agree and have revised this as follows “For the meta-analysis, we only included studies that either reported a basic set of TUS stimulation parameters or those sufficient for estimating the required parameters or those sufficient for estimating the required parameters necessary for the meta-analysis” [Lines 256/258]

Figure 2 - What is the purpose of this figure? Did you carry out simulations for all the studies? It doesn't seem to be relevant to the data here.

This figure illustrates the TUS targeting approach and simulations, in this case conducted in k-plan. These were conducted to evaluate approximations to ISPPA in brain values from the studies that did not report these values [Lines 264/268]).  

Figure 4 - The data in these figures is nice (and therefore doesn't need to have a NICE curve) To me it clearly shows that the data in the literature does not obviously segment into enhancement vs suppression with DC. I suspect it is the same with PRF. I think it would have been better if C and D had PRF on the horizontal axis for on-line and off-line so that effect could be seen more clearly.

We have kept the NICE curve only for a reference that some readers familiar with the NICE model might want to see overlaid in the figure, but have ensured that the text throughout makes clear that the NICE model predictions are not as statistically robust as initially anecdotally thought. PRF results are not significant but we do show a panel with the PRF measures on one axis (Fig. 4D). Figure 5 also shows box plot results with PRF as well as the other key TUS parameters. Moreover, in the inTUS resource we have provided an app for users to explore the data (https://benslaterneuro.shinyapps.io/Caffaratti_inTUS_Resource/).

Figure 5 - The text on the axes is too small to read. Was the DC significant for both on-line and offline? What about ISPPA for off-line. At least by eye, it looks as different as DC. Figure 5C doesn't add anything.

We have boosted the font for Figure 5 and have cut panel 5C since it was not adding much. We have also checked whether DC parameter was significant separately for on-line and off-line effects, but the sample sizes were too small for significance, and the statistical test was not significantly different for Online and Offline effects even in the 12025 database. Therefore they might look stronger for Offline effects in some of the plots in Figure 5, but are currently statistically indistinguishable [Lines 347/348].

Table 1 - There is a typo in the 3rd column. FF should have units of kHz, not KHz. In addition, SD should have units of s as that is the SI symbol for seconds. I would swap columns 9 and 10 so that ISPPA in water and ISPPA in the brain are next to each other.

We have corrected the typo in the 3rd column and ensured that units are kHz. SD in the tables has units of ‘s’ for seconds and have put ISPPA in water and in brain next to each other in the data tables.

Line 767 - "M.K. was supported..." There are TWO MKs in the author list.

We have changed this to M.Ka. for Marcus Kaiser.

Briefing : L'augmentation des coûts de la cantine scolaire en France

Ce briefing examine les défis croissants liés à l'augmentation des coûts de la restauration scolaire en France, explorant les répercussions sur les familles, les collectivités locales et la qualité des repas, ainsi que les stratégies mises en œuvre pour y faire face.

1. Un fardeau financier croissant pour les familles

L'augmentation du coût des repas à la cantine scolaire est devenue une source de préoccupation majeure pour de nombreuses familles, qui voient leur budget mensuel significativement impacté.

• Impact direct sur le budget des familles : Des exemples concrets illustrent l'ampleur de l'augmentation. Élodie et Éric, un couple avec un enfant, ont vu le prix du repas passer de 4,50 € à 7 €, soit une augmentation de 2,50 € par repas. Pour un enfant mangeant 4 jours par semaine, cela représente une hausse de 126 € par mois, et "sur un an, ça fait 1200 euros". Cette dépense supplémentaire est qualifiée de "fichue" et "énorme".
• Répercussions sur les activités familiales : Face à cette hausse, les familles sont contraintes de réduire d'autres dépenses. Élodie confie qu'il y aura "moins d'activités pour les enfants". Par exemple, les sorties au restaurant ou dans les parcs d'attractions seront moins fréquentes, au profit de pique-niques avec des sandwichs.
• Impact généralisé : Des "millions de parents" devraient recevoir des factures de cantine "un peu plus salée que l'an dernier", avec une augmentation moyenne de "10% de plus en moyenne en France".
• Recherche d'alternatives : Certains parents, comme Vanessa, cherchent des solutions pour contourner ces coûts élevés. Elle a décidé que sa fille mangerait à la maison deux jours par semaine lorsqu'elle est en télétravail. Cela lui permet d'économiser "32 euros par mois", soit "320 euros sur 10 mois", une "économie non négligeable".
• Mécontentement et actions : Le mécontentement est tel que des actions sont envisagées. Vanessa et d'autres parents envisagent de "boycotter la cantine pendant une semaine pour que la mairie prenne en charge sur son budget une partie de la hausse des tarifs".
• Préférences des enfants : Les enfants eux-mêmes peuvent préférer manger à la maison, comme en témoigne la fille de Vanessa : "Je peux manger des trucs que j'aime mais que je suis sûr que je pourrais manger au moins alors qu'à la cantine des fois bah j'ai pas envie de manger parce que c'est des trucs que je n'aime pas."

2. Les collectivités face à un casse-tête financier

L'explosion des coûts des denrées alimentaires et de l'énergie met les collectivités locales dans une situation financière délicate.

Un repas de cantine coûte en moyenne "environ 14 euros si on compte les coûts de production, le transport et le personnel encadrant."

• Hausse des prix des matières premières : Les prix de certains ingrédients ont grimpé en flèche. Un repas qui coûtait 2,55 € en ingrédients il y a un an coûte aujourd'hui "37% de plus".
• Moutarde : Le prix du seau de moutarde est passé de "5 euros" à "15 euros".
• Huile de tournesol : Le bidon, négocié à "7 euros 48", est désormais payé "16 euros", soit "quasi 2 fois plus".
• Viandes : La saucisse de veau est à "12-13 € le kilo", et le gigot à "18 € le kilo".
• Rationnement et contrôle des stocks : Les responsables des stocks, comme Samir, doivent rationner les produits devenus chers et rares. Il conserve ces denrées "précieuses" dans un local dont il est le seul à avoir la clé, pour "réfréner les ardeurs des cuisiniers" et éviter le gaspillage ou l'utilisation excessive.
• Contraintes des marchés publics : Bien que les fournisseurs aient obtenu une "autorisation exceptionnelle de l'État" pour augmenter les prix en dehors des tarifs fixes négociés annuellement, les collectivités subissent ces augmentations. "On n'a pas le choix que de subir cette augmentation."
• Déficit budgétaire : Le budget alimentaire de la cuisine centrale de Fontenay-sous-Bois est déficitaire de "3,46%", et c'est "la première année où on va dépasser le budget alimentaire de la caisse des écoles". Pour continuer à servir 50% de bio, il faudrait "environ 60 000 euros supplémentaires" par rapport à 2022.

3. Ajustements et compromis sur la qualité des repas

Pour compenser les augmentations, les cuisines centrales et les villes sont contraintes de revoir leurs pratiques, ce qui a des implications sur les menus et la gestion des stocks.

• Modification des menus : Pour maîtriser les coûts, les nutritionnistes et chefs de production doivent faire des compromis sur les ingrédients.
• La saucisse de veau et le gigot d'agneau sont les "deux protéines animales" que la nutritionniste doit "défendre" face aux impératifs budgétaires.
• Le gigot d'agneau à "18 € le kilo" est remplacé par du jambon "deux fois moins cher". Cette décision impacte "l'éveil au goût des enfants", mais l'équipe doit "s'adapter aux contraintes de production financière etc."
• Les potages, peu coûteux, sont privilégiés pour les "grosses journées".
• Réduction des quantités et chasse au gaspillage : Les chefs cuisiniers doivent être extrêmement précis dans les quantités.
• Le chef Léo a réduit sa consommation d'huile "de la moitié", passant de 5 litres à "2 litres et demi, 3 litres pas plus" pour la réalisation d'omelettes.
• Les cuisiniers doivent respecter un grammage strict, par exemple "3 kg maximum" pour la ratatouille pour 15 enfants, avec une marge de seulement "50 g". "Faut pas qu'il y en ait trop parce que trop puisse trop plus trop va nous à la fin ça fait moins."
• Défi de l'anticipation des effectifs : La gestion des repas est complexifiée par l'imprévisibilité du nombre d'enfants mangeant à la cantine. Claude, le chef de production, reçoit des rectifications quotidiennes des écoles.
• Livrer trop de repas entraîne des pertes importantes, comme les "soixantaine de repas" gaspillés un lundi, représentant "267 euros d'aliments gâchés", car "pour des raisons sanitaires Claude ne peut pas récupérer les repas".
• À l'inverse, des prévisions trop basses peuvent entraîner un surcroît de travail urgent pour les cuisiniers, comme les "249 repas supplémentaires" à préparer en urgence pour une seule école.

4. Options et décisions politiques

Les maires et les responsables financiers sont confrontés à un choix difficile pour maintenir la qualité des services sans surcharger les contribuables ou les parents.

• Les deux options du maire : Le maire de Fontenay-sous-Bois, Jean-Philippe Gautret, n'a que "deux options" pour compenser le déficit : "Est-ce qu'on augmente les tarifs ? Est-ce qu'on augmente les impôts ?"
• Inflation galopante : Il souligne l'impossibilité économique de faire face à une telle situation : "L'inflation égale aupante, c'est économiquement en termes de gestion impossible à tenir pour personne, aucune organisation normalement constituée ne peut faire face à ce type d'inflation galopante."
• Décisions contrastées des municipalités :À Fontenay-sous-Bois, le maire a décidé de "ne pas augmenter les tarifs de cantine" jusqu'à la fin de l'année scolaire, malgré le déficit.
• À Chennevières-sur-Marne, à l'inverse, "les parents doivent déjà payer plus cher", ce qui pousse certains à trouver des solutions alternatives.
• En conclusion, l'augmentation des coûts de la cantine scolaire est une problématique complexe qui impacte lourdement les budgets familiaux et municipaux, forçant des ajustements dans la gestion, les menus et, potentiellement, la qualité des repas, tout en soulevant des questions sur l'équité d'accès à la restauration scolaire.

Briefing sur la production et la consommation des repas en cantine scolaire

Ce briefing examine en détail le processus de préparation, de distribution et de consommation des repas dans les cantines scolaires françaises, en se basant sur l'exemple de la cuisine centrale d'Antony.

Il met en lumière les défis industriels, les contraintes sanitaires, les efforts pour promouvoir une alimentation équilibrée et la réalité du gaspillage alimentaire.

1. La complexité de la production industrielle

La préparation des repas pour les cantines scolaires est une opération logistique et industrielle de grande envergure, impliquant de nombreux acteurs et processus.

A. La cuisine centrale : une véritable usine alimentaire

• Capacité de production massive : La cuisine centrale d'Antony, employée par une entreprise de restauration collective, prépare 1,3 million de repas par an pour 42 écoles. Un seul plat comme le bœuf bourguignon représente 800 kg de viande pour 5500 enfants.
• Contraintes sanitaires strictes : L'ingestion de bactéries telles que la salmonelle ou le staphylocoque entraîne une centaine d'intoxications alimentaires par an dans les écoles françaises. Pour prévenir cela, des mesures drastiques sont appliquées :
• Décontamination : "Passer cette porte, la chasse aux bactéries est ouverte".
• Contrôle de la chaîne du froid : Les livraisons, comme les 7200 yaourts, sont systématiquement contrôlées pour s'assurer que la température à cœur du produit ne dépasse pas 6 degrés. Au-delà, la marchandise est renvoyée.
• Cellule de refroidissement : Cette machine est "impossible" de s'en passer. Elle réduit la température de 63°C à 10°C en moins de 2 heures pour freiner la prolifération bactérienne et étendre la durée de vie des produits à 5 jours. Les repas sont ensuite stockés entre 0 et 3 degrés.
• Interdiction des œufs en coquille : Pour la pâtisserie, les œufs liquides sont utilisés car les œufs en coquille sont "interdits en centrale [car] c'est quand même plein de bactéries".
• Optimisation des processus et des coûts :Surgelés et prédécoupés : Pour gagner du temps et réduire le besoin en main-d'œuvre, une grande partie des ingrédients, comme les garnitures aromatiques et les légumes (haricots verts, oignons, ail), sont surgelés et prédécoupés : "Tout le reste est surgelé... tout faire sur place demande beaucoup de temps et énormément de main d'œuvre".
• Recettes adaptées : Les recettes sont conçues pour des productions de masse, utilisant par exemple de la margarine à la place du beurre (moins cher) et du chocolat en pistole (fond plus facilement).

B. Des exigences de qualité variables et des coûts cachés

• Exigences des municipalités : La mairie d'Antony exige des produits de qualité supérieure, tels que de la viande d'origine française ("50% plus cher que la viande européenne"), de la volaille Label Rouge et un produit bio par jour. Le prix payé par la mairie est de 8,95€ par repas, "plus que la moyenne nationale".
• Secret industriel : Le prix de revient d'un repas pour l'entreprise de restauration collective reste un "secret industriel".
• Réglementation des apports nutritionnels : Pour lutter contre l'obésité, les apports protéiques quotidiens sont réglementés (80g pour un élève de primaire, 60g en maternelle).

2. L'innovation au service de l'équilibre alimentaire (et de la ruse)

Face à la réticence des enfants envers certains aliments, les entreprises de restauration collective développent des stratégies innovantes pour masquer les légumes et les fruits.

A. "Monsieur Innovation" et les recettes "mystères"

• Disguiser les légumes : Un "Monsieur innovation", ancien pâtissier, est chargé d'intégrer des légumes dans des plats appréciés des enfants. L'exemple le plus frappant est le "cake mystère" au chocolat : "si le cake s'appelle mystère c'est parce qu'il y a un ingrédient surprise : de la courgette noyée dans le chocolat". L'idée est de le dire "en fin de repas : vous avez mangé la courgette".
• Jouer sur les noms : La "brunoise de légumes agrémentée d'un crumble aux épices" est rebaptisée "crumble du jardin" car "si je dis brunoise de légumes avec un crumble, l'enfant quand il va entendre brunoise de légumes, ça va être compliqué. Moi j'ai pris un nom un peu plus poétique, j'ai mis crumble du jardin".
• Adapter les classiques : Les pizzas peuvent être garnies de "poivrons", la "terreur des enfants", mais sous une forme attractive.
• Intégrer les fruits : Un challenge est de faire manger des fruits aux enfants, qui ont une "certaine réticence aux fruits entiers", en les intégrant dans des gâteaux comme le cake à la mirabelle.

B. Les enfants, juges ultimes des recettes

• Tests de validation : Des recettes sont testées auprès d'enfants dans une cantine scolaire. Si "70% des testeurs valident une recette, elle sera distribuée 6 mois plus tard dans les écoles à environ 1 million d'enfants".
• Verdict mitigé : Le "crumble du jardin" est un échec ("sans appel") et le cake mirabelle aussi ("décidément les fruits ont du mal à passer"). En revanche, la pizza végétale avec les poivrons est "validée".

3. La réalité de la distribution et du gaspillage alimentaire

Malgré les efforts de production et d'innovation, la consommation des repas en cantine est confrontée à des défis logistiques et comportementaux, entraînant un gaspillage significatif.

A. La gestion des effectifs et l'imprévisibilité

• Prévisions complexes : Lucy, responsable de la logistique, doit prévoir le nombre de repas à servir, un véritable casse-tête influencé par des facteurs imprévisibles comme les épidémies de grippe, les veilles de vacances, ou même la météo.
• Flexibilité des parents : Dans cette ville, les parents peuvent décider le matin même si leur enfant déjeune ou non à la cantine, ce qui rend les prévisions d'autant plus difficiles.
• Pertes financières : Lors d'une journée pédagogique, 200 repas sont perdus, représentant "1500€ de manque à gagner pour le groupe de restauration collective". Ces repas "vont à la poubelle".

