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el capital ve a la naturaleza como recurso ilimitado y explotable, pero es todo lo contrario, destruyendo la bases de los materiales. el capital depende de la naturaleza pero a la vez la destruye

cada innovación técnica busca ahorrar costos de trabajo humano y aumentar la productividad, esto provoca desempleo, inseguridad laboral. en vez de usarse como herramientas, se ocupa mas en la sustitución en el trabajo humano.

el capitalismo impulsa la competencia para crear innovación y eficiencia, esto mismo genera monopolios y concentración de mercado que eliminan la misma competencia

el capital depende del trabajo para generar plusvalia, pero el mismo capital buscar pagar menos por el trabajo. trabajador crea riqueza, el capitalista, se apropia la riqueza.

aqui el trabajo es el origen del valor, pero existe solamente si se expresa en dinero. es un mediador universal y eso es mas importante que el trabajo mismo.

lo humano queda subordinado a lo abstracto, lo cual genera una distancia bruta entre la riqueza y su reconocimiento.

valor de uso y valor de cambio. el valor como objetivo de satisfacer necesidades, solo lo que se domina es que tenga precio y se pueda vender

significa literalmente "en el estado en que" y se refiere al estado o condición existente de un asunto o situación en un momento determinado

contribute your own thinking

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Reply to the reviewers

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

The manuscript by Xu et al. investigated split gene drive systems by targeting multiple female essential genes involved in fertility and viability in Drosophila. The authors evaluate the suppression efficiency through individual corsses and cage trials. Resistance allele formation and fitness costs are explored by examining the sterility and fertility of each line. Overall, the experimental design is sound and methods are feasible. The work is comprehensive, and conclusions are well supported by the data. This work offers informative insights that could guide the design of suppression gene drive systems in other invasive disease vectors or agricultural pests.

However, several points requiring clarification or improvement:

1 Methodological clarity: Some experimental details are indufficiently described, for example, regarding the setup of genetic crosses involving different Cas9 derivatives. In line 197-198, "the mated females, together with females that were mated with Cas9 only males", it is unclear whether the latter group refers to gRNA-females.

-We thank the reviewer for pointing out this ambiguity. The latter group refers to Cas9 females crossed to Cas9 males. We have clarified this both in the methods (line 207) and results (line 505-509).

2.Regarding the inheritance rates, you included the reverse orientation of CG4415-Cas9, as I understood, it means this component is in reverse orientation with fluorescent marker. Since it is standard to design adjacent components in opposite direction to avoid transcriptional interference, the rationale for including this comparison should be better justified.

• In our construct, ‘CG4415 (reverse orientation)’ indicates that Cas9 was oriented in the same direction as the fluorescent marker, while the other Cas9 constructs (nanos-Cas9 and CG4415-Cas9) places them in opposite directions. “reverse” just indicates a change from a “standard” in another study. Our previous publication showed that Cas9 orientation relative to the marker had little apparent effect on drive performance at the yellow-G locus. In this study, we compared both orientations in a fertility gene and again observed similar results, suggesting that orientation relative to the marker does not substantially affect drive efficiency in our system. We have clarified this in the figure legend text.

Embryo resistance is inferred from the percentage of sterile drive females derived from drive mothers. How many female individuals were analysed per line and why deep sequencing was not employed to directly detect resistance alleles.

-Embryo resistance can mean slightly different things for different applications. The most important is probably the fraction of females that have little to no fertility due to embryo resistance. Some of these may not have complete embryo resistance alleles, but instead, have mosaicism, with a sufficient level of resistance to still cause sterility. It is unclear exactly what proportion of resistance to wild-type may cause this, and thus, proportions from pooled sequencing, which could include both complete and all levels of mosaicism, may not be sufficient to measure this parameter. Another relevant parameter that we did not measure is the fraction of males rendered unable to do drive conversion (this value should be closer to the complete resistance rate, but probably still lower because of the multiple gRNAs). Even in this case, deep sequencing would not allow us to determine exactly what is happening in males, making individual sequencing a preferred approached. It is very nice, of course, for characterizing which resistance alleles are present overall, but in this study, we wanted to put a bit more emphasis on the effect of resistance, rather than its sequence characterizing.

We analyzed 30 females per line for lines targeting nox, oct, dec and stl, 9 females for ndl and 276 individuals for line tra-v2 (Data Set S4). We believe such individual analyses sufficiently detected embryo resistance causing sterility within reasonable error. Note that we did also randomly genotype several sterile females and found mutations at target sites that disrupted gene functions.

In response to this comment, we have added some text to justify our measurement of resistance alleles and include some of this discussion:

“Note also that this defines embryo resistance as sufficient to induce sterility, but these may be mosaic rather than complete resistance. Further, note that the multiplex gRNA design in males may allow for continued drive conversion with a complete (as opposed to mosaic) embryo resistance allele, if some sites remain wild-type.”

Masculinisation phenotypes were observed upon disruption of tra gene. How strong intersexes were distinguished from males? What molecular markers were used to determine genetic sex. This information should be clearly provided.

-We observed two types of strong masculinisation phenotypes (Figure S2), one with bigger body size than wildtype males, and the other was identical to wildtype males. The homozygosity of the drive allele could be assessed by the brightness of red fluorescence in the eyes. However, we also randomly genotyped these masculinized females (as part of a batch that included males) to confirm their sex using primers for the Y-linked gene PP1Y2. A specific band was detected in wild-type males but not in masculinized females, confirming their genetic sex. This information has been added to the manuscript (lines 477-480).

It would be more appropriate to use "hatchability"rather than "fertility" when referring to egg-to-larva viability.

-Thank you for the suggestion. We used egg-to-adult survival rates as a proxy for the fertility of their parents because they usually laid similar number of eggs. However, it still technically incorrect language. We have fixed this in line 582 and elsewhere in the section.

In cage trials, a complete gene drive is mimicked by introducing Cas9 to the background population, but this differs from actual complete gene drive, due to potential effects from separate insertion sites (different chromosome or loci). These difference could impact the system's performance and should be discussed.

-We appreciate this point and have added discussion on the limitations of mimicking a complete gene drive using split components (line 766-779).

7.Given the large amount of data presented, it would improve readability and interpretation if each result section concluded with a concise summary highlighting the key findings and implications.

-Thank you for the suggestion. We have added brief summaries at the end of each results section to highlight the key findings and their significance.

Reviewer #1 (Significance (Required)):

The authors evaluate suppression efficiency through individual courses and cage trials. Resistance allele formation and fitness costs are explored by examining the sterility and fertility of each line. Overall, the experimental design is sound and methods are feasible. The work is comprehensive, and conclusions are well supported by the data. This work offers informative insights that could guide the design of suppression gene drive systems in other invasive disease vectors or agricultural pests.

-We appreciate the reviewer’s positive assessment of our work.

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

Paper summary

The manuscript by Xu. et al presents an insightful and valuable contribution to the field of gene drive research. The manuscript by Xu et al. presents an insightful and valuable contribution to the field of gene drive research. The strategy of targeting and disrupting female fertility genes using selfish homing genetic elements was first proposed by Burt in 2003. However, for this approach to be effective, the phenotypic constraints associated with gene disruption have meant that the pool of suitable target genes remains relatively small - notwithstanding the significant expansion in accessible targets enabled by CRISPR-based genome editing nucleases. Population suppression gene drives are well developed as proof-of-principle systems, with some now in the late stages of development as genetic control strains. However, advancing the pipeline will require a broader set of validated target genes - both to ensure effectiveness across diverse species and to build redundancy into control strategies, reducing reliance on any single genetic target.

In their paper, the authors conduct a systematic review of nine female fertility genes in Drosophila melanogaster to assess their potential as targets for homing-based suppression gene drives. The authors first conduct a thorough bioinformatic review to select candidate target genes before empirically testing candidates through microinjection and subsequent in vivo analyses of drive efficiency, population dynamics, and fitness costs relating to fecundity and fertility. After finalising their results, the authors identify two promising candidate target genes - oct and stl - which both demonstrate high gene conversion rates and, regarding the latter, can successfully suppress a cage population at a high release frequency. However, the manuscript suffers from a lack of in-depth discussion of a key limitation in its experimental design - namely, that the authors utilise a split-drive design to assess population dynamics and fitness effects when such a drive will not reflect release scenarios in the field. The review below highlights some major strengths and weaknesses of the paper, with suggestions for improvement.

Key strengths

The study's most significant strength is in its systematic selection and empirical testing of nine distinct genes as targets for homing-based gene drive, hence providing a valuable resource that substantially expands the pool of potential targets beyond the more commonly studied target genes (e.g. nudel, doublesex, among others). The identification of suitable target genes presents a significant bottleneck in the development of gene drives and the work presented here provides a foundational dataset for future research. The authors bolster the utility of their results by assessing the conservation of candidate genes across a range of pest species, suggesting the potential for broader application.

A key finding in the paper is the successful suppression of a cage population using a stl-targeting gene drive (albeit at a high release frequency). This provides a critical proof-of-principal result demonstrating that stl is a viable target for a suppression drive. While in the paper suppression was not possible at lower release frequencies, together, the results provide evidence for complex population dynamics and threshold effects that may govern the success or failure of a gene drive release strategy - hence moving the conversation from a technical perspective ("can it work") to how a gene drive may be implemented. Moreover, the authors also employ a multiplexed gRNA strategy for all their gene drive designs and in particular their population suppressive gene drive targeting stl. This provides further proof-of-principal evidence for multiplexed gRNAs in order to combat the evolution of functional resistance following gene drive deployment.

Finally, a further strength of this paper is in the clever dissection of fitness effects resulting from maternal Cas9 deposition. The authors design and perform a robust set of crosses to elucidate the parental source of fitness effects (i.e. maternally, paternally, or biparentally derived Cas9), finding (as they and others have before) that embryonic fitness was significantly reduced when Cas9 was inherited from a maternal source. As discussed, the authors conclude that maternal deposition is particularly pronounced in the context of split drives as opposed to complete drives, with the implication being that a complete drive might succeed where a split-drive has failed; thus providing a key directive for future study.

Concerns

The manuscript's central weakness lies in its interpretation of the results from the cage experiments - namely that a split-drive system was used to "mimic the release of a complete drive". In the study, mosquitoes carrying the drive element (i.e. the gRNA) were introduced into a population homozygous for the Cas9 element over several generations. This design is likely not representative of a real-world scenario and, as the authors state, likely exaggerates fitness costs. This is because the females carrying Cas9 will maternally deposit Cas9 protein into her eggs, with activity spanning several generations. When mated with a drive-carrying male the gRNA will immediately co-exist with maternally deposited Cas9, leading to early somatic cleavage and significant fitness costs (reflected in the author's own fertility crosses). This is fundamentally different to how a complete drive would function in a real-world release, where complete-drive males would mate with wild-type females not carrying Cas9. Their offspring would carry the drive element but would not be exposed to maternally deposited cas9, thus deleterious maternal effects would only begin to appear in the subsequent generation from females carrying the drive. Fitness costs measured from split-drive designs are therefore likely substantially overestimated compared to what would occur during the initial but critical release phase of a complete drive. This flaw weakens the paper's ability to predict the failure or success of the screened targets in a complete drive design, thus weakening the interpretation of the results from the cage trials. As a suggestion for improvement, the authors should explicitly and more prominently discuss the limitations of their split-drive model compared to complete drive models, both in the Results and Discussion. It is also recommended to include a schematic for both strategies that contrasts the experimental setup design (i.e. release of the drive into a Cas9 homozygous background) with a complete-drive release, clearly illustrating differences in maternal deposition pathways. This will not only contextualise the results and support the author's conclusion that observed fitness costs are likely an overestimate but will further strengthen the arguments that the candidate target genes found in this study may still be viable in a complete-drive system.

-We sincerely appreciate the thoughtful review and the valuable comments and suggestions provided, which have helped improve both the clarity and readability of this study. We have revised several parts in the discussion of the manuscript and hope that these changes adequately address the concerns raised. We have also made Figure S5 to illustrate the differences between two release strategies (biparental-Cas9 split drive in our study and complete drive in real release).

Please note that this type of fitness cost may have partially undermined our cage study (the fitness effect is notable, but still small compared to total fitness costs), but this is also among the first studies to propose and investigate this phenomenon in the first place (it is also noted in another preprint from our lab but to our knowledge not proposed elsewhere). Thus, part of the impact of our manuscript is showing that this is important, which may inform future cage studies in our lab and elsewhere.

A second weakness in the manuscript relates to its limited explanation and discussion of key concepts. For example, the manuscript reports a stark difference in outcome of the two stl-targeting drives, where a high initial release in cage 1 led to population elimination versus a failure of the drive to spread in cage 2. The authors attribute this to vague "allele effects" and stochastic factors such as larval competition; however the results appear reminiscent of the Allee effect, which is a well-characterised phenomenon describing the correlation of population size (or density) and individual fitness (or per capita population growth rate). Using their results as an example, is it plausible that the high-frequency initial release in cage 1 imposed enough genetic load to quickly drive the population density below the Allee threshold thus quickly leading to population eradication. In cage 2, the low-frequency at initial release was insufficient to cross the Allee threshold. Omitting mention of this ecological principal greatly weakens the Discussion, and further presents a missed opportunity to discuss one of the more crucial strengths of the paper - that is, in providing a deeper insight into the practical requirements for successful field implementation.

-While we do indeed mention this Allee effect (the “allele effect” noted above is a misspelling that we have corrected), we were hesitant to give it much discussion, considering that the specific Allee effect in our cages is likely of a very different nature than one would find in nature (we explain that it is likely due to bacterial growth that occurs when fewer larvae are present). However, it is perhaps still a good excuse to cover it in the discussion, while still noting that the specific Allee effect in our cage may not be representative. We have added the following text: “Nonetheless, the successful result in the cage with high release study may point to a potential field strategy for a drive that is less efficient (perhaps even one found to be less efficient in initial field tests compared to laboratory tests). If the initial release frequency of the drive is sufficiently high and widespread, then short-term high genetic load may substantially reduce the population, perhaps enough for Allee effects to become important. At this point, even if average genetic load is slowly declining without additional drive releases, persistent moderate genetic load coupled with the Allee effect may be sufficient to ensure population elimination.”

In a similar vein, the authors provide only a superficial mechanistic discussion into the fitness costs associated with drives targeting key candidate genes. The paper would benefit from a deeper discussion regarding the specific molecular functions of top-performing genes (stl, oct, nox) and how unintended Cas9 activity could disrupt their activity, integrating known molecular functions with observed fitness costs. For instance, oct encodes a G-protein coupled receptor essential for ovulation and oviduct muscle relaxation, thus disruption to the oct gene would directly impair egg-laying which would account for the observed phenotypic effects. A deeper discussion linking unintended Cas9 activity to the specific, sensitive functions of target genes would elevate the paper from a descriptive screen to a more insightful mechanistic study.

-We appreciate the reviewer’s comment. We have added a discussion to further explain fitness cost caused by unintended Cas9 activity disrupting target gene functions. However, keep in mind that the exact timing of Cas9 cleavage and the exact timing of these gene’s essential functions is still somewhat uncertain, which may limit insights from this line of analysis compared to a situation where ideal, high quality data is available for both of these. Here is the new material in the discussion:

“The functions of the top-performing genes suggests a mechanistic basis for the observed fitness costs. Aside from germline cells, nanos has expression in other ovary cells as well. CG4415 lacks this expression, but our Cas9 construct with this promoter may have a different expression pattern that the native gene, as evidenced by its support for good drive conversion in females. stl is essential for ovarian follicle development, and its disruption likely in non-germline ovary cells could compromise egg chamber development and fertility. oct encodes the octopamine β2 receptor, a G-protein coupled receptor critical for ovulation and fertilization, so if it were similarly lost, egg-laying would be directly impaired. nox, which encodes NADPH oxidase, contributes to calcium flux and smooth muscle contraction during ovulation, so its disruption may prevent egg laying. tra is needed in the whole body for sexual development, but may also play an important role in ovary function. Thus, unintended Cas9 activity at these non-germline ovary cells can directly interfere with sensitive reproductive functions, potentially explaining the fertility costs observed in drive carriers. This issue could potentially be overcome if promoters were available that were truly restricted to germline cells rather than other reproductive cells, though it remains unclear if such promoters both exist and would retain their expression pattern at a non-native locus.”

It is curious that the authors chose two genes on the X chromosome as targets. In insects (such as Drosophila here) that have heterogametic sex chromosomes, homing is not possible in the heterogametic sex as there is no chromosome to home to - so there will be no homing in males. On top of that, there is usually some fitness effect in carrier (heterozygous) females, so in a population these are nearly always bad targets for drives - unless there is some other compelling reason to choose that target?

-Our rationale for testing X-linked targets is twofold. First, these genes are likely to play important roles in sex-specific functions and may have a different expression pattern (which is why specifically Dec was included), potentially reducing fitness costs. Although homing cannot occur in males, if drive conversion at these sites in females is very high and fitness costs are minimal, the resulting genetic load could still be sufficient to suppress populations (thus, such candidates could be superior even in diploids if they happen to have a lower fitness costs). Second, X-linked targets may have broader relevance for suppression drives in haplodiploid pests (e.g., fire ants), which has the same population dynamics as an X-linked target in a diploid populations. Our results therefore could have provided useful insights for such scenarios (such as for fire ants: Liu et al., bioRxiv 2025) if drive performance was sufficient for followup testing.

Minor comments

• Enhanced clarity in the Figures and data presentation would greatly improve readability. For example, Figure 5 is critical yet difficult to interpret; consider changing x-axis labels from icons to explicit text (e.g. "biparental Cas9", "maternal cas9", "paternal Cas9"). Similarly, Figure 4 is difficult to read and the y-axis label "population size" is ambiguous; consider adding shapes or dashes (rather than relying solely on colour) and clarifying the y-axis (e.g. no. adults collected) in the legend.

-We appreciate the reviewer’s comment and have revised Figure 4 as suggested. Regarding Figure 5, we attempted to replace the icons with text labels; however, this was not possible because there is very little horizontal space and two generations to specify. Instead, we have revised the figure legend to provide a clearer explanation, which can hopefully improve clarity..

• Expand on or include a schematic to show the differences in construction between the tra-v1 and tra-v2 constructs to better contextualise the discrepancies in results (e.g. inheritance rates of 61%-66% for tra-v1 and 81%-83% for tra-v2 between the two.

-We have expanded Figure 2 to compare the constructs of tra-v1 and tra-v2. The further explanation of these two constructs was added into the result section: ‘When targeting tra, we originally tested the 4-gRNA construct tra-v1. However, the drive inheritance rate was relatively low (61%-66%), and sequencing revealed that only the middle two gRNAs were active (Table S3). Lack of cleavage at the outmost sites is particularly detrimental to achieving high drive conversion. Therefore, a second construct tra-v2 was tested that retained the two active gRNAs and included two new gRNAs. It showed substantially improved drive inheritance (81%-83%). ’

• Minor typos e.g.:

o Line 87: "form" to "from"

o Line 484: "expended" to "expanded

o Line 560: "foor" to "for"

o Line 732: "conversed" to "conserved

-We have revised these typos.

• Clarify the split drive system: the authors introduce split drive for the first time in Line 118. They should at least give a clear definition and explanation of split drive and complete drive in the introduction.

-We have included an introduction of split drive and complete drive in the introduction (line 47-53).

• Line 237-238., The fitness evaluation lacks a clear description of controls. How were non-drive flies generated and validated as controls?

-Drive heterozygotes were crossed with Cas9 homozygotes to generate the flies used for fitness evaluation. From the same cross, non-drive progeny were obtained and used as controls, ensuring they shared a comparable genetic background and rearing conditions with the drive-carrying individuals. We have now clarified in the manuscript results that “these served as the controls because they had the same environment and parents as the drive flies”.

• Line 409-412.,line 423.,The high inheritance rates of stl and oct drives are impressive; however, variation in results across Cas9 promoters should be explained further in the discussion.

-In the discussion section (lines 751-765), we included a dedicated paragraph addressing the variation observed between the nanos and CG4415 promoters. We have now expanded it to briefly note some differences:

“Our previous works showed that both nanos and CG4415 have high drive conversion rates8, but nanos failed to suppress target populations in a homing drive targeting the female fertility gene yellow-G due to its fitness cost in drive females27. CG4415 had much lower maternal deposition, which allowed the elimination of cage populations by targeting yellow-G8. Here, we tested both promoters with drives targeting oct and stl, with both showing slightly higher drive efficiency than the drive targeting yellow-G in small-scale crosses. CG4415 has slightly worse though still good performance in females, likely due to male-biased expression compared to nanos.”

• Line 414: The CG4415 promoter yielded reduced drive conversion rates in females, yet is still referred to as a promising promoter. This conclusion seems optimistic and should be clarified/more justified.

-Based on our previous study cited in this context, CG4415 shows relatively lower germline conversion rates compared to nanos, although still remaining at a high level. Importantly, CG4415 also exhibits reduced maternal deposition relative to nanos, which could help mitigate fitness costs associated with maternal deposition—an important consideration for suppression systems. Taken together, while its conversion efficiency is lower (but only slightly), the potential benefits of reduced maternal deposition and perhaps even fitness costs provide a rationale for regarding CG4415 as a promising promoter. We state this when first introducing the promoter in the “Drive efficiency assessment” results subsection.

• Specify the number of flies released, sex ratio, and cage size per generation (Line 466). This is essential for reproducibility.

-We appreciate the reviewer’s comment and have revised the text to clarify our release approach, which differed from that used in other studies (which tend to have substantial fitness differences between lines in the first generation that can complicate analysis and change results). Rather than directly releasing drive males or females into cages, we first crossed drive males with non-drive females and then mixed them with non-drive females mated to non-drive males. The offspring (including males and females) from these crosses were recorded as the G0 generation, and their ratios were recorded as release frequency. We have specified the release ratio adult numbers in the following paragraph and supplementary file.

Reviewer #2 (Significance (Required)):

Overall the manuscript presents a valuable and timely resource for gene drive research, in particular for its systematic appraisal of potential target genes for population suppression drives and its rigorous assessment of the impact of maternal Cas9 deposition. The value in the generation and empirical testing of a novel multiplexed stl-targeting gene drive that led to population eradication in a cage trial should not be understated. While several key aspects of the discussion of the manuscript should be strengthened, the study presents a meaningful contribution to the field, extending previous work and and outlines important considerations for the design and implementation of effective gene drive systems.

-We thank the reviewer for their encouraging and constructive comments. We are pleased that the systematic evaluation of target genes, the analysis of maternal Cas9 deposition, and the multiplexed stl-targeting drive were recognized as valuable contributions. We have strengthened the discussion as suggested, and we believe these revisions further enhance the manuscript as an aid for the design and implementation of future gene drive systems.

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

In this study, Xu and colleagues explored how CRISPR-based homing gene drives could be used to suppress insect populations by targeting female fertility genes in Drosophila melanogaster. They engineered split gene drives with multiplexed guide RNAs to target nine candidate genes, seeking to prevent functional resistance and achieve high drive conversion with minimal fitness costs.

Here my comments about this work:

Abstract: While the stated aim of the study on line 16 is to "maintain high drive conversion efficiency with low fitness costs in female drive carriers," the conclusion in lines 29-31 shifts focus toward the broader challenges and future optimization of gene drive systems. This conclusion does not clearly highlight the specific results of the study or how they relate directly to the original objective. It would be more effective to emphasize the actual findings, such as which target genes performed best and under what conditions, and how these findings support or contradict the stated goals. The study primarily aimed to assess the efficiency of specific female fertility genes and to evaluate strategies for minimizing the formation of functional resistance alleles, rather than proposing a protocol for optimization. Therefore, better alignment is needed between the study's aim, experimental design, and concluding statements. Clarifying this alignment would also help refine the paper's focus and more accurately communicate its contribution, including whether it is exploratory, comparative, or methodologically driven.

-We have revised the abstract to clarify the alignment as suggested by the reviewer. We note that this discrepancy is due to the initial aim of our study being different than some of the important lessons learned along the way regarding fitness effects from Cas9 deposition in split drives. Still, we agree that it would be better to be more consistent in our wording and conclusions.

Introduction: One of the key design elements in this study is the use of multiplexed gRNAs. It is reasonable to assume that this strategy may influence fitness costs, potentially in more than one way. Given that assessing fitness cost is a major focus of the study, it would be helpful to include a brief discussion of previous research examining how multiplexed gRNAs may impact fitness in gene drive systems. A short review of relevant studies, if available, would provide important context for interpreting the results and could help clarify whether any observed fitness costs might be attributed, at least in part, to the multiplexing strategy itself. This addition could be appropriately placed around line 102, where gRNA design is discussed.

-We have added an explanation in the Discussion to mention this. However, it has not been conclusively shown that multiplexed gRNAs have any effect on fitness. Indeed, there have been some multiplexed constructs that seem to have no fitness effect, and some that have high fitness costs. This doesn’t rule out the potential for multiplexed gRNAs to influence fitness itself, but it means that the mechanism may be complex. The new text reads:

“Another potential though unconfirmed source of fitness cost arises from increased cleavage events associated with multiplexed gRNAs, where the greater number of gRNAs can enhance the overall cut rate compared to single-gRNA designs.”

Line 42: Cas12a also showed efficacy using gene drives in yeast and Drosophila.

-We now mention Cas12a at the beginning of the introduction.

Line 133: The paragraph begins by stating that homologs of the target genes were identified and aligned. To improve clarity, especially for readers who are new to gene drive research, it would be helpful to begin the paragraph with a brief introductory sentence explaining the purpose of this step. For example, you could state the importance of identifying and aligning homologs to assess the conservation of target sites across species, which is critical for evaluating the broader applicability of gene drive strategies. This context would guide the reader and clarify the relevance of the analysis.

-We have added the explanation as suggested.

Lines 144-145: You mention that "the exception was tra, for which two constructs containing different gRNA sets were generated." For clarity, it would be helpful to provide a brief explanation of why two different gRNA sets were used for tra, and whether this differs from the approach taken with the other target genes. It's currently unclear whether all other genes were targeted using a single, standardized set of gRNAs, and this should be explicitly stated here for consistency, even though it is mentioned later in the plasmid construction section. Additionally, I suggest combining the sections on gRNA target design and plasmid construction. Since these components are closely related and sequential in the experimental workflow, presenting them together would improve the logical flow and help readers follow the methodology more smoothly.

-We have combined both the gRNA target design and plasmid construction sections. We also discuss the two tra constructs early in the results section (see response to reviewer 2).

Line 210: The analysis of the cage experiments was based on models from previous studies that used a simplified assumption of a single gRNA at the target site. While I understand this approach has precedent, it raises important questions about potential limitations. Specifically, could simplifying the analysis to one gRNA affect the conclusions of this study, given that the experimental design involves multiplexed gRNAs with four distinct target sites? The implications of using this simplified model should be clearly addressed, as the dynamics of drive efficiency, resistance formation, and fitness effects may differ when multiple gRNAs are employed. Additionally, while I am not a statistician, it is worth asking whether more sophisticated modeling approaches could be applied to account for all four gRNAs, rather than reducing the system to a single-gRNA framework. A discussion of the modeling choices and their potential consequences would strengthen the interpretation of the results.