B. Le moment du repas : entre incitation et frustration

• Personnel limité : Dans les écoles, le personnel est réduit (trois employées pour servir 152 repas).
• Rôle des animateurs : Les animateurs, comme Tatiana, ont pour rôle de s'assurer que les enfants "mangent bien équilibrés", mais leur "marge de manœuvre est assez limitée".
• Goûter, mais sans forcer : L'obligation n'est pas de "tout manger", mais de "goûter un tout petit peu". "On peut pas non plus les forcer à manger les enfants, mais faut juste qu'ils goûtent, ça, le plus important".
• Succès et échecs à table : Les carottes bio et les haricots verts surgelés ont "visiblement pas beaucoup de succès". Le cake mystère, en revanche, s'en sort "plutôt bien", son ingrédient secret n'ayant pas été démasqué, bien que certains enfants puissent deviner ou ne pas apprécier la courgette une fois révélée.

C. Le gaspillage, une douloureuse réalité

• Assiettes à peine entamées : Les employés "jettent des assiettes à peine entamées", ce qui leur "fait mal".
• Le "baromètre" de la poubelle : Le niveau de gaspillage est un indicateur de la qualité du repas. "Moyen moins et du BFI moyen cela sous-entend qu'on en a jeté la moitié".
• Chiffres alarmants : Selon des études, une école de 500 élèves jette "150 kg de nourriture par jour". Pour la cantine d'Antony, sur 152 repas servis, "entre 100 et 120 kg ont été jetés à la poubelle", soulignant l'ampleur du problème.
• En conclusion, la restauration scolaire est un système complexe, régi par des normes strictes et des objectifs ambitieux d'équilibre nutritionnel. Si l'industrialisation permet de servir un grand nombre d'enfants, elle se heurte à la réalité des goûts des enfants et à des défis logistiques qui mènent à un gaspillage alimentaire conséquent, malgré les efforts et les innovations mises en place.

Analyse Détaillée des Repas en Cantine Scolaire et d'Entreprise : Enjeux et Perspectives

Ce documentaire analyse les défis et les innovations liés à la production et la distribution des repas en cantine scolaire et d'entreprise en France, en s'appuyant sur des extraits de l'émission "Cantines scolaires : ce que pensent vraiment nos enfants de ces repas | 750GTV".

Il met en lumière les pressions financières, les contraintes réglementaires, les préoccupations sanitaires, les efforts pour réduire le gaspillage et l'évolution des attentes des consommateurs.

1. La Crise des Cantines Scolaires : Augmentation des Coûts et Impact sur les Familles

L'inflation actuelle, notamment l'explosion du coût des denrées alimentaires et de l'énergie, pèse lourdement sur les budgets des cantines scolaires et des ménages.

1.1. L'Augmentation des Tarifs : Un Fardeau pour les Parents

• Impact Financier Direct : Des familles comme celle de Mélodie et Éric voient le prix du repas passer de 4,50 € à 7 €, soit une augmentation de 2,50 € par repas. Pour leurs deux enfants, cela représente "126 € 80" par mois, soit "1200 €" supplémentaires par an.
• Répercussions sur le Budget Familial : Cette hausse force les parents à "restreindre" leurs activités familiales. Mélodie et Éric déclarent : "on ira pas au Center Park où on ira mais on ira avec nos sandwichs". Vanessa, mère célibataire, a vu ses dépenses augmenter en cascade (carburant, gaz, électricité, alimentation) et l'augmentation de 50 centimes par repas à la cantine a été "la goutte d'eau qui l'a amené à prendre une décision radicale".
• Stratégies d'Économie des Familles : Face à l'augmentation, certains parents retirent leurs enfants de la cantine pour les faire manger à la maison. Vanessa prépare des repas à moins de 2 € par enfant, contre 5 € à la cantine, réalisant un gain de "30-32 € en fait par mois", soit "320 €" sur 10 mois.
• Boycott en Vue : Certains parents envisagent de "boycotter la cantine pendant une semaine pour que la mairie prenne en charge sur son budget une partie de la hausse des tarifs".

1.2. Les Collectivités Locales sous Pression

• Coût Réel d'un Repas : Un repas de cantine coûte environ "14 €" si l'on inclut les coûts de production, le transport et le personnel encadrant.
• Défi pour les Communes : Les collectivités doivent faire face à un "casse-tête financier" et cherchent "par tous les moyens à faire des économies". Le budget de la cuisine centrale de Fontenay-sous-Bois est déficitaire de "3,46 %", la première année où il "va dépasser le budget alimentaire de la Caisse des écoles".
• Choix Difficiles pour les Maires : Le maire de Fontenay-sous-Bois, Jean-Philippe Gautier, se retrouve face à deux options : "Est-ce qu'on augmente les tarifs ? Euh, est-ce qu'on augmente les impôts ?". Il souligne que "si l'inflation est galopante c'est économiquement en terme de gestion impossible à tenir pour personne". Pour l'année scolaire en cours, il a décidé de ne pas augmenter les tarifs.
• 1. Les Enjeux de la Production en Cantine Scolaire : Entre Contraintes et Innovations
• La production des repas en cantine scolaire est un exercice d'équilibriste entre respect des normes, contraintes budgétaires et efforts pour améliorer la qualité.

2.1. L'Explosion des Prix des Matières Premières

• Rationnement et Nouveaux Produits de Luxe : Samir, responsable des stocks, constate que "Certains produits sont devenus très chers et très rares". Il donne l'exemple de la moutarde dont le prix est passé de "5 € le seau" à "15 €". L'huile de tournesol, un "nouveau produit de luxe", est passée de "7,48 € le bidon" à "16 €", "quasi deux fois plus". Samir doit rationner ces denrées.
• Impact sur la Variété des Menus : Pour compenser, certains aliments sont retirés de l'assiette des enfants. Le "gigot d'agneau à 18 € le kilo" est remplacé par le jambon, "deux fois moins cher".

2.2. Respect des Normes Nutritionnelles et Légales

• Obligations Légales : Aurélie, la nutritionniste, veille à ce que les menus contiennent "au moins quatre poissons, huit viandes, 10 fruits et 10 légumes" par mois, une "obligation légale". Les diététiciennes Laura et Magalie du CESCO doivent également respecter ces normes, tout en maintenant un coût moyen des matières premières "inférieur à 2 €".
• Lutte contre l'Obésité : Les apports protidiques quotidiens sont réglementés : "80 g pour un élève de primaire, 60 ans en maternel".

2.3. La Complexité de la Gestion des Stocks et du Gaspillage

• Prévisions Incertaines : Claude, alias "monsieur Chiffre", est obsédé par la question : "Combien d'enfants vont manger à la cantine ?". Chaque matin, il doit ajuster ses prévisions, mais les rectifications peuvent entraîner des "pertes".
• Gaspillage Alimentaire Important : Des repas entiers peuvent finir à la poubelle faute de pouvoir être récupérés pour des raisons sanitaires. Lors d'une "journée pédagogique", 242 repas ont été perdus, soit "1500 € de manque à gagner".
• Chiffres Alarmants : Selon des études, "une école de 500 élèves gaspille 100 kg de nourriture par jour". Lors d'un service avec seulement 152 élèves, "entre 100 et 120 kg ont été jetés à la poubelle".
• Causes du Gaspillage : Le chou-fleur, par exemple, a un taux de gâchis de "50 %", et le plat végétarien de "45 %". Les enfants n'apprécient pas certains légumes, souvent "baignés dans de la sauce à cause de la liaison froide", ce qui altère le goût.
• Solution : la Chasse au Gaspillage : Le CESCO estime qu'en ajustant les quantités, il y aurait "une trentaine de centimes par repas à gagner".

2.4. La Qualité des Repas et l'Industrialisation

• Cuisines Centrales : De nombreuses cantines, notamment dans les écoles, ne font que réchauffer et portionner des plats préparés dans des cuisines centrales. Le bœuf bourguignon et le cake courgette chocolat de l'école Pasquier d'Antony sont préparés "quelques jours" avant et livrés la veille.
• Procédés Industriels : Pour produire "43 000 élèves par jour dans 19 communes", le CESCO utilise une production "quasi industrielle". Le chef cuisinier Daniel décrit : "Avant que j'arrive on prenait de l'eau on mettait de la poudre et on obtenait des sauces qui avaient le goût de la poudre". Désormais, il essaie de revenir à une cuisine plus "comme à la maison".
• Liaison Froide : Les plats subissent un refroidissement express pour être conservés "jusqu'à 5 jours". Cependant, il y a une "perte de goût" avec ce procédé.
• Contenants Plastiques et Risques Sanitaires : Le réchauffage des barquettes en plastique jetables dans le four fait "gondoler" les contenants.

Selon l'ANSES, "des molécules de plastique se mélangeraient alors avec la nourriture", ce qui pourrait être "responsable de pubertés précoces et de cancers infantiles".

Sabrina, une mère, exprime son inquiétude : "on empoisonne ses enfants à long terme".

2.5. Les Efforts pour l'Amélioration de la Qualité

• Loi de 2025 : Fin du Plastique et Plus de Bio : La loi impose "la fin du plastique à la cantine en 2025" et de "nouvelles contraintes" : "chaque année plus de bio, de produits de qualité labellisés et de circuits courts".
• Recherche de Fournisseurs Locaux : Le CESCO cherche de nouveaux fournisseurs, comme Hotman Berou, producteur de yaourt bio artisanal. Il est le "seul producteur local capable de répondre à des critères de qualité draconiens". Cependant, ses yaourts sont "deux fois plus cher que l'industrie agroalimentaire".
• Coûts de la Transition : Le passage au bio et à l'élimination du plastique représente un coût énorme. Le directeur de la régie du CESCO estime que les contenants réutilisables seuls représenteraient "5 millions d'euros" pour 100 000 pièces.

Cela nécessiterait "une nouvelle cuisine", une laverie et des camions supplémentaires. Il demande aux mairies "180 000 € par an en fonctionnement" en plus.

• Implication des Enfants dans le Choix des Menus : Des chefs tentent de faire valider de nouvelles recettes par des groupes d'enfants. Si "70 % des testeurs valident une recette elle sera distribuée 6 mois plus tard dans les écoles à environ 1 million d'enfants". L'objectif est de leur faire manger des légumes, parfois en les "cachant" (comme la courgette dans le cake mystère).

3. Les Cantines d'Entreprise : Un Modèle en Évolution Les cantines d'entreprise connaissent également une transformation, s'adaptant aux nouvelles exigences des salariés.

3.1. Attentes Élevées des Salariés

Qualité et Équilibre : "Fini les plats industriels et bas de gamme avaler sur le pouce ils veulent prendre le temps de manger sain et équilibré". Les salariés sont "de plus en plus exigeants".

Expérience Culinaire : Le groupe Accord propose plus d'une "dizaine de stands" avec des plats variés (Asian, burger, Méditerranée, paella, poisson, végétarien, rôti de sanglier, plateau de fromage avec truffe). Les assiettes et desserts sont dressés "devant le client à la commande", donnant l'impression d'un "vrai restaurant en mode cafétéria".

Prix Abordables : Un plateau coûte 11 €, mais le salarié ne paie que 6 €, le reste étant pris en charge par l'employeur.

3.2. Innovations des Grands Groupes de Restauration Collective

• Service et Logistique Optimisés : Le groupe Accord utilise une application qui indique la fréquentation en temps réel, permettant aux employés d'éviter les files d'attente.

• Valorisation du Fait Maison et des Produits Locaux : Christophe, chef pour Elior, un leader de la restauration collective, met l'accent sur le "fait maison" et les produits frais. Il s'approvisionne chez des producteurs à moins de "200 km". Par exemple, les frites sont produites localement et livrées crues "sous vide", sans congélation, offrant une "fraîcheur extrême".

• Créativité Culinaire : Les chefs comme Mathieu, pâtissier chez Elior, ont conservé leur créativité malgré le volume : "J'ai même apporter des recettes que je faisais dans des restaurants auquels je travaillais avant et que je propose aux clients ici et qui sont super contents".

• Réduction du Gaspillage : Christophe vise des "pertes infimes" en commandant au plus juste et en ajustant les quantités en fonction de l'historique des ventes. "Moi je préfère produire moins Quitte à perdre du temps un petit peu en fin de service pour refaire et voilà on arrive au résultat où les pertes sont infimes".

3.3. Un Cadre de Travail Amélioré

• Lieu de Vie : Les grandes entreprises conçoivent leur siège comme des "start-up de la Silicone Valley", où la cantine est un "lieu design et confortable".
• Bien-être des Salariés : Des espaces de détente avec WiFi, iPad, jeux et même des "fauteuils massants" pour la sieste sont mis à disposition. "une bonne sieste c'est bien 15 20 minutes il y a pas besoin de ce soit plus longtemps Je pense pas que ce soit abusé de faire 15 20 minutes de sieste dans une journée".
• Restauration Haut de Gamme : Au 27ème étage de la tour Accord, un "restaurant gastronomique avec vue sur tout Paris" propose des menus à "32 € élaboré par des grands chefs", attirant les cadres pour leurs rendez-vous d'affaires.

Conclusion

• Le secteur de la restauration collective, qu'elle soit scolaire ou d'entreprise, est en pleine mutation.

Confronté à des pressions économiques inédites, il doit jongler entre l'augmentation des coûts, les exigences réglementaires croissantes en matière de qualité et de durabilité (fin du plastique, plus de bio), et les attentes grandissantes des consommateurs.

Si les cantines scolaires peinent à trouver l'équilibre financier et à réduire le gaspillage, les cantines d'entreprise semblent s'adapter plus rapidement, transformant le repas en un véritable service d'entreprise et un levier de bien-être au travail.

Les innovations et les efforts pour une alimentation plus saine et locale sont palpables, mais leur généralisation dépendra en grande partie de la capacité des collectivités et des entreprises à investir dans ces transformations coûteuses, tout en protégeant le pouvoir d'achat des familles.

How were these identified?

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The following is the authors’ response to the original reviews

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Summary:

The authors aimed to enhance the effectiveness of PARP inhibitors (PARPi) in treating high-grade serous ovarian cancer (HGSOC) and triple-negative breast cancer (TNBC) by inhibiting PRMT1/5 enzymes. They conducted a drug screen combining PARPi with 74 epigenetic modulators to identify promising combinations.

Zhang et al. reported that protein arginine methyltransferase (PRMT) 1/5 inhibition acts synergistically to enhance the sensitivity of Poly (ADP-ribose) polymerase inhibitors (PARPi) in high-grade serous ovarian cancer (HGSOC) and triple-negative breast cancer (TNBC) cells. The authors are the first to perform a drug screen by combining PARPi with 74 well-characterized epigenetic modulators that target five major classes of epigenetic enzymes. Their drug screen identified both PRMT1/5 inhibitors with high combination and clinical priority scores in PARPi treatment. Notably, PRMT1/5 inhibitors significantly enhance PARPi treatment-induced DNA damage in HR-proficient HGSOC and TNBC cells through enhanced maintenance of gene expression associated with DNA damage repair, BRCAness, and intrinsic innate immune pathways in cancer cells. Additionally, bioinformatic analysis of large-scale genomic and functional profiles from TCGA and DepMap further supports that PRMT1/5 are potential therapeutic targets in oncology, including HGSOC and TNBC. These results provide a strong rationale for the clinical application of a combination of PRMT and PARP inhibitors in patients with HR-proficient ovarian and breast cancer. Thus, this discovery has a high impact on developing novel therapeutic approaches to overcome resistance to PARPi in clinical cancer therapy. The data and presentation in this manuscript are straightforward and reliable.

Strengths:

(1) Innovative Approach: First to screen PARPi with a large panel of epigenetic modulators.

(2) Significant Results: Found that PRMT1/5 inhibitors significantly boost PARPi effectiveness in HR-proficient HGSOC and TNBC cells.

(3) Mechanistic Insights: Showed how PRMT1/5 inhibitors enhance DNA damage repair and immune pathways.

(4) Robust Data: Supported by extensive bioinformatic analysis from large genomic databases.

Weaknesses:

(1) Novelty Clarification: Needs clearer comparison to existing studies showing similar effects.

(2) Unclear Mechanisms: More investigation is needed on how MYC targets correlate with PRMT1/5.

(3) Inconsistent Data: ERCC1 expression results varied across cell lines.

(4) Limited Immune Study: Using immunodeficient mice does not fully explore immune responses.

(5) Statistical Methods: Should use one-way ANOVA instead of a two-tailed Student's t-test for multiple comparisons.

We sincerely thank Reviewer #1 for the insightful and constructive feedback, as well as for the kind acknowledgment of the significance of our work: “These results provide a strong rationale for the clinical application of a combination of PRMT and PARP inhibitors in patients with HR-proficient ovarian and breast cancer. Thus, this discovery has a high impact on developing novel therapeutic approaches to overcome resistance to PARPi in clinical cancer therapy. The data and presentation in this manuscript are straightforward and reliable.” We greatly appreciate the reviewer #1’s thoughtful comments, which have significantly improved the quality of our manuscript. In response, we conducted additional experiments and analyses, and made comprehensive revisions to the text, figures, and supplementary materials. In the “Recommendations for the authors” sections, we have provided point-by-point responses to each of the reviewer’s comments, which were immensely helpful in guiding our revisions. We believe these updates have substantially strengthened the manuscript and have fully addressed all reviewer concerns.

Reviewer #2 (Public Review):

Summary:

The authors show that a combination of arginine methyltransferase inhibitors synergize with PARP inhibitors to kill ovarian and triple-negative cancer cell lines in vitro and in vivo using preclinical mouse models.

PARP inhibitors have been the common targeted-therapy options to treat high-grade serous ovarian cancer (HGSOC) and triple-negative breast cancer (TNBC). PRMTs are oncological therapeutic targets and specific inhibitors have been developed. However, due to the insufficiency of PRMTi or PARPi single treatment for HGSOC and TNBC, designing novel combinations of existing inhibitors is necessary. In previous studies, the authors and others developed an "induced PARPi sensitivity by epigenetic modulation" strategy to target resistant tumors. In this study, the authors presented a triple combination of PRMT1i, PRMT5i and PARPi that synergistically kills TNBC cells. A drug screen and RNA-seq analysis were performed to indicate cancer cell growth dependency of PRMT1 and PRMT5, and their CRISPR/Cas9 knockout sensitizes cancer cells to PARPi treatment. It was shown that the cells accumulate DNA damage and have increased caspase 3/7 activity. RNA-seq analysis identified BRCAness genes, and the authors closely studied a top hit ERCC1 as a downregulated DNA damage protein in PRMT inhibitor treatments. ERCC1 is known to be synthetic lethal with PARP inhibitors. Thus, the authors add back ERCC1 and reduce the effects of PRMT inhibitors suggesting PRMT inhibitors mediate, in part, their effect via ERCC1 downregulation. The combination therapy (PRMT/PARP) is validated in 2D cultures of cell lines (OVCAR3, 8 and MDA-MB-231) and has shown to be effective in nude mice with MDA-MB-231 xenograph models.

Strengths and weaknesses:

Overall, the data is well-presented. The experiments are well-performed, convincing, and have the appropriate controls (using inhibitors and genetic deletions) and statistics.

They identify the DNA damage protein ERCC1 to be reduced in expression with PRMT inhibitors. As ERCC1 is known to be synthetic lethal with PARPi, this provides a mechanism for the synergy. They use cell lines only for their study in 2D as well as xenograph models.

We sincerely thank Reviewer #2 for the insightful and constructive feedback, as well as for the kind acknowledgment of the significance of our work: “Overall, the data are well-presented. The experiments are well-performed, convincing, and supported by appropriate controls (using inhibitors and genetic deletions) and statistics.” We greatly appreciate the reviewer #2’s thoughtful comments, which have significantly improved the quality of our manuscript. In response, we conducted additional experiments and analyses, and made comprehensive revisions to the text, figures, and supplementary materials. In the “Recommendations for the authors” sections, we have provided point-by-point responses to each of the reviewer’s comments, which were immensely helpful in guiding our revisions. We believe these updates have substantially strengthened the manuscript and have fully addressed all reviewer concerns.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

(1) Recent studies have revealed promising synergistic effects between PRMT inhibitors and chemotherapy, as well as DDR-targeting drugs (ref. 89-92). In the discussion, the authors should highlight what is novel in this study compared to the reported studies.

We thank the reviewer for this important comment and fully agree that prior studies have demonstrated the potential of PRMT inhibitors to enhance the efficacy of DNA damage-targeting agents and certain chemotherapies[1-4]. In response to the reviewer’s constructive suggestion, we have now revised the discussion to highlight the novel aspects of our study compared to previously reported findings. Specifically, our work presents several key advances that go beyond prior studies. Below, we would like to emphasize the novelty of our current study as follows:

In the clinic, a strategy termed “induced PARP inhibitor (PARPi) sensitivity by epigenetic modulation” is being evaluated to sensitize homologous recombination (HR)-proficient tumors to PARPi treatments. Together with other groups, we reported that repression of BET activity significantly reduces the expression levels of essential HR genes by inhibiting their super-enhancers[5]. This preclinical discovery is now being assessed in a Phase 1b/2 clinical trial combining the BET inhibitor ZEN-3694 with the PARPi talazoparib for the treatment of patients with metastatic triple-negative breast cancer (TNBC) who do not carry germline BRCA1/2 mutations. Promising anti-tumor activity has been observed in this ongoing trial[6]. Importantly, gene expression profiles from paired tumor biopsies demonstrated robust target engagement, evidenced by repression of BRCA1 and RAD51 mRNA expression, consistent with our preclinical findings in xenograft models. Based on these encouraging results, the trial is being expanded to a Phase 2b stage to enroll additional TNBC patients. Moreover, other combination strategies[7-13] based on this “induced PARPi sensitivity by epigenetic modulation” approach have also shown promising clinical responses in both intrinsic and acquired HR-proficient settings. Notably, these clinical studies indicate that the strategy is well-tolerated, likely due to cancer cells being particularly sensitive to epigenetic repression of DNA damage response (DDR) genes, compared with normal cells.

However, two key clinical challenges remain for broader application of this strategy in oncology: 1) which clinically actionable epigenetic drugs can produce the strongest synergistic effects with PARPi? and 2) can a BRCA-independent approach be developed? To address these questions, we performed a drug screen combining the FDA-approved PARPi olaparib with a panel of clinically relevant epigenetic drugs. This panel includes 74 well-characterized epigenetic modulators targeting five major classes of epigenetic enzymes, comprising 7 FDA-approved drugs, 14 agents in clinical trials, and 54 in preclinical development. Notably, both type I PRMT inhibitors (PRMTi) and PRMT5 inhibitors (PRMT5i) achieved high combination and clinical prioritization scores in the screen. Functional assays demonstrated that PRMT inhibition markedly enhances PARPi-induced DNA damage in HR-proficient cancer cell lines. In line with a strong positive correlation between PRMT and DDR gene expression across primary tumors, we observed that PRMT activity supports the transcription of DDR genes and maintains a BRCAness-like phenotype in cancer cells. These findings provide strong rationale for clinical development of PRMT/PARPi combinations in patients with HR-proficient ovarian or breast cancers. Mechanistic characterization from our study further supports PRMTi clinical development by elucidating mechanisms of action, identifying rational combinations, defining predictive biomarkers, and guiding dosing strategies.

We believe our studies will be of significant interest to the cancer research community for several reasons. First, they address major clinical challenges in women’s cancers, specifically, high-grade serous ovarian cancer (HGSOC) and TNBC, both of which are aggressive malignancies with limited therapeutic options. Second, they offer a novel solution to overcome PARPi resistance. Our earlier discovery of “induced PARPi sensitivity by epigenetic modulation” has already shown promising clinical results and represents a new path to overcome both primary and acquired resistance to PARPi and platinum therapies. Third, they focus on a clinically translatable drug class. Selective and potent PRMT inhibitors have been developed by leading pharmaceutical companies, with more than ten currently in advanced clinical trials. Fourth, they support mechanism-driven combination strategies. Preclinical evaluation of PRMTi-based combinations with other therapeutic agents is urgently needed for future clinical success. Finally, our work highlights understudied but therapeutically relevant mechanisms in cancer biology. In-depth mechanistic analysis of the PRMT regulome is essential, and our studies provide important new insights into how PRMTs regulate transcription, RNA splicing, DNA damage repair, and anti-tumor immune responses in the context of HGSOC and TNBC.

In summary, our study identifies PRMT1 and PRMT5 as key epigenetic regulators of DNA damage repair and shows that their inhibition sensitizes HR-proficient tumors to PARP inhibitors by repressing transcription and altering splicing of BRCAness genes. Distinct from prior strategies, dual inhibition of type I PRMT and PRMT5 exhibits strong synergy, allowing for lower-dose combination treatments that may reduce toxicity. Our findings also nominate ERCC1 as a potential predictive biomarker and suggest that MYC-driven tumors may be particularly responsive to this approach. Collectively, these results offer a mechanistic rationale and translational framework to broaden the clinical application of PARP inhibitors.

(2) In Figures 3H-J, MYC targets were likely to correlate with the expression levels of PRMT1/PRMT5 in various public datasets, supporting previous reports that the Myc-PRMT loop plays critical roles during tumorigenesis (ref. 45). "Myc-targets" signatures were also the most significant signatures correlated with the expression of PRMT1 and PRMT5. The authors suggest that under MYC-hyperactivated conditions, tumors may be extremely sensitive to PRMT inhibitors or PRMTi/PARPi combination. However, the underlying mechanism remains unclear.

We sincerely thank the reviewer for the critical and insightful comments. We fully agree that more direct evidence is needed to establish the regulatory relationship between MYC and PRMT1/5. To investigate the effect of c-Myc on PRMT1 and PRMT5 expression, we analyzed RNA-seq data from P493-6 Burkitt lymphoma cells, which harbor a tetracycline (Tet)-repressible MYC transgene. In this system, MYC expression can be suppressed to very low levels and then reactivated, enabling a gradual increase in c-Myc protein levels[14]. Upon Tet removal to induce MYC expression, we observed a robust upregulation of both PRMT1 (4.3-fold) and PRMT5 (3.6-fold) RNA levels within 24 hours, as measured by RNA-seq. These findings indicate that MYC activation can transcriptionally upregulate PRMT1 and PRMT5. To determine whether this regulation is directly driven by MYC, we further analyzed MYC ChIP-seq profiles from the same cell line following 24 hours of MYC induction. Consistently, we observed remarkably increased MYC binding at the promoter regions of both PRMT1 and PRMT5 genes. Interestingly, MYC’s regulatory influence was not limited to PRMT1 and PRMT5, we also observed transcriptional upregulation of other PRMT family members, including PRMT3, PRMT4, and PRMT6, in response to MYC activation. Together with the data presented in Figure 3H, these new results strongly suggest that MYC directly upregulates the expression of PRMT family genes by binding to their promoter regions. Consequently, increased PRMT expression may facilitate MYC’s regulation of target gene expression and splicing in cancer cells. In cancers with MYC hyperactivation, this feed-forward loop may be amplified, creating a potential therapeutic vulnerability. In response to the reviewer’s insightful suggestion, we have further explored how MYC regulates PRMT1/5 and whether this regulation modulates the efficacy of PRMT inhibitors in oncology. These unpublished observations are currently being prepared for a separate manuscript, and we have now incorporated a discussion of these unpublished findings into the revised version of this manuscript. We thank the reviewer again for the thoughtful and constructive comments regarding the MYC–PRMT regulatory axis.

(3) In Figure 5F, ERCC1 expression was unlikely to be reduced in cells treated with GSK025, especially in OVCAR8 cells, although other cells, including TNBC cells, are dramatically changed after treatment.

We sincerely thank the reviewer for the critical and insightful comments. We agree with the reviewer that in Figure 5F, although GSK025 treatment reduced ERCC1 expression, the loading control Tubulin also showed a notable decrease in the OVCAR8 cell line. This may be because Tubulin expression is not specifically affected by the chemical inhibitor GSK025 in this particular cell line, or it may be secondarily reduced as a consequence of PRMT inhibitor-induced cell death. As the reviewer pointed out, this phenomenon was not observed in other cell lines, suggesting that the effect on Tubulin is not specific to PRMT inhibition. To further investigate, we employed CRISPR/Cas9-mediated knockout of PRMT1 or PRMT5 in OVCAR8 cells, a more specific genetic approach to inhibit PRMT activity. In both cases, ERCC1 expression was significantly reduced, whereas Tubulin levels remained stable (Figure 5G). These results support the conclusion that PRMT1 and PRMT5 specifically regulate ERCC1 expression in OVCAR8 cells. The inconsistent effect on Tubulin is likely due to nonspecific cellular responses to chemical inhibition, which are generally more variable and less precise than those induced by genetic perturbation.

(4) In Figure 7H-L, MDA-MB-231 cells were implanted subcutaneously in nude immunodeficient mice to confirm the synergistic therapeutic action of the PRMTi/PARPi combination in vivo. Although PRMT inhibition activates intrinsic innate immune pathways in cancer cells, suggesting that PRMTi treatments may enhance intrinsic immune reactions in tumor cells, the use of nude immune deficient mice means that changes in the tumor immune microenvironment remain unknown.

We sincerely thank the reviewer for the critical and insightful comments. We fully agree with the reviewer that our in vivo experiments using the human cancer cell line MDA-MB-231 in immunodeficient nude mice limit our ability to assess changes in the tumor immune microenvironment. We thank the reviewer for highlighting this important limitation. While the primary goal of the current study was to investigate the therapeutic synergy between PRMT inhibition and PARP inhibition in cancer cells, we would like to take this opportunity to share additional unpublished data that further support and extend the reviewer’s point regarding the immunomodulatory effects of PRMT inhibitors. In syngeneic mouse tumor models, we have observed that the combination of PRMT inhibition and PARP inhibition leads to a more robust anti-tumor immune response compared to either treatment alone. Specifically, we found increased infiltration of CD8⁺ cytotoxic T cells within the tumor microenvironment, suggesting enhanced immune activation and tumor immunogenicity. Furthermore, we have also obtained preliminary evidence that PRMT inhibition can potentiate immune checkpoint blockade therapy. Mechanistically, this may be mediated through the activation of the STING1 pathway and the upregulation of splicing-derived neoantigens, both of which have been implicated in promoting tumor immune visibility. These findings indicate that beyond enhancing DNA damage response, PRMT inhibition may have a broader impact on tumor-immune interactions and could serve as a promising strategy to sensitize tumors to immunotherapy. A separate manuscript detailing these results is currently in preparation and will be submitted for publication as an independent research article. In light of the reviewer’s thoughtful suggestions and in consideration of feedback from Reviewer #2, who recommended removing Figure 6 from the manuscript, we have carefully reevaluated the overall organization of the manuscript. Given the scope and focus of the current work, as well as the desire to maintain a concise and coherent narrative, we decided to move the content originally presented in Figure 6 to the supplementary materials. This figure is now included as Supplementary Figure S5 in the revised version of the manuscript. We believe this change helps streamline the main text while still making the additional data available for interested readers.

(5) In Figures 6-7, a two-tailed Student's t-test was used to determine the statistical differences among multiple comparisons, which should be performed by one-way ANOVA followed by a post hoc test.