-We have clarified this. While we have modeled multiple gRNAs with high fidelity in SLiM, the maximum likelihood method is not very amenable to such treatment. It may cause our fitness estimate to be a small overestimate, but give the low fitness inferences, would certainly not have a large enough effect to fundamentally change any conclusion (and should be of a consistent level across all cages). We now discuss this in the methods section.

Lines 297-300: Your results show that the expression of all target genes was higher in females, except for oct, which had higher expression in males. Additionally, oct expression decreased in adults. Given that oct is functionally important for ovulation and fertilization, processes that are primarily required in adult females, this pattern is somewhat unexpected. Could there be a possible explanation for the lower expression of oct, particularly in females and especially in adults, where its function would presumably be most critical? A brief discussion or hypothesis addressing this discrepancy would help clarify the biological relevance and interpretation of the expression data.

-Based on transcriptome data from FlyBase, derived from Graveley et al. (2011), Oct is indeed expressed slightly higher in adult males than in adult females. This difference may be attributed to the fact that the female flies used in the study were virgins; Oct expression could be upregulated post-mating to mediate ovulation. Additionally, Oct is expressed not only in reproductive tissues but also in other organs such as the nervous system, where sex-specific differences in cell type composition or neural activity may contribute to the observed expression bias. However, high expression does not necessarily correlate with essential expression. Though Oct could have multiple functions, it’s still possible that the only apparent phenotype upon knockout is female sterility. We have added the following text: “This male-biased expression may result from the use of virgin females in the dataset, as oct is likely upregulated after mating. Moreover, oct is also expressed in non-reproductive tissues such as the nervous system, which may contribute to sex-specific differences in expression38. While oct may have multiple functions, it is possible that it is only essential for female fertility.”

Lines 346-347: What is the distance between the gRNA target sites within each gene? Are all of the gRNAs confirmed to be active? It would be valuable to include a table summarizing the distance between target sites for each gene, the activity levels of the individual gRNAs, and the corresponding homing rates. This would help determine whether there is a correlation between gRNA spacing and drive efficiency. For example, Lopez del Amo et al. (Nature Communications, 2020) demonstrated that even a 20-nucleotide mismatch at each homology arm can significantly reduce drive conversion. Including such a comparative analysis in your study could provide important insights into how gRNA arrangement influences overall drive performance and would be incredibly helpful for future multiplexing designs.

-We have showed previously that close spacing of gRNAs should help maintain high drive conversion efficiency, and this is alluded to indirectly in the introduction (we now mention it more directly). In our study, gRNAs were positioned in close proximity without overlap, with the general distance between the outermost cut sites within each gene being We have added a summary table (Table S3) presenting the sequencing results, which also showed gRNA activity levels. Notably, most but not all gRNAs were active, at least for embryo resistance (low to moderate activity may still be present in the germline). Coupled with varying activity levels for those that were active, this likely contributed to reduced drive conversion due to mismatches at the homology arms. This observation supports the notion that drive performance could be optimized by selecting and arranging more active gRNAs. Consistent with this, our second construct targeting tra (tra-v2) exhibited a higher inheritance rate than the original construct, suggesting that gRNA arrangement and activity critically influence drive efficiency. Testing the activity of every single gRNA requires the construction of multiple gRNA lines, since in vitro or ex vivo tests will not be accurate as in vivo transformation test. However, in our study, as long as drive conversion rates were reasonably high, further optimization was not needed. Therefore, the multiplexing gRNA design can not only maximize drive conversion, but also reduce labor filtering an increased number of 1-gRNA designs with lower performance.

Line 434: I was not able to find any sequencing data. This is important to evaluate gRNA activities and establish correlations with drive efficiency.

-We have added a summary of the sequencing results in Table S3, though these are for embryo resistance alleles. Note that while high gRNA activity is correlated with high drive inheritance, these are not directly related. For suppression drives, germline resistance rates are usually of low importance compared to drive inheritance, so we did not assess these in detail (and pessimistically assumed complete germline resistance in our cage models).

Line 482: Did the authors test Cas9-only individuals (without the drive) against a wild-type population? This would help determine whether Cas9 alone has any unintended fitness effects. Additionally, is Cas9 expression stable over time and across generations? It would be helpful to include any observations or thoughts on the long-term stability and potential fitness impact of Cas9 in the absence of the drive element.

-We did not perform a direct comparison of Cas9-only individuals and wild-type flies in this study. However, previous studies (Champer et al., Nature Communications, 2020 - Langmuller et al., eLife 2022), which we now cite in the discussion, found no significant fitness difference between very similar Cas9-expressing lines and wild type in the absence of a drive element, indicating no significant fitness impact from Cas9 alone (though we cannot exclude a small effect, it certainly could not come close to explaining our results). In our experiments, Cas9 expression was generally stable across generations, as indicated by consistent drive inheritance and fertility test results obtained from independent batches. Separate from this study, we did observe rare instability in one nanos-Cas9 line, which had remained stable for over five years but recently became inactive (low population maintenance size may have caused stochastic removal of the functional allele). It is something to watch out for, but probably not on the timescale of a single study.

Discussion: I would appreciate a more direct and clearly stated conclusion that summarizes the key findings of the study. While the discussion addresses the main outcomes in depth, presenting a concise concluding paragraph, either at the end of the discussion or as a standalone conclusion section, would provide a stronger and more definitive closing statement. This would help reinforce what the study ultimately achieved and ensure the main takeaways are clearly communicated to the reader.

-We have revised and expanded the last paragraph of the discussion section to make our findings more direct and clear.

Overall, I believe this is an important study that offers valuable insights for advancing the design of CRISPR-based gene drives. The findings contribute to the development of more efficient and practical gene drive prototypes, bringing the field closer to real-world applications.

Reviewer #3 (Significance (Required)):

In this study, Xu and colleagues explored how CRISPR-based homing gene drives could be used to suppress insect populations by targeting female fertility genes in Drosophila melanogaster. They engineered split gene drives with multiplexed guide RNAs to target nine candidate genes, seeking to prevent functional resistance and achieve high drive conversion with minimal fitness costs. Among the targets, the stall (stl) and octopamine β2 receptor (oct) genes performed better, showing the highest inheritance rates in lab crosses. When tested in population cages, the stl drive was able to completely eliminate a fly population, but only when released at a high enough frequency, while other cages failed. These failures were traced and explained by fitness cost in drive-carrying females, caused largely by maternally deposited Cas9, which led to embryo resistance and reduced fertility. Through additional fertility assays and modeling, the team confirmed that the origin and timing of Cas9 expression, particularly from mothers, significantly impacted drive success. Surprisingly, even when Cas9 was driven by promoters with supposedly low somatic activity, such as nanos, fitness still persisted. The study revealed that while gene drives can be powerful, their effectiveness relies on finely balanced factors like promoter choice, drive architecture, and gene function. Overall, the research offers valuable lessons for designing robust, next-generation gene drives aimed at ecological pest control.

-We sincerely appreciate the reviewer’s positive and thoughtful comments. We agree that the points raised highlight the importance of our findings and hope that our revisions have further improved both the clarity and overall content of the manuscript.

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

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Referee #2

Evidence, reproducibility and clarity

Paper summary

The manuscript by Xu. et al presents an insightful and valuable contribution to the field of gene drive research. The manuscript by Xu et al. presents an insightful and valuable contribution to the field of gene drive research. The strategy of targeting and disrupting female fertility genes using selfish homing genetic elements was first proposed by Burt in 2003. However, for this approach to be effective, the phenotypic constraints associated with gene disruption have meant that the pool of suitable target genes remains relatively small - notwithstanding the significant expansion in accessible targets enabled by CRISPR-based genome editing nucleases. Population suppression gene drives are well developed as proof-of-principle systems, with some now in the late stages of development as genetic control strains. However, advancing the pipeline will require a broader set of validated target genes - both to ensure effectiveness across diverse species and to build redundancy into control strategies, reducing reliance on any single genetic target. In their paper, the authors conduct a systematic review of nine female fertility genes in Drosophila melanogaster to assess their potential as targets for homing-based suppression gene drives. The authors first conduct a thorough bioinformatic review to select candidate target genes before empirically testing candidates through microinjection and subsequent in vivo analyses of drive efficiency, population dynamics, and fitness costs relating to fecundity and fertility. After finalising their results, the authors identify two promising candidate target genes - oct and stl - which both demonstrate high gene conversion rates and, regarding the latter, can successfully suppress a cage population at a high release frequency. However, the manuscript suffers from a lack of in-depth discussion of a key limitation in its experimental design - namely, that the authors utilise a split-drive design to assess population dynamics and fitness effects when such a drive will not reflect release scenarios in the field. The review below highlights some major strengths and weaknesses of the paper, with suggestions for improvement.

Key strengths

The study's most significant strength is in its systematic selection and empirical testing of nine distinct genes as targets for homing-based gene drive, hence providing a valuable resource that substantially expands the pool of potential targets beyond the more commonly studied target genes (e.g. nudel, doublesex, among others). The identification of suitable target genes presents a significant bottleneck in the development of gene drives and the work presented here provides a foundational dataset for future research. The authors bolster the utility of their results by assessing the conservation of candidate genes across a range of pest species, suggesting the potential for broader application. A key finding in the paper is the successful suppression of a cage population using a stl-targeting gene drive (albeit at a high release frequency). This provides a critical proof-of-principal result demonstrating that stl is a viable target for a suppression drive. While in the paper suppression was not possible at lower release frequencies, together, the results provide evidence for complex population dynamics and threshold effects that may govern the success or failure of a gene drive release strategy - hence moving the conversation from a technical perspective ("can it work") to how a gene drive may be implemented. Moreover, the authors also employ a multiplexed gRNA strategy for all their gene drive designs and in particular their population suppressive gene drive targeting stl. This provides further proof-of-principal evidence for multiplexed gRNAs in order to combat the evolution of functional resistance following gene drive deployment. Finally, a further strength of this paper is in the clever dissection of fitness effects resulting from maternal Cas9 deposition. The authors design and perform a robust set of crosses to elucidate the parental source of fitness effects (i.e. maternally, paternally, or biparentally derived Cas9), finding (as they and others have before) that embryonic fitness was significantly reduced when Cas9 was inherited from a maternal source. As discussed, the authors conclude that maternal deposition is particularly pronounced in the context of split drives as opposed to complete drives, with the implication being that a complete drive might succeed where a split-drive has failed; thus providing a key directive for future study.

Concerns

The manuscript's central weakness lies in its interpretation of the results from the cage experiments - namely that a split-drive system was used to "mimic the release of a complete drive". In the study, mosquitoes carrying the drive element (i.e. the gRNA) were introduced into a population homozygous for the Cas9 element over several generations. This design is likely not representative of a real-world scenario and, as the authors state, likely exaggerates fitness costs. This is because the females carrying Cas9 will maternally deposit Cas9 protein into her eggs, with activity spanning several generations. When mated with a drive-carrying male the gRNA will immediately co-exist with maternally deposited Cas9, leading to early somatic cleavage and significant fitness costs (reflected in the author's own fertility crosses). This is fundamentally different to how a complete drive would function in a real-world release, where complete-drive males would mate with wild-type females not carrying Cas9. Their offspring would carry the drive element but would not be exposed to maternally deposited cas9, thus deleterious maternal effects would only begin to appear in the subsequent generation from females carrying the drive. Fitness costs measured from split-drive designs are therefore likely substantially overestimated compared to what would occur during the initial but critical release phase of a complete drive. This flaw weakens the paper's ability to predict the failure or success of the screened targets in a complete drive design, thus weakening the interpretation of the results from the cage trials. As a suggestion for improvement, the authors should explicitly and more prominently discuss the limitations of their split-drive model compared to complete drive models, both in the Results and Discussion. It is also recommended to include a schematic for both strategies that contrasts the experimental setup design (i.e. release of the drive into a Cas9 homozygous background) with a complete-drive release, clearly illustrating differences in maternal deposition pathways. This will not only contextualise the results and support the author's conclusion that observed fitness costs are likely an overestimate but will further strengthen the arguments that the candidate target genes found in this study may still be viable in a complete-drive system.

A second weakness in the manuscript relates to its limited explanation and discussion of key concepts. For example, the manuscript reports a stark difference in outcome of the two stl-targeting drives, where a high initial release in cage 1 led to population elimination versus a failure of the drive to spread in cage 2. The authors attribute this to vague "allele effects" and stochastic factors such as larval competition; however the results appear reminiscent of the Allee effect, which is a well-characterised phenomenon describing the correlation of population size (or density) and individual fitness (or per capita population growth rate). Using their results as an example, is it plausible that the high-frequency initial release in cage 1 imposed enough genetic load to quickly drive the population density below the Allee threshold thus quickly leading to population eradication. In cage 2, the low-frequency at initial release was insufficient to cross the Allee threshold. Omitting mention of this ecological principal greatly weakens the Discussion, and further presents a missed opportunity to discuss one of the more crucial strengths of the paper - that is, in providing a deeper insight into the practical requirements for successful field implementation. In a similar vein, the authors provide only a superficial mechanistic discussion into the fitness costs associated with drives targeting key candidate genes. The paper would benefit from a deeper discussion regarding the specific molecular functions of top-performing genes (stl, oct, nox) and how unintended Cas9 activity could disrupt their activity, integrating known molecular functions with observed fitness costs. For instance, oct encodes a G-protein coupled receptor essential for ovulation and oviduct muscle relaxation, thus disruption to the oct gene would directly impair egg-laying which would account for the observed phenotypic effects. A deeper discussion linking unintended Cas9 activity to the specific, sensitive functions of target genes would elevate the paper from a descriptive screen to a more insightful mechanistic study.

It is curious that the authors chose two genes on the X chromosome as targets. In insects (such as Drosophila here) that have heterogametic sex chromosomes, homing is not possible in the heterogametic sex as there is no chromosome to home to - so there will be no homing in males. On top of that, there is usually some fitness effect in carrier (heterozygous) females, so in a population these are nearly always bad targets for drives - unless there is some other compelling reason to choose that target?

Minor comments

• Enhanced clarity in the Figures and data presentation would greatly improve readability. For example, Figure 5 is critical yet difficult to interpret; consider changing x-axis labels from icons to explicit text (e.g. "biparental Cas9", "maternal cas9", "paternal Cas9"). Similarly, Figure 4 is difficult to read and the y-axis label "population size" is ambiguous; consider adding shapes or dashes (rather than relying solely on colour) and clarifying the y-axis (e.g. no. adults collected) in the legend.
• Expand on or include a schematic to show the differences in construction between the tra-v1 and tra-v2 constructs to better contextualise the discrepancies in results (e.g. inheritance rates of 61%-66% for tra-v1 and 81%-83% for tra-v2 between the two.
• Minor typos e.g.:
  • Line 87: "form" to "from"
  • Line 484: "expended" to "expanded
  • Line 560: "foor" to "for"
  • Line 732: "conversed" to "conserved
• Clarify the split drive system: the authors introduce split drive for the first time in Line 118. They should at least give a clear definition and explanation of split drive and complete drive in the introduction.
• Line 237-238., The fitness evaluation lacks a clear description of controls. How were non-drive flies generated and validated as controls?
• Line 409-412.,line 423.,The high inheritance rates of stl and oct drives are impressive; however, variation in results across Cas9 promoters should be explained further in the discussion.
• Line 414: The CG4415 promoter yielded reduced drive conversion rates in females, yet is still referred to as a promising promoter. This conclusion seems optimistic and should be clarified/more justified.
• Specify the number of flies released, sex ratio, and cage size per generation (Line 466). This is essential for reproducibility.

Significance

Overall the manuscript presents a valuable and timely resource for gene drive research, in particular for its systematic appraisal of potential target genes for population suppression drives and its rigorous assessment of the impact of maternal Cas9 deposition. The value in the generation and empirical testing of a novel multiplexed stl-targeting gene drive that led to population eradication in a cage trial should not be understated. While several key aspects of the discussion of the manuscript should be strengthened, the study presents a meaningful contribution to the field, extending previous work and and outlines important considerations for the design and implementation of effective gene drive systems.

Reviewer #1 (Public review):

Summary:

The authors state the study's goal clearly: "The goal of our study was to understand to what extent animal individuality is influenced by situational changes in the environment, i.e., how much of an animal's individuality remains after one or more environmental features change." They use visually guided behavioral features to examine the extent of correlation over time and in a variety of contexts. They develop new behavioral instrumentation and software to measure behavior in Buridan's paradigm (and variations thereof), the Y-maze, and a flight simulator. Using these assays, they examine the correlations between conditions for a panel of locomotion parameters. They propose that inter-assay correlations will determine the persistence of locomotion individuality.

Strengths:

The OED defines individuality as "the sum of the attributes which distinguish a person or thing from others of the same kind," a definition mirrored by other dictionaries and the scientific literature on the topic. The concept of behavioral individuality can be characterized as: (1) a large set of behavioral attributes, (2) with inter-individual variability, that are (3) stable over time. A previous study examined walking parameters in Buridan's paradigm, finding that several parameters were variable between individuals, and that these showed stability over separate days and up to 4 weeks (DOI: 10.1126/science.aaw718). The present study replicates some of those findings, and extends the experiments from temporal stability to examining correlation of locomotion features between different contexts.

The major strength of the study is using a range of different behavioral assays to examine the correlations of several different behavior parameters. It shows clearly that the inter-individual variability of some parameters is at least partially preserved between some contexts, and not preserved between others. The development of high-throughput behavior assays and sharing the information on how to make the assays is a commendable contribution.

Weaknesses:

The definition of individuality considers a comprehensive or large set of attributes, but the authors consider only a handful. In Supplemental Fig. S8, the authors show a large correlation matrix of many behavioral parameters, but these are illegible and are only mentioned briefly in Results. Why were five or so parameters selected from the full set? How were these selected? Do the correlation trends hold true across all parameters? For assays in which only a subset of parameters can be directly compared, were all of these included in the analysis, or only a subset?

The correlation analysis is used to establish stability between assays. For temporal re-testing, "stability" is certainly the appropriate word, but between contexts it implies that there could be 'instability'. Rather, instead of the 'instability' of a single brain process, a different behavior in a different context could arise from engaging largely (or entirely?) distinct context-dependent internal processes, and have nothing to do with process stability per se. For inter-context similarities, perhaps a better word would be "consistency".

The parameters are considered one-by-one, not in aggregate. This focuses on the stability/consistency of the variability of a single parameter at a time, rather than holistic individuality. It would appear that an appropriate measure of individuality stability (or individuality consistency) that accounts for the high-dimensional nature of individuality would somehow summarize correlations across all parameters. Why was a multivariate approach (e.g. multiple regression/correlation) not used? Treating the data with a multivariate or averaged approach would allow the authors to directly address 'individuality stability', along with the analyses of single-parameter variability stability.

The correlation coefficients are sometimes quite low, though highly significant, and are deemed to indicate stability. For example, in Figure 4C top left, the % of time walked at 23{degree sign}C and 32{degree sign}C are correlated by 0.263, which corresponds to an R2 of 0.069 i.e. just 7% of the 32{degree sign}C variance is predictable by the 23{degree sign}C variance. Is it fair to say that 7% determination indicates parameter stability? Another example: "Vector strength was the most correlated attention parameter... correlations ranged... to -0.197," which implies that 96% (1 - R2) of Y-maze variance is not predicted by Buridan variance. At what level does an r value not represent stability?

The authors describe a dissociation between inter-group differences and inter-individual variation stability, i.e. sometimes large mean differences between contexts, but significant correlation between individual test and retest data. Given that correlation is sensitive to slope, this might be expected to underestimate the variability stability (or consistency). Is there a way to adjust for the group differences before examining correlation? For example, would it be possible to transform the values to in-group ranks prior to correlation analysis?

What is gained by classifying the five parameters into exploration, attention, and anxiety? To what extent have these classifications been validated, both in general, and with regard to these specific parameters? Is increased walking speed at higher temperature necessarily due to increased 'explorative' nature, or could it be attributed to increased metabolism, dehydration stress, or a heat-pain response? To what extent are these categories subjective?

The legends are quite brief and do not link to descriptions of specific experiments. For example, Figure 4a depicts a graphical overview of the procedure, but I could not find a detailed description of this experiment's protocol.

Using the current single-correlation analysis approach, the aims would benefit from re-wording to appropriately address single-parameter variability stability/consistency (as distinct from holistic individuality). Alternatively, the analysis could be adjusted to address the multivariate nature of individuality, so that the claims and the analysis are in concordance with each other.

The study presents a bounty of new technology to study visually guided behaviors. The Github link to the software was not available. To verify successful transfer or open-hardware and open-software, a report would demonstrate transfer by collaboration with one or more other laboratories, which the present manuscript does not appear to do. Nevertheless, making the technology available to readers is commendable.

The study discusses a number of interesting, stimulating ideas about inter-individual variability, and presents intriguing data that speaks to those ideas, albeit with the issues outlined above.

While the current work does not present any mechanistic analysis of inter-individual variability, the implementation of high-throughput assays sets up the field to more systematically investigate fly visual behaviors, their variability, and their underlying mechanisms.

Comments on revisions:

While the incorporation of a hierarchical mixed model (HMM) appears to represent an improvement over their prior single-parameter correlation approach, it's not clear to me that this is a multivariate analysis. They write that "For each trait, we fitted a hierarchical linear mixed-effects model in Matlab (using the fit lme function) with environmental context as a fixed effect and fly identity (ID) as a random intercept... We computed the intraclass correlation coefficient (ICC) from each model as the between-fly variance divided by total variance. ICC, therefore, quantified repeatability across environmental contexts."

Does this indicate that HMM was used in a univariate approach? Can an analysis of only five metrics of several dozen total metrics be characterized as 'holistic'?

Within Figure 10a, some of the metrics show high ICC scores, but others do not. This suggests that the authors are overstating the overall persistence and/or consistency of behavioral individuality. It is clear from Figure S8 that a large number of metrics were calculated for each fly, but it remains unclear, at least to me, why the five metrics in Figure 10a are justified for selection. One is left wondering how rare or common is the 0.6 repeatability of % time walked among all the other behavioral metrics. It appears that a holistic analysis of this large data set remains impossible.

The authors write: "...fly individuality persists across different contexts, and individual differences shape behavior across variable environments, thereby making the underlying developmental and functional mechanisms amenable to genetic dissection." However, presumably the various behavioral features (and their variability) are governed by different brain regions, so some metrics (high ICC) would be amenable to the genetic dissection of individuality/variability, while others (low ICC) would not. It would be useful to know which are which, to define which behavioral domains express individuality, and could be targets for genetic analysis, and which do not. At the very least, the Abstract might like to acknowledge that inter-context consistency is not a major property of all or most behavioral metrics.

I hold that inter-trial repeatability should rightly be called "stability" while inter-context repeatability should be called "consistency". In the current manuscript, "consistency" is used throughout the manuscript, except for the new edits, which use "stability". If the authors are going to use both terms, it would be preferable if they could explain precisely how they define and use these terms.

Author response:

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

Reviewer #1 (Public review):  

Summary:  

The authors state the study's goal clearly: "The goal of our study was to understand to what extent animal individuality is influenced by situational changes in the environment, i.e., how much of an animal's individuality remains after one or more environmental features change." They use visually guided behavioral features to examine the extent of correlation over time and in a variety of contexts. They develop new behavioral instrumentation and software to measure behavior in Buridan's paradigm (and variations thereof), the Y-maze, and a flight simulator. Using these assays, they examine the correlations between conditions for a panel of locomotion parameters. They propose that inter-assay correlations will determine the persistence of locomotion individuality.

Strengths:  

The OED defines individuality as "the sum of the attributes which distinguish a person or thing from others of the same kind," a definition mirrored by other dictionaries and the scientific literature on the topic. The concept of behavioral individuality can be characterized as: (1) a large set of behavioral attributes, (2) with inter-individual variability, that are (3) stable over time. A previous study examined walking parameters in Buridan's paradigm, finding that several parameters were variable between individuals, and that these showed stability over separate days and up to 4 weeks (DOI: 10.1126/science.aaw718). The present study replicates some of those findings and extends the experiments from temporal stability to examining correlation of locomotion features between different contexts.  

The major strength of the study is using a range of different behavioral assays to examine the correlations of several different behavior parameters. It shows clearly that the inter-individual variability of some parameters is at least partially preserved between some contexts, and not preserved between others. The development of high-throughput behavior assays and sharing the information on how to make the assays is a commendable contribution.

Weaknesses:  

The definition of individuality considers a comprehensive or large set of attributes, but the authors consider only a handful. In Supplemental Fig. S8, the authors show a large correlation matrix of many behavioral parameters, but these are illegible and are only mentioned briefly in Results. Why were five or so parameters selected from the full set? How were these selected? Do the correlation trends hold true across all parameters? For assays in which only a subset of parameters can be directly compared, were all of these included in the analysis, or only a subset?  

The correlation analysis is used to establish stability between assays. For temporal re-testing, "stability" is certainly the appropriate word, but between contexts it implies that there could be 'instability'. Rather, instead of the 'instability' of a single brain process, a different behavior in a different context could arise from engaging largely (or entirely?) distinct context-dependent internal processes, and have nothing to do with process stability per se. For inter-context similarities, perhaps a better word would be "consistency".  

The parameters are considered one-by-one, not in aggregate. This focuses on the stability/consistency of the variability of a single parameter at a time, rather than holistic individuality. It would appear that an appropriate measure of individuality stability (or individuality consistency) that accounts for the high-dimensional nature of individuality would somehow summarize correlations across all parameters. Why was a multivariate approach (e.g. multiple regression/correlation) not used? Treating the data with a multivariate or averaged approach would allow the authors to directly address 'individuality stability', along with the analyses of single-parameter variability stability.

The correlation coefficients are sometimes quite low, though highly significant, and are deemed to indicate stability. For example, in Figure 4C top left, the % of time walked at 23{degree sign}C and 32{degree sign}C are correlated by 0.263, which corresponds to an R2 of 0.069 i.e. just 7% of the 32{degree sign}C variance is predictable by the 23{degree sign}C variance. Is it fair to say that 7% determination indicates parameter stability? Another example: "Vector strength was the most correlated attention parameter... correlations ranged... to -0.197," which implies that 96% (1 - R2) of Y-maze variance is not predicted by Buridan variance. At what level does an r value not represent stability?

The authors describe a dissociation between inter-group differences and inter-individual variation stability, i.e. sometimes large mean differences between contexts, but significant correlation between individual test and retest data. Given that correlation is sensitive to slope, this might be expected to underestimate the variability stability (or consistency). Is there a way to adjust for the group differences before examining correlation? For example, would it be possible to transform the values to in-group ranks prior to correlation analysis?

What is gained by classifying the five parameters into exploration, attention, and anxiety? To what extent have these classifications been validated, both in general, and with regard to these specific parameters? Is increased walking speed at higher temperature necessarily due to increased 'explorative' nature, or could it be attributed to increased metabolism, dehydration stress, or a heat-pain response? To what extent are these categories subjective?

The legends are quite brief and do not link to descriptions of specific experiments. For example, Figure 4a depicts a graphical overview of the procedure, but I could not find a detailed description of this experiment's protocol.

Using the current single-correlation analysis approach, the aims would benefit from re-wording to appropriately address single-parameter variability stability/consistency (as distinct from holistic individuality). Alternatively, the analysis could be adjusted to address the multivariate nature of individuality, so that the claims and the analysis are in concordance with each other.

The study presents a bounty of new technology to study visually guided behaviors. The Github link to the software was not available. To verify successful transfer or open-hardware and open-software, a report would demonstrate transfer by collaboration with one or more other laboratories, which the present manuscript does not appear to do. Nevertheless, making the technology available to readers is commendable.

The study discusses a number of interesting, stimulating ideas about interindividual variability and presents intriguing data that speaks to those ideas, albeit with the issues outlined above.

While the current work does not present any mechanistic analysis of interindividual variability, the implementation of high-throughput assays sets up the field to more systematically investigate fly visual behaviors, their variability, and their underlying mechanisms.  

Comments on revisions:  

I want to express my appreciation for the authors' responsiveness to the reviewer feedback. They appear to have addressed my previous concerns through various modifications including GLM analysis, however, some areas still require clarification for the benefit of an audience that includes geneticists.  

(1) GLM Analysis Explanation (Figure 9)  

While the authors state that their new GLM results support their original conclusions, the explanation of these results in the text is insufficient. Specifically:

The interpretation of coefficients and their statistical significance needs more detailed explanation. The audience includes geneticists and other nonstatistical people, so the GLM should be explained in terms of the criteria or quantities used to assess how well the results conform with the hypothesis, and to what extent they diverge.

The criteria used to judge how well the GLM results support their hypothesis are not clearly stated.

The relationship between the GLM findings and their original correlationbased conclusions needs better integration and connection, leading the reader through your reasoning.

We thank the reviewer for highlighting this important point. We have revised the Results section in the reviseed manuscript to include a more detailed explanation of the GLM analysis. Specifically, we now clarify the interpretation of the model coefficients, including the direction and statistical significance, in relation to the hypothesized effects. We also outline the criteria we used to assess how well the GLM supports our original correlation-based conclusions—namely, whether the sign and significance of the coefficients align with the expected relationships derived from our prior analysis. Finally, we explicitly describe how the GLM results confirm or extend the patterns observed in the correlation-based analysis, to guide readers through our reasoning and the integration of both approaches.

(2) Documentation of Changes  

One struggle with the revised manuscript is that no "tracked changes" version was included, so it is hard to know exactly what was done. Without access to the previous version of the manuscript, it is difficult to fully assess the extent of revisions made. The authors should provide a more comprehensive summary of the specific changes implemented, particularly regarding:

We thank the reviewer for bringing this to our attention. We were equally confused to learn that the tracked-changes version was not visible, despite having submitted one to eLife as part of our revision. 

Upon contacting the editorial office, they confirmed that we did submit a trackedchanges version, but clarified that it did not contain embedded figures (as they were added manually to the clean version).  The editorial response said in detail: “Regarding the tracked-changes file: it appears the version with markup lacked figures, while the figure-complete PDF had markup removed, which likely caused the confusion mentioned by the reviewers.” We hope this answer from eLife clarifies the reviewers’ concern.

(2)  Statistical Method Selection  

The authors mention using "ridge regression to mitigate collinearity among predictors" but do not adequately justify this choice over other approaches. They should explain:

Why ridge regression was selected as the optimal method  

How the regularization parameter (λ) was determined  

How this choice affects the interpretation of environmental parameters' influence on individuality

We appreciate the reviewer’s thoughtful question regarding our choice of statistical method. In response, we have expanded the Methods section in the revised manuscript to provide a more detailed justification for the use of a GLM, including ridge regression. Specifically, we explain that ridge regression was selected to address collinearity and to control for overfitting.

We now also describe how the regularization parameter (λ) was selected: we used 5-fold cross-validation over a log-spaced grid (10^⁻⁶ - 10<sup>⁶</sup) to identify the optimal value that minimized the mean squared error (MSE).

Finally, we clarify in both the Methods and Results sections how this modeling choice affects the interpretation of our findings. 

Reviewer #2 (Public review):  

Summary:  

The authors repeatedly measured the behavior of individual flies across several environmental situations in custom-made behavioral phenotyping rigs.

Strengths:  

The study uses several different behavioral phenotyping devices to quantify individual behavior in a number of different situations and over time. It seems to be a very impressive amount of data. The authors also make all their behavioral phenotyping rig design and tracking software available, which I think is great, and I'm sure other folks will be interested in using and adapting to their own needs.

Weaknesses/Limitations:  

I think an important limitation is that while the authors measured the flies under different environmental scenarios (i.e. with different lighting, temperature) they didn't really alter the "context" of the environment. At least within behavioral ecology, context would refer to the potential functionality of the expressed behaviors so for example, an anti-predator context, or a mating context, or foraging. Here, the authors seem to really just be measuring aspects of locomotion under benign (relatively low risk perception) contexts. This is not a flaw of the study, but rather a limitation to how strongly the authors can really say that this demonstrates that individuality is generalized across many different contexts. It's quite possible that rank-order of locomotor (or other) behaviors may shift when the flies are in a mating or risky context.  

I think the authors are missing an opportunity to use much more robust statistical methods It appears as though the authors used pearson correlations across time/situations to estimate individual variation; however far more sophisticated and elegant methods exist. The problem is that pearson correlation coefficients can be anti-conservative and additionally, the authors have thus had to perform many many tests to correlate behaviors across the different trials/scenarios. I don't see any evidence that the authors are controlling for multiple testing which I think would also help. Alternatively, though, the paper would be a lot stronger, and my guess is, much more streamlined if the authors employ hierarchical mixed models to analyse these data, which are the standard analytical tools in the study of individual behavioral variation. In this way, the authors could partition the behavioral variance into its among- and within-individual components and quantify repeatability of different behaviors across trials/scenarios simultaneously. This would remove the need to estimate 3 different correlations for day 1 & day 2, day 1 & 3, day 2 & 3 (or stripe 0 & stripe 1, etc) and instead just report a single repeatability for e.g. the time spent walking among the different strip patterns (eg. figure 3). Additionally, the authors could then use multivariate models where the response variables are all the behaviors combined and the authors could estimate the among-individual covariance in these behaviors. I see that the authors state they include generalized linear mixed models in their updated MS, but I struggled a bit to understand exactly how these models were fit? What exactly was the response? what exactly were the predictors (I just don't understand what Line404 means "a GLM was trained using the environmental parameters as predictors (0 when the parameter was not changed, 1 if it was) and the resulting individual rank differences as the response"). So were different models run for each scenario? for different behaviors? Across scenarios? What exactly? I just harp on this because I'm actually really interested in these data and think that updating these methods can really help clarify the results and make the main messages much clearer!

I appreciate that the authors now included their sample sizes in the main body of text (as opposed to the supplement) but I think that it would still help if the authors included a brief overview of their design at the start of the methods. It is still unclear to me how many rigs each individual fly was run through? Were the same individuals measured in multiple different rigs/scenarios? Or just one?

I really think a variance partitioning modeling framework could certainly improve their statistical inference and likely highlight some other cool patterns as these methods could better estimate stability and covariance in individual intercepts (and potentially slopes) across time and situation. I also genuinely think that this will improve the impact and reach of this paper as they'll be using methods that are standard in the study of individual behavioral variation

Reviewer #3 (Public review):  

This manuscript is a continuation of past work by the last author where they looked at stochasticity in developmental processes leading to inter-individual behavioural differences. In that work, the focus was on a specific behaviour under specific conditions while probing the neural basis of the variability. In this work, the authors set out to describe in detail how stable individuality of animal behaviours is in the context of various external and internal influences. They identify a few behaviours to monitor (read outs of attention, exploration, and 'anxiety'); some external stimuli (temperature, contrast, nature of visual cues, and spatial environment); and two internal states (walking and flying).

They then use high-throughput behavioural arenas - most of which they have built and made plans available for others to replicate - to quantify and compare combinations of these behaviours, stimuli, and internal states. This detailed analysis reveals that:

(1) Many individualistic behaviours remain stable over the course of many days.  

(2) That some of these (walking speed) remain stable over changing visual cues. Others (walking speed and centrophobicity) remain stable at different temperatures.

(3) All the behaviours they tested fail to remain stable over spatially varying environment (arena shape).

(4) and only angular velocity (a read out of attention) remains stable across varying internal states (walking and flying)

Thus, the authors conclude that there is a hierarchy in the influence of external stimuli and internal states on the stability of individual behaviours.

The manuscript is a technical feat with the authors having built many new high-throughput assays. The number of animals are large and many variables have been tested - different types of behavioural paradigms, flying vs walking, varying visual stimuli, different temperature among others.  

Comments on revisions:'  

The authors have addressed my previous concerns.  

We thank the reviewer for the positive feedback and are glad our revisions have satisfactorily addressed the previous concerns. We appreciate the thoughtful input that helped us improve the clarity and rigor of the manuscript.

Reviewer #1 (Recommendations for the authors):  

Comment on Revised Manuscript  

Recommendations for Improvement  

(1) Expand the Results section for Figure 9 with a more detailed interpretation of the GLM coefficients and their biological significance

(2) Provide explicit criteria (or at least explain in detail) for how the GLM results confirm or undermine their original hypothesis about environmental context hierarchy

While the claims are interesting, the additional statistical analysis appears promising. However, clearer explanation of these new results would strengthen the paper and ensure that readers from diverse backgrounds can fully understand how the evidence supports the authors' conclusions about individuality across environmental contexts. 

We thank the reviewer for these constructive suggestions. In response to these suggestions, we have expanded both the Methods and Results sections to provide a more detailed explanation of the GLM coefficients, including their interpretation and how they relate to our original correlation-based findings.

We now clarify how the direction, magnitude, and statistical significance of specific coefficients reflect the influence of different environmental factors on the persistence of individual behavioral traits. To make this accessible to readers from diverse backgrounds, we explicitly outline the criteria we used to evaluate whether the GLM results support our hypothesis about the hierarchical influence of environmental context, namely, whether the structure and strength of effects align with the patterns predicted from our prior correlation analysis.

These additions improve clarity and help readers understand how the new statistical results reinforce our conclusions about the context-dependence of behavioral individuality.

Reviewer #2 (Recommendations for the authors):  

Thanks for the revision of the paper! I updated my review to try and provide a little more guidance by what I mean about updating your analyses. I really think this is a super cool data set and I genuinely wish this were MY dataset so that way I could really dig into it to partition the variance. These variance partitioning methods are standard in my particular subfield (study of individual behavioral variation in ecology and evolution) and so I think employing them is 1) going to offer a MUCH more elegant and holistic view of the behavioral variation (e.g. you can report a single repeatability estimate for each behavior rather than 3 different correlations) and 2) improve the impact and readership for your paper as now you'll be using methods that a whole community of researchers are very familiar with. It's just a suggestion, but I hope you consider it!

We sincerely thank the reviewer for the insightful and encouraging feedback and for introducing us to this modeling approach. In response to this suggestion, we have incorporated a hierarchical linear mixed-effects model into our analysis (now presented in Figure 10), accompanied by a new supplementary table (Table T3). We also updated the Methods, Results, and Discussion sections to describe the rationale, implementation, and implications of the mixed-model analysis.

We agree with the reviewer that this approach provides a more elegant way to quantify behavioral variation and individual consistency across contexts. In particular, the ability to estimate repeatability directly aligns well with the core questions of our study. It facilitates improved communication of our findings to ecology, evolution, and behavior researchers. We greatly appreciate the suggestion; it has significantly strengthened both the analytical framework and the interpretability of the manuscript.

Reviewer #1 (Public review):

This is an interesting and timely computational study using molecular dynamics simulation as well as quantum mechanical calculation to address why tyrosine (Y), as part of an intrinsically disordered protein (IDP) sequence, has been observed experimentally to be stronger than phenylalanine (F) as a promoter for biomolecular phase separation. Notably, the authors identified the aqueous nature of the condensate environment and the corresponding dielectric and hydrogen bonding effects as a key to understand the experimentally observed difference. This principle is illustrated by the difference in computed transfer free energy of Y- and F-containing pentapeptides into solvent with various degrees of polarity. The elucidation offered by this work is important. The computation appears to be carefully executed, the results are valuable, and the discussion is generally insightful. However, there is room for improvement in some parts of the presentation in terms of accuracy and clarity, including, e.g., the logic of the narrative should be clarified with additional information (and possibly additional computation), and the current effort should be better placed in the context of prior relevant theoretical and experimental works on cation-π interactions in biomolecules and dielectric properties of biomolecular condensates. Accordingly, this manuscript should be revised to address the following, with added discussion as well as inclusion of references mentioned below.

(1) Page 2, line 61: "Coarse-grained simulation models have failed to account for the greater propensity of arginine to promote phase separation in Ddx4 variants with Arg to Lys mutations (Das et al., 2020)". As it stands, this statement is not accurate, because the cited reference to Das et al. showed that although some coarse-grained model, namely the HPS model of Dignon et al., 2018 PLoS Comput did not capture the Arg to Lys trend, the KH model described in the same Dignon et al. paper was demonstrated by Das et al. (2020) to be capable of mimicking the greater propensity of Arg to promote phase separation than Lys. Accordingly, a possible minimal change that would correct the inaccuracy of this statement in the manuscript would be to add the word "Some" in front of "coarse-grained simulation models ...", i.e., it should read "Some coarse-grained simulation models have failed ...". In fact, a subsequent work [Wessén et al., J Phys Chem B 126: 9222-9245 (2022)] that applied the Mpipi interaction parameters (Joseph et al., 2021, already cited in the manuscript) showed that Mpipi is capable of capturing the rank ordering of phase separation propensity of Ddx4 variants, including a charge scrambled variant as well as both the Arg to Lys and the Phe to Ala variants (see Fig.11a of the above-cited Wessén et al. 2022 reference). The authors may wish to qualify their statements in the introduction to take note of these prior results. For example, they may consider adding a note immediately after the next sentence in the manuscript "However, by replacing the hydrophobicity scales ... (Das et al., 2020)" to refer to these subsequent findings in 2021-2022.

(2) Page 8, lines 285-290 (as well as the preceding discussion under the same subheading & Fig.4): "These findings suggest that ... is not primarily driven by differences in protein-protein interaction patterns ..." The authors' logic in terms of physical explanation is somewhat problematic here. In this regard, "Protein-protein interaction patterns" appears to be a straw man, so to speak. Indeed, who (reference?) has argued that the difference in the capability of Y and F in promoting phase separation should be reflected in the pairwise amino acid interaction pattern in a condensate that contains either only Y (and G, S) and only F (and G, S) but not both Y and F? Also, this paragraph in the manuscript seems to suggest that the authors' observation of similar contact patterns in the GSY and GSF condensates is "counterintuitive" given the difference in Y-Y and F-F potentials of mean force (Joseph et al., 2021); but there is nothing particularly counterintuitive about that. The two sets of observations are not mutually exclusive. For instance, consider two different homopolymers, one with a significantly stronger monomer-monomer attraction than the other. The condensates for the two different homopolymers will have essentially the same contact pattern but very different stabilities (different critical temperatures), and there is nothing surprising about it. In other words, phase separation propensity is not "driven" by contact pattern in general, it's driven by interaction (free) energy. The relevant issue here is total interaction energy or critical point of the phase separation. If it is computationally feasible, the authors should attempt to determine the critical temperatures for the GSY condensate versus the GSF condensate to verify that the GSY condensate has a higher critical temperature than the GSF condensate. That would be the most relevant piece of information for the question at hand.

(3) Page 9, lines 315-316: "...Our ε [relative permittivity] values ... are surprisingly close to that derived from experiment on Ddx4 condensates (45{plus minus}13) (Nott et al., 2015)". For accuracy, it should be noted here that the relative permittivity provided in the supplementary information of Nott et al. was not a direct experimental measurement but based on a fit using Flory-Huggins (FH), but FH is not the most appropriate theory for polymer with long-spatial-range Coulomb interactions. To this reviewer's knowledge, no direct measurement of relative permittivity in biomolecular condensates has been made to date. Explicit-water simulation suggests that relative permittivity of Ddx4 condensate with protein volume fraction ≈ 0.4 can have relative permittivity ≈ 35-50 (Das et al., PNAS 2020, Fig.7A), which happens to agree with the ε = 45{plus minus}13 estimate. This information should be useful to include in the authors' manuscript.

(4) As for the dielectric environment within biomolecular condensates, coarse-grained simulation has suggested that whereas condensates formed by essentially electric neutral polymers (as in the authors' model systems) have relative permittivities intermediate between that of bulk water and that of pure protein (ε = 2-4, or at most 15), condensates formed by highly charge polymers can have relative permittivity higher than that of bulk water [Wessén et al., J Phys Chem B 125:4337-4358 (2021), Fig.14 of this reference]. In view of the role of aromatic residues (mainly Y and F) in the phase separation of IDPs such as A1-LCD and LAF-1 that contain positively and negatively charged residues (Martin et al., 2020; Schuster et al., 2020, already cited in the manuscript), it should be useful to address briefly how the relationship between the relative phase-separation promotion strength of Y vs F and dielectric environment of the condensate may or may not be change with higher relative permittivities.

(5) The authors applied the dipole moment fluctuation formula (Eq.2 in the manuscript) to calculate relative permittivity in their model condensates. Does this formula apply only to an isotropic environment? The authors' model condensates were obtained from a "slab" approach (p.4) and thus the simulation box has a rectangular geometry. Did the authors apply their Eq.2 to the entire simulation box or only to the central part of the box with the condensate (see, e.g., Fig.3C in the manuscript). If the latter is the case, is it necessary to use a different dipole moment formula that distinguishes between the "parallel" and "perpendicular" components of the dipole moment (see, e.g., Eq.16 in the above-cited Wessén et al. 2021 paper). A brief added comments will be useful.

(6) With regard to the general role of Y and F in the phase separation of biomolecules containing positively charged Arg and Lys residues, the relative strength of cation-π interactions (cation-Y vs cation-F) should be addressed (in view of the generality implied by the title of the manuscript), or at least discussed briefly in the authors' manuscript if a detailed study is beyond the scope of their current effort. It has long been known that in the biomolecular context, cation-Y is slightly stronger than cation-F, whereas cation-tryptophan (W) is significantly stronger than either cation-Y and cation-F [Wu & McMahon, JACS 130:12554-12555 (2008)]. Experimental data from a study of EWS (Ewing sarcoma) transactivation domains indicated that Y is a slightly stronger promoter than F for transcription, whereas W is significantly stronger than either Y or F [Song et al., PLoS Comput Biol 9:e1003239 (2013)]. In view of the subsequent general recognition that "transcription factors activate genes through the phase-separation capacity of their activation domain" [Boija et al., Cell 175:1842-1855.e16 (2018)] which is applicable to EWS in particular [Johnson et al., JACS 146:8071-8085 (2024)], the experimental data in Song et al. 2013 (see Fig.3A of this reference) suggests that cation-Y interactions are stronger than cation-F interactions in promoting phase separation, thus generalizing the authors' observations (which focus primarily on Y-Y, Y-F and F-F interactions) to most situations in which cation-Y and cation-F interactions are relevant to biomolecular condensation.

(7) Page 9: The observation of a weaker effective F-F (and a few other nonpolar-nonpolar) interaction in a largely aqueous environment (as in an IDP condensate) than in a nonpolar environment (as in the core of a folded protein) is intimately related to (and expected from) the long-recognized distinction between "bulk" and "pair" as well as size dependence of hydrophobic effects that have been addressed in the context of protein folding [Wood & Thompson, PNAS 87:8921-8927 (1990); Shimizu & Chan, JACS 123:2083-2084 (2001); Proteins 49:560-566 (2002)]. It will be useful to add a brief pointer in the current manuscript to this body of relevant resource in protein science.

Comments on revisions:

The authors have largely addressed my previous concerns and the manuscript has been substantially improved. Nonetheless, it will benefit the readers more if the authors had included more of the relevant references provided in my previous review so as to afford a broader and more accurate context to the authors' effort. This deficiency is particularly pertinent for point number 6 in my previous report about cation-pi interactions. The authors have now added a brief discussion but with no references on the rank ordering of Y, F, and W interactions. I cannot see how providing additional information about a few related works could hurt. Quite the contrary, having the references will help readers establish scientific connections and contribute to conceptual advance.

Author response:

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

Reviewer #1 (Public review):

This is an interesting and timely computational study using molecular dynamics simulation as well as quantum mechanical calculation to address why tyrosine (Y), as part of an intrinsically disordered protein (IDP) sequence, has been observed experimentally to be stronger than phenylalanine (F) as a promoter for biomolecular phase separation. Notably, the authors identified the aqueous nature of the condensate environment and the corresponding dielectric and hydrogen bonding effects as a key to understanding the experimentally observed difference. This principle is illustrated by the difference in computed transfer free energy of Y- and F-containing pentapeptides into a solvent with various degrees of polarity. The elucidation offered by this work is important. The computation appears to be carefully executed, the results are valuable, and the discussion is generally insightful. However, there is room for improvement in some parts of the presentation in terms of accuracy and clarity, including, e.g., the logic of the narrative should be clarified with additional information (and possibly additional computation), and the current effort should be better placed in the context of prior relevant theoretical and experimental works on cation-π interactions in biomolecules and dielectric properties of biomolecular condensates. Accordingly, this manuscript should be revised to address the following, with added discussion as well as inclusion of references mentioned below.

We are grateful for the referee’s assessment of our work and insightful suggestions, which we address point by point below.

(1) Page 2, line 61: "Coarse-grained simulation models have failed to account for the greater propensity of arginine to promote phase separation in Ddx4 variants with Arg to Lys mutations (Das et al., 2020)". As it stands, this statement is not accurate, because the cited reference to Das et al. showed that although some coarse-grained models, namely the HPS model of Dignon et al., 2018 PLoS Comput did not capture the Arg to Lys trend, the KH model described in the same Dignon et al. paper was demonstrated by Das et al. (2020) to be capable of mimicking the greater propensity of Arg to promote phase separation than Lys. Accordingly, a possible minimal change that would correct the inaccuracy of this statement in the manuscript would be to add the word "Some" in front of "coarse-grained simulation models ...", i.e., it should read "Some coarse-grained simulation models have failed ...". In fact, a subsequent work [Wessén et al., J Phys Chem B 126: 9222-9245 (2022)] that applied the Mpipi interaction parameters (Joseph et al., 2021, already cited in the manuscript) showed that Mpipi is capable of capturing the rank ordering of phase separation propensity of Ddx4 variants, including a charge scrambled variant as well as both the Arg to Lys and the Phe to Ala variants (see Figure 11a of the above-cited Wessén et al. 2022 reference). The authors may wish to qualify their statements in the introduction to take note of these prior results. For example, they may consider adding a note immediately after the next sentence in the manuscript "However, by replacing the hydrophobicity scales ... (Das et al., 2020)" to refer to these subsequent findings in 2021-2022.

We agree with the referee that the wording used in the original version was inaccurate. We did not want to expand too much on the previous results on Lys/Arg, to avoid overwhelming our readers with background information that was not directly relevant to the aromatic residues Phe and Tyr. We have now introduced some of the missing details in the hope that this will provide a more accurate account of what has been achieved with different versions of coarse-grained models. In the revised version, we say the following:

Das and co-workers attempted to explain arginine’s greater propensity to phase separate in Ddx4 variants using coarse-grained simulations with two different energy functions (Das et al., 2020). The model was first parametrized using a hydrophobicity scale, aimed to capture the “stickiness” of different amino acids (Dignon et al., 2018), but this did not recapitulate the correct rank order in the stability of the simulated condensates (Das et al., 2020). By replacing the hydrophobicity scale with interaction energies from amino acid contact matrices —derived from a statistical analysis of the PDB (Dignon et al., 2018; Miyazawa and Jernigan, 1996; Kim and Hummer, 2008)— they recovered the correct trends (Das et al., 2020). A key to the greater propensity for LLPS in the case of Arg may derive from the pseudo-aromaticity of this residue, which results in a greater stabilization relative to the more purely cationic character of Lys (Gobbi and Frenking, 1993; Wang et al., 2018; Hong et al., 2022).

(2) Page 8, lines 285-290 (as well as the preceding discussion under the same subheading & Figure 4): "These findings suggest that ... is not primarily driven by differences in protein-protein interaction patterns ..." The authors' logic in terms of physical explanation is somewhat problematic here. In this regard, "Protein-protein interaction patterns" appear to be a straw man, so to speak. Indeed, who (reference?) has argued that the difference in the capability of Y and F in promoting phase separation should be reflected in the pairwise amino acid interaction pattern in a condensate that contains either only Y (and G, S) and only F (and G, S) but not both Y and F? Also, this paragraph in the manuscript seems to suggest that the authors' observation of similar contact patterns in the GSY and GSF condensates is "counterintuitive" given the difference in Y-Y and F-F potentials of mean force (Joseph et al., 2021); but there is nothing particularly counterintuitive about that. The two sets of observations are not mutually exclusive. For instance, consider two different homopolymers, one with a significantly stronger monomer-monomer attraction than the other. The condensates for the two different homopolymers will have essentially the same contact pattern but very different stabilities (different critical temperatures), and there is nothing surprising about it. In other words, phase separation propensity is not "driven" by contact pattern in general, it's driven by interaction (free) energy. The relevant issue here is total interaction energy or the critical point of the phase separation. If it is computationally feasible, the authors should attempt to determine the critical temperatures for the GSY condensate versus the GSF condensate to verify that the GSY condensate has a higher critical temperature than the GSF condensate. That would be the most relevant piece of information for the question at hand.

We are grateful for this very insightful comment by the referee. We have followed this suggestion to address whether, despite similar interaction patterns in GSY and GSF condensates, their stabilities are different. As in our previous work (De Sancho, 2022), we have run replica exchange MD simulations for both condensates and derived their phase diagrams. Our results, shown in the new Figure 5 and supplementary Figs. S6-S7, clearly indicate that the GSY condensate has a lower saturation density than the GSF condensate. This result is consistent with the trends observed in experiments on mutants of the low-complexity domain of hnRNPA1, where the relative amounts of F and Y determine the saturation concentration (Bremer et al., 2022).

(3) Page 9, lines 315-316: "...Our ε [relative permittivity] values ... are surprisingly close to that derived from experiment on Ddx4 condensates (45{plus minus}13) (Nott et al., 2015)".  For accuracy, it should be noted here that the relative permittivity provided in the supplementary information of Nott et al. was not a direct experimental measurement but based on a fit using Flory-Huggins (FH), but FH is not the most appropriate theory for a polymer with long-spatial-range Coulomb interactions. To this reviewer's knowledge, no direct measurement of relative permittivity in biomolecular condensates has been made to date. Explicit-water simulation suggests that the relative permittivity of Ddx4 condensate with protein volume fraction ≈ 0.4 can have a relative permittivity ≈ 35-50 (Das et al., PNAS 2020, Fig.7A), which happens to agree with the ε = 45{plus minus}13 estimate. This information should be useful to include in the authors' manuscript.

We thank the referee for this useful comment. We are aware that the estimate we mentioned is not direct. We have now clarified this point and added the additional estimate from Das et al. In the new version of the manuscript, we say:

Our 𝜀 values for the condensates (39 ± 5 for GSY and 47 ± 3 for GSF) are surprisingly close to that derived from experiments on Ddx condensates using Flory-Huggins theory (45±13) (Nott et al., 2015) and from atomistic simulations of Ddx4 (∼35−50 at a volume fraction of 𝜙 = 0.4) (Das et al., 2020).