We thank the reviewer for this thoughtful and important comment regarding the choice of statistical method. We fully agree with the reviewer that one-way ANOVA followed by a post hoc test is one of the standard approaches for multiple group comparisons. In response to the suggestion, we have performed one-way ANOVA on our data and found that the statistical conclusions are consistent with those obtained from the two-tailed Student’s t-tests. For example, in the first panel of Figure 6A (OVCAR8 treated with GSK715), one-way ANOVA (p = 1.1 × 10^-6), followed by Tukey’s HSD test, confirmed significant differences between control and Olaparib (p = 0.000165), control and GSK715 (p = 0.000145), control and combination (p = 6.067 × 10^-7), Olaparib and combination (p = 0.0003523), and GSK715 and combination (p = 0.0004015), consistent with the conclusions from the two-tailed t-test shown in Figure 6H. Additionally, we would like to explain why two-tailed Student’s t-tests were used in our current study. When comparisons are predefined and conducted pairwise (i.e., two groups at a time), a two-tailed Student’s t-test is statistically equivalent to one-way ANOVA for those comparisons. In our study, each comparison involved only two groups, and we therefore chose t-tests for hypothesis-driven, specific comparisons rather than exploratory multiple testing. This approach aligns with valid statistical principles. All statistical analyses presented in Figures 6-7 were designed to evaluate specific, biologically meaningful comparisons (e.g., treatment vs. control or treatment A vs treatment B). The study was hypothesis-driven, not exploratory, and did not involve simultaneous comparisons across multiple groups. In such cases, the t-test provides a more direct and interpretable result for targeted comparisons. The use of Student’s t-tests reflects the focused nature of the analysis, where each test directly addresses a specific biological question rather than a global group comparison. We sincerely appreciate the reviewer’s thoughtful comments on the statistical methods.

Reviewer #2 (Recommendations for the authors):

(1) If the authors kept the tumors of various sizes in Figure 7I, it would be important to assess the protein and/or mRNA level of ERCC1 to further support their mechanism.

We sincerely thank the reviewer for the insightful comments. We fully agree that evaluating ERCC1 expression in drug-treated tumor samples is critical to support the proposed mechanism. Due to the limited volume of tumor specimens and extensive necrosis observed after three weeks of treatment in the condition used for Figure 7I, we were unable to obtain sufficient material for expression analysis in the original cohort. To address this, we conducted an additional experiment using xenograft-bearing mice (MDA-MB-231 model), initiating treatment when tumors reached approximately 200 mm³ to ensure adequate tissue collection. We also shortened the treatment duration to 7 days to assess early molecular responses to therapy, rather than downstream effects. Consistent with our in vitro results, both GSK715 and GSK025 significantly reduced ERCC1 RNA expression (0.79 ± 0.17, p = 0.03; 0.82 ± 0.11, p = 0.02, respectively), and the combination treatment further decreased ERCC1 expression (0.49 ± 0.20, p = 0.0003), as determined by qRT-PCR. A two-tailed Student’s t-test was used for statistical analysis. In this experiment, we used the same dosing regimen as in the three-week treatment shown in Figure 7I. Importantly, the shorter treatment period and moderate tumor size at treatment initiation minimized necrosis and did not significantly affect tumor growth, allowing for reliable molecular evaluation. We sincerely thank the reviewer for highlighting this important point.

(2) Figure 2G: please explain why two bands remain for sgPRMT1.

We greatly appreciate the reviewer for raising this insightful and important question. As the reviewer pointed out, an additional band appeared after PRMT1 knockdown in OVCAR8 cells using two sequence-independent gRNAs. Notably, this band was not observed in MDA-MB-231 cells. The antibody used to detect PRMT1 (clone A33, #2449, Cell Signaling Technology) is widely adopted in PRMT1 research, with over 65 citations supporting its specificity. Interestingly, previous studies[15] have identified seven PRMT1 isoforms (v1–v7), generated through alternative splicing and exhibiting tissue-specific expression patterns. Of these, three isoforms are detectable using the A33 antibody. We believe the additional band observed upon sgRNA treatment likely represents a PRMT1 isoform that is normally expressed at low levels in OVCAR8 cells. Upon knockdown of the major isoforms by CRISPR/Cas9, expression of this minor isoform may have increased as part of a compensatory feedback mechanism, rendering it detectable by immunoblotting. Because PRMT1 isoform expression is largely tissue-type specific, it is not surprising that the same band was absent in MDA-MB-231 cells, which are derived from a different lineage than OVCAR8 cells. The reviewer raised an important question regarding the role of PRMT1 isoforms in regulating DNA damage response in cancer. We agree this is an intriguing direction and will investigate it further in future studies.

(3) Figure 4D: Please correct the figure legend so the description matches the color in the figure. Red and blue are absent.

We sincerely thank the reviewer for the critical and insightful comments. The figure legend for Figure 4D has been corrected in the revised version of the manuscript to accurately match the colors shown in the figure. We thank the reviewer for pointing out this issue.

(4) Figure 7A and B: please indicate the cell lines used.

We sincerely thank the reviewer for the critical and insightful comments. In Figure 7A and 7B, human embryonic kidney 293T (HEK293T) cells were used due to their high transfection efficiency and widespread application in reporter assays. This information has been incorporated into the figure legend for Figures 7A and 7B.

(5) What is the link with ERCC1 splicing because reduced overall ERCC1 expression is clear?

We sincerely thank the reviewer for the critical and insightful comments. As the reviewer pointed out, although the direct impact of ERCC1 alternative splicing on its protein expression remains to be fully elucidated, it is likely that PRMT inhibition induces aberrant splicing events that result in the production of alternative ERCC1 isoforms with impaired or altered function. These splicing changes may compromise ERCC1’s role in DNA repair pathways. Furthermore, as shown in Figure 4G, we observed a reduction in the total ERCC1 mRNA reads following PRMTi treatment. This decrease may be attributed, at least in part, to the instability of the alternatively spliced ERCC1 transcripts, which could be more prone to degradation. In combination with the transcriptional downregulation of ERCC1 induced by PRMT inhibition, these alternative splicing events may lead to a further reduction in functional ERCC1 protein levels. This dual impact on ERCC1 expression, through both decreased transcription and the generation of unstable or non-functional isoforms, likely contributes to the enhanced cellular sensitivity to PARP inhibitors observed in our study. We believe this represents an important mechanistic insight into how PRMT inhibition modulates the DNA damage response in cancer cells, and further studies are warranted to investigate the precise role of ERCC1 splicing regulation in this context. We thank the reviewer for pointing out this interesting future research direction.

(6) Figure 7J: From the graph, it seems like Olaparib+G715 and G715+G025 have a similar effect on tumor volume (two curves overlap). Please discuss.

We sincerely thank the reviewer for the critical and insightful comments. In the current study, the doses used for single-agent treatments were selected based on prior publications. For example, the dose of GSK715 was guided by a recent study from the GSK group[16]. Our in vitro and in vivo findings, together with previously published data, consistently demonstrate that GSK715 is more potent than both GSK025 and Olaparib. Notably, treatment with GSK715 alone led to significantly greater inhibition of tumor growth compared to either GSK025 or Olaparib administered individually. This higher potency of GSK715 also explains the comparable levels of tumor suppression observed in the combination groups, including GSK715 plus Olaparib and GSK715 plus GSK025. These results suggest that GSK715 is likely the primary driver of efficacy in the two drug combination settings. Importantly, this observation provides a valuable opportunity to further refine and optimize the dosing strategy for GSK715. Specifically, because GSK715 is highly potent, its dose may be reduced when used in combination regimens without compromising therapeutic efficacy. This approach could significantly improve the safety profile of GSK715 by minimizing potential dose-related toxicities, thereby enhancing its suitability for future clinical development in combination therapy contexts.

(7) Discussion: "PRMT5i increased global sDMA levels"-> "... aDMA levels.".

We sincerely thank the reviewer for the critical and insightful comments. In response, we have corrected the sentence in the discussion from “PRMT5i increased global sDMA levels, which suggested that type I PRMT and PRMT5 share a substrate (i.e., MMA) and/or their functions are compensatory” to “PRMT1i increased global sDMA levels, which suggested that type I PRMT and PRMT5 share a substrate (i.e., MMA) and/or their functions are compensatory.” We apologize for the misstatement and have corrected this error in the revised version of the manuscript.

(8) In addition to the methods, add that nude mice were used in the body of the results and the figure legend for Figure 7J.

We sincerely thank the reviewer for the critical and insightful comments. In the revised version of the manuscript, we have added that immunodeficient nude mice were used in both the body of the Results section and the figure legend for Figure 7J, in addition to the Methods section. We thank the reviewer for this helpful suggestion.

(9) Figure 6 can be deleted to focus the manuscript. It does not add to the PARP inhibition story, but only suggests a link to immunotherapy where this has been reported previously PMID: 35578032 and 32641491.

We sincerely thank the reviewer for the critical and insightful comments. Reviewer #1 also raised a related concern regarding the relevance of this section to the main focus of the manuscript. In consideration of both reviewers’ comments, we have decided to move the data previously shown in Figure 6 to the supplementary section as Supplementary Figure S5. This revision allows us to streamline the main text and maintain a clear focus on the core findings related to PARP inhibition. At the same time, we believe the immunotherapy-related observation may still be of interest to some readers. By presenting these results in the supplementary materials, we ensure that this potentially relevant link remains accessible without distracting from the primary narrative of the manuscript. We greatly appreciate the reviewers’ guidance in helping us improve the clarity and focus of our work. We thank the reviewer for the thoughtful suggestion.

References

(1) Dominici, C., et al. Synergistic effects of type I PRMT and PARP inhibitors against non-small cell lung cancer cells. Clin Epigenetics 13, 54 (2021).

(2) O'Brien, S., et al. Inhibiting PRMT5 induces DNA damage and increases anti-proliferative activity of Niraparib, a PARP inhibitor, in models of breast and ovarian cancer. BMC Cancer 23, 775 (2023).

(3) Carter, J., et al. PRMT5 Inhibitors Regulate DNA Damage Repair Pathways in Cancer Cells and Improve Response to PARP Inhibition and Chemotherapies. Cancer Res Commun 3, 2233-2243 (2023).

(4) Li, Y., et al. PRMT blockade induces defective DNA replication stress response and synergizes with PARP inhibition. Cell Rep Med 4, 101326 (2023).

(5) Yang, L., et al. Repression of BET activity sensitizes homologous recombination-proficient cancers to PARP inhibition. Sci Transl Med 9(2017).

(6) Aftimos, P.G., et al. A phase 1b/2 study of the BET inhibitor ZEN-3694 in combination with talazoparib for treatment of patients with TNBC without gBRCA1/2 mutations. Journal of Clinical Oncology 40, 1023-1023 (2022).

(7) Karakashev, S., et al. BET Bromodomain Inhibition Synergizes with PARP Inhibitor in Epithelial Ovarian Cancer. Cell Rep 21, 3398-3405 (2017).

(8) Sun, C., et al. BRD4 Inhibition Is Synthetic Lethal with PARP Inhibitors through the Induction of Homologous Recombination Deficiency. Cancer Cell 33, 401-416 e408 (2018).

(9) Johnson, S.F., et al. CDK12 Inhibition Reverses De Novo and Acquired PARP Inhibitor Resistance in BRCA Wild-Type and Mutated Models of Triple-Negative Breast Cancer. Cell Rep 17, 2367-2381 (2016).

(10) Iniguez, A.B., et al. EWS/FLI Confers Tumor Cell Synthetic Lethality to CDK12 Inhibition in Ewing Sarcoma. Cancer Cell 33, 202-216 e206 (2018).

(11) Shan, W., et al. Systematic Characterization of Recurrent Genomic Alterations in Cyclin-Dependent Kinases Reveals Potential Therapeutic Strategies for Cancer Treatment. Cell Rep 32, 107884 (2020).

(12) Muvarak, N.E., et al. Enhancing the Cytotoxic Effects of PARP Inhibitors with DNA Demethylating Agents - A Potential Therapy for Cancer. Cancer Cell 30, 637-650 (2016).

(13) Abbotts, R., et al. DNA methyltransferase inhibitors induce a BRCAness phenotype that sensitizes NSCLC to PARP inhibitor and ionizing radiation. Proc Natl Acad Sci U S A 116, 22609-22618 (2019).

(14) Lin, C.Y., et al. Transcriptional amplification in tumor cells with elevated c-Myc. Cell 151, 56-67 (2012).

(15) Goulet, I., Gauvin, G., Boisvenue, S. & Cote, J. Alternative splicing yields protein arginine methyltransferase 1 isoforms with distinct activity, substrate specificity, and subcellular localization. J Biol Chem 282, 33009-33021 (2007).

(16) Fedoriw, A., et al. Anti-tumor Activity of the Type I PRMT Inhibitor, GSK3368715, Synergizes with PRMT5 Inhibition through MTAP Loss. Cancer Cell 36, 100-114 e125 (2019).

This work has been peer reviewed in GigaScience (see https://doi.org/10.1093/gigascience/giaf051), which carries out open, named peer-review. These reviews are published under a CC-BY 4.0 license and were as follows:

Reviewer: Abhishek Gogna

Thank you for the submission. The dataset surely holds value for the plant breeding community but my major concerns are (1) the availability of genetic data, (2) non-conformity to MIAPPE standards (https://www.miappe.org/). These restrict value of the otherwise excellent publication. I would welcome a submission addressing these major points. In addition, I have some minor points for specific sections. Please use the strings in quotation marks ("") to locate the specific sections.

1. Context Change of Equipment: Please indicate how the change of equipment from TLS to drone affects data interoperability. "Figure 2, gray bars": Kindly update Figure 2 to clarify the representation of the gray bars.* "Heads were annotated": Does this mean that not all relevant images were annotated? If so, please modify the title to avoid confusion.

2. Description of FAIR: Please revise this section. Both links listed under "Findable" and "Accessible" are eligible for these tags. Please modify "Interoperability" with reference to the publication listed in the "Re-use Potential."

3. Reference measurements "Senescence was": Was this measurement done for all relevant images? Please include this information. "Adjusted genotype means with year calculation": Please add variance decomposition data for traits.

3. Compilation as Data set* "pure GABI-WHEAT set for the extended set": Please revise this sentence for clarity.

1. Heritabilities of intermediate and target traits* "y of the public marker" - Please revise the sentence for clarity.

2. Genomic prediction ability of unseen multi-environment trial* Is the CDC data part of the data publication? Please add this information.6. Example 1 to

6* Please revise all code for consistency and updated results. Also, include the necessary packages required to run the code.7. Availability of Source code and RequirementPlease create connectivity between repositories and add descriptive README files outlining their usage. Additionally, please provide instructions on how individual repositories may be used.I appreciate your attention to these points and believe that addressing them will strengthen your manuscript

This work has been peer reviewed in GigaScience (see https://doi.org/10.1093/gigascience/giaf030 ), which carries out open, named peer-review. These reviews are published under a CC-BY 4.0 license and were as follows:

Reviewer Hardip Patel

Dear Authors, thank you for compiling this resource and the manuscript. I apologise for the delay in my review. I have read the manuscript with great interest. I have some major concerns that need be addressed and a lot of minor concerns. Without line numbers, it was difficult to provide comments. I have chosen to write the part of the sentence that my comment refers to for you to consider for improvements.

Major concerns:

Abstract can include quantitative values for some key results such as the genome size, contiguity (e.g.N50, L90) and quality metrics (e.g. BUSCO) of the genome assembly among other result claims listed in the abstract. Venom as the keyword can perhaps be described/defined. Authors interchangeably use "venom", "toxin", "venom toxin", genes coding venom proteins. I strongly suggest the use of consistent terminologies that are well defined in the manuscript. Methods need elaborate descriptions about reagents, procedures including for library preparations, sequencing machines, library kits and versions, etc. These are relevant for downstream analyses. For all software, list parameters used, even if default, then explicitly state that "default parameters were used". For all software, list version numbers used for analyses. Authors are urged to change "macorsynteny" and "microsynteny" terms to chromosome level and local synteny analyses. This is to avoid confusion related to macro/microchromosomes. "Genomic diversity" analyses use cross-species alignments and variant calling using software and methods developed for same species data. This can introduce significant bias in downstream interpretation and use of the variant data (heterozygosity measure may be). I suggest removal of this section because of lack of accuracy. Discussion of new discovery is largely lacking. I would appreciate if authors contextualized their results with other discoveries in the field. Section headings in Results and Discussions can be changed to reflect main findings instead of "transcriptomics" or "genomic diversity". One of the main findings is about SVMP gene family expansion. However, due to the lack of evidence about assembly accuracy in the region, accurate annotation of copies, and the effect of studying "primary assembly" instead of "haplotype assembly" at this region, I am not convinced of claims made in the paper. Appropriate justification is required for this section. The nomenclature of SVMP genes is confusing. For example, In Figure 4A, they are all labelled as SVMPs with different colours, but then they are labelled as MDCs and MADs in Figure 4b and Supp Figure 6. Please label each gene in each species with consistent names that can reflect orthologous relationship. This is hard to discern, especially without appropriate species labels in Supp Figure 6. Provide MSA files and trees used to infer evolutionary history. In the absence of the sequence alignments, and raw tree file, I am unable to evaluate this section of the manuscript. Please provide all required details for reviewers and readers. ??: It is not clear what authors mean by the word, term, phrase. Please correct them to convey accurate meaning using established and accepted scientific terminologies and English conventions. Minor concerns:

Abstract:

"compousing" ?? "highly expressed toxin genes": in what tissues? "genome-wide diversity" ?? "toxin gene families in venomous species" -> "toxin gene families in venomous snake species" Background: "Such advances in sequencing technologies": remove "Such" "depending on their type, interactions, and the organism": interactions with what? "proteomic (and transcriptomic) approaches": remove parenthesis "to new therapies for human illnesses including but not": since the title contains "medically important", it would be great to include some specific examples here from the literature. "However, venomous snakes are one": remove "However" "therefore, the fundamental model system": change "fundamental" to "useful" "of medical importance by the World Health Organization (WHO) due to their": provide citation "Within venomous snakes, the most medically": restructure the sentence for brevity and clarity. "cytotoxic effects (among others)": remove "(among others)" "conducted using a proteomic approach": clarify what proteomic approach mean here. "Hirst et al., (in review);" : remove this citation "within the Viperidae family posses an available reference": change the word "posses" to something meaningful "Moreover, employing several -omics techniques": be specific about techniques "We deciphered numerous genomic attributes": be specific Methods: Describe how blood was extracted from animals with all details including animal handling techniques, body part etc. "was stored in RNAlater until RNA extraction": source for RNAlater "We extracted gDNA from the blood of a female individual": provide additional details such as the quantity of blood used, thawing process, qty of reagents, especially elution buffer etc. Manufacturer protocols may be suited best for mammalian blood (humans, mice) without nucleus in RBCs unlike snakes. "Then, we sequenced a total of two 8M SMRT HiFi cells, aiming for a ∼30x of coverage, at the University of Leiden": provide details of library preparation, sequencing machine etc. "(including venom glands, tongue, liver and pancreas, among others": Either list all or refer to the table. "RNA libraries were prepared with the VAHTS": Was the library and sequencing strand specific? Provide complete details on these processes. "8M SMRT HiFi cell containing two Iso-seq HiFi libraries": use correct names of these and also include sequencing machine details. "Quality control on HiFi and Illumina reads was assessed using FastQC": correct the phrasing of this sentence "To make an initial exploration of the genome, …..we generated a k-mer profile with Meryl": Explicitly state the purpose of this analysis. "Manual curation was performed with Pretext": cite Pretext properly. Explain decisions of this manual curation. i.e. what evidence was used to join or break contigs. "Then, we ran three iterative rounds of RepeatMasker to annotate the known and unknown elements identified by RepeatModeler and soft-masked the genome for simple repeats": break this sentence into two and explain reasons for running RepeatMasker three times. "We used GeMoMa v.1.9": Include all details about the annotations. This sentence is not sufficient for reproducibility. Were the RNAseq data assembled or provided as raw files to GeMoMa. How were they mapped to the genome assembly f "published: Anolis carolinensis from Alföldi": Remove the word "from" here as citation is sufficient. Provide details of assembly versions, annotation version, database of annotations etc. "Crotalus ruber from Hirst et al., (in review)": remove this citation or list it as personal communication "We previously quality checked and removed the adapters of the RNA-seq data": remove "previously" and provide details on how adapters were removed from RNAseq data "also removed the adapters for the Iso-seq data": Explain how this was performed. "We blast our ..": Change all occurrence of "blast" to "BLAST" and specify parameters, if it was BLASTN or BLASTP or something else. This is not clear at all. "we performed additional annotation steps for venom genes.": Details are not complete for reproducibility. State explicitly what decisions were made and how gene structure was determined. This is the main part of the paper and does require accurate details. "Whole-genome synteny was explored between": synteny by definition refers to being on the same string/chromosome. Therefore whole-genome synteny as a term doesn't make sense given that genome is divided into chromosomes. Revise it to say "chromosomal synteny" "chromosomes assembled in the reverse complement, which were corrected using SAMtools faidx": samtools faidx cannot do this. Explain how this was done. "After adapter trimming and quality control, we mapped our RNA-seq reads": how were adapters trimmed and QC implemented. "Gene counts per gene": change gene counts to read counts "Differential expression analyses were carried out": requires additional details such as filters applied for the count, groups compared, statistical model, multiple testing correction methods. "characterize the venom arsenal of Cerastes gasperettii": change the arsenal word. "Fragmentation spectra were matched against a customized database including the bony vertebrates taxonomy dataset of the NCBI non-redundant database": revise for accuracy "Unmatched MS/MS spectra were de novo sequenced": spectra were sequenced how?? "we used blast, incorporating both toxin and non-toxin paralogs": change blast to BLAST and provide additional details about the tool used "Then, we aligned those regions using Mafft (Katoh": provide coordinates of these regions for future research in each assembly "history for the main groups of toxins (i.e.,": parenthesis is not closed. Close it or remove it. "we also included other non-toxin paralogous genes from nontoxic species (for details about this see Supplementary Information": where do I look into the supplementary information? Be very clear. Provide coordinates of regions that were compared. "When needed, we translated CDS": when was this needed? Explain. "built a phylogeny for each of the toxin groups using Phyml": I presume that this is done with translated CDS sequences in toxin genomic regions. Please clarify. "Heterozygous positions were obtained from bam files with Samtools v1.9": provide details as to how this was done. Samtools doesn't have features to operate at a site level and therefore I am confused. "Filtered reads were mapped against the new reference genome of Cerastes gasperettii using the bwa mem algorithm": bwa mem is designed for same species comparisons. Here you have used it for crossspecies. Provide justification and perhaps biases it may have introduced for distantly related species. "SNP calling was carried out …": This is not appropriate as models assume same species data. You have used cross-species alignments, which can be highly biased. Results and Discussion: "PacBio HiFi (~40x), Hi-C (~60x) and Illumina data (~78x)": change to number of base pairs. 40x for a genome of 2GB is 80GB data and for genome of 1GB size, it is 40GB data. Before sequencing and assembly, the genome size cannot be known. "After manual curation, we enhanced the scaffolding parameters of our genome": what was done as manual curation. Please specify. "∼228 times more contiguous than the Anolis sagrei genome": how is 228 more measured. How is this useful as a metric without the known ground truth. Assemblies can and do have errors. "27,158 different protein-coding genes within our assembly": this seems large compared to other species. Can you elaborate or compare these numbers with other species. "Toxin genes usually found in venomous snakes (see proteome results below) were mainly found on macrochromosomes, although major toxin groups were found on microchromosomes (SVMPs, SVSPs and PLA2; Fig. 1)." : please revise this statement. Two part of the sentence are saying opposite things. Second provide coordinates of these genes as GFF/BED file as supplementary file with their exon structure annotations for others to reuse this information. "showed a great level of similarity between Cerastes gasperettii and Crotalus adamanteus": provide quantitative metrics for "great" level of similarity. "we found several fission events in the A. sagrei genome,": Since A. sagrei genome is not contiguous and chromosome scale, you cannot infer fissions as it may be artefact of non-contiguous assembly. If that is not the case, provide evidence of this. "The last four…": Belongs in methods "Macrosyntenic differences between lizards and snakes": this is very superficial discussion point. Please remove it or strengthen it with evidence. "Heatmap analyses with the most 2,000": Revise this statement. It doesn't make sense. E.g. Heatmap is a visualisation technique and not analyses method. "We studied venom evolution within the most abundant toxin groups": rewrite the sentence for clarity and brevity. "After a thorough manual curation": Explain what was this manual curation process clearly and the purpose of it. "contiguous tandem repeat SVMPs for": Change "repeat" to "array" because tandem repeat has a different meaning in genomics research context. "flanked by the NEFL and NEFM": Unclear if they are both 5' or 3' of toxin genes. Clarify "Microsyntenic analyses showed": change to local synteny "gene copy number variation between": Since these are duplicate copies, clearly state how gene copies were identified. Include details of open reading frames, exon structures, pseudogene status, etc "we can see an expansion in": Describe number of new copies, their status as intact or not, and sequence similarity between copies. Provide evidence that there is no false duplication due to heterozygous allele collapse in the assembly. "More genomic data will indicate if SVMP12": Did you mean SVMP13? "This difference may be expected, as PLA2 only represents around 5% of the proteome for Cerastes gasperettii": This is not true. Proteome doesn't equal to genome in some cases and superficial inference such as this is not warranted. For PSMC analyses, please discuss the effect of mutation rate and generation time. Figures: Figure 1: Add y-axis scales to the circos plot. Figure 1b legend says it is a linkage map, but looks more like HiC contact map. Please edit. Figure 1b legend also says "including the sex chromosomes", which is not consistent with the circos plot. Figure 3A refers to transcriptome and 3b to proteome. Please make this very clear. Figure 4A, C and E, label genes consistent with the phylogenetic trees in supplementary figures so readers can know their genomic arrangements. Figure S4: Discuss why CG1 sample separates from rest of the samples. Seems like a batch effect.

This work has been peer reviewed in GigaScience (see https://doi.org/10.1093/gigascience/giaf049), which carries out open, named peer-review. These reviews are published under a CC-BY 4.0 license and were as follows:

Reviewer: Zexuan Zhu

The paper presents an encoding of the VCF data using Zarr to enable fast retrieving subsets of the data. A vcf2arr conversion was provided and validated on both simulated and real-world data sets. The topic of this work is interesting and of good values, however, the experimental studies and contributions should be considerable improved.1. The proposed method is simply a conversion from VCF to Zarr format. Since both are existing formats, the contributions and originality of this work are not impressive.2. The compression and query performance is the main concern of this work. The method should be compared with other state-of-the-art queriable VCF compressors like GTC, GBC, and GSC.Danek A, Deorowicz S. GTC: how to maintain huge genotype collections in a compressed form. Bioinformatics, 2018;34(11):1834-1840.Zhang L, Yuan Y, Peng W, Tang B, Li MJ, Gui H,etal. GBC: a parallel toolkit based on highly addressable byte-encoding blocks for extremely large-scale genotypes of species. Genome Biology, 2023;24(1):1-22.Luo X, Chen Y, Liu L, Ding L, Li Y, Li S, Zhang Y, Zhu Z. GSC: efficient lossless compression of VCF files with fast query. Gigascience, 2024; 2;13:giae046.3. The method should be evaluated on more real VCF data sets.

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DOCUMENT DE SYNTHÈSE DETAILLE

Objet : Évaluation des psychologues de l'éducation nationale (PsyEN) spécialité « éducation, développement et conseil en orientation scolaire et professionnelle » (EDO).

Source : Extrait du rapport "igesr-rapport-22-23-253b-psychologues-education-nationale-specialite-edo-pdf-194106.pdf", daté de mars 2024, de l'Inspection générale de l’éducation, du sport et de la recherche.

Date : 15 mai 2024

1. Résumé

Le rapport évalue le corps des psychologues de l'éducation nationale (PsyEN), créé en 2017. Ce corps unique regroupe deux spécialités distinctes : « éducation, développement et apprentissages » (EDA) et « éducation, développement et conseil en orientation scolaire et professionnelle » (EDO), cette dernière étant l'objet principal du rapport.

La création de ce corps visait à unifier les psychologues scolaires et les conseillers d'orientation psychologues, mais le rapport constate un manque d'unité professionnelle et un cloisonnement persistant entre les deux spécialités.

Les PsyEN EDO sont confrontés à une tension entre leurs missions d'orientation et la demande croissante d'aide psychologique pour les élèves, notamment en raison de l'augmentation des problèmes de santé mentale chez les jeunes.

Leur expertise de psychologue est inégalement sollicitée, et le pilotage actuel ne couvre pas suffisamment cet aspect de leurs missions.

Le rapport souligne la nécessité de redéfinir clairement leurs rôches, de renforcer leur positionnement en tant qu'experts psychologues, et de repenser la gouvernance et la formation pour mieux répondre aux besoins des élèves et des équipes éducatives.

2. Constats Principaux

• Manque d'Unité du Corps des PsyEN (1. Un corps unique de psychologues sans réelle unité… ; Synthèse ; Conclusion)
• Le corps des PsyEN, créé en 2017, a réuni deux métiers très différents (psychologue scolaire et conseiller d'orientation psychologue) sous un même statut, mais "le corps unique manque singulièrement d’unité, et les agents relevant des deux spécialités travaillent très peu ensemble." (Synthèse).
• Les deux spécialités (EDA et EDO) opèrent à des niveaux scolaires différents (premier degré pour EDA, second degré pour EDO) et selon des organisations indépendantes.

Il n'y a "aucune activité commune ou conjointe, et parfois même on constate un défaut de continuité dans le suivi des élèves entre le premier et le second degré". (1.1. … pour des métiers différents et cloisonnés …).

• Le cadre statutaire de 2017, qui visait à créer un corps de psychologues au service du développement psychologique, social et cognitif des enfants et adolescents, n'a pas été pleinement accompagné par l'institution dans son évolution, en dehors des aspects de recrutement, formation initiale et évaluation. (Synthèse ; Conclusion).

• Évolution des Missions et Faible Lisibilité (2. Une profession en mutation devenue peu lisible ; Synthèse)

• Le statut de PsyEN EDO de 2017 a explicitement intégré la dimension psychologique de leur métier, ce qui n'était pas le cas pour les anciens Conseillers d'Orientation Psychologues (COP) où l'expertise psychologique était implicitement au service de l'orientation.

"Ainsi, les PsyEN « mobilisent leurs compétences professionnelles au service des enfants et des adolescents pour leur développement psychologique, cognitif et social. »" (2.1. De COP à PsyEN EDO, une évolution statutaire des missions).

• Malgré cette évolution statutaire, "Pour la plupart des interlocuteurs de la mission, les PsyEN sont vus comme des conseillers d’orientation psychologues qui ont changé de nom, avec toutefois une compétence de psychologue qui est davantage identifiée, notamment par les personnels santé-sociaux." (2.2.2. Des représentations sociales et des organisations qui n’ont pas évolué). Les enseignants, craignant que le titre de psychologue n'effraie les élèves, continuent d'utiliser la dénomination "conseiller d'orientation".