(4) As for the dielectric environment within biomolecular condensates, coarse-grained simulation has suggested that whereas condensates formed by essentially electric neutral polymers (as in the authors' model systems) have relative permittivities intermediate between that of bulk water and that of pure protein (ε=2-4, or at most 15), condensates formed by highly charged polymers can have relative permittivity higher than that of bulk water [Wessén et al., J Phys Chem B 125:4337-4358 (2021), Fig.14 of this reference]. In view of the role of aromatic residues (mainly Y and F) in the phase separation of IDPs such as A1-LCD and LAF-1 that contain positively and negatively charged residues (Martin et al., 2020; Schuster et al., 2020, already cited in the manuscript), it should be useful to address briefly how the relationship between the relative phase-separation promotion strength of Y vs F and dielectric environment of the condensate may or may not be change with higher relative permittivities.

We thank the referee for their comment regarding highly charged polymers. However, we have chosen not to address these systems in our manuscript, as they are significantly different from the GSY/GSF peptide condensates under investigation. In polyelectrolyte systems, condensate formation is primarily driven by electrostatic interactions and counterion release, while we highlight the role of transfer free energies. At high dielectric constants (and dielectrics even higher than that of water), the strength of electrostatic interactions will be greatly reduced. In our approach to estimate differences between Y and F, the transfer free energy should plateau at a value of ΔΔG=0 in water. At greater values of ε>80, it becomes difficult to predict whether additional effects might become relevant. As this lies beyond the scope of our current study, we prefer not to speculate further.

(5) The authors applied the dipole moment fluctuation formula (Eq.2 in the manuscript) to calculate relative permittivity in their model condensates. Does this formula apply only to an isotropic environment? The authors' model condensates were obtained from a "slab" approach (page 4 and thus the simulation box has a rectangular geometry. Did the authors apply Equation 2 to the entire simulation box or only to the central part of the box with the condensate (see, e.g., Figure 3C in the manuscript). If the latter is the case, is it necessary to use a different dipole moment formula that distinguishes between the "parallel" and "perpendicular" components of the dipole moment (see, e.g., Equation 16 in the above-cited Wessén et al. 2021 paper). A brief added comment will be useful.

We have calculated the relative permittivity from dense phases only. These dense phases were sliced from the slab geometry and then re-equilibrated. Long simulations were then run to converge the calculation of the dielectric constant. We have clarified this in the Methods section of the paper. We say:

For the calculation of the dielectric constant of condensates, we used the simulations of isolated dense phases mentioned above.

(6) Concerning the general role of Y and F in the phase separation of biomolecules containing positively charged Arg and Lys residues, the relative strength of cation-π interactions (cation-Y vs cation-F) should be addressed (in view of the generality implied by the title of the manuscript), or at least discussed briefly in the authors' manuscript if a detailed study is beyond the scope of their current effort. It has long been known that in the biomolecular context, cation-Y is slightly stronger than cation-F, whereas cation-tryptophan (W) is significantly stronger than either cation-Y and cation-F [Wu & McMahon, JACS 130:12554-12555 (2008)]. Experimental data from a study of EWS (Ewing sarcoma) transactivation domains indicated that Y is a slightly stronger promoter than F for transcription, whereas W is significantly stronger than either Y or F [Song et al., PLoS Comput Biol 9:e1003239 (2013)]. In view of the subsequent general recognition that "transcription factors activate genes through the phase-separation capacity of their activation domain" [Boija et al., Cell 175:1842-1855.e16 (2018)] which is applicable to EWS in particular [Johnson et al., JACS 146:8071-8085 (2024)], the experimental data in Song et al. 2013 (see Figure 3A of this reference) suggests that cation-Y interactions are stronger than cation-F interactions in promoting phase separation, thus generalizing the authors' observations (which focus primarily on Y-Y, Y-F and F-F interactions) to most situations in which cation-Y and cation-F interactions are relevant to biomolecular condensation.

We thank our referee for this insightful comment. While we restrict our analysis to aromatic pairs in this work, the observed crossover will certainly affect other pairs where tyrosine or phenylalanine are involved. We now comment on this point in the discussions section of the revised manuscript. This topic will be explored in detail in a follow-up manuscript we are currently completing. We say:

We note that, although we have not included in our analysis positively charged residues that form cation-π interactions with aromatics, the observed crossover will also be relevant to Arg/Lys contacts with Phe and Tyr. Following the rationale of our findings, within condensates, cation-Tyr interactions are expected to promote phase separation more strongly than cation-Phe pairs.

(7) Page 9: The observation of weaker effective F-F (and a few other nonpolar-nonpolar) interactions in a largely aqueous environment (as in an IDP condensate) than in a nonpolar environment (as in the core of a folded protein) is intimately related to (and expected from) the long-recognized distinction between "bulk" and "pair" as well as size dependence of hydrophobic effects that have been addressed in the context of protein folding [Wood & Thompson, PNAS 87:8921-8927 (1990); Shimizu & Chan, JACS 123:2083-2084 (2001); Proteins 49:560-566 (2002)]. It will be useful to add a brief pointer in the current manuscript to this body of relevant resources in protein science.

We thank the referee for bringing this body of work to our attention. In the revised version of our work, we briefly mention how it relates to our results. We also note that the suggested references have pointed to another of the limitations of our study, that of chain connectivity, addressed in the work by Shimizu and Chan. While we were well aware of these limitations, we had not mentioned them in our manuscript. Concerning the distinction between pair and bulk hydrophobicities, we include the following in the concluding lines of our work:

The observed context dependence has deep roots in the concepts of “pair” and “bulk” hydrophobicity (Wood and Thompson, 1990; Shimizu and Chan, 2002). While pair hydrophobicity is connected to dimerisation equilibria (i.e. the second step in Figure 2B), bulk hydrophobicity is related to transfer processes (the first step). Our work stresses the importance of considering both the pair contribution that dominates at high solvation, and the transfer free energy contribution, which overwhelms the interaction strength at low dielectrics.

Reviewer #2 (Public review):

Summary:

In this preprint, De Sancho and López use alchemical molecular dynamics simulations and quantum mechanical calculations to elucidate the origin of the observed preference of Tyr over Phe in phase separation. The paper is well written, and the simulations conducted are rigorous and provide good insight into the origin of the differences between the two aromatic amino acids considered.

We thank the referee for his/her positive assessment of our work. Below, we address all the questions raised one by one.

Strengths:

The study addresses a fundamental discrepancy in the field of phase separation where the predicted ranking of aromatic amino acids observed experimentally is different from their anticipated rankings when considering contact statistics of folded proteins. While the hypothesis that the difference in the microenvironment of the condensed phase and hydrophobic core of folded proteins underlies the different observations, this study provides a quantification of this effect. Further, the demonstration of the crossover between Phe and Tyr as a function of the dielectric is interesting and provides further support for the hypothesis that the differing microenvironments within the condensed phase and the core of folded proteins is the origin of the difference between contact statistics and experimental observations in phase separation literature. The simulations performed in this work systematically investigate several possible explanations and therefore provide depth to the paper.

Weaknesses:

While the study is quite comprehensive and the paper well written, there are a few instances that would benefit from additional details. In the methods section, it is unclear as to whether the GGXGG peptides upon which the alchemical transforms are conducted are positioned restrained within the condensed/dilute phase or not. If they are not, how would the position of the peptides within the condensate alter the calculated free energies reported? 

The peptides are not restrained in our simulations and can therefore diffuse out of the condensate given sufficient time. Although the GGXGG peptide can, given sufficient time, leave the peptide condensate, we did not observe any escape event in the trajectories we used to generate starting points for switching. Hence, the peptide environment captured in our calculations reflects, on average, the protein-protein and protein-solvent interactions inside the model condensate. We believe this is the right way of performing the calculation of transfer free energy differences into the condensate. We have clarified this point when we describe the equilibrium simulation results in the revised manuscript. We say:

Also, the peptide that experiences the transformation, which is not restrained, must remain buried within the condensate for all the snapshots that we use as initial frames, to avoid averaging the work in the dilute and dense phases.

On the referee’s second point of whether there would be differences if the peptide visited the dilute phase, the answer is that, indeed, we would. We expect that the behaviour of the peptide would approach ΔΔG=0, considering the low protein concentration in the dilute phase. For mixed trajectories with sampling in both dilute and dense phases, our expectation would be a bimodal distribution in the free energy estimates from switching (see e.g. Fig. 8 in DOI:10.1021/acs.jpcb.0c10263). Because we are exclusively interested in the transfer free energies into the condensate, we do not pursue such calculations in this work.

It would also be interesting to see what the variation in the transfer of free energy is across multiple independent replicates of the transform to assess the convergence of the simulations. 

Upon submission of our manuscript, we were confident that the results we had obtained would pass the test of statistical significance. We had, after all, done many more simulations than those reported, plus the comparable values of ΔΔG_Transfer for both GSY and GSF pointed in the right direction. However, we acknowledge that the more thorough test of running replicates recommended by the referee is important, considering the slow diffusion within the Tyr peptide condensates due to its stickiness. Also, the non-equilibrium switching method had not been tested before for dense phases like the ones considered here.

We have hence followed our referee's suggestion and done three different replicates, 1 μs each, of the equilibrium runs starting from independent slab configurations, for both the GSY and GSF condensates (see the new supporting figures Fig. S1, S2 and S5). We now report the errors from the three replicates as the standard error of the mean (bootstrapping errors remain for the rest of the solvents). Our results are entirely consistent with the values reported originally, confirming the validity of our estimates.

Additionally, since the authors use a slab for the calculation of these free energies, are the transfer free energies from the dilute phase to the interface significantly different from those calculated from the dilute phase to the interior of the condensate? 

We thank the referee for this valuable comment, as it has pointed us in the direction of a rapidly increasing body of work on condensate interfaces, for example, as mediators of aggregation, that we may consider for future study with the same methodology. However, as discussed above, we have not considered this possibility in our work, as we decided to focus on the condensate environment, rather than its interface.

The authors mention that the contact statistics of Phe and Tyr do not show significant difference and thereby conclude that the more favorable transfer of Tyr primarily originates from the dielectric of the condensate. However, the calculation of contacts neglects the differences in the strength of interactions involving Phe vs. Tyr. Though the authors consider the calculation of energy contact formation later in the manuscript, the scope of these interactions are quite limited (Phe-Phe, Tyr-Tyr, Tyr-Amide, Phe-Amide) which is not sufficient to make a universal conclusion regarding the underlying driving forces. A more appropriate statement would be that in the context of the minimal peptide investigated the driving force seems to be the difference in dielectric. However, it is worth mentioning that the authors do a good job of mentioning some of these caveats in the discussion section.

We thank the referee for this important comment. Indeed, the similar contact statistics and interaction patterns that we reported originally do not necessarily imply identical interaction energies. In other words, similar statistics and patterns can still result in different stabilities for the Phe and Tyr condensates if the energetics are different. Hence, we cannot conclude that the GSF and GSY condensate environments are equivalent.

To address this point, we have run new simulations for the revised version of our paper, using the temperature-replica exchange method, as before. From the new datasets, we derive the phase diagrams for both the GSF and GSY condensates (see the new Fig. 5). We find that the tyrosine-containing condensate is more stable than that of phenylalanine, as can be inferred from the lower saturation density in the low-density branch of the phase diagram. In consequence, despite the similar contact statistics, the energetics differ, making the saturation density of the GSY slightly lower than that of GSF. This result is consistent with experimental data by Bremer et al (Nat. Chem. 2022). 

Reviewer #3 (Public review):

Summary:

In this study, the authors address the paradox of how tyrosine can act as a stronger sticker for phase separation than phenylalanine, despite phenylalanine being higher on the hydrophobicity scale and exhibiting more prominent pairwise contact statistics in folded protein structures compared to tyrosine.

We are grateful for the referee’s favourable opinion on the paper. Below, we address all of the issues raised.

Strengths:

This is a fascinating problem for the protein science community with special relevance for the biophysical condensate community. Using atomistic simulations of simple model peptides and condensates as well as quantum calculations, the authors provide an explanation that relies on the dielectric constant of the medium and the hydration level that either tyrosine or phenylalanine can achieve in highly hydrophobic vs. hydrophilic media. The authors find that as the dielectric constant decreases, phenylalanine becomes a stronger sticker than tyrosine. The conclusions of the paper seem to be solid, it is well-written and it also recognises the limitations of the study. Overall, the paper represents an important contribution to the field.

Weaknesses:

How can the authors ensure that a condensate of GSY or GSF peptides is a representative environment of a protein condensate? First, the composition in terms of amino acids is highly limited, second the effect of peptide/protein length compared to real protein sequences is also an issue, and third, the water concentration within these condensates is really low as compared to real experimental condensates. Hence, how can we rely on the extracted conclusions from these condensates to be representative for real protein sequences with a much more complex composition and structural behaviour?

We agree with the main weakness identified by the referee. In fact, all these limitations had already been stated in our original submission. Our ternary peptide condensates are just a minimal model system that bears reasonable analogies with condensates, but definitely is not identical to true LCR condensates. The analogies between peptide and protein condensates are, however, worth restating: 

(1) The limited composition of the peptide condensates is inspired by LCR sequences (see Fig. 4 in Martin & Mittag, 2018).

(2) The equilibrium phase diagram, showing a UCST, is consistent with that of LCRs from Ddx4 or hnRNPA1.

(3) The dynamical behaviour is intermediate between liquid and solid (De Sancho, 2022). 

(4) The contact patterns are comparable to those observed for FUS and LAF1 (Zheng et al, 2020).

The third issue pointed out by the referee requires particular attention. Indeed, the water content in the model condensates is low (~200 mg/mL for GSY) relative to the experiment (e.g. ~600 mg/mL for FUS and LAF-1 from simulations). Considering that both interaction patterns and solvation contribute to the favorability of Tyr relative to Phe, we speculate that a greater degree of solvation in the true protein condensates will further reinforce the trends we observe.

In any case, in the revised version of the manuscript, we have made an effort to insist on the limitations of our results, some of which we plan to address in future work.

Reviewer #3 (Recommendations for the authors):

(1) The fact that protein density is so high within GSY or GSF peptide condensates may significantly alter the conclusions of the paper. Can the authors show that for condensates in which the protein density is ~0.2-0.3 g/cm3, the same conclusions hold? Could the authors use a different peptide sequence that establishes a more realistic protein concentration/density inside the condensate?

Unfortunately, recent work with a variety of peptide sequences suggests that finding peptides in the density range proposed by the referee may be very challenging. For example, Pettit and his co-workers have extensively studied the behaviour of GGXGG peptides. In a recent work, using the CHARMM36m force field and TIP3P water, they report densities of ~1.2-1.3 g/mL for capped pentapeptide condensates (Workman et al, Biophys. J. 2024; DOI: 10.1016/j.bpj.2024.05.009). Brown and Potoyan have recently run simulations of zwitterionic GXG tripeptides with the Amber99sb-ILDNQ force field and TIP3P water, starting with a homogenous distribution in cubic simulation boxes (Biophys. J. 2024, DOI: 10.1016/j.bpj.2023.12.027). In a box with an initial concentration of 0.25 g/mL, upon phase separation, the peptide ends up occupying what would seem to be ~1/3 of the box, although we could not find exact numbers. This would imply densities of ~0.75 g/mL in the dense phase, with the additional problem of many charges. Finally, Joseph and her co-workers have recently simulated a set of hexapeptide condensates with varied compositions using a combination of atomistic and coarse-grained simulations. For the atomistic simulations, the Amber03ws force field and TIP4P water were used (see BioRxiv reference 10.1101/2025.03.04.641530). They have found values of the protein density in the dense phase ranging between 0.8 and 1.2 g/mL.  The consistency in the range of densities reported in these studies suggests that short peptides, at least up to 7-residues long, tend to form quite dense condensates, akin to those investigated in our work. While the examples mentioned do not comprehensively span the full range of peptide lengths, sequences, and force fields, they nonetheless support the general behaviour we observe. A systematic exploration of all these variables would require an extensive search in parameter space, which we believe falls outside the scope of the present study.

(2) Do the conclusions hold for phase-separating systems that mostly rely on electrostatic interactions to undergo LLPS, like protein-RNA complex coacervates? In other words, could the authors try the same calculations for a binary mixture composed of polyR-polyE, or polyK-polyE?

This is an excellent idea that we may attempt in future work, but the remit of the current work is aromatic amino acids Phe and Tyr only. Hence, we do not include calculations or discussion on polyR-polyE systems in our revised manuscript.

(3) One of the major approximations made by the authors is the length of the peptides within the condensates, which is not realistic, or their density. Specifically, could they double or triple the length of these peptides while maintaining their composition so it can be quantified the impact of sequence length in the transfer of free energies?

We thank the referee for this comment and agree with the main point, which was stated as a limitation in our original submission. The suggested calculations anticipate research that we are planning but will not include in the current work. One of the advantages of our model systems is that the small size of the peptides allows for small simulation boxes and relatively rapid sampling. Longer peptide sequences would require conformational sampling beyond our current capabilities, if done systematically. An example of these limitations is the amount of data that we had to discard from the new simulations we report, which amounts to up to 200 ns of our replica exchange runs in smaller simulation boxes (i.e. >19 μs in total for the 48 replicas of the two condensates!). As stated in the answer to point 1, we have found in the literature work on peptides in the range of 1-7 residues with consistent densities. Additionally, a recent report using alchemical transformations using equilibrium techniques with tetrapeptide condensates, pointing to the role of transfer free energy as driving force for condensate formation, further supports the observations from our work.

Minor issues:

(1) The caption of Figure 3B is not clear. It can only be understood what is depicted there once you read the main text a couple of times. I encourage the authors to clarify the caption.

We have rewritten the caption for greater clarity. Now it reads as follows:

Time evolution of the density profiles calculated across the longest dimension of the simulation box (L) in the coexistence simulations. In blue we show the density of all the peptides, and in dark red that of the F/Y residue in the GGXGG peptide.

(2) Why was the RDF from Figure 5A cut at such a short distance? Can the authors expand the figure to clearly show that it has converged?

In the updated Figure 5 (now Fig. 6), we have extended the g(r) up to r=1.75 nm so that it clearly plateaus at a value of 1.

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What is this?

with their son in law

Los nuevos significados de la familia le han permitido a la mujer trabajar, sin descuidar sus responsabilidades familiares.

Me llama la atencion estas oraciones, porque resaltan que la familia no solo consiste en una casa y las personas que viven en ella.

Creo que los adultos mayores están empezando a ser más independientes más pronto que tarde debido a la época en la que vivimos. Con esto me refiero a cómo las redes sociales han persuadido a la gente a ser más abierta (explorar), mientras que antes solo contábamos con nuestra familia de sangre y nos creíamos la mayoría de lo que decían. No sé si me explico, es un poco confuso. Y sí, puede haber desafíos al volverse más independiente, ¡pero esto es bueno! Nos ayuda a crecer y aprender de nuestros errores, y eso es lo que nos prepara para la edad adulta.

No me identifico personalmente con esto, pero tengo un hermano que se fue a tener hijos y siguió viviendo en casa de mis padres con su conyuge. Les llevó un tiempo conseguir su propio lugar, pero al final lo consiguieron. Dicho esto, entiendo que esto sea cierto en cierto modo.

El texto muestra que los padres que no viven con sus hijos y la ayuda entre generaciones son importantes para el desarrollo infantil. Tambien senala que los programas sociales para ninos vulnerables ayudan a mejorar su bienestar.

El texto senala que, a diferencia de lo que ocurre en Estados Unidos, en America Latina y el Caribe el aumento del nivel educativo de las mujeres no disminuye la probabilidad de que sean madres solteras.

El informe Pulso Social 2016 muestra que en America Latina y el Caribe casa vez mas adultos mayores viven solos so solo con su conyuge an lugar de con la familia extendida. Aunque este cambio puede favorecer la independencia, talmbien tiene retos que podrian poner los cuidados de la vejez en riesgo.

conjunto de uno o mas hijos y uno o dos padres.

Dans le corrigé, vous mettez cette balise dans le "header", mais dans le cours, on voit qu'elle vient après le "header". Du coup, je n'ai pas le même rendu que vous. De plus, vous ajoutez un lien "à propos" dans la page "à propos". Pour moi, cela est inutile : on ne va pas cliquer sur le lien d'une page où on y est déjà !

Mucho de la población de Los Estados Unions son hispanohablantes. Entonces, cuando los etudiantes aprenden inglés necesita se esfuerza como preservar español tambien y mezcla los culturas.

IMPORTANTE, MIRAR

OJO

OJO

OJO

glándulas mamarias presentes en hombres y mujeres en mujeres secretan leche para el neo-nato a través de los pezones Irrigado a través de arteria interna torácica, lateral torácica y posterior intercostal

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What is this?

jhfhjghj

A nivel general, considero que es una propuesta interesante, pero como bien señala la autora, quizá no sea para todos, no obstante no está demás intentarla para empezar a ser más conscientes de cómo empleamos nuestro tiempo, de como fluctúan nuestros pensamiento y emociones, e ir finalmente encontrando estrategias que nos ayuden a enfocarnos en este mundo en el que todo el tiempo nos vemos tentados a desviarnos por caminos que nos roban nuestro tiempo y atención.

Esto también me resulta muy interesante ya que por lo general no ponemos atención a nuestro estado de ánimo al momento de desarrollar actividades o en el día a día en general, y esto impacta directamente nuestro desempeño y capacidad. Muchas veces solo actuamos en automático y esperamos dar resultados similares día a día sin considerar cómo nos estamos sintiendo.

Estos dos aspectos sobre el contenido y las ideas es interesante, pero creo que si lo hacemos en un ambiente digital, mientras desarrollamos un proyecto con herramientas digitales, se pueden volver solo listas de cosas a las que nunca vamos a volver porque podrían resultar demasiadas opciones para revisar en un futuro hipotético con tiempo libre para dedicar a nuestros intereses.

Creo que con las pausas de procrastinación podría ocurrir lo mismo que con las pausas proactivas. Si nos damos cuenta de los desvíos de atención que nos hemos permitido, con base en mi experiencia, nos puede costar más retomar la tarea inicial al desanimarnos y sentir culpa de nuestra falta de atención.

Veo que las pausas proactivas son una herramienta que invita a la reflexión, a mirar el avance que se ha logrado y prepararse para continuar planeando anticipadamente. También veo que su fin es liberar la mente de aquellas distracciones que van surgiendo con el fin de enfocarnos en el desarrollo de la tarea en que nos hemos embarcado. Personalmente lo encuentro útil y lógico pero un poco complicado por la tendencia que tengo a sobre pensar y a abrumarme fácilmente. Planear nos supone la necesidad de cumplir el paso a paso que hemos diseñado y puede ser fácil desanimarse y bloquearse mentalmente cuando nos damos cuenta de que no hemos podido seguir lo que nos planteamos.

A pesar de las ventajas que podría tener el interstitial journaling para la productividad y el aprendizaje autónomo, a nivel personal me parece necesario reconocer que esta estrategia quizá no se ajusta a las necesidades y a las formas de todas y todos, y que en algunos casos podría resultar siendo un obstáculo o una carga adicional para llevar a cabo las tareas que se espera desarrollar.

Creo que este es un buen punto de partida para reflexionar sobre el interstitial journaling, porque más que su utilidad en relación con la productividad a la que tanto estamos llamados hoy en día, me interesa la idea de poder hacernos más consciente del tiempo y cómo hacemos uso de el, sobretodo cuando estamos buscando desarrollar una tarea en específico, pero también como herramienta para entendernos y conocernos mejor cuando tenemos dificultades para manejar nuestro tiempo y para enfocarnos en desarrollar tareas que nos proponemos o que debemos realizar.

Excelentes consejos, estos descansos son muy importantes para darle un respiro a nuestro cerebro y hacerlos significativos por medio del interstital journaling. Es necesario separar los descansos proactivos a la procrastinación.

Document d'Information : Synthèse sur le TDAH et l'Anxiété

Résumé Analytique

• Ce document synthétise les liens complexes entre le trouble du déficit de l'attention avec ou sans hyperactivité (TDAH) et l'anxiété, en se basant sur l'idée que de nombreux symptômes anxieux peuvent être mieux expliqués par des déficits sous-jacents de la fonction exécutive liés au TDAH. Jusqu'à 50 % des personnes atteintes de TDAH souffrent également d'un trouble anxieux, et le chevauchement des symptômes, tels que les pensées rapides, les troubles du sommeil et les problèmes de concentration, rend le diagnostic complexe.
• La distinction fondamentale réside dans la cause première des symptômes.

L'anxiété est généralement enracinée dans une réponse de peur exacerbée et des comportements d'évitement habituels, souvent liés à une suractivité de l'amygdale.

En revanche, lorsque les symptômes de type anxieux proviennent du TDAH, ils découlent principalement d'un déficit prédisposé de la fonction exécutive — la capacité du cerveau à planifier, organiser, réguler les émotions et contrôler les impulsions. Ce déficit est souvent lié à des différences dans le cortex préfrontal. * Un concept neurologique clé, la "dispersion du profil cognitif", illustre cette différence. Les personnes atteintes de TDAH présentent souvent des écarts importants entre leurs différentes capacités cognitives, comme une vitesse de traitement très élevée associée à une mémoire de travail faible.

Cette disparité, décrite comme un "moteur de voiture de course avec des freins de vélo", crée des difficultés de régulation émotionnelle qui alimentent l'anxiété. * Par conséquent, une approche efficace pour traiter l'anxiété chez les personnes atteintes de TDAH consiste à soutenir et à renforcer la fonction exécutive. Cela implique des stratégies pratiques visant à "ralentir la voiture de course" (améliorer l'inhibition), "renforcer les freins" (soutenir la mémoire de travail) et "obtenir une équipe de soutien" (utiliser des ressources externes), offrant ainsi une voie pour gérer l'anxiété en s'attaquant à sa racine neurodéveloppementale.

1. Le Chevauchement Symptomatique entre TDAH et Anxiété

• La relation entre le TDAH et l'anxiété peut être illustrée par la métaphore d'un arbre qui semble unique mais possède en réalité deux systèmes racinaires distincts. Bien que les "feuilles" — les symptômes visibles — soient identiques (inquiétude, surmenage, troubles du sommeil), les "racines" — les causes sous-jacentes — diffèrent. Une racine représente l'anxiété découlant d'une réponse de peur, tandis que l'autre représente des symptômes de type anxieux alimentés par un déficit de la fonction exécutive lié au TDAH.

De nombreux symptômes sont communs aux deux conditions, mais leur origine et leur mécanisme diffèrent.

• Symptôme
• Origine dans l'Anxiété
• Origine dans le TDAH
• Inquiétude
• Une habitude inadaptée visant à prévenir les événements négatifs, renforçant un cycle où l'anxiété mène à l'inquiétude, qui prévient (par coïncidence) le résultat redouté, ce qui renforce l'anxiété.
• Le cerveau active son "réseau du mode par défaut" en l'absence de stimulation, se tournant vers l'inquiétude. L'hyperfocalisation rend difficile le désengagement des pensées anxiogènes.

Hyperactivité * Une manifestation de la réponse combat-fuite-figer (fight/flight/freeze). Le corps est inondé d'hormones de stress, le préparant à fuir un danger perçu (par exemple, des e-mails). * Décrite comme être "propulsé par un moteur", résultant d'un surplus d'énergie physique combiné à une faible inhibition.