• Les PsyEN ressentent un "sentiment d’émiettement de leur activité, voire de saupoudrage" et souffrent d'être constamment en réaction aux urgences, ne parvenant pas à "prendre le contrôle de leur agenda et inscrire leur action dans l’anticipation et la construction d’une vision davantage stratégique." (3.1. Des compétences appréciées dans les établissements). Ils souhaitent une clarification de leurs attentes.

• Recul du Volet Orientation et Tension des Missions (4.3. Des composantes professionnelles en tension avec un recul du volet accompagnement à l’orientation)

• Les PsyEN EDO sont "moins disponibles pour la mission générale de conseil en orientation" (Synthèse), en partie en raison d'un paysage institutionnel complexifié par le rôle des régions et l'intégration de temps dédiés à l'orientation dans les emplois du temps des élèves.

• La majorité de leur temps est consacrée aux bilans psychologiques et aux entretiens individuels, en réponse aux demandes des élèves, familles ou signalements des équipes pédagogiques. (4.3.

Des composantes professionnelles en tension avec un recul du volet accompagnement à l’orientation).

• Ils reconnaissent traiter "beaucoup moins la partie dédiée à l’information générale de tous les élèves, d’une part, par manque de temps au regard des choix qu’ils doivent opérer, d’autre part, car ce rôle est dévolu à d’autres acteurs, notamment les régions". (4.3. Des composantes professionnelles en tension avec un recul du volet accompagnement à l’orientation).

• Besoins Croissants en Aide Psychologique et Santé Mentale (4.2. Des besoins croissants en matière d’aide psychologique ; Synthèse)

• Le rapport met en évidence "l’augmentation du mal-être des élèves, notamment des filles, au fur et à mesure de l’avancée dans leur scolarité". (4.2.1. Des chiffres qui confirment la priorité à accorder à la santé mentale et au bien-être à l’école).

• Il y a une "nécessité de développer les compétences psychosociales des élèves et de mettre en place des protocoles et du secourisme en santé mentale, toutes dispositions qui appellent une expertise de psychologue et des interactions coordonnées avec les acteurs santé-sociaux de l’éducation nationale" (Synthèse).

• Les PsyEN sont de plus en plus sollicités pour des situations de mal-être, de souffrance psychique, de décrochage ou de handicap, car leur expertise est unique pour "repérer, évaluer et prévenir les problématiques de santé mentale et favoriser le bien-être des élèves et leur réussite". (4.2.1. Des chiffres qui confirment la priorité à accorder à la santé mentale et au bien-être à l’école).

• Expertise Psychologique Inégalement Sollicitée et Manque de Pilotage (5. Une expertise de psychologue inégalement sollicitée ; 7. Des missions et une gouvernance à repenser pour répondre aux besoins des usagers)

• Bien que l'expertise de psychologue des PsyEN soit "davantage identifiée dans le second degré" depuis 2017, elle n'est pas "sollicitée à hauteur de ce qu’elle pourrait être" (5.1. Des compétences de psychologue qui s’affirment localement).

• Les PsyEN ne sont pas toujours cités par les parents, élèves ou professeurs comme acteurs identifiés pour le mal-être, en raison de leur faible temps de présence en établissement et de leur identité de conseiller d'orientation. (5.1. Des compétences de psychologue qui s’affirment localement).

• "La dimension psychologique des missions des PsyEN ne figurant pas dans les attributions de ces services [académiques d'information et d'orientation], sinon pour les situations d’orientation relevant de la grande difficulté scolaire, elle n’est pas vraiment pilotée" (5.3. Un pilotage qui ne porte pas sur le volet psychologique des missions).

• Les PsyEN regrettent de ne pas être suffisamment sollicités ou d'être "tête de file des équipes opérationnelles" dans les dispositifs de santé mentale ou de développement des compétences psychosociales, alors que cela relève de leur expertise première. (5.4. Un système de prévention en santé mentale et de développement des compétences psychosociales qui sollicite peu les PsyEN).

• Problèmes d'Organisation et de Gouvernance (4.4. Une activité professionnelle essentiellement itinérante ; 4.5. Des missions qui relèvent de domaines partagés avec d’autres ; 7.2. Des collectifs de travail à organiser aux différents niveaux de pilotage et de mise en œuvre)

• L'activité des PsyEN est "essentiellement itinérante" (4.4.), ce qui entraîne un "sentiment de dispersion avec des quotités horaires par établissement qu’ils jugent insuffisantes". Ils refusent d'être affectés en établissement sous l'autorité du chef d'établissement, arguant de la nécessité d'indépendance du conseil, bien que le rapport estime que l'autorité fonctionnelle porterait sur l'organisation et non sur l'exercice professionnel.

• La répartition des rôles avec d'autres acteurs (santé-sociaux, partenaires externes) n'est pas toujours claire, "ce qui nuit à une vision précise du périmètre d’intervention propre aux PsyEN". (4.5.).

• Les différents acteurs (équipes pédagogiques, santé-sociaux, régions, partenaires) appartiennent à des organisations qui ne travaillent pas toujours en synergie, et leurs cartographies d'intervention sont souvent incompatibles. (7.2.).

3. Idées et Faits Importants

• Le corps des PsyEN est en tension : Créé pour l'unité, il fonctionne en réalité comme deux corps distincts, avec des métiers "cloisonnés aux plans statutaire, hiérarchique et fonctionnel." (1.1.).

• Reconnaissance et sous-utilisation de l'expertise psychologique : Si la compétence psychologique est de plus en plus identifiée, elle n'est pas pleinement exploitée, notamment dans les domaines de la santé mentale et du bien-être des élèves, souvent pris en charge par d'autres acteurs non psychologues. "l’expertise de psychologue, que les PsyEN sont les seuls à avoir dans l’éducation nationale, est davantage identifiée... sans toutefois être sollicitée à hauteur de ce qu’elle pourrait être" (5.1.).

• Évolution des besoins des élèves : Une "augmentation du mal-être des élèves" est confirmée par diverses enquêtes nationales et internationales. Les PsyEN sont essentiels pour identifier, évaluer et prévenir ces problèmes, mais aussi pour accompagner les élèves à "besoins éducatifs particuliers" (4.2.).

• Recentrage nécessaire des missions : Le rapport suggère de recentrer l'activité des PsyEN sur "l’accompagnement des publics rencontrant des problématiques particulières, passagères ou durables, de scolarité, d’apprentissage, d’orientation ou de mal-être." (Synthèse ; Recommandation n° 8).

• L'orientation : une mission partagée : L'accompagnement à l'orientation est désormais largement dévolu aux équipes pédagogiques et aux régions, ce qui libère du temps pour les PsyEN sur des missions à plus forte valeur ajoutée psychologique. Cependant, les enseignants ne s'en saisissent pas toujours par manque de formation. (6.2. et 6.3.).

• Rôle central des CIO pour les publics fragiles : Les Centres d'Information et d'Orientation (CIO) sont devenus des maillons essentiels pour l'accès à la scolarité ou à la formation des publics non scolarisés, en rupture ou sans affectation. (6.6. Des CIO centrés sur l’accompagnement des publics fragiles ou sans solution).

• Importance du pilotage et de la coordination : La complexité de l'exercice professionnel des PsyEN est aggravée par un manque de pilotage cohérent et de coordination des acteurs.

Le niveau du bassin (ou équivalent) est proposé comme le niveau pertinent pour la mise en cohérence des actions et des acteurs. (7.2.2. Le niveau infra-départemental : le bassin).

• La formation initiale doit être repensée : Elle doit être plus modulaire, tenir compte de l'hétérogénéité des stagiaires et inclure davantage de mise en situation responsable, en lien avec les besoins du terrain et les dispositifs d'actualité comme la santé mentale et les compétences psychosociales. (Recommandation n° 14 ; 7.5. La formation initiale : des évolutions à envisager).

• Revalorisation du rôle de DCIO : Le rapport recommande de revaloriser le système indemnitaire des directeurs de CIO (Recommandation n°1), dont la rémunération n'est pas à la hauteur de leur engagement et de leurs responsabilités de management.

• Inquiétudes de la profession : Les PsyEN sont inquiets de leur devenir, face à de multiples rapports qui interrogent leur rôle et positionnement, et redoutent toute évolution qui réduirait leurs missions ou affecterait leur indépendance. (3.3. Une profession inquiète sur son devenir).

4. Recommandations Clés (Liste des préconisations)

Le rapport propose plusieurs recommandations pour réformer le corps des PsyEN EDO et optimiser leur contribution :

• Recentrage des missions sur l'expertise psychologique : Recentrer l'activité des PsyEN sur "l’accompagnement des publics rencontrant des problématiques particulières, passagères ou durables, de scolarité, d’apprentissage, d’orientation ou de mal-être." (R8).

• Renforcement de l'expertise en santé mentale et compétences psychosociales : Mobiliser l’expertise des PsyEN dans les protocoles santé mentale et pour les formations aux compétences psychosociales (R4). Les solliciter pour sensibiliser et former les personnels enseignants et éducatifs aux vulnérabilités des élèves (R2).

• Développement de l'accompagnement à l'orientation par les équipes éducatives : Mettre en place une certification pour le conseil en orientation à destination des enseignants, CPE ou PsyEN EDA (R5). Désigner dans chaque établissement un "référent orientation" (R6).

• Amélioration de la synergie et de la gouvernance :Organiser le travail des PsyEN dans un service coordonné par un PsyEN ayant une compétence en orientation (équivalent de l'actuel DCIO), sous l'autorité fonctionnelle de l'IEN-IO (R11).

• Organiser les coopérations des personnels du secteur santé-social avec les PsyEN des deux spécialités à tous les niveaux fonctionnels (R10).

• Repenser le pilotage de l’orientation et de la santé mentale des élèves en mobilisant les chefs d’établissement et les IEN, et faire du bassin le niveau de cohérence des actions (R13).

• Mettre en synergie les acteurs du SPRO (services régionaux, CIO, services jeunesse) pour l'information et le conseil en orientation (R7).

• Modernisation des outils et de la formation :Instituer des temps de passation de dossiers entre PsyEN EDA et EDO et créer une plateforme numérique sécurisée d'archivage des dossiers (R9).

• Repenser la formation initiale des PsyEN avec davantage de modularité, une implication conjointe d'universitaires, INSPÉ et PsyEN, et un stage en responsabilité dans l'académie d'affectation définitive (R14).

• Revalorisation : Revaloriser le système indemnitaire des directeurs de CIO (R1). Indemniser les PsyEN intervenant en formation des équipes pédagogiques via le "pacte" (R3).

5. Conclusion

Le rapport souligne l'urgence d'une "acte 2" après le décret de 2017, pour clarifier les missions et faire évoluer la gouvernance des PsyEN.

Il appelle à une meilleure exploitation de l'expertise psychologique unique des PsyEN au sein de l'Éducation nationale, en les recentrant sur les besoins spécifiques des élèves vulnérables.

La fusion à terme des deux spécialités (EDA et EDO) est envisagée comme un objectif à long terme pour parvenir à une réelle unité professionnelle, essentielle pour la continuité et la cohérence du suivi des parcours scolaires.

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Rapport IGESR 2024 : La Découverte des Métiers au Collège

Ce rapport, émanant d'une mission d'expertise de l'Inspection Générale de l'Éducation, du Sport et de la Recherche (IGÉSR), examine la mise en œuvre de la "découverte des métiers" en France, en particulier de la cinquième à la troisième.

Il s'inscrit dans un contexte de généralisation de ce dispositif depuis la rentrée 2023, suite à une phase d'expérimentation.

Le document souligne l'importance de cette découverte pour la construction du projet d'orientation des élèves, la lutte contre les déterminismes sociaux et de genre, et le renforcement de l'égalité des chances.

Thèmes Principaux et Idées Clés :

1. L'Impératif d'une Découverte Précoce et Généralisée des Métiers :

Réajustement aux instructions de la loi de juillet 2015 :

Le rapport met en évidence un "réajustement qui tend à s’inscrire dans les instructions de la loi de juillet 2015" pour la mise en place de la découverte des métiers dès la classe de cinquième.

Bien que la généralisation actuelle ne rende pas cette mise en œuvre obligatoire dès la cinquième, la loi impose d'amorcer les parcours dès la sixième.

Les pratiques vertueuses observées débutent effectivement en cinquième ou sixième.

Lutte contre les déterminismes sociaux et de genre : La découverte précoce des métiers est présentée comme "un atout à court et à long terme pour lutter contre les déterminismes sociaux et genrés de l’orientation".

Des études internationales, bien que peu fréquentes en France, montrent que cette introduction précoce "limite de façon significative l’impact de l’origine sociale et du genre sur les parcours scolaires des élèves et les métiers qu’ils exercent à l’âge adulte."

Limiter l'impact de l'origine sociale et du genre : Il s'agit d'un "enjeu majeur de justice sociale" en lien avec la découverte de secteurs d'activités offrant de fortes opportunités, y compris les "métiers d’avenir ainsi que les métiers en tension identifiés par « France stratégie »."

La note de service n° 21 du 25 mai 2023 "fait un lien explicite [...] pour limiter l’impact de l’origine sociale et de genre des élèves dans les choix opérés."

Renforcer l'égalité des chances par l'information des parents : Le rapport souligne le rôle fondamental des "responsables légaux" et la nécessité de les informer et de les impliquer pour "renforcer l’égalité des chances dans les processus d’orientation."

Des exemples d'autres pays (Australie) sont cités pour leur politique de soutien aux parents via des sites internet dédiés.

Lever les inhibitions et renforcer l'ambition : Le processus doit être engagé "tôt dans le parcours des élèves pour réussir à lever les inhibitions" et "modifier significativement l’ambition des parents des milieux sociaux les plus fragiles pour leurs enfants."

Des approches ludiques sont encouragées dès le cycle 3 (CM1/CM2) pour sensibiliser les enfants.

2. Modalités de Mise en Œuvre et Bonnes Pratiques :

• Approches diversifiées, progressives et raisonnées :

Le rapport identifie des "approches diversifiées qui rejoignent les pratiques identifiées comme vertueuses par les résultats de recherches essentiellement internationales."

Elles doivent être "progressives et raisonnées," alternant "exposition, exploration, expérience ou expérimentation."

Diversité des actions : Les actions courantes incluent :

• Fiches métiers : Exercices individuels et partagés, souvent complétés suite à des rencontres avec des professionnels.
• Événements thématiques : "Midis de…", "journée de…", "forums des métiers," "semaine de l’industrie."

L'efficacité des forums est accrue par une préparation et une exploitation pédagogique systématiques.

• Bureaux de Documentation et d'Information (BDI) : Espaces dédiés, souvent animés par des documentalistes, parents d'élèves ou professeurs.

• Intervenants extérieurs : Associations, entreprises, branches professionnelles.

• Visites d'entreprises : Permettent d'appréhender l'organisation de l'activité, l'environnement de travail, les contraintes et les avantages.

Elles doivent être "préparées en amont avec les élèves concernés et font l’objet d’une exploitation pédagogique spécifique."

• Stages d'observation : Le stage obligatoire de cinq jours en troisième est jugé "bienfondé."

Le rapport recommande de "préparer les stages d’observation de la classe de troisième dès la classe de quatrième et donner la possibilité de les réaliser dans plusieurs organisations selon le projet de découverte des métiers du collégien."

Les stages pendant les vacances sont également encouragés, notamment pour les élèves se destinant aux filières professionnelles.

• Lien avec les formations : Les équipes pédagogiques soulignent l'importance de "parler aux élèves des parcours de formation et des voies de formation" une fois qu'ils ont découvert les métiers associés.

Des immersions dans les lycées professionnels, généraux et technologiques, et CFA sont développées pour aider les élèves à "tester in situ à la fois la formation envisageable et l’environnement de l’établissement d’accueil."

3. Le Rôle des Acteurs et la Nécessité d'une Coordination Renforcée :

• Cadre national à formaliser : Il est "fondamental de poser un cadre national énonçant les principes de la découverte des métiers et de l’orientation."

Actuellement, le "cadre national de mise en œuvre du parcours avenir et de la découverte des métiers n’est pas formalisé en France."

Une "stratégie nationale de l’orientation" est préconisée, avec des objectifs pluriannuels et une évaluation de leurs effets.

• Coordination des acteurs : La coordination des "nombreux acteurs censés intervenir auprès des élèves et des étudiants nécessite de préciser ce que les ministères attendent précisément de chacun d’entre eux."

• Rôle des enseignants : Ils constituent le "plus grand nombre" d'acteurs.

Le rapport préconise d'étendre la formation des enseignants à la connaissance des entreprises et à l'accompagnement à l'orientation.

Un "stage obligatoire en entreprise pour tous les étudiants qui suivent les masters qui conduisent aux métiers d’enseignant" est recommandé.

• Psychologues de l'Éducation Nationale (Psy-EN) : Leur engagement est "très inégal" malgré leur expertise. Leur participation active à l'accompagnement des équipes est jugée essentielle.

• Régions : Détentrices de la "compétence d’information" depuis 2018, elles sont des "premiers partenaires pour fournir des informations pertinentes sur les métiers."

Le rapport note cependant une "diversité des situations" et des "malentendus parmi les acteurs de terrain," voire des "tensions" entre CIO et services régionaux.

• Campus des Métiers et des Qualifications (CMQ) : Acteurs clés pour la découverte des métiers, ils "développent des modèles économiques qui permettent ensuite d’étendre leurs offres avec des fonds privés."

Un "appel à projets" est recommandé pour "assurer une meilleure couverture thématique et territoriale."

• Associations et Startups : De nombreuses entités externes, comme AJE, C’Génial, ou JobIRL, interviennent et contribuent à réduire les inégalités sociales dans l'orientation.

• Parents : Leur implication est une "opportunité pour les collèges" mais l'offre doit être complétée pour éviter la "reproduction sociale."

Pilotage académique et au niveau des collèges :

Pilotage académique : Fortement impliqué dans l'expérimentation, avec des "comités académiques" et des formations en cascade.

Cependant, les inspecteurs territoriaux disciplinaires ne sont "pas encore pleinement engagés."

• Pilotage au niveau des collèges : Doit être "renforcé." Il est crucial d'intégrer la découverte des métiers dans la "politique générale du collège," de "mobiliser largement les équipes pédagogiques," et d'"organiser et planifier les temps consacrés au dispositif."

• Référent ou coordinateur : Tendance à se généraliser pour la découverte des métiers au collège.

4. Intégration Pédagogique et Outils :

• Intégration aux programmes disciplinaires : Les pratiques de découverte des métiers intégrées aux programmes sont "encore rares."

Le rapport préconise de "prévoir dans l’écriture de tous les programmes d’enseignement des temps spécifiques sur les métiers, y encourager les approches transversales."

L'objectif est de ne pas ajouter une charge supplémentaire, mais d'intégrer la découverte dans les cours existants.

• Référentiel de compétences à s'orienter : Le développement d'un référentiel spécifique au collège est jugé favorable, mais son usage doit être "accompagné au plus près des usagers" car les professeurs sont "perplexes quant à l’investissement supplémentaire nécessaire."

Ce référentiel devrait permettre de développer des compétences clés, notamment les "compétences psychosociales (CPS)."

• Outils numériques : L'Onisep propose des outils comme FOLIOS et la nouvelle plateforme "Avenir," qui visent à "développer les connaissances et les compétences qui sont nécessaires aux élèves pour construire progressivement un projet d’orientation scolaire et professionnelle."

La plateforme est conçue pour l'éducation progressive au choix, s'appuyant sur trois types de compétences : "les compétences à s’orienter (savoir devenir), les compétences techniques propres à un domaine d’études et d’expérience ainsi que les compétences du XXIe siècle (devenir soi)."

• Évaluation de la progression : L'évaluation des compétences en matière d'orientation "n’est pas encore une priorité."

Il est "essentiel de pouvoir mesurer le niveau de maîtrise des compétences et de suivre la progression des élèves, comme on le fait dans tout autre domaine d’apprentissage."

5. Défis et Recommandations Spécifiques :

• Élèves en risque de décrochage : Dès la classe de quatrième, il est recommandé de "permettre à des élèves en risque de décrochage des immersions en entreprise" et de mettre en place un "dispositif d’accompagnement renforcé."

Ces expériences ont des "effets très positifs [...] dans la remotivation et l’engagement des élèves."

• Financement et mobilité : L'organisation des sorties et visites nécessite des moyens pour les déplacements, ce qui peut se faire "au détriment de leur préparation indispensable."

• Transversalité et cohérence : La stratégie de formation doit être "explicite pour tous" afin de favoriser la "mise en synergie et en cohérence de ce qui se fait au sein des enseignements disciplinaires et dans d’autres espaces d’enseignement."

• Enfants de l'aide sociale à l'enfance : Une recommandation spécifique vise à "préparer un document à destination des services d’aide sociale à l’enfance donnant des conseils pour les décisions d’orientation des élèves qui leur sont confiés" afin de garantir une "orientation juste aussi pour ces élèves, qui sont probablement ceux qui en ont le plus besoin."

• Amélioration de la formation des enseignants : Mettre en place une "certification ou une habilitation pour le conseil en orientation à destination des enseignants, des CPE ou des Psy-EN."

En synthèse, le rapport insiste sur la nécessité d'une approche systémique et coordonnée pour la découverte des métiers, impliquant une généralisation précoce, une intégration pédagogique accrue, une collaboration renforcée entre tous les acteurs (éducation nationale, régions, entreprises, associations, parents), et un soutien ciblé pour les élèves les plus vulnérables, le tout encadré par une stratégie nationale claire et évaluée.

je conseille de présenter l'image d'origine si elle peut être retrouvée, car il semble y avoir désormais beaucoup de version et bruit autour

Note de Synthèse : Relations Police/Population en France – Constats 2024 et Évolutions

Source: Extraits de "https://www.defenseurdesdroits.fr/sites/default/files/2025-06/ddd_EAD-2024_volume-1_relations-police-population.pdf" (Défenseur des droits, "Relations police/population : contrôles d’identité et dépôts de plainte", Juin 2025).

Introduction et Contexte

Le Défenseur des droits, en tant qu'organe externe de contrôle de la déontologie des forces de sécurité, a publié la deuxième édition de son enquête "Accès aux droits" (EAD 2024), actualisant une étude menée initialement en 2016.

L'objectif est d'approfondir la connaissance des atteintes aux droits, notamment en matière de déontologie des forces de sécurité et des relations police-population.

Cette publication se concentre sur trois aspects clés : l'expérience des contrôles d'identité, l'expérience du dépôt de plainte ou de main courante, et la confiance envers l'institution policière.

L'étude de 2016 avait déjà mis en évidence des relations généralement satisfaisantes, mais notait des expériences plus contrastées pour certains groupes sociaux, notamment les jeunes hommes perçus comme noirs, arabes ou maghrébins, qui subissaient des contrôles plus fréquents et souvent dégradés.

Ces expériences négatives étaient corrélées à une faible confiance envers les forces de sécurité.

Une recommandation clé du Défenseur des droits en 2016 était la mise en place d'une traçabilité des contrôles d'identité pour lutter contre les discriminations.

L'édition 2024, menée entre octobre 2024 et janvier 2025 auprès de 5 030 personnes représentatives de la population de France métropolitaine (18-79 ans), utilise une méthodologie comparable à 2016, mais enrichie de nouvelles thématiques (notamment sur le dépôt de plainte).

Elle intègre des variables sociodémographiques détaillées (âge, sexe, origine perçue, religion, orientation sexuelle, handicap) pour une analyse intersectionnelle des discriminations.

Thèmes Principaux et Idées Clés

1. L'Expérience des Contrôles d'Identité

Les contrôles d'identité sont un point de contact majeur entre la police et la population, avec environ 47 millions estimés en 2021.

Leur cadre juridique est jugé "complexe et flou", laissant une "large marge d'interprétation aux forces de sécurité, ouvrant la voie à des usages divers, et parfois controversés".

L'existence de discriminations dans ce cadre a été reconnue à plusieurs reprises par la justice.

• Augmentation significative de la fréquence des contrôles :La proportion de personnes ayant été contrôlées au moins une fois au cours des 5 dernières années est passée de 16 % en 2016 à 26 % en 2024, soit une augmentation de 63 %.

• Cette hausse touche toutes les catégories de population, y compris celles "auparavant peu contrôlées" : +81 % pour les cadres, +148 % pour les 55-64 ans, et +79 % pour les personnes perçues "comme blanches exclusivement".

• En 2024, les contrôles multiples (plusieurs fois sur les 5 dernières années) sont majoritaires (15 % de la population contre 11 % pour un contrôle unique).

• Modalités et justifications des contrôles :90 % des contrôles rapportés en 2024 ont impliqué une vérification des titres d'identité (contre 68 % en 2016).

• Cependant, une part significative des contrôles est "poussée" : 22 % ont fait l'objet d'une fouille, 11 % ont reçu l'ordre de quitter les lieux, 6 % ont été plaquées contre un mur ou une voiture et 3 % ont été emmenées au poste.

• Pour plus d’une personne contrôlée sur deux, le motif du contrôle n’est pas explicité par les forces de sécurité. Seules 42 % des personnes ayant subi un contrôle "poussé" ont bénéficié d'une justification.

• Comportements inappropriés :19 % des personnes contrôlées déclarent avoir été confrontées à des comportements inappropriés (tutoiement, provocation, insultes, brutalité), une proportion qui était de 28 % en 2016 (bien que les questions aient pu évoluer).

• 14 % ont été tutoyées, 7 % provoquées ou insultées, et 7 % ont subi des comportements brutaux.

• Disparités socio-démographiques et discriminations :Les jeunes hommes perçus comme noirs, arabes ou maghrébins sont 4 fois plus à risque d’avoir été contrôlés que le reste de la population, et 12 fois plus à risque de faire l’objet d’un contrôle « poussé » (fouille, palpation, conduite au poste, injonction à quitter les lieux).

• Ils rapportent également plus fréquemment des comportements inappropriés : 30 % d'entre eux contre 15 % des personnes perçues comme blanches uniquement.

• Les personnes financièrement précaires (32 %) sont également plus contrôlées que celles à l'aise financièrement (22 %).

• Les personnes non hétérosexuelles ont 50 % de risque en plus d'être confrontées à des comportements inappropriés lors d'un contrôle d'identité.

• La "marge d’appréciation offerte par le droit actuel laisse les policiers et les gendarmes seuls avec leur propre instinct et leurs éventuels préjugés", ce qui "peut induire des comportements discriminatoires, volontaires ou non, et faire peser une suspicion sur l’ensemble des contrôles".

• Le manque de traçabilité des contrôles d'identité est un obstacle persistant à la preuve des discriminations et à l'effectivité du droit au recours.

• Réactions aux comportements inappropriés :Seules 8 % des personnes ayant subi des comportements inappropriés ont tenté de faire reconnaître la situation (via une association, avocat, Défenseur des droits, police/gendarmerie).

• La majorité (73 %) en a parlé à des proches.

2. L'Expérience du Dépôt de Plainte ou de Main Courante

Le dépôt de plainte est une autre modalité cruciale d'interaction avec les forces de sécurité.

• Fréquence et profil des plaignants :35 % des personnes interrogées se sont rendues dans un commissariat ou une gendarmerie pour déposer une plainte ou une main courante au cours des 5 dernières années.

• Les personnes en difficultés financières, en situation de handicap, ou atteintes de maladies chroniques ont une propension plus élevée à porter plainte.

Comportements non déontologiques lors du dépôt de plainte :21 % des personnes ayant souhaité déposer une plainte se sont heurtées à un refus, alors que le refus de dépôt de plainte est interdit par la loi (Article 15-3 du code de procédure pénale).

• Les refus de plainte touchent plus fréquemment les personnes en situation de handicap (37 %), celles portant un signe religieux (33 %), au chômage (30 %), résidant dans un quartier prioritaire de la politique de la ville (30 %), ou perçues comme noires, arabes ou maghrébines (28 %).

• 10 % des personnes ayant voulu déposer plainte rapportent des comportements inappropriés des forces de sécurité (tutoiement, insultes, humiliation, intimidation).

• Les personnes en situation de handicap ont un risque double d'être exposées à des comportements inappropriés lors d'un dépôt de plainte.

• Les jeunes (18-24 ans) et les personnes perçues comme non-blanches ont également un risque 80 % plus élevé d'y être confrontées.

• Expériences négatives multicontextuelles :Certains facteurs, comme l'origine perçue (noir, arabe, maghrébin), l'âge (jeunes 18-24 ans) et le chômage, surexposent aux comportements inappropriés "aussi bien lors d’un contrôle que lors d’un dépôt de plainte".

Cela "suggère l’existence de comportements discriminatoires car ciblés sur certains groupes sociaux plutôt que d’autres."

3. La Confiance en l'Institution Policière

La confiance se distingue en une confiance "diffuse" (missions générales de la police) et un soutien "spécifique" (évaluation basée sur des expériences concrètes).

L'enquête s'intéresse au soutien spécifique.

• Niveaux de confiance :50 % de la population se dit confiante ou rassurée en présence d'un policier ou d'un gendarme sur la voie publique.

• 28 % sont indifférents et 22 % se sentent méfiants ou inquiets.

• Lien avec les expériences concrètes :La confiance est "étroitement liée" aux expériences vécues : 51 % des personnes ayant pu enregistrer leur plainte sans incident se déclarent confiantes, contre seulement 37 % de celles confrontées à un refus.

• 59 % des personnes ayant vécu des discriminations lors d'un contrôle de police se sentent inquiètes ou méfiantes, contre 21 % de celles qui pensent que les discriminations existent mais ne les ont pas vécues personnellement, et 5 % de celles qui ne reconnaissent pas leur existence.

• Les personnes ayant fait l'expérience de comportements inappropriés (que ce soit lors d'un contrôle ou d'un dépôt de plainte) se déclarent plus fréquemment méfiantes ou inquiètes (respectivement 61 % et 51 %).

• Conséquences du manque de confiance :Le manque de confiance entraîne plus fréquemment une remise en question de la légitimité de l'intervention policière : 16 % des personnes méfiantes protestent lors d'un contrôle, contre 4 % des confiantes.

• Les personnes méfiantes sont plus nombreuses à percevoir le contrôle comme injustifié (59 % contre 18 % des confiantes).

• Une corrélation négative existe entre confiance et recours à la police : 21 % des personnes méfiantes déclarent ne pas avoir contacté les forces de sécurité par manque de confiance suite à une discrimination ou un harcèlement, contre 3 % des personnes confiantes.

• Cela crée une "dynamique délétère" qui "nourrit une défiance mutuelle lors des interactions police/population" et "peut conduire à une escalade des tensions en contexte d’intervention".

Conclusion Générale

L'enquête "Accès aux droits" de 2024 met en évidence une "dualisation des relations" entre les citoyens et les forces de sécurité en France.

Alors que l'expérience du contrôle d'identité s'est généralisée à une plus grande partie de la population, les modalités de ces interactions varient considérablement selon les caractéristiques sociales des individus.

Les catégories de population "traditionnellement" moins contrôlées (femmes, cadres, personnes âgées) sont désormais plus souvent contrôlées, mais généralement via des "simples contrôles d’identité, généralement ponctuels, courtois et perçus comme justifiés."