Difficulté de Concentration

• Le cerveau est constamment en état d'alerte, balayant l'environnement à la recherche de dangers. Il est difficile de se concentrer sur des tâches (par exemple, les devoirs) lorsqu'on se sent menacé.
• Une difficulté inhérente à maintenir l'attention sur des tâches qui ne fournissent pas le niveau de stimulation adéquat pour le cerveau TDAH.

Troubles du Sommeil

• Les pensées anxieuses et l'inquiétude préventive maintiennent le système nerveux sympathique actif (état d'alerte élevé), empêchant le corps et l'esprit de se détendre.
• Le cerveau a des difficultés avec le contrôle des impulsions et la régulation comportementale. Il déteste l'ennui et génère donc un flot d'idées aléatoires au moment de s'endormir pour se stimuler.

2. La Différence Fondamentale : La Fonction Exécutive

• La distinction la plus cruciale entre l'anxiété et les symptômes de type anxieux liés au TDAH réside dans le rôle de la fonction exécutive. Il s'agit de la capacité du cerveau à gérer les pensées, les émotions et les actions pour atteindre un objectif, englobant des compétences comme la planification, la concentration, la mémorisation et la régulation des impulses.
• • Racine du TDAH : Déficit de la Fonction Exécutive
• ◦ Le TDAH est caractérisé par un déficit prédisposé de la fonction exécutive, localisée principalement dans le cortex préfrontal.
• ◦ Ce déficit rend difficile le traitement des émotions. Par exemple, face à une grande quantité de devoirs, une faible fonction exécutive entrave la priorisation des tâches, diminue la motivation et rend difficile l'initiation du travail. Ce cycle peut mener au stress, au manque de sommeil, à l'anxiété concernant les notes et à un sentiment de surmenage.
• • Racine de l'Anxiété : Réponse de Peur et Évitement
• ◦ Les troubles anxieux sont souvent associés à une amygdale ou un axe HPA (hypothalamo-hypophyso-surrénalien) suractif, créant une réponse de peur accrue face à un danger perçu.
• ◦ Lorsque le cerveau est en mode survie, il détourne l'énergie du cortex préfrontal (la pensée rationnelle) vers des réponses réactives. Cela mène à des comportements d'évitement (par exemple, éviter les devoirs par peur de l'échec ou, à l'inverse, viser la perfection pour éviter l'échec), qui alimentent et renforcent le cycle de l'anxiété.
• Ce lien crée un cercle vicieux : la faible fonction exécutive (TDAH) alimente la dérégulation émotionnelle et l'anxiété. En retour, l'état émotionnel intense de l'anxiété inhibe davantage la fonction exécutive, rendant la pensée claire encore plus difficile.

3. Les Bases Neurologiques : La "Dispersion du Profil Cognitif"

• La "dispersion du profil cognitif" est un phénomène fréquemment observé chez les personnes atteintes de TDAH qui explique la déconnexion entre l'intelligence apparente et les difficultés fonctionnelles.
• Un score de QI est une moyenne de plusieurs sous-tests mesurant différentes compétences (par exemple, raisonnement verbal, mémoire de travail, vitesse de traitement). Chez une personne neurotypique, ces scores sont généralement groupés. Chez une personne atteinte de TDAH, il peut y avoir un écart significatif entre les scores.
• • L'Analogie de la Voiture de Course : Le Dr Russell Barkley décrit un schéma courant dans le TDAH comme ayant "un moteur de voiture de course avec des freins de vélo".
• ◦ Moteur de voiture de course : Vitesse de traitement élevée. Le cerveau génère des idées et des émotions à un rythme extrêmement rapide.
• ◦ Freins de vélo : Faible mémoire de travail. La mémoire de travail est la capacité de retenir et de manipuler plusieurs informations simultanément. Elle est essentielle pour la régulation émotionnelle, car elle permet de comparer l'expérience actuelle à des souvenirs passés ("J'ai déjà traversé cela, ça va aller") et de se calmer.
• Lorsque la vitesse de traitement dépasse de loin la capacité de la mémoire de travail, le cerveau est inondé d'émotions et de pensées catastrophiques sans avoir les outils internes pour ralentir, organiser ou apaiser ces sentiments. Cela peut entraîner des explosions émotionnelles impulsives, un sentiment de surmenage et une anxiété chronique.
• Cette disparité conduit souvent à des malentendus. Un enfant ou un adulte peut paraître "très intelligent" en raison de sa fluidité verbale (vitesse de traitement élevée), mais avoir du mal à initier des tâches ou à se réguler (faible mémoire de travail). Ils sont alors perçus à tort comme "non motivés" ou "paresseux", ce qui peut entraîner des punitions, une faible estime de soi et une peur de l'échec.

4. Stratégies Pratiques : Aborder l'Anxiété via la Fonction Exécutive

Si l'anxiété est alimentée par un déficit de la fonction exécutive, la solution consiste à construire des systèmes de soutien ("échafaudages") pour ces fonctions. L'objectif n'est pas d'appliquer plus de "discipline", mais de fournir des outils adaptés.

Stratégie 1 : Ralentir la Voiture de Course (Améliorer l'Inhibition)

• • Exercice physique : Soutient directement la fonction exécutive et l'inhibition.
• • Méditation : Considérée comme une compétence d'entraînement pour le cerveau afin de ralentir et d'améliorer l'attention, plutôt qu'une simple technique de relaxation.
• • Médicaments : Les stimulants pour le TDAH activent les parties du cerveau responsables de la fonction exécutive, aidant à ralentir les processus de pensée.
• • Écriture : Journaliser ou simplement écrire ses sentiments est un moyen efficace de ralentir le cerveau, de clarifier le chaos mental et d'activer les parties organisatrices du cerveau.
• ◦ Exemple : Le témoignage de Sean, coach en fonction exécutive, qui gère le surmenage en écrivant chaque jour : "Si je ne faisais qu'une seule chose aujourd'hui, quelle serait-elle ?" Cette approche minimise la pression et définit un critère de réussite unique et réalisable.
• • Faire une Pause : Pour contrer l'impulsivité, il est utile de s'entraîner à faire une pause avant de réagir.
• ◦ Exemple : Mettre en place une "règle de 48 heures" avant de prendre des décisions importantes (par exemple, démissionner, faire un achat coûteux).
• • Faire Moins de Choses : Le surbooking est une cause majeure de désorganisation et d'anxiété. Il est essentiel de planifier activement du temps pour le repos et l'organisation.

Stratégie 2 : Renforcer les Freins (Soutenir la Mémoire de Travail et l'Organisation)

• • Rappels Visuels : Pour les personnes atteintes de TDAH, "loin des yeux, loin du cœur" est une réalité. Utiliser des post-it, des tableaux blancs et des listes visibles pour les routines, les stratégies d'adaptation et les objectifs.
• • Systèmes et Routines : Les systèmes de planification (calendriers visuels, listes de contrôle) sont souvent plus efficaces que les approches cognitives comme la TCC pour gérer l'anxiété liée au TDAH.
• • Rappels Numériques : Utiliser des alarmes et des rappels sur son téléphone pour les tâches importantes, les pauses ou même les gestes relationnels (par exemple, "faire un câlin à son conjoint").
• • Traitement Physique des Émotions : Au lieu de traiter les émotions uniquement mentalement, utiliser des supports physiques comme dessiner un schéma de ses sentiments, faire un "brain dump" (décharge mentale) ou une carte mentale.

Stratégie 3 : Obtenir une Équipe de Soutien (Support Externe)

• • Aide Professionnelle : Un thérapeute ou un coach spécialisé en fonction exécutive peut aider à ralentir, à être moins impulsif et à renforcer les stratégies de régulation.
• • "Body Doubling" : Accomplir des tâches difficiles en présence d'une autre personne. La présence d'un ami peut fournir la structure et la motivation nécessaires.
• • Verbaliser ses Pensées : Exprimer ses pensées à voix haute — que ce soit à un ami, dans un mémo vocal ou même à une IA — les empêche de tourner en boucle et les rend plus faciles à analyser.

En conclusion, lorsque l'anxiété et le TDAH sont liés, soutenir la fonction exécutive en premier lieu permet de mieux résoudre les problèmes et d'apaiser les émotions.

Cette approche permet de construire les fondations nécessaires pour que la pensée claire et les choix alignés avec ses valeurs deviennent plus accessibles.

Further discussion on the reliance/use of AI but by students and the tone of AI writing. Connects to the “flattens your voice” statement that Caplan made.

IDEAL

OJO, IMPORTANTE LA PENÚLTIMA ORACIÓN. No sé hasta que punto es viable que ciertos titulares tienen efectos retardados o combinados.

MUY IMPORTANTE ESTA CARACTERÍSTICA.

MUCHO OJO Y MUY IMPORTANTE, COMPRENDER BIEN

OJO

OJO, MUY IMPORTANTE ESTE PASO; ES CLAVE.

OJO, Dataset

Proposition : Ajouter une intro en amont de cette section sur le projet, qui devient ainsi une première partie. On peut ainsi y mettre davantage de notions théoriques. Ci-dessous un premier jet.

On sait que les bibliothèques brûlent, les musées aussi. La disparition de la documentation numérique, tout aussi réelle, est moins perceptible ; elle a longtemps été un impensé pour les professionnels du domaine. Les musées, chargés de conserver des collections physiques, sont aussi producteurs de recherche et de savoirs, qu’ils diffusent sous diverses formes. La plus évidente en est l’exposition, pensée et perçue comme temporaire ou évolutive, et son prolongement édité, durable qu’en sont les catalogues. Depuis les années 1980 apparaissent de nouveaux formats numériques, en ligne ou non : bases de données sur Minitel telles que 3615 Joconde, sites internet et expositions virtuelles dans les années 1990, CDROM et vidéodisques. Ces nouvelles productions échappent aux habitudes précédentes, le contenu n’étant accessible que via un dispositif technique. (RTP DOC) Contrairement aux catalogues papiers, qui trouvent durablement leur place dans les bibliothèques indépendamment de l’action de leur producteur, les sites internet n’ont pas de pérennité en eux-mêmes : leur contenu ne reste accessible qu’aussi longtemps que l’institution productrice maintient serveurs, noms de domaines, et technologies. Pour autant, le public peut s’en être emparé, sans que l’institution n’en ait conscience, jusqu’au jour où les accès sont coupés. Se pose alors la question de restituer, sous une forme ou une autre, ces ressources documentaires. L’archivage du web en est une solution privilégiée, mais ne répond pas à tous les besoins ni à toutes les questions. Comment garder l’accès à un site développé avec Flash ? Pourquoi redonner accès à un site dont le contenu paraît daté ? Comment compléter les creux de ces archives ? Le Mucem a été amené à se poser ces questions lorsque des chercheurs ont fait part de leur déception à la mise hors ligne fin 2020 d’un site sur les cornemuses, produit en 2007. Ce site a été le point de départ d’une réflexion plus large, portée dans le cadre d’un appel à projet bnf Data Lab. Du fait su contexte singulier de production de ce site, la question technique du départ s’est élargie et déplacée, conduisant à retisser une histoire plus large, touchant à celle de l’institution comme des pratiques de médiation numérique des années 2000.

I. L’origine du projet Très rapidement, il apparaît que ce site est intégré à une collection, les « Recherches ethnologiques », produite durant les années de préfiguration du Mucem. Les archives numériques de cette période sont parcellaires et peu structurées : les sites en eux-mêmes n’avaient pas été archivés, et la documentation du projet dispersée dans un « vrac numérique ». Pour répondre aux attentes de MBLG, le Mucem s’est donc tourné vers d’autres partenaires.

Author response:

Reviewer #1 (Public review): 

Summary: 

The authors analyzed the expression of ATAD2 protein in post-meiotic stages and characterized the localization of various testis-specific proteins in the testis of the Atad2 knockout (KO). By cytological analysis as well as the ATAC sequencing, the study showed that increased levels of HIRA histone chaperone, accumulation of histone H3.3 on post-meiotic nuclei, defective chromatin accessibility and also delayed deposition of protamines. Sperm from the Atad2 KO mice reduces the success of in vitro fertilization. The work was performed well, and most of the results are convincing. However, this manuscript does not suggest a molecular mechanism for how ATAD2 promotes the formation of testis-specific chromatin. 

We would like to take this opportunity to highlight that the present study builds on our previously published work, which examined the function of ATAD2 in both yeast S. pombe and mouse embryonic stem (ES) cells (Wang et al., 2021). In yeast, using genetic analysis we showed that inactivation of HIRA rescues defective cell growth caused by the absence of ATAD2. This rescue could also be achieved by reducing histone dosage, indicating that the toxicity depends on histone over-dosage, and that HIRA toxicity, in the absence of ATAD2, is linked to this imbalance.

Furthermore, HIRA ChIP-seq performed in mouse ES cells revealed increased nucleosome-bound HIRA, particularly around transcription start sites (TSS) of active genes, along with the appearance of HIRA-bound nucleosomes within normally nucleosome-free regions (NFRs). These findings pointed to ATAD2 as a major factor responsible for unloading HIRA from nucleosomes. This unloading function may also apply to other histone chaperones, such as FACT (see Wang et al., 2021, Fig. 4C).

In the present study, our investigations converge on the same ATAD2 function in the context of a physiologically integrated mammalian system—spermatogenesis. Indeed, in the absence of ATAD2, we observed H3.3 accumulation and enhanced H3.3-mediated gene expression. Consistent with this functional model of ATAD2— unloading chaperones from histone- and non-histone-bound chromatin—we also observed defects in histone-toprotamine replacement.

Together, the results presented here and in Wang et al. (2021) reveal an underappreciated regulatory layer of histone chaperone activity. Previously, histone chaperones were primarily understood as factors that load histones. Our findings demonstrate that we must also consider a previously unrecognized regulatory mechanism that controls assembled histone-bound chaperones. This key point was clearly captured and emphasized by Reviewer #2 (see below).

Strengths: 

The paper describes the role of ATAD2 AAA+ ATPase in the proper localization of sperm-specific chromatin proteins such as protamine, suggesting the importance of the DNA replication-independent histone exchanges with the HIRA-histone H3.3 axis. 

Weaknesses: 

(1) Some results lack quantification. 

We will consider all the data and add appropriate quantifications where necessary.

(2) The work was performed well, and most of the results are convincing. However, this manuscript does not suggest a molecular mechanism for how ATAD2 promotes the formation of testis-specific chromatin. 

Please see our comments above.

Reviewer #2 (Public review): 

Summary: 

This manuscript by Liakopoulou et al. presents a comprehensive investigation into the role of ATAD2 in regulating chromatin dynamics during spermatogenesis. The authors elegantly demonstrate that ATAD2, via its control of histone chaperone HIRA turnover, ensures proper H3.3 localization, chromatin accessibility, and histone-toprotamine transition in post-meiotic male germ cells. Using a new well-characterized Atad2 KO mouse model, they show that ATAD2 deficiency disrupts HIRA dynamics, leading to aberrant H3.3 deposition, impaired transcriptional regulation, delayed protamine assembly, and defective sperm genome compaction. The study bridges ATAD2's conserved functions in embryonic stem cells and cancer to spermatogenesis, revealing a novel layer of epigenetic regulation critical for male fertility. 

Strengths: 

The MS first demonstration of ATAD2's essential role in spermatogenesis, linking its expression in haploid spermatids to histone chaperone regulation by connecting ATAD2-dependent chromatin dynamics to gene accessibility (ATAC-seq), H3.3-mediated transcription, and histone eviction. Interestingly and surprisingly, sperm chromatin defects in Atad2 KO mice impair only in vitro fertilization but not natural fertility, suggesting unknown compensatory mechanisms in vivo. 

Weaknesses:

The MS is robust and there are not big weaknesses 

Reviewer #3 (Public review): 

Summary: 

The authors generated knockout mice for Atad2, a conserved bromodomain-containing factor expressed during spermatogenesis. In Atad2 KO mice, HIRA, a chaperone for histone variant H3.3, was upregulated in round spermatids, accompanied by an apparent increase in H3.3 levels. Furthermore, the sequential incorporation and removal of TH2B and PRM1 during spermiogenesis were partially disrupted in the absence of ATAD2, possibly due to delayed histone removal. Despite these abnormalities, Atad2 KO male mice were able to produce offspring normally. 

Strengths: 

The manuscript addresses the biological role of ATAD2 in spermatogenesis using a knockout mouse model, providing a valuable in vivo framework to study chromatin regulation during male germ cell development. The observed redistribution of H3.3 in round spermatids is clearly presented and suggests a previously unappreciated role of ATAD2 in histone variant dynamics. The authors also document defects in the sequential incorporation and removal of TH2B and PRM1 during spermiogenesis, providing phenotypic insight into chromatin transitions in late spermatogenic stages. Overall, the study presents a solid foundation for further mechanistic investigation into ATAD2 function. 

Weaknesses:

While the manuscript reports the gross phenotype of Atad2 KO mice, the findings remain largely superficial and do not convincingly demonstrate how ATAD2 deficiency affects chromatin dynamics. Moreover, the phenotype appears too mild to elucidate the functional significance of ATAD2 during spermatogenesis. 

We respectfully disagree with the statement that our findings are largely superficial. Based on our investigations of this factor over the years, it has become evident that ATAD2 functions as an auxiliary factor that facilitates mechanisms controlling chromatin dynamics (see, for example, Morozumi et al., 2015). These mechanisms can still occur in the absence of ATAD2, but with reduced efficiency, which explains the mild phenotype we observed.

This function, while not essential, is nonetheless an integral part of the cell’s molecular biology and should be studied and brought to the attention of the broader biological community, just as we study essential factors. Unfortunately, the field has tended to focus primarily on core functional actors, often overlooking auxiliary factors. As a result, our decade-long investigations into the subtle yet important roles of ATAD2 have repeatedly been met with skepticism regarding its functional significance, which has in turn influenced editorial decisions.

We chose eLife as the venue for this work specifically to avoid such editorial barriers and to emphasize that facilitators of essential functions do exist. They deserve to be investigated, and the underlying molecular regulatory mechanisms must be understood.

(1) Figures 4-5: The analyses of differential gene expression and chromatin organization should be more comprehensive. First, Venn diagrams comparing the sets of significantly differentially expressed genes between this study and previous work should be shown for each developmental stage. Second, given the established role of H3.3 in MSCI, the effect of Atad2 knockout on sex chromosome gene expression should be analyzed. Third, integrated analysis of RNA-seq and ATAC-seq data is needed to evaluate how ATAD2 loss affects gene expression. Finally, H3.3 ChIP-seq should be performed to directly assess changes in H3.3 distribution following Atad2 knockout.  

(1) In the revised version, we will include Venn diagrams to illustrate the overlap in significantly differentially expressed genes between this study and previous work. However, we believe that the GSEAs presented here provide stronger evidence, as they indicate the statistical significance of this overlap (p-values). In our case, we observed p-value < 0.01 (**) and p < 0.001 (***).

(2) Sex chromosome gene expression was analyzed and is presented in Fig. 5C.

(3) The effect of ATAD2 loss on gene expression is shown in Fig. 4A, B, and C as histograms, with statistical significance indicated in the middle panels.

(4) Although mapping H3.3 incorporation across the genome in wild-type and Atad2 KO cells would have been informative, the available anti-H3.3 antibody did not work for ChIP-seq, at least in our hands. The authors of Fontaine et al., 2022, who studied H3.3 during spermatogenesis in mice, must have encountered the same problem, since they tagged the endogenous H3.3 gene to perform their ChIP experiments.

(2) Figure 3: The altered distribution of H3.3 is compelling. This raises the possibility that histone marks associated with H3.3 may also be affected, although this has not been investigated. It would therefore be important to examine the distribution of histone modifications typically associated with H3.3. If any alterations are observed, ChIP-seq analyses should be performed to explore them further.  

Based on our understanding of ATAD2’s function—specifically its role in releasing chromatin-bound HIRA—in the absence of ATAD2 the residence time of both HIRA and H3.3 on chromatin increases. This results in the detection of H3.3 not only on sex chromosomes but across the genome. Our data provide clear evidence of this phenomenon. The reviewer is correct in suggesting that the accumulated H3.3 would carry H3.3-associated histone PTMs; however, we are unsure what additional insights could be gained by further demonstrating this point.

(3) Figure 7: While the authors suggest that pre-PRM2 processing is impaired in Atad2 KO, no direct evidence is provided. It is essential to conduct acid-urea polyacrylamide gel electrophoresis (AU-PAGE) followed by western blotting, or a comparable experiment, to substantiate this claim. 

Figure 7 does not suggest that pre-PRM2 processing is affected in Atad2 KO; rather, this figure—particularly Fig. 7B—specifically demonstrates that pre-PRM2 processing is impaired, as shown using an antibody that recognizes the processed portion of pre-PRM2. ELISA was used to provide a more quantitative assessment; however, in the revised manuscript we will also include a western blot image.

(4) HIRA and ATAD2: Does the upregulation of HIRA fully account for the phenotypes observed in Atad2 KO? If so, would overexpression of HIRA alone be sufficient to phenocopy the Atad2 KO phenotype? Alternatively, would partial reduction of HIRA (e.g., through heterozygous deletion) in the Atad2 KO background be sufficient to rescue the phenotype? 

These are interesting experiments that require the creation of appropriate mouse models, which are not currently available.

(5)The mechanism by which ATAD2 regulates HIRA turnover on chromatin and the deposition of H3.3 remains unclear from the manuscript and warrants further investigation. 

The Reviewer is absolutely correct. In addition to the points addressed in response to Reviewer #1’s general comments (see above), it would indeed have been very interesting to test the segregase activity of ATAD2 (likely driven by its AAA ATPase activity) through in vitro experiments using the Xenopus egg extract system described by Tagami et al., 2004. This system can be applied both in the presence and absence (via immunodepletion) of ATAD2 and would also allow the use of ATAD2 mutants, particularly those with inactive AAA ATPase or bromodomains. However, such experiments go well beyond the scope of this study, which focuses on the role of ATAD2 in chromatin dynamics during spermatogenesis

Reference

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DOI: 10.1016/j.celrep.2025.116300

Resource: (EnCor Biotechnology Cat# CPCA-MAP2, RRID:AB_2138173)

Curator: @scibot

SciCrunch record: RRID:AB_2138173

What is this?

il n'ya pas d'art du jugement mais pratique natty du jugement Kant rattache jugement au bon à l'esprit matternel =/ esprit ecole pr Kant c'est jugeote =/ rien avoir avec ce qu'on apprend à l'ecole

Hi, I found this study to be interesting and potentially very impactful for diagnostic needs, as you mention. I have a few questions: 1. Do you have any insights into which timepoints or periods were the most useful (or most noisy) for your training purposes? 2. Do you know whether introducing imaging steps every 30 minutes may have had any effects on colony growth patterns? 3. For the species/strains that were most difficult to predict, do you have any idea what factors may have contributed to this, i.e. technical difficulties in holography, heterogeneity within or across colonies, etc Thank you!

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

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Reply to the reviewers

• *

1. General Statements [optional]

This section is optional. Insert here any general statements you wish to make about the goal of the study or about the reviews.

• *We appreciate the reviewers' assessment of the significance of our work and would like to highlight where we believe the novelty of this study lies. Our findings identify E4BP4 as a key transcription factor that maintains mitochondrial homeostasis by restraining the overactivation of biological pathways - such as de novo ceramide synthesis - that are known to drive mitochondrial oxidative dysfunction in the context of obesity. We fully acknowledge that the link between C16:0 ceramide and mitochondrial fragmentation has been previously established. However, to our knowledge, our study is the first to connect this phenomenon to a transcriptional safeguard mechanism, thereby providing a new layer of understanding of how transcription factors preserve mitochondrial integrity and function in brown adipocytes. We believe this conceptual advance adds significant value to the field by framing E4BP4 as a transcriptional "guardian" of mitochondrial homeostasis.

2. Description of the planned revisions

Insert here a point-by-point reply that explains what revisions, additional experimentations and analyses are planned to address the points raised by the referees.

• *Reviewer #1 comment:

Figures B: Sample size of EE experiments is too low to draw any meaningful conclusions or to know for certain if the data are reproducible. Small sample sizes, likely coming from one litter and one batch of AAV are prone to type I error.

Response: We agree with reviewer observation that increasing sample size is essential to confirm reproducibility and robustness. We have therefore planned to repeat the EE experiments with a larger number of mice per group, derived from independent litters and AAV preparations, in order to strengthen the statistical power and validate the phenotype observed in the current study.

Reviewer #1 comment:

Figure 3I: Why do cells (none of the groups) show no response to NE stimulation? Please clarify or provide potential mechanistic insight. Perhaps the cells were not differentiated well.

__ ____Response:__ We agree that the absence of a robust NE response in Figure 3I requires further clarification. To address this, we have planned to repeat the in vitro oxygen consumption assay to confirm the phenotype presented in the study.

Reviewer #1 comment: Figures 3I vs 5N. There is a striking discrepancy between these panels. In both, cells were treated with palmitate for 6 h, yet the NE and CCCP responses differ significantly. Are these the same cell types and conditions? Please reconcile the differences.

Response: We would like to clarify that Figures 3I and 5N represent different experimental systems: Figure 3I shows data from primary brown adipocytes with E4bp4 transgene overexpression, whereas Figure 5N shows data from immortalized brown adipocytes with Cas9-mediated mutation of a 65 kb Cers6 enhancer site. Given the distinct cell types and genetic manipulations, a direct comparison between these two panels is not appropriate. Nevertheless, we agree that confirming the consistency of the phenotype across systems is important. To address this, we have planned to repeat oxygen consumption assays in both models to further validate the reproducibility of the observed effects.

Reviewer #2 comment: A key experiment is missing: does adding C16:0 block the mitochondrial benefits of E4BP4-OE?

Response: We thank the reviewer for this excellent suggestion. We agree that a rescue experiment is important to directly test whether C16:0 affects the mitochondrial benefits of E4BP4. To address this, we have planned to perform a co-overexpression of E4bp4 and Cers6 in brown adipocytes. The readouts will include mitochondrial morphology and oxygen consumption, enabling us to determine whether restoration of C16:0 production mitigates the protective mitochondria effects of E4BP4 overexpression. This experiment will provide direct mechanistic confirmation of the proposed model.__ __

__Reviewer #2 comment: __Whether PRDM16-OE mimics the effects of E4BP4 to induce p-Drp1 is not shown.

__Response: __We thank the reviewer for this valuable suggestion. We agree that testing whether PRDM16 overexpression mimics the effects of E4BP4 on p-Drp1 is important to strengthen the mechanistic link between these transcription factors in terms of regulation of mitochondrial fragmentation. To address this, we have planned to include a Western blot analysis of p-Drp1 in the PRDM16-OE in brown adipocytes.

3. Description of the revisions that have already been incorporated in the transferred manuscript

Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. If no revisions have been carried out yet, please leave this section empty.

• *Reviewer #1 comment:

Figure 1F: There is an unexpected dip in gene expression at cold exposure days 3 and 7, followed by a rebound at day 14. Is this fluctuation biologically meaningful or technical?

Response: We thank the reviewer for this thoughtful observation. A previous study demonstrated that E4bp4 (Nfil3) expression displays an early increase (at 2 hours), followed by a decrease in magnitude - while still remaining significantly higher than control - during beige adipocyte differentiation in response to forskolin treatment (DOI: 10.1016/j.molmet.2022.101619). The authors of that study suggested that E4bp4 may contribute to a second wave of cAMP-driven beige adipocyte differentiation. However, in the context of our work, further discussion on whether the fluctuations in BAT E4bp4 expression observed during cold exposure reflect biological regulation would be speculative. Importantly, despite these oscillations across time points, E4bp4 expression remained statistically significant compared with control, supporting the robustness of our findings. We have now introduced this observation in the Results section of the revised manuscript.

Reviewer #1 comment: Figures 2H and 2I (GTT): How was the AUC calculated? The GTT and ITT curves appear largely parallel aside from fasting differences. If total AUC was used instead of incremental AUC, it may overstate group differences. The recommended method is outlined in [DOI: 10.1038/s42255-021-00414-7]. Also, since insulin's half-life is ~10 minutes, later differences in the ITT curve likely reflect counterregulatory responses driven by hepatic gluconeogenesis.

Response: We would like to clarify that in our original manuscript we had already calculated the area of the curve (AOC) rather than the area under the curve (AUC), following the recommended approach (DOI: 10.1038/s42255-021-00414-7). Specifically, the AOC was derived by subtracting the baseline glucose value from each subsequent time point, ensuring that the analysis reflects incremental changes rather than absolute glucose levels. We have now made this description more explicit in the revised version to avoid any ambiguity.

__Reviewer #1 comment: __Figure 4F: How was mitochondrial fragmentation quantified? Please ensure that the ROI boxes shown in zoomed panels match the same region in size and shape - this applies throughout the manuscript.

__ _Response: _We thank the reviewer for this valuable comment. To improve the quality and interpretation of the data, we have now included a quantitative analysis of mitochondrial morphology parameters associated with Figure 4F (Figure S4B)__. Specifically, we analyzed:

• Mitochondrial volume (µm³): reflecting overall mitochondrial size.
• Surface area (µm²): reflecting membrane expansion.
• Sphericity index: indicating morphological rounding, which increases with fragmentation.
• Number of branches and branch junctions per mitochondrion: reflecting mitochondrial networking and fusion. Myriocin treatment preserved mitochondrial volume and surface area, reduced sphericity, and increased both the number of branches and branch junctions, reflecting maintenance of a more interconnected mitochondrial network.

Additionally, we verified that the ROI boxes shown in the zoomed panels are consistent in both size and shape across groups, as requested. We have now introduced this observation in the Methods section of the revised manuscript.

__ ____Reviewer #1 comment: __Figure 3A: The claim that one group contains smaller mitochondria is not convincing. Both small and elongated mitochondria appear in each group. Moreover, it is unclear whether these minor differences are of any physiological relevance or whether they drive phenotypes.

Response: We respectfully disagree with this observation and would like to clarify a few points.

1. We have already demonstrated a statistically significant difference in mitochondrial length between E4bp4-OE and control groups (Figures 3B and 3C). This was based on a random, unbiased analysis, which consistently confirmed longer mitochondria in E4bp4-OE compared with control.

Some degree of variability in mitochondrial length is expected in electron microscopy analyses, particularly because mitochondria from multiple cell types within iBAT are captured. It is important to note that the protective action of E4bp4 against mitochondrial fragmentation occurs specifically in brown adipocytes, where the transgene is expressed under the control of the adiponectin promoter.

To address the potential confounding heterogeneity of iBAT mitochondria, we performed complementary cell-autonomous analyses in vitro, allowing us to directly compare mitochondrial dynamics in E4bp4-OE versus control brown adipocytes. This analysis further confirmed that E4bp4-OE prevents lipid overload - induced mitochondrial fragmentation in brown adipocytes.

Finally, we emphasize that several studies have demonstrated that changes in mitochondrial dynamics, particularly under high-fat diet conditions, disrupt systemic energy homeostasis (DOI: 10.1016/j.cmet.2017.05.010; DOI: 10.1016/j.cell.2019.05.008; DOI: 10.1038/s42255-024-00978-0). Therefore, the differences we report are biologically meaningful in the broader context of mitochondrial dynamics and metabolic disease.

__Reviewer #1 comment: __Figure 3E: The claim that confocal microscopy reveals palmitate-induced mitochondrial fragmentation is difficult to discern. The images lack clear morphological differences.

__ _Response: _We thank the reviewer for this observation. To improve the interpretation of these results, we have now included a quantitative analysis of mitochondrial morphology parameters associated with Figure 3E.__ Specifically, we measured:

• __Mitochondrial volume (µm³): __reflecting overall mitochondrial size.
• __Surface area (µm²): __reflecting membrane expansion.
• __Sphericity index: __indicating morphological rounding, which increases with fragmentation.
• Number of branches and branch junctions per mitochondrion: __reflecting mitochondrial networking and fusion. __ __As shown in the new analysis (Figure S4A)__, palmitate treatment reduced mitochondrial volume, surface area, branches, and branch junctions, while increasing sphericity, consistent with a more fragmented phenotype in control cells. In contrast, these effects were significantly attenuated in E4bp4-OE cells, supporting our conclusion that E4BP4 overexpression protects against lipid overload-induced mitochondrial fragmentation. This text was added in the Results section of the revised manuscript.

We believe this additional analysis strengthens the robustness of our findings and provides clear quantitative evidence for the morphological changes that were less apparent from qualitative image inspection alone.

__Reviewer #1 comment: __Figure 3G: Dendra2-labeled mitochondria appear unaffected by palmitate, raising concern about the robustness of the effect across readouts.

__ _Response: _We respectfully disagree with this observation. As shown in Figure 3G__ (bar graphs), palmitate-treated brown adipocytes exhibited a clear reduction in mitochondrial co-localization, which reflects lower levels of fused mitochondria, in the control group compared with E4bp4-OE. Importantly, no difference in mitochondrial co-localization was observed between the two groups under vehicle-treated conditions. This indicates that E4bp4 overexpression does not promote mitochondrial fusion per se, but rather prevents lipid overload - induced mitochondrial fragmentation. We also note that the representative images presented in Figure 3G are single snapshots taken from a time-lapse assay of mitochondrial dynamics. To further illustrate this effect, we direct the reviewer to the supplementary video accompanying this experiment, which clearly demonstrates the differences in mitochondrial behavior over time.

__ ____Reviewer #1 comment: __Figure 5H: Were E4BP4 expression levels equivalent between WT and mutant cells? Quantification should be shown. Figure 5H: Were E4BP4 expression levels equivalent between WT and mutant cells? Quantification should be shown.

Response: __We thank the reviewer for this important point. We have now added the quantification of E4bp4 mRNA levels in cells transduced with either the non-mutated vector (control) and the vector carrying a mutation in the E4bp4 DNA-binding domain (Figure S5)__. The data show no significant difference in E4bp4 expression between the two groups.

__Reviewer #2 comment: __The evidence of mitochondrial fragmentation is not convincing. In the reviewer's opinion, Figures 3E, 3G, 4F, and 5M demonstrated a decrease in mitochondrial quantity, but not fragmentation.

Response: __We thank the reviewer for this observation. We have already addressed the comments from reviewer #1 (above) regarding Figures 3E, 3G and 4F related to measurements of mitochondria fragmentation. To strengthen the interpretation of these results, we have also performed a quantitative analysis of mitochondrial morphology parameters associated with __Figure 5M. Specifically, we measured:

• __Mitochondrial volume (µm³): __reflecting overall mitochondrial size.
• __Surface area (µm²): __reflecting membrane expansion.
• __Sphericity index: __indicating morphological rounding, which increases with fragmentation.
• Number of branches and branch junctions per mitochondrion: __reflecting mitochondrial networking and fusion. As shown in the new analysis (Figure S4C), palmitate treatment significantly reduced mitochondrial volume, surface area, and branching, while increasing sphericity, consistent with enhanced mitochondrial fragmentation in control cells. Notably, these changes were significantly blunted in the Cers6 enhancer edited cells (EΔ), supporting our conclusion that disruption of Cers6 protects against lipid overload-induced mitochondrial fragmentation. __This text was added in the Results section of the revised manuscript.

Regarding the reviewer's understanding of a "decrease in mitochondrial quantity, but not fragmentation," we respectfully disagree. The analyses performed for Figures 3E, 3G, 4F, and 5M clearly demonstrate that E4bp4 overexpression (E4bp4-OE) prevents lipid overload -induced mitochondrial fragmentation.

In relation to mitochondrial quantity, our data do not support differences in mitochondrial biogenesis between groups. Specifically, the expression of thermogenic and mitochondrial biogenesis genes (Figure S2G) as well as the mitochondrial-to-nuclear DNA ratio (Figure S3D) showed no significant changes, indicating that mitochondrial biogenesis is not altered.

Alternatively, it is possible that E4bp4 prevents mitophagy, as our results (Figure 3H) show that E4bp4-OE protects against lipid overload-induced mitochondrial depolarization. In this regard, previous studies have demonstrated that fragmented and depolarized mitochondria are targeted for degradation through mitophagy (DOI: 10.2337/db07-1781; DOI: 10.1074/jbc.M111.242412). While this explanation is consistent with our findings, we acknowledge that it remains speculative at this stage and, although interesting, is beyond the scope of the current study.

__Reviewer #2 comment: __It is confusing whether the association shown in Figure 1C is a positive or an inverse association.

Response: __We thank the reviewer for pointing out this source of confusion. __Figure 1C represents common variant associations for E4BP4, where the y-axis indicates the strength of association (-log10 p-value) rather than the direction (positive or inverse) of the effect. We have clarified this in the revised manuscript to avoid misinterpretation. The associations indicate that genetic variants in E4bp4 are positively linked with anthropometric traits such as weight, BMI, and waist-hip ratio.

5. Description of analyses that authors prefer not to carry out

*Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. *

This can be due to time or resource limitations or in case of disagreement about the necessity of such additional data given the scope of the study. Please leave empty if not applicable.

• *Reviewer #2 comment:

It would be worthwhile to investigate whether in vivo knockdown of E4BP4 blunts the Cers6-suppressing effects of PRDM16-OE.

Response: We agree that assessing in vivo loss-of-function of E4bp4 in the context of Prdm16 overexpression would be highly informative. At present, this experiment is technically not feasible, as it would require the generation and characterization of complex in vivo models beyond the scope of the current study. Nevertheless, we are actively considering this as a future direction. In the meantime, we believe that the in vitro experiments in brown adipocytes provided here are sufficient to establish the mechanistic relationship between E4BP4 and PRDM16 in the regulation of Cers6 expression.

__Reviewer #2 comment: __Whether E4BP4-OE affects cold tolerance in mice is now shown.

__Response: __We thank the reviewer for this thoughtful comment. In our study, we performed an iBAT-specific E4bp4 gain-of-function assay because we observed a downregulation of E4bp4 expression in the context of obesity. The rationale for this approach was to rescue E4bp4 expression in iBAT and thereby evaluate its systemic and mechanistic effects under obesogenic conditions. We recognize that a gain-of-function assay during cold challenge would further enhance E4bp4 expression and, while interesting, this would more directly address the role of E4bp4 in thermogenic regulation rather than in obesity-related metabolic dysfunction. For this reason, we believe that a detailed investigation of E4bp4 in cold-induced thermogenesis is an important but separate question that lies beyond the scope of the current study.

IMPORTANTE ACLARACIÓN

Reviewer #1 (Public review):

Summary:

This study addresses the important question of how top-down cognitive processes affect tactile perception in autism - specifically, in the Fmr1-/y genetic mouse model of autism. Using a 2AFC tactile task in behaving mice, the study investigated multiple aspects of perceptual processing, including perceptual learning, stimulus categorization and discrimination, as well as the influence of prior experience and attention.

Strengths:

The experiments seem well performed, with interesting results. Thus, this study can/will advance our understanding of atypical tactile perception and its relation to cognitive factors in autism.

Weaknesses:

Certain aspects of the analyses (and therefore the results) are unclear, which makes the manuscript difficult to understand. Clearer presentation, with the addition of more standard psychometric analyses, and/or other useful models (like logistic regression) would improve this aspect. The use of d' needs better explanation, both in terms of how and why these analyses are appropriate (and perhaps it should be applied for more specific needs rather than as a ubiquitous measure).

Reviewer #3 (Public review):

Summary:

Developing consistent and reliable biomarkers is critically important for developing new pharmacological therapies in autism spectrum disorders (ASDs). Altered sensory perception is one of the hallmarks of autism and has been recently added to DSM-5 as one of the core symptoms of autism. Touch is one of the fundamental sensory modalities, yet it is currently understudied. Furthermore, there seems to be a discrepancy between different studies from different groups focusing on tactile discrimination. It is not clear if this discrepancy can be explained by different experimental setups, inconsistent terminology, or the heterogeneity of sensory processing alterations in ASDs. The authors aim to investigate the interplay between tactile discrimination and cognitive processes during perceptual decisions. They have developed a forepaw-based 2-alternative choice task for mice and investigated tactile perception and learning in Fmr1-/y mice

Strengths:

There are several strengths of this task: translational relevance to human psychophysical protocols, including controlled vibrotactile stimulation. In addition to the experimental setup, there are also several interesting findings: Fmr1-/y mice demonstrated choice consistency bias, which may result in impaired perceptual learning, and enhanced tactile discrimination in low-salience conditions, as well as attentional deficits with increased cognitive load. The increase in the error rates for low salience stimuli is interesting. These observations, together with the behavioral design, may have a promising translational potential and, if confirmed in humans, may be potentially used as biomarkers in ASD.

Weaknesses:

Some weaknesses are related to the lack of the original raster plots and density plots of licks under different conditions, learning rate vs time, and evaluation of the learning rate at different stages of learning. Overall, these data would help to answer the question of whether there are differences in learning strategies or neural circuit compensation in Fmr1-/y mice. It is also not clear if reversal learning is impaired in Fmr1-/y mice.

Author response:

Reviewer #1 (Public review): 

Summary: 

This study addresses the important question of how top-down cognitive processes affect tactile perception in autism - specifically, in the Fmr1-/y genetic mouse model of autism. Using a 2AFC tactile task in behaving mice, the study investigated multiple aspects of perceptual processing, including perceptual learning, stimulus categorization and discrimination, as well as the influence of prior experience and attention.  

We appreciate the reviewer’s statement highlighting the importance of our study. 

Strengths: 

The experiments seem well performed, with interesting results. Thus, this study can/will advance our understanding of atypical tactile perception and its relation to cognitive factors in autism. 

We thank the reviewer for recognizing the quality of our experiments and the relevance of our findings for understanding tactile perception and cognition in autism.

Weaknesses: 

Certain aspects of the analyses (and therefore the results) are unclear, which makes the manuscript difficult to understand. Clearer presentation, with the addition of more standard psychometric analyses, and/or other useful models (like logistic regression) would improve this aspect. The use of d' needs better explanation, both in terms of how and why these analyses are appropriate (and perhaps it should be applied for more specific needs rather than as a ubiquitous measure). 

We thank the reviewer for the helpful comments. We understand that the analyses were difficult to follow, and we will work on the clarity of the Results section. However, we would like to emphasize that every d′ measure is accompanied by analyses of response rates (i.e., correct and incorrect choice rates). In addition, we applied standard psychometric analyses whenever possible. Specifically, psychometric functions were fitted to the data using logistic regression. We will rework the text to clarify these points.

During training, only two stimulus amplitudes were presented, which precluded the construction of psychometric curves. For the categorization task, however, psychometric analyses were feasible and conducted (Figure 2). These analyses revealed no evidence of categorization bias (as measured by threshold) or accuracy (as measured by the slope) across stimulus strengths.

The calculation of d’ is included in the Methods, but we will also report and explain its use in each part of the Results section where it has been included.

Reviewer #2 (Public review): 

Summary: 

This manuscript presents a tactile categorization task in head-fixed mice to test whether Fmr1 knockout mice display differences in vibrotactile discrimination using the forepaw. Tactile discrimination differences have been previously observed in humans with Fragile X Syndrome, autistic individuals, as well as mice with loss of Fmr1 across multiple studies. The authors show that during training, Fmr1 mutant mice display subtle deficits in perceptual learning of "low salience" stimuli, but not "high salience" stimuli, during the task. Following training, Fmr1 mutant mice displayed an enhanced tactile sensitivity under low-salience conditions but not high-salience stimulus conditions. The authors suggest that, under 'high cognitive load' conditions, Fmr1 mutant mouse performance during the lowest indentation stimuli presentations was affected, proposing an interplay of sensory and cognitive system disruptions that dynamically affect behavioral performance during the task. 

Strengths: 

The study employs a well-controlled vibrotactile discrimination task for head-fixed mice, which could serve as a platform for future mechanistic investigations. By examining performance across both training stages and stimulus "salience/difficulty" levels, the study provides a more nuanced view of how tactile processing deficits may emerge under different cognitive and sensory demands. 

We thank the reviewer for emphasizing the strengths of our task design and analysis approach, and we appreciate that the potential of this platform for future mechanistic investigations is recognized.

Weaknesses: 

The study is primarily descriptive. The authors collect behavioral data and fit simple psychometric functions, but provide no neural recordings, causal manipulations, or computational modeling. Without mechanistic evidence, the conclusions remain speculative. 

We thank the reviewer for the careful reading of our manuscript and for the constructive feedback. The reviewer raises a valid point. We agree that our study is primarily descriptive and focused on behavioral data, and we appreciate the opportunity to clarify the scope and interpretation of our findings. Our primary goal was to characterize behavioral patterns during tactile discrimination and categorization, and the psychometric analyses were intended to provide a detailed description of these patterns. We do not claim to provide direct neural, causal, or computational evidence. 

Second, the authors repeatedly make strong claims about "categorical priors," "attention deficits," and "choice biases," but these constructs are inferred indirectly from secondary behavioral measures. Many of the effects are based on non-significant trends, and alternative explanations (such as differences in motivation, fatigue, satiety, stereotyped licking, and/or reward valuation) are not considered. 

Alternative explanations of our findings, such as differences in motivation, fatigue, satiety, stereotyped licking, and reward valuation have indeed been considered. We will revise the manuscript to present these points more clearly. 

Third, the mapping of the behavioral results onto high-level cognitive constructs is tenuous and overstated. The authors' interpretations suggest that they directly tested cognitive theories such as Load Theory, Adaptive Resonance Theory, or Weak Central Coherence. However, the experiments do not manipulate or measure variables that would allow such theories to be tested. More specific comments are included below.

This was not done intentionally. We do not claim to have tested the Load Theory; rather, inspired by it, we assessed behavioral patterns in our tactile categorization task. We agree that referring to the Adaptive Resonance Theory, which is based on artificial neural network models, might be misleading since we focus on behavioral results, and we will revise the text accordingly. However, our task allowed us to examine the impact of categorization on discrimination, confirming that Fmr1^-/yation can amplify perceptual differences between stimuli belonging to different categories and reduce perceived differences within a category in WT mice but not in the mice when low-salience stimuli were experienced. Finally, we do not claim to have tested the Weak Central Coherence theory, although our results suggest reduced use of categories in low-salience tactile discrimination. 

(1) The authors employ a two-choice behavioral task to assess forepaw tactile sensitivity in Fmr1 knockout mice. The data provide an interesting behavioral observation, but it is a descriptive study. Without mechanistic experiments, it is difficult to draw any conclusions, especially regarding top-down or bottom-up pathway dysfunctions. While the task design is elegant, the data remain correlational and do not advance our mechanistic understanding of Fmr1-related sensory and/or cognitive alterations. 

We agree with the reviewer that our current experiments are behavioral in nature and do not provide direct mechanistic evidence for top-down pathway dysfunction. Our goal was to carefully characterize tactile responses and behavioral patterns in Fmr1^-/y mice. The notion of “top-down” is used at the behavioral level, referring to the influence of higher-level cognitive processes (e.g., categorization, attention) on perception, rather than to underlying neural circuits. We will revise the manuscript to more clearly emphasize that our conclusions are based on behavioral observations, and we will frame mechanistic inferences as hypotheses rather than established findings. We will also explicitly note that future work using neural recordings or causal manipulations will be required to directly test these hypotheses.

We also note that identifying the precise top-down circuits involved will require extensive additional experimentation. For example, one would first need to pinpoint the specific top-down pathway that modulates the influence of categorization on discrimination without directly altering categorization itself. After such a circuit is identified, further work would then be needed to rescue or manipulate this pathway in the Fmr1^-/y model. These steps represent a substantial program of mechanistic research that, while important, goes well beyond the scope of the present study.

(2) The conclusions hinge on speculative inferences about "reduced top-down categorization influence" or "choice consistency bias," but no neural, circuit-level, or causal manipulations (e.g., optogenetics, pharmacology, targeted lesions, modeling) are used to support these claims. Without mechanistic data, the translational impact is limited. 

We recognize that “reduced top-down categorization influence” and “choice consistency bias” are based on behavioral observations. However, we respectfully disagree that this makes these constructs inherently speculative. Similar behavioral inferences have been applied in previous clinical studies to characterize cognitive tendencies (Soulières et al., 2007; Feigin et al., 2021). The translational impact of our work lies in the highly translational platform we have developed – and in highlighting the complexity of tactile measures and additional analyses that can be conducted in clinical studies.

We agree with the reviewer that the neural-based experiments would indeed provide valuable mechanistic insight into our observed behavioral alterations, and we believe future studies should therefore focus on their underlying neurobiological substrate.

We will revise the language throughout the manuscript to clarify that all conclusions are based on behavioral measures.  

(3) Statistical analysis: 

(a) Several central claims are based on "trends" rather than statistically significant effects (e.g., reduced task sensitivity, reduced across-category facilitation). Building major interpretive arguments on nonsignificant findings undermines confidence in the conclusions.  

Several trends are evident in complex measures, such as d’ analyses on task sensitivity or responses pooled across different amplitudes. Additional analyses revealed which component of these measures showed a statistically significant difference across genotypes, namely the low-salience incorrect choices accounting for low task sensitivity. We chose to present all analyses to be transparent and to highlight that commonly used complex measures (like d’ analyses) may mask important findings. In the text, we described p-values between 0.05 and 0.1 as observed trends without over-interpreting their significance. 

(b) The n number for both genotypes should be increased. In several experiments (e.g., Figure 1D, 2E), one animal appears to be an outlier. Considering the subtle differences between genotypes, such an outlier could affect the statistical results and subsequent interpretations. 

The number of mice used in each genotype group is consistent with standard practices in behavioral studies using mice and sensory tasks. We have performed effect size measures (e.g., Cohen’s d) alongside some of the statistical comparisons, showing a medium effect size (>0.5). 

As the reviewer correctly noted, no mice were excluded based on outlier analyses, since the observed variability reflects true biological differences rather than experimental or technical errors. We will reexamine our dataset for potential outliers. If any are identified, we will perform analyses both with and without the outlier and report any effects that are sensitive to single animals. These procedures and results will be explicitly described in the Methods and Results sections.

(c) The large number of comparisons across salience levels, categories, and trial histories raises concern for false positives. The manuscript does not clearly state how multiple comparisons were controlled.  

We thank the reviewer for raising this important point and we will include a clear statement on multiple comparisons in the Methods section. 

(d) The data in Figure 5, shown as separate panels per indentation value, are analyzed separately as ttests or Mann-Whitney tests. However, individual comparisons are inappropriate for this type of data, as these are repeated stimulus applications across a given session. The data should be analyzed together and post-hoc comparisons reported. Given the very subtle difference in miss rates across control and mutant mice for 'low-salience' stimulus trials, this is unlikely to be a statistically meaningful difference when analyzed using a more appropriate test. 

We thank the reviewer for raising this point. This was not done intentionally. A repeated-measures ANOVA on miss rates for low-salience stimuli during categorization confirmed that there are statistically significant differences both across stimulus amplitudes and between genotypes. Additional correction for multiple comparisons will be performed and explained in the Methods section.  

(4) Emphasis on theoretical models: The paper leans heavily on theories such as Adaptive Resonance Theory, Load Theory of Attention, and Weak Central Coherence, but the data do not actually test these frameworks in a rigorous way. The discussion should be reframed to highlight the potential relevance of these frameworks while acknowledging that the current data do not allow them to be assessed. 

As mentioned above, our goal was not to directly test these theories but rather to apply them within our translational framework. The Discussion section will be reframed to highlight that our findings are consistent with predictions from certain cognitive theories rather than implying that these frameworks were directly tested.

Reviewer #3 (Public review): 

Summary: 

Developing consistent and reliable biomarkers is critically important for developing new pharmacological therapies in autism spectrum disorders (ASDs). Altered sensory perception is one of the hallmarks of autism and has been recently added to DSM-5 as one of the core symptoms of autism. Touch is one of the fundamental sensory modalities, yet it is currently understudied. Furthermore, there seems to be a discrepancy between different studies from different groups focusing on tactile discrimination. It is not clear if this discrepancy can be explained by different experimental setups, inconsistent terminology, or the heterogeneity of sensory processing alterations in ASDs. The authors aim to investigate the interplay between tactile discrimination and cognitive processes during perceptual decisions. They have developed a forepaw-based 2-alternative choice task for mice and investigated tactile perception and learning in Fmr1-/y mice 

Strengths: 

There are several strengths of this task: translational relevance to human psychophysical protocols, including controlled vibrotactile stimulation. In addition to the experimental setup, there are also several interesting findings: Fmr1-/y mice demonstrated choice consistency bias, which may result in impaired perceptual learning, and enhanced tactile discrimination in low-salience conditions, as well as attentional deficits with increased cognitive load. The increase in the error rates for low salience stimuli is interesting. These observations, together with the behavioral design, may have a promising translational potential and, if confirmed in humans, may be potentially used as biomarkers in ASD. 

We appreciate the reviewer’s positive assessment of our study’s translational value and the importance of our behavioral findings.

Weaknesses: 

Some weaknesses are related to the lack of the original raster plots and density plots of licks under different conditions, learning rate vs time, and evaluation of the learning rate at different stages of learning. Overall, these data would help to answer the question of whether there are differences in learning strategies or neural circuit compensation in Fmr1-/y mice. It is also not clear if reversal learning is impaired in Fmr1-/y mice.  

We thank the reviewer for these helpful suggestions. We agree that visualizing behavioral patterns, such as raster and density plots of licks, as well as learning rate over time, could provide additional insights into learning dynamics. This analysis will be conducted and added into the revised manuscript.

There was no assessment of reversal learning in Fmr1^-/y mice in this study. While it is an interesting and important question based on previous findings in preclinical and clinical studies, it falls outside the scope of the current manuscript.    

Feigin H, Shalom-Sperber S, Zachor DA, Zaidel A (2021) Increased influence of prior choices on perceptual decisions in autism. Elife 10.

Soulières I, Mottron L, Saumier D, Larochelle S (2007) At ypical categorical perception in autism: Autonomy of discrimination? J Autism Dev Disord 37:481–490.

RELEVANTE

MUY IMPORTANTE, MUCHO OJO

OJO

MUY IMPORTANTE

Exactamente como se normalizó?

Añadir aquí las datos sobre sondeos y noticias? Pensar muy bien.

OJO

DATOS

Relevante

IMPORTANTE

OJO

¿Es acaso esto lo que se logra? ¿Está el Festival no sólo pensado sino además gestionado físicamente para que esto ocurra? ¿Qué es "sacar a la calle", "protagonizar", "encontrarse", "debatir" y "escuchar"?