En revanche, pour les personnes perçues comme noires, arabes ou maghrébines, les jeunes, les hommes et les personnes précaires, on observe une persistance de contrôles plus fréquents, plus intrusifs ("poussés"), et accompagnés de comportements contraires à la déontologie.

Ces groupes sont également plus exposés aux refus de dépôt de plainte et aux comportements inappropriés lors de ces démarches.

Ces expériences négatives et discriminatoires ont un impact direct et significatif sur la confiance envers les forces de sécurité, conduisant à une méfiance accrue, une remise en question de la légitimité des actions policières, et une diminution du recours à la police.

L'étude souligne que cette "érosion de la confiance" peut "nourrir les crispations entre la population et les forces de sécurité et, in fine, peut conduire à une escalade des tensions en contexte d’intervention."

Le Défenseur des droits souhaite que ce rapport "favorise la réflexion pour établir des relations plus apaisées" entre la police et la population.

Programme JADE (Jeunes Ambassadeurs des Droits) 2024-2025 : Synthèse et Thèmes Principaux

Introduction au Programme JADE

• Le programme des Jeunes Ambassadeurs et Ambassadrices des Droits (JADE) est un dispositif d'éducation aux droits, créé en 2006 par le Défenseur des Enfants et piloté par le Défenseur des Droits depuis 2011.

Son objectif principal est de promouvoir les droits de l'enfant, l'égalité et la lutte contre les discriminations, en particulier auprès des jeunes les plus vulnérables.

Le programme s'appuie sur une approche de "pair à pair", où de jeunes volontaires en service civique sensibilisent d'autres enfants et jeunes à leurs droits.

Comme le souligne le rapport, "Trop souvent, la parole et les droits des enfants sont délaissés, alors que c’est avec eux que les droits des générations actuelles et futures doivent se renforcer, se solidifier, se développer."

En 2024-2025, le programme a mobilisé 82 JADE qui ont bénéficié de près de 150 heures de formation et ont effectué 2646 interventions, sensibilisant et formant près de 40 722 enfants à leurs droits.

Le programme JADE incarne la volonté de l'institution d'« aller vers » les enfants et les jeunes, et vise un triple objectif :

Favoriser l'accès aux droits des plus jeunes.

• Informer les jeunes sur le rôle et les missions du Défenseur des Droits.
• Sensibiliser les jeunes à leurs droits et développer leur esprit de citoyenneté.
• Le programme se décline en deux missions principales : les JADE Enfants (sensibilisation aux droits fondamentaux de l'enfant selon la CIDE) et les JADE Égalité (sensibilisation à l'égalité et à la lutte contre les discriminations).

Qui sont les JADE ?

• Les JADE sont des volontaires en service civique âgés de 16 à 25 ans (jusqu'à 30 ans pour les personnes en situation de handicap), issus d'horizons variés.

Le rapport mentionne que leur "richesse de parcours, cette diversité des profils et cette envie partagée de faire bouger les lignes qui font la force du programme JADE."

Ils peuvent être étudiants, en reconversion ou en recherche de leur voie, tous partageant un engagement commun à sensibiliser les jeunes à leurs droits partout en France.

Profil Démographique (Promotion 2024-2025) :

• Niveau de diplôme : 25% Bac, 25% Bac+5, 15% Bac+1, 13% Bac+2, 13% Bac+3, 6% Secondaire, 3% Bac+4.

• Expérience en animation : 77% non, 23% oui.

• Leurs domaines d'études et projets professionnels sont divers, incluant la promotion des droits, le médico-social, l'humanitaire/solidarité et l'éducation spécialisée.

• La mission JADE est formatrice et exigeante, demandant de travailler en collectif, de s'adapter à des publics variés et de transmettre des valeurs fondamentales, tout en développant leurs propres compétences.

Elle attire des volontaires intéressés par les questions sociales et éducatives, leur permettant de rencontrer des professionnels et d'améliorer leurs compétences pédagogiques.

Elle est également attractive pour les profils juridiques souhaitant vulgariser des concepts juridiques et défendre les droits humains.

Principales Évolutions du Programme en 2024-2025 Le programme JADE continue de se consolider et de s'adapter. Plusieurs évolutions marquent l'année 2024-2025 :

Redéploiement Territorial

• Le programme est implanté dans de nombreux départements et régions, avec un total de 82 JADE recrutés (42 JADE Droits des Enfants, 25 JADE Égalité, et 15 JADE Droits des Enfants et Égalité à Mayotte et la Réunion).

Cependant, des difficultés financières dues à la réduction des dépenses publiques ont conduit à des désengagements de certaines collectivités territoriales.

Par exemple, des Conseils départementaux se sont partiellement ou totalement retirés. Pour pallier cela, le Défenseur des Droits a exceptionnellement financé un binôme JADE Enfant en Seine-Maritime et pris en charge la moitié des coûts de l'équipe de Gironde.

Malheureusement, le programme n'a pas pu être maintenu en Île-et-Vilaine malgré son succès.

• À l'inverse, certains territoires renforcent leur engagement.

La Métropole Rouen-Normandie a augmenté son financement, et la DILCRAH (Délégation interministérielle à la lutte contre le racisme, l'antisémitisme et la haine anti-LGBT) a alloué des fonds complémentaires, permettant le recrutement d'une équipe JADE Égalité de quatre volontaires en Seine-Maritime.

Le rapport met également en lumière le "partenariat exemplaire" en Côte d’Or, où le programme JADE fête ses 10 ans, ayant sensibilisé plus de 20 000 enfants et jeunes dans l'ensemble des collèges du département et des structures spécialisées (ASE, PJJ, CHU).

Équipe de Coordination Renforcée

• L'équipe de coordination JADE a été renforcée pour faire face à la croissance du programme, qui accueille désormais une centaine de volontaires en moyenne chaque année.

Ce renforcement vise à "assurer un accompagnement des volontaires de qualité dans leur mission de service civique et de répondre aux attentes des partenaires désireux de mettre en place une équipe JADE sur leur territoire." Alexandra Frontali, chargée de mission JADE, souligne son souhait de s'impliquer dans un projet qui "valorise la sensibilisation entre pairs et qui accorde une attention particulière à la diversité des profils des jeunes sensibilisés : jeunes de l’Aide sociale à l’enfance et de la Protection judiciaire de la jeunesse, jeunes hospitalisés, etc."

Outils de Sensibilisation Retravaillés

Le programme innove constamment dans ses outils. Pour les JADE Égalité, de nouvelles animations ont été développées, telles que :

• "Indiscri" : un jeu d'enquête pour identifier la victime, le critère et le domaine d'une discrimination.
• "La permanence" : une simulation de permanence d'un délégué du Défenseur des Droits pour orienter les réclamants. Pour les JADE Enfants, cinq nouvelles animations ont été créées, ciblant des droits moins abordés et favorisant des formats adaptables :
• Des activités sur le droit à une justice adaptée à l'âge.
• "Les apprentis sorciers de la santé" : un jeu sur le droit au meilleur état de santé possible et la santé mentale.
• "Les maîtres du temps" : une activité sur l'équilibre entre les droits à l'éducation, aux loisirs et au repos.
• Formation Unique et Approche "Pair à Pair"
• Le programme JADE se distingue par une "offre de formation unique dans le champ des services civiques". Sur 9 mois de mission, les JADE bénéficient de 150 heures de formation, un volume et une qualité de contenu qui le démarquent des autres services civiques.

Formation Initiale et Complémentaire La formation initiale se déroule sur trois semaines à Paris (ou localement pour l'Outre-Mer).

Elle vise à doter les volontaires des connaissances et savoir-faire essentiels, tout en créant un esprit de promotion. Les modules couvrent :

• Le rôle et les missions du Défenseur des Droits, avec des échanges directs avec la Défenseure des Droits, Claire HÉDON, et le Défenseur des Enfants, Éric DELEMAR.
• Le contenu juridique spécifique aux missions JADE (droits de l'enfant et lutte contre les discriminations).
• Des rencontres avec des professionnels de la protection de l'enfance (juges des enfants, psychologues, services de l'Éducation nationale, 119, etc.).
• Des techniques d'animation, de prise de parole en public, de gestion de groupe, et l'appropriation du catalogue d'animations, souvent animées par d'anciens JADE.
• L'utilisation d'outils informatiques professionnels.
• Les formations complémentaires et locales permettent d'approfondir des sujets spécifiques aux réalités des territoires. Un exemple notable est la "formation commune aux Jade de Mayotte et de La Réunion" pour la première fois, favorisant les échanges interculturels et une compréhension mutuelle des spécificités locales, avec une double formation sur les droits de l'enfant et la lutte contre les discriminations.

L'Apport de l'Approche "Pair à Pair"

Le modèle "pair à pair" est central au programme. Les JADE, étant eux-mêmes jeunes, peuvent établir un dialogue de confiance plus facilement avec les publics qu'ils sensibilisent.

Cette proximité en termes d'âge et d'expérience facilite les échanges et la transmission des messages. Lilou BENANIBA (JADE Occitanie Ouest - Toulouse) témoigne de l'importance des formations pour "créer un esprit d’équipe et de promotion au cours des nombreux temps favorisant l’échange et le partage entre JADE." Gwenaël MENAN (JADE Île-de-France) souligne comment la mission l'a aidé à "gagner en aisance à l’oral", notamment grâce au soutien du binôme lors des interventions.

Une Vision Universelle : Atteindre les Enfants et les Jeunes dans leur Diversité

Le programme JADE s'efforce d'atteindre le plus grand nombre d'enfants et de jeunes, quelles que soient leur situation ou leur cadre de vie.

Types d'Interventions et Publics Ciblés

Les JADE interviennent dans diverses structures :

• Milieu scolaire : Collèges (6ème et 5ème pour JADE Enfants, 3ème pour JADE Égalité), lycées et apprentis. Les interventions abordent la construction, la définition juridique de la discrimination et les solutions pour la combattre.

• Hors temps scolaire : Structures d'accueil périscolaires ou extrascolaires.

• Interventions spécialisées : Après une formation dédiée, les JADE interviennent auprès de jeunes hospitalisés (services pédiatriques et pédopsychiatriques), en situation de handicap (IME, ULIS, instituts thérapeutiques éducatifs et pédagogiques, services d'aide par le travail), sous la protection de l'Aide Sociale à l'Enfance (ASE), sous Protection Judiciaire de la Jeunesse (PJJ), et auprès de mineurs non accompagnés. Ces interventions sont cruciales pour "informer sur leurs droits et sensibiliser à la notion d’égalité" des jeunes en situation de vulnérabilité.

Participation à des Événements

Les JADE participent également à divers événements locaux et nationaux, représentant leur mission et l'institution, comme le Cross départemental du Calvados, la Journée de l'Engagement à Marseille, les Hauts de Seine Digital Games, ou le projet "jeunes contre le racisme et l'antisémitisme" en Seine-Saint-Denis.

La célébration du 20 novembre (adoption de la CIDE) est un moment fort, où les JADE sont mobilisés pour animer des séances et participer à des événements de promotion des droits de l'enfant.

L'Hôtel de Ville de Lyon a notamment sollicité les JADE pour des ateliers sur le droit à la protection de la vie privée.

Un Programme Piloté par le Défenseur des Droits dans une Logique Partenariale

Le succès du programme JADE repose sur une "logique partenariale" solide entre le Défenseur des Droits et de nombreux acteurs locaux.

Rôle du Défenseur des Droits

• L'institution du Défenseur des Droits assure la conception, le pilotage et le suivi du programme. De nombreux agents de l'institution sont mobilisés pour la formation et le bilan des JADE.

Les pôles régionaux du Défenseur des Droits, avec leurs chefs de pôles et chargés de mission, contribuent à l'organisation des rencontres et formations locales, et au développement du programme sur les territoires en assurant sa promotion auprès des collectivités.

Les "délégués référents JADE", bénévoles de l'institution, jouent un rôle primordial en apportant un "soutien personnalisé et de proximité" aux JADE, en les aidant à démarcher les établissements et en observant leurs interventions.

Partenaires Essentiels

• Collectivités Territoriales : Elles sont "investies" et soutiennent financièrement le dispositif, mettent à disposition des moyens (bureaux, matériel, véhicules) et aident à l'orientation du programme en identifiant les publics cibles et en diffusant le programme via leurs réseaux.

Elles contribuent aussi à la formation locale des JADE, par exemple en organisant des présentations sur le fonctionnement des conseils départementaux ou les services de l'Aide Sociale à l'Enfance.

• Structures Agréées de Service Civique : Quatre associations sont partenaires (Apprentis d'Auteuil, Concordia, Institut international des droits de l'Homme et de la Paix, et Unis-Cité).

Elles sont liées par une convention tripartite et assurent le suivi quotidien, administratif et l'accompagnement individuel des JADE dans leur projet d'avenir.

• Éducation Nationale : Elle participe à la formation locale des JADE, présentant les dispositifs spécifiques d'accueil des élèves (SEGPA, ULIS, UPE2A) et les procédures de lutte contre le harcèlement scolaire.

• Les Paroles Inquiétantes : Une Problématique Croissante

• Un aspect crucial du programme JADE est la gestion des "paroles inquiétantes" que les volontaires peuvent recueillir. Du fait de leur proximité d'âge avec les enfants et jeunes, les JADE sont parfois confrontés à des confidences ou des situations préoccupantes.

Le Défenseur des Droits a mis en place une procédure spécifique pour s'assurer que ces témoignages soient traités par des professionnels compétents. Véronique MAHL, chargée de mission au Pôle Défense des droits de l’enfant et référente Paroles inquiétantes, explique que le pôle "s’assure de la prise en compte de ces paroles par les services compétents pour y donner suite."

Elle ajoute que la juriste peut contacter les assistants sociaux et infirmiers scolaires, les conseillers techniques des académies, les responsables d'accueils de loisirs, et "même si cela est rare, de procéder lui-même à une information préoccupante à la Cellule de Recueil des Informations Préoccupantes (CRIP) ou un signalement au parquet lorsque la situation le justifie."

Le rapport souligne une "augmentation très significative du nombre de paroles inquiétantes reçues" en 2025, avec 173 alertes comptabilisées en mai 2025, contre une moyenne stable de 137 à 151 les années précédentes.

La majorité de ces situations n'était pas connue des professionnels. Cette hausse réaffirme "l’utilité de l’action des JADE et la nécessité de sensibiliser les enfants à leurs droits."

En conséquence, l'équipe de coordination JADE a adapté la procédure, abordant les paroles inquiétantes dès les entretiens de recrutement et renforçant le module dédié lors de la formation initiale.

Conclusion

Le programme JADE du Défenseur des Droits est une initiative éducative vitale qui contribue activement à la promotion et à la défense des droits de l'enfant et à la lutte contre les discriminations en France.

En s'appuyant sur l'engagement de jeunes volontaires et une approche de "pair à pair", il parvient à sensibiliser des dizaines de milliers d'enfants et de jeunes chaque année, y compris les plus vulnérables.

Malgré les défis financiers et la problématique croissante des paroles inquiétantes, le programme continue de se renforcer grâce à des formations de qualité, des outils innovants et un réseau de partenariats solides.

L'implication et l'énergie des JADE constituent "une fierté" pour l'institution, qui s'engage à œuvrer "vers un futur plus juste pour nos enfants et nos jeunes : ils prendront la relève de la défense des droits."

O objetivo deste exercício é ler atentamente o texto proposto e identificar os conceitos que considerar mais relevantes para a análise da arquitectura e do urbanismo. Cada conceito selecionado deve ser comentado de forma crítica, justificando a sua escolha e explicando qual a relação que estabelece com a prática arquitectónica e urbanística. Reflicta sobre a forma como a compreensão desse conceito pode contribuir para o desenvolvimento do pensamento projectual, para a interpretação do espaço construído ou para a abordagem de problemas concretos no exercício da profissão.