DOI: 10.1038/s42255-025-01366-y

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DOI: 10.1038/s42255-025-01366-y

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DOI: 10.1038/s42255-025-01366-y

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DOI: 10.1007/s12272-025-01564-y

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DOI: 10.1007/s12272-025-01564-y

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DOI: 10.1007/s12272-025-01564-y

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What is this?

In March 1948, Newsweek said TV was spreading quickly like a serious illness called scarlet fever. Even though TV started to grow a lot in late 1947, the early TV industry had many problems to overcome.

Document de Synthèse : L'Emprise du Numérique et les Dangers des Réseaux Sociaux

Introduction : Une Lutte "David contre Goliath"

Ce briefing expose la problématique alarmante de l'impact des réseaux sociaux sur la santé mentale et la sécurité des enfants et adolescents.

Il met en lumière les témoignages poignants de victimes et de leurs familles, les actions en justice, le manque de régulation et les tactiques des géants de la technologie.

La lutte est présentée comme un combat "David contre Goliath" entre des familles endeuillées et des entreprises multimillionnaires.

Thèmes Principaux et Faits Importants :

1. Addiction et Impact sur la Santé Mentale des Adolescents :

Témoignage d'Alexis Spence : Alexis a développé de l'anorexie, de la dépression et s'est scarifiée à partir de 11 ans après avoir téléchargé Instagram.

L'algorithme l'a submergée de contenus sur la minceur, puis de photos de personnes anorexiques, de contenus tristes et déprimants.

Elle décrit comment elle s'est enfermée dans sa souffrance, devenant "une personne qu'on ne reconnaissait plus".

Citation : "J'avais 11 ans quand j'ai téléchargé Instagram pour la première fois et c'est là que tout a commencé. [...]

À force de regarder de la fitness, l'application a commencé à me montrer des mannequins. [...] Les mannequins étaient de plus en plus minces jusqu'à ce que ce ne soient plus des mannequins mais des personnes anorexiques."

Citation : "Mon compte est devenu rempli de ces contenus. C'était des photos tristes en noir et blanc avec des textes déprimants."

Citation : "Je pense vraiment qu'Instagram a une grande part de responsabilité dans les problèmes de santé mentale dont j'ai souffert, surtout si on prend en compte mon Je n'avais que 13 ans."

Idées Suicidaires et Automutilation : Plusieurs témoignages de parents évoquent les scarifications et les tentatives de suicide de leurs enfants, directement liées aux contenus diffusés par les algorithmes.

Citation : "J'ai posté une photo qui disait que j'avais l'intention de me suicider ce soir-là. [...] J'ai reçu un appel de l'assistante sociale. Vous devez venir à l'école immédiatement. Votre fille a tenté de se suicider."

Citation : "On avait mis en place des scarifications un peu contrôlées. Donc lorsqu'il allait pas bien, il me demandait ses lames. J'attendais derrière la porte de sa chambre et voir se scarifier."

Déni des Plateformes : Les dirigeants des Big Tech ont longtemps nié le lien entre leurs plateformes et les problèmes de santé mentale.

Citation d’un sénateur interrogeant Mark Zuckerberg : "everyone knows that kids who spend a lot of time too much time on your platforms are at risk and it's not just the mental health issues. I mean let me ask you a question is your platform safe for kids I believe it is but there's a difference between country if we don't start honest."

2. Cyberpédocriminalité et Manque de Sécurité :

Prolifération de Contenus Dangereux : Les plateformes sont des vecteurs de cyberpédocriminalité, avec des prédateurs sexuels qui exploitent les algorithmes et les fonctionnalités pour cibler les enfants. Interpol Europe est "débordé par la cyberpédocriminalité".

Citation : "on est quand même un moment assez crucial où Interpol Europe on est débordé par la cyberpédocriminalité et les plateformes elles sont vraiment utilisées par les prédateurs sexuels."

Citation : "Plus de 80 % des cas de sextorsion, c'est sur Instagram et Snapchat. Urgence à ce qu'elles fassent le ménage."

Algorithmes Complices : Une expérience avec un avatar de 13 ans, "Lili", démontre que les algorithmes proposent très rapidement des contenus sombres, des scènes d'automutilation, du vampirisme, des scènes sexualisées, et même l'apologie du suicide, même sans recherche préalable de l'utilisateur.

Citation : "Sur TikTok, l'algorithme est encore plus rapide. En moins de 5 minutes, la plateforme met en avant des vidéos faisant l'apologie du suicide."

Citation : "En quelques clics, la petite Lili se retrouve témoin de plusieurs viols sur mineurs."

Techniques de Manipulation des Prédateurs : Des modes d'emploi pour piéger les enfants sont disponibles en ligne. Les prédateurs utilisent des tactiques psychologiques comme le "love bombing" et la sexualisation progressive des conversations, détournant des codes familiers (personnages de dessins animés) pour normaliser des comportements abusifs.

Citation : "Ils vont vraiment jouer sur plein de ressorts psychologiques différents au niveau des enfants."

Citation : "Le fait de reprendre des codes par exemple de la Reine des Neiges, enfin des des différents personnages comme ça, il y a il y a des choses qui sont familières qui font pas forcément heurté comme un cohite frontal de de pornographie."

Réponse Insuffisante des Plateformes : Malgré les signalements, les plateformes ne suppriment pas toujours les contenus illicites et les comptes de prédateurs. Leurs efforts de sécurité sont jugés insuffisants.

Citation d’un sénateur : "Mr. Zuckerberg, what the hell were you thinking? [...] In what I understand get resources in what saying universe is there a link for se results anyway?" (concernant un message d'avertissement offrant l'option "voir les résultats quand même" pour des contenus problématiques).

Citation d’un représentant de l’office de lutte contre la cyberpédocriminalité : "On a très très peu de signalement qui parviennent par exemple WhatsApp."

3. Le Rôle des Entreprises de Technologie et leur Responsabilité :

Le "Business Model" des Big Tech : Les documents internes de Meta révélés par Frances Haugen (une lanceuse d'alerte) montrent que l'entreprise était consciente des vulnérabilités des enfants et des impacts négatifs, mais a privilégié les profits.

Citation : "Ces documents montrent que depuis 20 ans mett à enquête sur les vulnérabilités des enfants."

Citation : "Facebook repeatedly encounter conflicts between its own profits and our safety."

Citation d’un sénateur : "Children are not your priority. Children are your product. Children you see as a way to make money."

L'Article 230 comme Bouclier : Les entreprises se cachent derrière l'article 230 du droit américain, qui leur confère une immunité en tant qu'hébergeurs de contenu, les protégeant des poursuites judiciaires pour le contenu publié par leurs utilisateurs.

Citation : "Ces entreprises se cachent derrière l'article 230 qui est vraiment archaïque. Ils utilisent cette loi comme bouclier pour dire vous ne pouvez pas nous attaquer."

Citation d’un sénateur : "It's an astonishing benefit that your industry has that no other industry has. They just don't have to worry about being held in court if they're negligent."

Lobbying Intense : Pour contrer les projets de loi visant à lever leur immunité et à les responsabiliser, les Big Five ont dépensé près de 100 millions de dollars en lobbying, plus de la moitié provenant du groupe Meta.

Citation : "Ils ont dépensé près de 100 millions de dollars pour faire renoncer les députés et les sénateur, plus de la moitié de cette somme provient du seul groupe métablill."

4. Mobilisation Collective et Actions en Justice :

Mouvement Mondial des Parents : Des parents et des familles du monde entier se mobilisent pour exiger des changements et une meilleure protection des enfants.

Citation d’un père : "Nous en tant que père Tant que mer nous ne faisons rien, personne ne le fera à notre place. C'est notre lutte."

Citation d’une mère : "Nous sommes des milliers de pères et de mères qui pensons que les smartphones et les réseaux sociaux ne sont pas bons pour nos fils et nos filles."

Collectif Algos Victima : Fondé par l'avocate Maître Laure Bouttron Marmion, ce collectif rassemble des familles d'adolescents dont le suicide est lié aux réseaux sociaux, notamment l'affaire de Marie, une jeune fille décédée en 2021.

Le collectif vise à faire reconnaître la responsabilité des entreprises.

Citation de Maître Bouttron Marmion : "On souhaite la régulation cette plateforme qui aujourd'hui est au degré zéro de la régulation."

Citation de Maître Bouttron Marmion : "On ne peut pas ne pas considérer que le réseau social n'a pas sa part de responsabilité dans le suicide de Marie."

Actions Judiciaires aux États-Unis et en Europe : Plus de 1000 familles et 44 États américains sur 50 poursuivent les géants de la technologie. Des avocats cherchent des bases juridiques solides pour les attaquer.

Citation d’Alexis : "Depuis, plus de 1000 familles nous ont rejoint et maintenant 44 États américains sur 50 attaquent en justice les grandes entreprises technologiques pour qu'ils soient tenu responsable." Initiatives de Réglementation : Des projets de loi comme le "Kids Online Safety Act", le "EARN IT Act" et le "STOP CSAM Act" visent à rendre les entreprises responsables de l'exploitation des enfants et à supprimer leur immunité.

Citation d’un sénateur : "We have bills that have passed through this incredibly diverse committee when it comes to our political views. Kids online safety act earned act stopam act."

5. Solutions et Espoirs :

Interdiction des Smartphones avant un certain âge : En Espagne, un mouvement de parents a réussi à réglementer l'utilisation des téléphones portables dans les collèges et milite pour une interdiction totale avant 16 ans.

Citation d’une mère : "Nous souhaitons que les smartphones ne puissent pas être utilisés avant 16 ans."

Citation : "Maintenant, dans les classes et dans la cour, ils ne peuvent plus utiliser leur téléphone portable, sauf si le professeur le demande à un moment précis."

Désactivation des Algorithmes pour les Mineurs : Une demande clé est la désactivation des algorithmes pour les mineurs afin de les protéger des contenus inappropriés.

Citation : "Nous devons veiller à ce que l'algorithme soit désactivé pour les mineurs."

Espoir dans la Lutte "d'en bas" : L'espoir réside dans la mobilisation des familles et des citoyens face à l'inaction des entreprises et des législateurs.

Citation : "J'ai beaucoup plus d'espoir dans les familles, dans la lutte qui vient d'en bas plutôt que d'en haut."

L'excuse de Zuckerberg : Lors d'une audition au Sénat, Mark Zuckerberg a été contraint de s'excuser devant les victimes, bien que ses excuses aient été perçues comme insincères et non liées à la nature de son produit.

Citation de Mark Zuckerberg : "I'm sorry for everything that you all gone through terrible. No one should have to go through the things that your families have have suffered."

Citation d’Alexis : "Ses excuses n'étaient pas sincères. Il s'est excusé mais il ne s'est pas excusé à cause de son produit qu'il appelle lui-même un produit et qui fait du mal."

Conclusion : Un Monde Post-Écran pour les Enfants ?

Le briefing souligne que le consensus sur la menace profonde que représentent les réseaux sociaux pour la santé mentale et la sécurité des enfants est désormais établi.

La persévérance des victimes et des familles est cruciale pour obliger les entreprises et les législateurs à agir, avec l'espoir qu'un jour, "ça nous semblera tout aussi horrible qu'un enfant possède un téléphone portable et soit déconnecté de la vie".

Briefing : L'Alcoolisme au Féminin – Briser le Tabou

Objectif : Ce briefing vise à synthétiser les principaux thèmes, idées et faits marquants concernant l'alcoolisme au féminin, tels qu'abordés dans les extraits sonores de "Alcool au féminin, elles brisent le tabou".

Résumé Exécutif

L'alcoolisme au féminin est une maladie complexe, souvent invisible et entourée d'une honte et d'une culpabilité profondes, rendant sa détection et sa prise en charge plus difficiles que chez les hommes.

Les femmes sont physiologiquement plus vulnérables à l'alcool et l'utilisent souvent comme une "béquille" pour gérer un mal-être, une anxiété, une dépression, ou des traumatismes passés, notamment des violences sexuelles.

L'industrie de l'alcool cible activement les femmes avec des produits et des stratégies marketing spécifiques.

Le chemin vers la sobriété est long, marqué par des rechutes possibles, et nécessite un soutien indispensable de l'entourage, des groupes de parole, et des structures spécialisées.

Briser le tabou et reconnaître l'alcoolisme comme une maladie est crucial pour aider les femmes à s'en sortir.

Thèmes et Idées Principales

1. L'Alcoolisme Féminin : Une Réalité Invisible et Sous-Estimée

Prévalence incertaine : Le nombre de femmes dépendantes à l'alcool est difficile à estimer, oscillant entre 100 000 et 1,5 million, en raison du silence imposé par la honte et la culpabilité.

Honte et Culpabilité Accrues pour les Femmes : "Une femme qui boit tout d'un coup, c'est une honte. C'est deux fois plus dur qu'un homme.

Une femme alcoolique, c'est vraiment on nous le pardonne pas." Ce jugement social conduit à l'isolement et au déni, retardant la consultation de 10 ans en moyenne par rapport aux hommes.

Stratégies de Dissimulation : Les femmes mettent souvent en place des stratagèmes pour cacher leur consommation, comme planquer des bouteilles dans des endroits inattendus (ex: "planquer la bouteille dans le landau de ma fille").

2. Vulnérabilité Physiologique et Conséquences Spécifiques

Métabolisme et Dilution : "Quand on donne la même quantité d'alcool à un homme et une femme du même poids, l'alcoolémie sera plus élevée chez la femme."

Cela est dû à un métabolisme plus lent et une moindre proportion d'eau dans le corps féminin.

Impacts Accrus sur la Santé : Les maladies (cirrhose, maladies cardiovasculaires, troubles cognitifs comme la mémoire et la concentration) se développent plus rapidement et sont plus violentes chez les femmes.

Un lien fort existe avec le risque de cancer du sein, "quelque chose qui est très peu connu".

Signes Visibles : L'alcool "abîme énormément et chez les femmes, ça se voit. Une femme alcoolique, ça se voit au visage, aux yeux. Les yeux sont tristes souvent. La peau est abîmée."

3. Les Racines Psychologiques de l'Addiction chez les Femmes

Alcool comme Béquille ou Auto-Médication : Contrairement aux hommes dont la consommation "part d'une consommation plus festive qui dérape", les femmes "le plus souvent consomment pour traiter quelque chose, pour traiter un mal-être, une dépression, une anxiété."

Noémilovski témoigne : "j'ai bu de l'alcool comme j'aurais pris des médicaments pour pour apaiser et l'angoisse et la dépression."

Traumatismes d'Enfance et Violences Sexuelles : Derrière l'addiction se cachent souvent des "traumatismes d'enfance, des drames intimes".

Le vécu d'une agression sexuelle peut multiplier "jusqu'à 36 le risque de développer une addiction".

L'alcool permet "d'économiser, d'avoir à se confronter à ces horreurs". Laurence, par exemple, a découvert que son alcoolisme masquait un inceste.

Sentiment de Solitude et Différence : Muriel Robin a ressenti : "je me sentais tellement différente que j'étais très seule. Donc j'étais en souffrance."

L'alcool est alors apparu comme une solution pour "masquer tout", "penser à rien" et "se perdre".

L'Illusion du Plaisir et du "Soi-Même" : Beaucoup croient que l'alcool est une source de plaisir ou qu'il permet d'être "soi-même".

Noémilovski réfute cette idée : "on n'est pas soi-même. On est l'alcool, on est l'effet de l'alcool."

L'alcool crée une "chaleur, une douceur, un calme", mais mène à un "cercle vicieux" où l'on est "encore plus déprimé que la veille, encore plus angoissé".

4. L'Influence de la Société et du Marketing de l'Alcool

Normalisation de la Consommation Féminine : Boire est devenu "courant" pour les femmes, une manière de "s'intégrer", de décompresser, ou de faire la fête.

Lucille Woodward souligne : "on a toujours eu l'impression que c'était cool de boire et normal et plutôt une démonstration de force de la femme et on se rend pas compte en fait que finalement ça nous affaiblit."

Ciblage Marketing Spécifique : L'industrie de l'alcool cible les femmes avec des produits et des packagings "ultra girly" (ex: "tube de rouge à lèvres géant qui en fait contient une bouteille de champagne") et des saveurs aromatisées (mangue, litchi, cerise, pamplemousse) pour des alcools "moins forts".

Ces stratégies "associent un univers positif à un produit qui est quand même problématique pour la santé."

La "Zone Grise" : De nombreuses femmes se situent dans une "zone grise" où elles dépassent les limites recommandées (10 verres/semaine) sans se considérer comme dépendantes.

Le critère n'est pas le nombre de verres, mais "quand on ne peut pas s'en séparer et quand on a le sentiment d'avoir perdu la liberté de s'abstenir" et l'impact sur la santé et l'environnement.

5. Le Chemin vers la Sobriété : Un Combat Difficile mais Possible

Reconnaître la Maladie : L'alcoolisme est une maladie, non un manque de volonté.

C'est "une maladie que l'on peut soigner à condition d'oser la regarder en face."

L'Importance du Soutien : "L'alcool, on ne peut pas s'en sortir seul. Il faut demander de l'aide."

Groupes de parole : Les Alcooliques Anonymes ont été une "révélation" pour Noémilovski grâce à l'absence de jugement.

Des groupes spécifiques aux femmes permettent de reconnaître une "consommation autothérapeutique" commune.

Entraide et Témoignages : Des initiatives comme celle de Sylvie, qui aide d'autres femmes via internet, sont cruciales. "À force d'en parler, de déculpabiliser, d'avoir moins honte, j'ai pu tomber le masque en fait."

L'Entourage Aimant : Le soutien du conjoint est fondamental, comme pour Fiona Géin et Muriel Robin. Leurs partenaires ont cessé de boire et ont posé des limites claires pour leur relation.

La Reconstruction Personnelle :Deuil de l'Alcool :

L'arrêt peut être vécu comme un deuil, "comme si ma meilleure amie était morte", laissant un sentiment de vide.

Accepter les Rechutes : Les rechutes sont fréquentes et "ne remettent pas tout en cause". La mémoire de l'alcool reste présente ("l'image de Pac-Man dans mon cerveau").

Se Réconcilier avec Soi-Même : Le processus de reconstruction inclut la réappropriation de son image, de son corps, et de son estime de soi, souvent perdus pendant l'addiction.

Des ateliers d'art-thérapie ou de socio-esthétique aident à "se redonner une dignité" et à "adoucir le regard sur soi-même".

Trouver de Nouveaux Plaisirs : Remplacer l'alcool par d'autres sources de joie, comme le thé pour Sylvie, est une stratégie efficace.

6. L'Impact sur l'Entourage, en Particulier les Enfants

Souffrance Familiale : Pour chaque personne alcoolique, "en moyenne sept personnes qui souffrent autour d'elle", les enfants étant souvent en première ligne.

Les Enfants Observateurs : Charlotte, fille d'une mère alcoolique, mesurait le niveau des bouteilles et comprenait l'ambiance "sordide" de la maison.

Le Paradoxe de l'Amour et de la Haine : Les enfants d'alcooliques doivent gérer un paradoxe : "Je pouvais beaucoup l'aimer mais je pouvais la haïr en même temps parce que je ne la reconnaissais pas quand elle était ivre."

Nécessité de se Sauver Soi-Même : Malgré les tentatives de "réparer" le parent, le chemin est souvent de "sauver notre peau" et "abandonner cette famille dysfonctionnelle".

Citations Clés

"J'ai senti que dans mon disque dur, il y avait quelque chose qui était là et que et boire était normal." – Muriel Robin, sur l'installation de sa dépendance.

"Moi je buvais je buvais un litre de champagne quand je quand j'étais dehors. Je buvais un litre de champagne tous les soirs minimum." – Muriel Robin, sur la quantité consommée.

"L'alcool, j'allais dire c'est la récompense. Ce n'est pas une récompense. C'est quelque c'est c'est quelque chose qui qui vous veut du mal." – Muriel Robin, sur la nature trompeuse de l'alcool.

"Oui, j'étais alcoolique. Ouais, j'étais alcoolique pendant 30 ans." – Muriel Robin, sur la durée de son addiction. "L'alcool dérobe des années de vie de manière insidieuse et pour les femmes en particulier de façon invisible. C'est un poison qui s'instille à l'abri des regards." – Narratrice.

"Une femme qui boit tout d'un coup, c'est une honte. C'est deux fois plus dur qu'un homme. Une femme alcoolique, c'est vraiment on nous le pardonne pas." – Témoignage.

"Les hommes, ça part d'une consommation plus festive qui dérape. Les femmes le plus souvent consomment pour traiter quelque chose, pour traiter un mal-être, une dépression, une anxiété." – Experte.

"J'ai commencé à boire suite à un viol." – Anaïs. "Mon engagement, j'ai un problème avec l'alcool. Je bois, je bois trop." – Lucille Woodward, brisant le tabou en ligne.

"On a un problème d'alcool lorsqu'on ne peut pas s'en séparer et quand on a le sentiment d'avoir perdu la liberté de s'abstenir." – Définition de l'addiction.

"J'ai pris de l'alcool comme on prendrait des anxiolytiques." – Noémilovski.

"Tu n'es pas toi-même quand tu bois et moi je veux être avec toi quand tu es toi-même." – Proche de Noémilovski. "L'alcool, c'est sans faim. Vous voyez le matin, vous vous dites, je vais arrêter de boire et puis le soir, vous remettez ça." – Sylvie.

"Le pire que j'ai fait, je crois que c'était dans le landau de ma fille. J'avais planqué la bouteille dans le landau de ma fille." – Sylvie, sur la dissimulation.

"Il y a un gros pourcentage de risque de cancer du sein lié à l'alcool et ça vraiment c'est quelque chose qui est très peu connu." – Dr. Sarah Coscas, psychiatre addictologue. "Ma petite me disait : 'Maman, tu sens la bière ?'" – Témoignage d'une mère.

"Le vécu d'une agression sexuelle par une femme pouvait multiplier jusqu'à 36 le risque de développer une addiction." – Dr. Sarah Coscas.

"La personne, elle préfère préfère dire non, j'ai pas bu pour ne pas passer la soirée à se disputer avec son conjoint ou sa conjointe alors que elle peut pas aligner trois mots parce que elle a passé sa soirée ou sa journée à à boire." – Richard Baudouin, compagnon de Fiona Géin.

"Écoute moi si tu veux boire une bouteille de champagne tous les soirs c'est ta vie mais moi je j'ai trop peur de te perdre et entre la cigarette et l'alcool je peux pas voir quelqu'un qui se détruit donc on arrête l'histoire." – Anne Le Nen à Muriel Robin, un ultimatum salvateur.

Conclusion

Le document met en lumière la spécificité de l'alcoolisme au féminin, caractérisé par une invisibilité sociale, une vulnérabilité physiologique accrue, et des origines souvent liées à des traumatismes ou un mal-être profond.

Il souligne l'importance cruciale de la reconnaissance de cette maladie, de la brisure du tabou, et du soutien collectif pour permettre aux femmes de se reconstruire et de retrouver une vie digne et sobre.

Le chemin est long, mais le témoignage de ces femmes courageuses montre que la sortie est possible.

Briefing Détaillé : La Relation des Français à l'Alcool – Entre Héritage Culturel et Lutte Personnelle

Ce document de briefing explore la relation complexe et souvent paradoxale des Français à l'alcool, à partir d'un enregistrement audio riche en témoignages et analyses.

Il met en lumière comment l'alcool est profondément ancré dans la culture française, ses différentes fonctions sociales et personnelles, les dangers sous-estimés, les défis de la sobriété et l'influence des lobbies.

Thèmes Principaux

L'Alcool comme Héritage Culturel et Art de Vivre Français : L'alcool est présenté comme une tradition séculaire, un "art de vivre" fait de rituels et de moments de convivialité.

L'Alcool, Rite de Passage et Quête d'Identité : De l'enfance à l'âge adulte, l'alcool marque les étapes de la vie, offrant un sentiment de liberté, de socialisation et de performance.

Les Illusions et Dangers de l'Alcool : Malgré sa valorisation, l'alcool est une drogue qui masque les problèmes, conduit à des comportements risqués (violences, blackouts) et a des conséquences dévastatrices sur la santé et les relations.

La Lutte pour la Sobriété : Le parcours vers l'abstinence est semé d'embûches, confronté à la pression sociale, au déni et à la nécessité d'une reconstruction profonde.

L'Influence des Lobbies et les Croyances Tenaces : Les campagnes de santé publique se heurtent à la puissante influence des lobbies de l'alcool et à des mythes persistants comme le "French Paradoxe".

Idées et Faits Importants

1. L'Alcool comme Héritage Culturel et Art de Vivre Français

Ancrage Profond : L'alcool est "une histoire profondément ancrée dans nos mémoires. C'est la France, une bonne bouteille." Il est omniprésent lors des rencontres entre amis ("on va boire un coup"), symbolisant la convivialité.

Rituels Sociaux : L'expression "il y a toujours une bonne bouteille sur la table" souligne l'aspect ritualisé de la consommation.

Initiation Précoce : De nombreux témoignages révèlent une initiation à l'alcool dès la petite enfance, souvent en famille.

Charlotte se souvient d'avoir "fini la soupe avec le vin" avec son grand-père à 6 ans, et David d'un "fond de Sauternes" à un repas de Noël. Cette initiation est vécue avec fierté, comme un partage du "patrimoine".

Traditions Institutionnelles : Jusqu'en novembre 1956, les écoles primaires servaient de l'eau coupée au vin à la cantine, ce qui témoigne de la normalisation de l'alcool dès le jeune âge.

2. L'Alcool, Rite de Passage et Quête d'Identité

Adolescence et Transgression : Pour les adolescents, "boire est alors un rite de passage pour rentrer dans l'âge adulte." C'est une manière de "faire comme les grands", de "faire partie d'un groupe", même si le goût n'est pas apprécié au début.

Libération et Communication : Le premier verre est un "déclic" qui permet de "vivre différemment", de "se libérer de quelque chose", de "communiquer avec les autres et avec soi-même".

Sentiment de Puissance et de Liberté : Baptiste décrit l'alcool comme des "super pouvoirs", de "l'essence dans [son] moteur", le "breuvage magique qui va [lui] permettre d'être pleinement [lui]-même".

Il procure un sentiment de "liberté" et de "rébellion", où "la nuit nous appartient, on est les rois du monde."

Performance et Compétition : L'ivresse est associée à des notions de "performance" et de "compète" : "bien tenir l'alcool", "accepter les défis", "pas savoir dire non". Le "binge drinking" (cinq verres en moins de deux heures) est courant chez les jeunes.

Séduction et Désirabilité : L'alcool est perçu comme un moyen de devenir "quelqu'un", de "plaire", d'avoir des "premières expériences avec les filles". Charlotte buvait "pour me sentir désirable.

Draguer sans alcool me paraissait inconcevable." Près d'un jeune sur trois confie avoir besoin de boire avant un rapport sexuel.

Valorisation de l'Excès : "Boire c'est rentrer dans la norme, boire c'est s'émanciper." La "valorisation de l'ivresse et de la transgression et des excès" est perçue comme faisant "partie de la jeunesse."

Désinhibition et Faux Courage : L'alcool "désinhibe les timides", "décoince les coincés" et sert de "petite dose de courage liquide" pour Charlotte, qui a du mal à aborder des inconnus sobre.

3. Les Illusions et Dangers de l'Alcool

Une "Drogue Plaisir" aux Conséquences Néfastes : L'alcool est une "molécule plaisir qui va dès le premier verre agir dans le cerveau et puis euh donner un petit peu d'effets euphorisant, plaisant, relaxant", mais il conduit à l'illusion. Perte de Mémoire et Blackouts :

L'alcool peut "détruire mes souvenirs" ("Je sais où je suis allé mais je me rappelle plus de ce que j'y ai fait"). Marie décrit des "trous noirs" fréquents où elle ne se souvenait de rien, y compris comment elle était rentrée chez elle.

Violences et Agressions Sexuelles : L'alcool est impliqué dans 40% des condamnations pour violence familiale en France. Plus d'un jeune sur cinq (18-24 ans) déclare avoir eu un rapport sexuel non consenti à cause de l'alcool.

Le témoignage de Marie, violée par un ami de son père pendant un confinement alcoolisé, est particulièrement frappant.

Elle affirme : "pour moi, le problème c'est pas l'alcool, c'est qu'il faut éduquer les garçons". Son père, dévasté, reconnaît : "Et l'alcool a une part de une part de responsabilité là-dedans".

Baptiste raconte son propre viol, "J'aurais aussi aimé qu'on me dise que qu'on peut me faire du mal quand je suis bourré. On peut aussi me faire du mal parce que je suis bourré."

La discussion met en évidence la culture du viol persistante : "encore aujourd'hui, si une femme a bu trop, qu'elle a perdu le contrôle, qu'en plus elle avait mis une mini jupe, bah s'il lui arrive quelque chose, elle a un petit peu cherché." Le responsable est l'agresseur.

Impact sur la Santé : L'alcool multiplie les risques de cancer, d'AVC hémorragique et de troubles du rythme cardiaque.

Il cause 41 000 décès par an en France, étant la deuxième cause de mort évitable après le tabac.

Détérioration des Relations : David explique comment l'alcool a détruit son couple et son lien avec ses enfants. Sa femme décrit son regard "dans le vide, fuyant, vitreux" et le sentiment qu'il ne faisait "pas d'efforts pour moi."

L'alcool le rend agressif et manipulateur verbalement, au point d'une altercation physique avec sa femme devant leurs enfants.

L'Alcool comme Béquille face à l'Ennui et l'Angoisse : Jean-François a commencé à boire seul après sa carrière sportive, pour combler "la petite mort du sportif" et "accompagner l'ennui".

Il buvait ses "émotions", son "salaire", "tout". Rose utilisait l'alcool pour "tuer l'ennui et défier la mort".

4. La Lutte pour la Sobriété Pression Sociale et Jugement : Refuser de boire, surtout jeune, expose à des jugements : "tu te crains", "celle qui l'emmerdeuse", "celle qui n'est pas marrante".

Pour Lou, qui ne boit pas du tout à 21 ans, la pression sociale est "quasiment en permanence sur [ses] épaules". Arrêter de boire suscite la réaction "Ah, vous êtes devenu chiant", ou "tu es malade".

Le Déni : Les personnes dépendantes sont souvent dans le déni, se croyant capables de contrôler leur consommation. Jean-François et Baptiste décrivent des "pauses" pour se rassurer, avant de reprendre de plus belle.

L'addiction est une "pathologie de la liberté", la "perte de la liberté de s'abstenir."

Le Chemin Difficile de la Reconstruction : La sobriété n'est pas un "glamour instantané". C'est un processus long et douloureux, car le système de plaisir est "endormi". Il faut "apprendre à vivre sans cette béquille là".

Le Rôle du Soutien : Le soutien des proches est crucial. La lettre des amis de Baptiste l'a aidé à "ouvrir les yeux". Coluche soulignait l'importance d'"avoir des copains qui vous aident".

Bénéfices de la Sobriété : Pour David, la sobriété lui a permis de "redevenir acteur de [sa] vie", d'"être présent" pour ses enfants et sa femme, de "répondre au téléphone quand les gens [l']appellent".

C'est une source de fierté et de bonheur retrouvé.

5. L'Influence des Lobbies et les Croyances Tenaces Publicités et Mythes : Pendant des décennies, l'alcool a été promu comme un bienfait, voire un médicament ("le bon grog picon chaud tue la grippe", "soignez-vous par le vin"). Un livre de 1974 "Soignez-vous par le vin" a connu un immense succès.

Le "French Paradoxe" : La croyance que le vin rouge protège des maladies cardiaques, popularisée par le "French Paradoxe" dans les années 90, a "fait du mal" car elle est "restée très ancrée".

Cette "corrélation" n'a jamais été réellement prouvée comme un lien de causalité.

Le slogan "consommer avec modération" a été judicieusement introduit par les lobbies pour atténuer les messages de prévention.

Loi Evain (1991) : Cette loi a marqué un tournant en interdisant la publicité à la télévision et au cinéma et en imposant un message de prévention. Cependant, elle a été rapidement "attaquée" par les lobbies.

Influence Politique : Le président de la République est "sous influence des lobbies de l'alcool", ce qui se traduit par des annulations de campagnes de prévention jugées trop "prohibitionnistes" par l'industrie.

La campagne de 2023 "C'est pas un peu absurde de se souhaiter une bonne santé avec de l'alcool ?" a provoqué une réaction virulente des lobbies.

L'attitude des hommes politiques, comme le président qui "va boire une bière avec les joueurs", "brouille vraiment tous les messages."

En conclusion, l'alcool en France est un phénomène culturel complexe, intriqué dans l'histoire, les rituels sociaux et les parcours individuels.

Si son rôle dans la convivialité et la libération est souvent mis en avant, les témoignages révèlent les dangers profonds sur la santé physique et mentale, les relations humaines, et la dignité individuelle.

La lutte pour la sobriété est un combat personnel et collectif, exacerbé par la pression sociale et la puissante influence des lobbies qui perpétuent des mythes favorables à la consommation.

Le défi est de reconsidérer une culture où "boire était aussi naturel que respirer" pour une société plus consciente et en meilleure santé.

Daniel 2:44,45

Daniel 2:23,24

Author response:

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

Reviewer #1 (Public review):

“Alternative possibilities are discussed regarding the prior and likelihood of the model. Given that the second case study inspired the introduction of the zero-inflation likelihood, it is not clear how applicable the general methodology is to various datasets. If every unique dataset requires a tailored prior or likelihood to produce the best results, the methodology will not easily replace more traditional statistical analyses that can be applied in a straightforward manner. Furthermore, the differences between the results produced by the two Bayesian models in case study 2 are not discussed. In specific regions, the models provide conflicting results (e.g., regions MH, VPMpc, RCH, SCH, etc.), which are not addressed by the authors. A third case study would have provided further evidence for the generalizability of the methodology.”

We hope in this paper to propose a ‘standard workflow’ for these data; this standard workflow uses the horseshoe prior and we propose that this is the approach used to describe cell count data instead of the better established, but to our thinking, inefficient, t-testing approach.

The horseshoe prior is robust and allows a partially-pooled model to used while weighing-up the contribution of different data points. This is an analogue of excluding outliers and, in any analysis it is normal to investigate further if there are points being excluded as outliers. Often this reveals a particular challenge with the data, in the case of the data here, there are a lot of zeros, indicating that some samples should be excluded because the preparation failed to tag cells rather than because there were no cells to tag. This idea behind the ZIP example is to show that the Bayesian method can allow for this sort of further investigation and, indeed, as the reviewer notes this sort of extended analysis is often bespoke, tailored to the data.

We have clearly failed to explain that the ‘standard workflow’ we propose replace the more traditional methods is the first one we describe, with the horseshoe prior; this produces better results on both datasets than the traditional approach. However, we also feel it is useful to show how a more tailored follow-on can be useful; we need to make it clear that this is intended as an illustration of an ‘optional extra’ rather than a part of the more straightforward ‘standard workflow’.

To make this clearer we have made altered the text in several locations:

• end of Introduction: added clarifying sentence “Here, our aim is to introduce a ‘standard’ Bayesian model for cell count data. We illustrate the application of this model to two datasets, one related to neural activation and the other to developmental lineage. For the second dataset, we also demonstrate a second example extension Bayesian model.”

• Section Hierarchical modeling: “Our goal in both cases is to quantify group differences in the data. We present a ‘standard’ hierarchical model. This model reflects the experimental features common to cell count experiments and reflects the hierarchical structure of cell count data; the standard model is designed to deal robustly and efficiently with noise. On some occasions, to reflect a specific hypotheses, the structure of a particular experiment or an observed source of noise, this model can be further refined or changed to target the analysis. We will give an example of this for our second dataset.”

• Section Horseshoe prior: “The alternative is via a flexible prior such as the horseshoe Carvalho et al., 2010; Piironen and Vehtari, 2017. This more generic option may be suitable as a default ‘standard’ approach in the typical case where outliers are poorly understood.”

• Discussion: word ‘standard’ added to sentence: “Our standard workflow uses a horseshoe prior, along with the partial pooling, this allows our model to deal effectively with outliers.”

• Discussion: modified sentence “The horseshoe prior model workflow we have exhibited here is intended as a standard approach.”

Indeed, because the horseshoe prior deals robustly with outliers, whereas the ZIP is intended to model the outliers, any substantial difference between the two should be examined carefully. The referee is right to point out that we have not explained this in any detail and has helpfully listed a few brain regions were there are differences. This is useful, particularly since the examples listed illustrate in a useful way the opportunities and hazards this sort of data presents. To address this, we have added a new version of Figure 6 to the revised manuscript

Previously Figure 6 showed two example brain regions: MPN and TMd. We have now added MH and SCH to the figure, and new text commenting on the insights the plots provide, both in the Results and Discussion.

Reviewer #2 (Public review):

“A clearer link between the experimental data and model-structure terminology would be a benefit to the non-expert reader.”

This is a very good point and we are acutely aware through our own work how difficult it can be moving between fields with different research goals, different scientific cultures and different technical vocabularies. Just as it can be difficult translating from one language to another without losing nuance and meaning, it can be a real challenge finding technical terms that are useful for the non-expert reader while retaining the precision the application requires! In the long run, we hope that, just as some of the very specialized vocabulary that surrounds frequentist statistics has become familiar to to the working experimental scientists, the precise terminology involved in Bayesian modelling will become familiar and transparent. However, in advance of that day, we have included a glossary of terms at the end of the main text, and have made numerous small tweaks to make sure that link between data and model terminology is clearer and better explained.

Reviewer #1 (Recommendations fro the authors):

(1) “I would strongly recommend that the authors include more case studies in the manuscript, and address the qualitative differences between the different versions of the model.”

We agree that our method will only become established when it is applied to more datasets, we hope to contribute to further analysis and we know other people are already using the approach on their own data. We do, however, feel that adding more datasets to this paper will make it longer and more complex; the plan, instead, is to use the method on novel datasets to test specific hypotheses, so that the results will include novel scientific findings as well as adding another illustration of the Bayesian approach applied to data that is already well studied.

(2) “Figure 6 is not discussed in the main text.”

We had discussed the results presented in Figure 6 in the second paragraph of the section “Case study two – Ontogeny of inhibitory interneurons of the mouse thalamus”, however the reviewer is right in that we did not directly refer to the Figure – this was an oversight. In any case, in the revised manuscript we present a new version of Figure 6 (in response to above comment), which is now explicitly cited in the text.

Revised Figure 6: Example data and inferences highlighting model discrepancies. On the left under ‘data’: boxplots with medians and interquartile ranges for the raw data for four example brain regions. The shape of each point pairs left and right hemisphere readings in each of the five animals. On the right under ‘inference’: HDIs and confidence intervals are plotted. Purple is the Bayesian horseshoe model, pink is the Bayesian ZIP model, and orange is the sample mean. The Bayesian estimates are not strongly influenced by the zero-valued observations (MPN, SCH, TMd) or large-valued outliers (MH) and have means close to the data median. This explains the advantage of the Bayesian results over the confidence interval.

Reviewer #2 (Recommendations from the authors):

(1) “This is a generally well-written methodology paper that also provides the underlying code as a resource. As a reviewer outside both cell-count modelling and hierarchical-Bayesian approaches (though with a general interest in the topics) I found the method a little difficult to follow and would have liked to have been left with a better understanding of how the method is applied to the data. For example, in Figure 1 we are introduced to brain region count, animal count, and “items”. Then in the next line: pooling, model, structure, population and etc in subsequent lines. It is not clear what the subscripts (the pools?) are referring to: are they different regions R or animals N? These terms need to be better linked to the data and/or trimmed. Having said that, the later results look like a solid contribution to the field with a significant reduction in uncertainty from the Bayesian approach over the frequentist one. A future version of the manuscript, therefore, would benefit from greater precision of language as well as an economy and greater focus of terms linking the method to the biology. This is particularly the case around the exposition parts in Figure 1, Figure 2, and the “Hierarchical modelling” section.”

This is another important point. We have now made numerous small changes to tighten up the text in the paper, in response to both this point and the next point.

(2) “Language throughout could be sharpened. Subjectivity like “surprising outliers” could be removed and quirky grammar like “often small, ten is a typical” improved. There are also typos “an rate” etc that should be tidied up.”

As per previous response, we have made numerous tweaks and small improvements and feel that the paper is stronger in this respect.

(3) “Figure 1 caption. “It is a spectrum that depends” Is spectrum the right word here? Also, “thicker stroke” what does this refer to? Wasn’t immediately clear. In A, why is the whole animal within the R bracket that signifies brain regions, and then the brain regions are within the N bracket that signifies whole animals? Apart from the teal colouring, what are the other coloured regions in the image referring to? Improving this first figure would greatly help a reader unfamiliar with the context of the approach.”

We have replaced the word “spectrum” with “continuum”. We have replaced “ Observed quantities have been highlighted with a thicker stroke in the graphical model.” with “The observed data quantities, y_i to y_n, are highlighted with a thick line in the model diagrams”. We have added the following text to describe the red and green lines in panel A: “green and red lines indicate regions labeled as damaged”.

(4) “On P2 there is no discussion of priors when running through the advantage of the Bayesian approach. Is this a choice or an oversight? Priors do have a role in the later analysis.”

A short additional paragraph has been added to the introduction outlining the advantage of having a prior, but also noting that the obligation to pick a prior can be intimidating and that suggesting priors is one of the contributions of our paper: “A Bayesian model also includes a set of probability distributions, referred to as the prior, which represent those beliefs it is reasonable to hold about the statistical model parameters before actually doing the experiment. The prior can be thought of as an advantage, it allows us to include in our analysis our understanding of the data based on previous experiments. The prior also makes explicit in a Bayesian model assumptions that are often implicit in other approaches. However, having to design priors is often considered a challenge and here we hope to make this more straightforward by suggesting priors that are suitable for this class of data.”

(5) “On P4 more explanation would help greatly. Formulas like 23*10*4 or 50*6+50*4 are presented without explanation. What are the various numbers being multiplied? Regions, animals? Again, a clearer link between biological data and model structure would be advantageous.”

We have now modified this line to clearly state the numbers’ sources: “The index i runs over the full set of samples, which in this case comprises 23 brain regions ×10 animals ×4 groups ≈920 datapoints in the first study, and 50 brain regions × 6 HET animals + 50 brain regions × 4 KO animals ≈500 datapoints in the second.”

(6) “P6 and Results. Is it possible to show examples of the data set sampled from? Perhaps an image or two for the two experiments. Both Figures 4 and 5 as they currently are could be made slightly smaller to provide space for a small explanatory sub-panel. This would help ground the results.”

This is a good idea. We have now added heatmap visualisations of both entire datasets to revised versions of Figures 4 and 5 (assuming that this is what the reviewer was suggesting).

Author response:

Reviewer #1 (Public review):

Weaknesses:

(1) Data:

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

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

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

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

We aimed to show that kinocilia in neonatal cochlear and vestibular hair cells are largely similar, except that neonatal cochlear hair cells lack key genes and proteins required for the motile apparatus. While these genes (e.g., Dynll1, Dynll2, Dynlrb1, Cetn2, and Mdh1) appear more highly expressed in P2 cochlear hair cells, they are not uniquely associated with the axoneme. For example, Dynll1/2 and Dynlrb1 are components of the cytoplasmic dynein-1 complex (Pfister et al., 2006), Cetn2 has multiple basic cellular functions beyond cilia (e.g., centrosome organization, DNA repair), and Mdh1 encodes a cytosolic malate dehydrogenase involved in central metabolic pathways such as the citric acid cycle and malate–aspartate shuttle. This contrasts with axonemal dyneins, which are uniquely required for cilia motility. To avoid ambiguity, we will mark such cytoplasmic or multifunctional genes with stars in both Figure 5G and Figure 6D together with legend in the revised manuscript.

Although those genes (i.e., Dynll1, Dynll2, Dynlrb1, Cetn2, and Mdh1) are highly expressed in neonatal cochlear hair cells, key genes for motile machinery are not detected. For example, Dnah6, Dnah5, and Wdr66 are not expressed in the P2 cochlear hair cells.  Dnah6 and Dnah5 encode axonemal dynein and are part of inner and outer dynein arms while Wdr66 is a component of radial spokes. Importantly, we did not detect the expression of CCDC39 and CCDC40 in kinocilia of P2 cochlear hair cells.  Axonemal CCDC39 and CCDC40 are the molecular rulers that organize the axonemal structure in the 96-nm repeating interactome and are required for the assembly of IDAs and N-DRC for ciliary motility (Becker-Heck et al., 2011; Merveille et al., 2011; Oda et al., 2014). We will modify Figure 6D to highlight the key difference between P2 cochlear and vestibular hair cells in the revised manuscript. We will also revise the text so that the key differences will clearly be described.

(2) Interpretation:

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

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

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

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

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

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

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

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

Joint Recommendations:

We will make changes in the revision based on the joint recommendations of the two reviewers.

References

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DOI: 10.1016/j.celrep.2025.116291

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DOI: 10.1016/j.celrep.2025.116291

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What is this?

1. Coincido totalmente en que lo importante no es si el título es corto o largo, sino que sea preciso y refleje tal cual el problema y el objetivo del estudio. Con las palabras justas se entiende el alcance de inmediato

2.Para poder realizar o empezar el proyecto se necesita pasar por varias etapas, ya que el planteamiento del título es lo primero, se necesita definir el problema y los objetivos que se planea investigar y con eso el título resulta más fácil de realizar.

1. El titulo de un articulo o investigación es como la portada de los libros ya que depende de su presentación si el lector estará interesado en leerlo, es por eso que es importante desarrollar un buen titulo que abarque el tema que se desarrollo a lo largo de la investigación y que además sea novedoso para que llame la atención de los lectores.

Bien, pero falta especificar un pequeño parrafo que diga que se construyen indices con los promedios de los indicadores de cada subdimension para simplidicar. Y darle formato tabla a la salida no como output

Mejorar el esquema y el caption

Caption? y hay que desmenuzar mucho más la figura, para saber qué indicadores componen esas subdimensiones (ej, justicia distributiva)

Y el esquema de operacionalización? ya sea figura o tabla?

pero eso es cohesión vertical

yo veo solo un indicador tanto en la figura como en el visualizador

square root of n law

Five years without public schools for black, poor white students.

DOI: 10.1038/s44161-025-00698-y

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DOI: 10.1038/s44161-025-00698-y

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What is this?

Reviewer #1 (Public review):

Summary:

The authors demonstrate with a simple stochastic model that the initial composition of the community is important in achieving a target frequency during the artificial selection of a community.

Strengths:

To my knowledge, the intra-collective selection during artificial selection has not been seriously theoretically considered. However, in many cases, the species dynamics during the incubation of each selection cycle is important and relevant to the outcome of the artificial selection experiment. Stochasticity from birth and death (demographic stochasticity) plays a big role in these species' abundance dynamics. This work uses a simple framework to tackle this idea meticulously.

This work may or may not be related to hysteresis (path dependency). If this is true, maybe it would be nice to have a discussion paragraph talking about how this may be the case. Then, this work would even attract the interest of people studying dynamical systems.

Weaknesses:

(1) Connecting structure and function.<br /> In typical artificial selection literature, most of them select the community based on collective function. Here in this paper, the authors are selecting a target composition. Although there is a schematic cartoon illustrating the relationship between collective function (y-axis) and the community composition in the main figure 1, there is no explicit explanation or justification of what may be the origin of this relationship. I think giving the readers a naïve idea about how this structure-function relationship arises in the introduction section would help. This is because the conclusion of this paper is that the intra-collective selection makes it hard to artificially select for a community that has an intermediate frequency of f (or s). If there is really evidence or theoretical derivation from this framework that indeed the highest function comes from the intermediate frequency of f, then the impact of this paper would increase because the conclusions of this stochastic model could allude to the reasons for the prevalent failures of artificial selection in literature.

(2) Explain intra-collective and inter-collective selection better for readers.<br /> The abstract, the introduction, and the result section use these terms or intra-collective and inter-collective selection without much explanation. For the wide readership of eLife, a clear definition in the beginning would help the audience grasp the importance of this paper, because these concepts are at the core of this work.

(3) Achievable target frequency strongly depending on the degree of demographic stochasticity.<br /> I would expect that the experimentalists would find these results interesting and would want to consider these results during their artificial selection experiments. The main figure 4 indicates that the Newborn size N0 is a very important factor to consider during the artificial selection experiment. This would be equivalent to how much bottleneck you impose on the artificial selection process in every iteration step (i.e., the ratio of serial dilution experiment). However, with a low population size, all target frequencies can be achieved, and therefore in these regimes, the initial frequency now does not matter much. It would be great for the authors to provide what the N0 parameter actually means during the artificial selection experiments. Maybe relative to some other parameter in the model. I know this could be very hard. But without this, the main result of this paper (initial frequency matters) cannot be taken advantage of by the experimentalists.

(4) Consideration of environmental stochasticity.<br /> The success (gold area of Figure 2d) in this framework mainly depends on the size of the demographic stochasticity (birth-only model) during the intra-collective selection. However, during experiments, a lot of environmental stochasticity appears to be occurring during artificial selection. This may be out of the scope of this study. But it would definitely be exciting to see how much environmental stochasticity relative to the demographic stochasticity (variation in the Gaussian distribution of F and S) matters in succeeding in achieving the target composition from artificial selection.

(5) Assumption about mutation rates<br /> If setting the mutation rates to zero does not change the result of the simulations and the conclusion, what is the purpose of having the mutation rates \mu? Also, is the unidirectional (S -> F -> FF) mutation realistic? I didn't quite understand how the mutations could fit into the story of this paper.

(6) Minor points<br /> In Figure 3b, it is not clear to me how the frequency difference for the Intra-collective and the Inter-collective selection is computed.<br /> In Figure 5b, the gold region (success) near the FF is not visible. Maybe increase the size of the figure or have an inset for zoom-in. Why is the region not as big as the bottom gold region?

Comments on revisions:

I thank the authors for addressing many points raised by the reviewers. Overall, the readability of the manuscript has improved with more context provided around why they were solving this specific problem. However, I've found many of the responses to be too terse. It would have been nicer if there had been more discussion and description of the thought process that led up to the conclusions they made for each comment or question. Instead, many of the responses only showed the screenshot of the text they added.

Most of my comments or questions were answered. Below are my comments on some of the authors' responses.

(2) Explain intra-collective and inter-collective selection better for readers.<br /> In the Abstract and Introduction, you've added more sentences about the intra-collective or inter-collective selection. However, these are either making analogies to the waterfall or just describing the result of the intra/inter-collective selection. I would still appreciate a proper definition of those terms, which is paramount for readers to understand the entire paper.

(4) Consideration of environmental stochasticity.<br /> I think providing the reason 'why' the paper focuses on demographic stochasticity and not environmental stochasticity will greatly justify the paper's work. For example, citing papers that actually performed artificial selection and pointing out that your model captures the stochasticity from those kinds of experiments would be great.

(5) Assumption about mutation rates.<br /> It would be great if you could add a citation in the added sentence to support your claim: "This scenario is encountered in biotechnology: .....".

DOI: 10.1007/s00411-025-01146-y

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DOI: 10.1007/s00401-025-02934-y

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What is this?

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

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Reply to the reviewers

Manuscript number: RC-2025-03098

Corresponding author: Pedro Escoll

1. General Statements

Our study investigates the interplay between the metabolism of host cells and the intracellular replication of Salmonella enterica serovar Typhimurium (ST). Type III Secretion Systems (T3SSs) are considered essential for ST to replicate within macrophages. However, we found that restricting macrophages to different bioenergetic contexts, such as supplementing them with glycerol, modulates bacterial replication and remarkably, enables a T3SS-deficient ST mutant (ΔprgH/ΔssaV) to replicate intracellularly. This T3SS-independent replication occurs within the Salmonella-containing vacuole (SCV) and is driven by the capacity of the host cell to provide these preferred nutrients, rather than by the host glycolytic activity itself.

2. Description of the planned revisions

__Reviewer #1 (Evidence, reproducibility and clarity): __

Summary:

In this manuscript, the authors investigate how host cell metabolic heterogeneity influences the intracellular replication of Salmonella enterica serovar Typhimurium. They use live-cell imaging of infected human primary macrophages to reveal that bacterial replication does not occur uniformly across infected cells. They demonstrate that supplementation with specific carbon sources-used by Salmonella during infection-promotes bacterial replication and increases the proportion of macrophages supporting intracellular growth. These effects are seen even in the absence of functional Type III Secretion Systems (T3SS), using a ΔprgH/ΔssaV double mutant. The authors further suggest that this replication enhancement is not strictly dependent on host glycolytic activity but rather on the host cell's ability to import nutrients. Their findings imply that intracellular Salmonella can exploit host cell metabolism to grow, even without its canonical virulence secretion systems, under nutrient-favorable conditions.

Major Concern:

While the topic is potentially interesting, the novelty is not fully clear. The concept that nutrient availability impacts intracellular Salmonella replication, largely via T3SS2 function, has been addressed previously (e.g., Liss et al., 2017). The finding that added exogenous carbon sources can enhance bacterial growth is thus not unexpected. The key claim-that Salmonella can replicate intracellularly even in the absence of T3SS function-would be significantly strengthened by demonstrating whether this is specific to Salmonella, or whether similar effects are seen with non-intracellular organisms such as E. coli K-12. If the phenomenon is unique to Salmonella, this would suggest a pathogen-specific mechanism beyond general metabolic support.

As acknowledged by the Reviewer, the novelty and key claim of our work is that Salmonella can replicate intracellularly even in the absence of T3SS. To experimentally sustain that claim, we showed evidence that providing macrophages with the preferred carbon sources used by Salmonella during infection, such as glycerol, bypass the requirement of both T3SS by Salmonella to grow, intravacuolarly, inside macrophages.

With respect to the article mentioned by the Reviewer (Liss et al. 2017, ref 36 in the manuscript), there are three important novel insights provided by our work: i) we show that Salmonella can replicate intracellularly in the SCV even in the absence of T3SS if certain carbon sources are provided; ii) we show the preference of Salmonella for certain carbon sources intracellularly such as glycerol and galactose (but not preferentially glucose); and iii) we have extended our observations to primary human macrophages in addition to RAW cells.

We are not convinced that the experiment suggested by the Reviewer to use E. coli K12 (ECK12) is necessary to support our findings for Salmonella, but we propose to add the requested experiment. Briefly, we will infect hMDMs and RAW macrophages with ST-WT-GFP, ST-ΔprgHΔssaV or ECK12-WT-GFP, while culturing macrophages on different carbon sources (glucose, glycerol, galactose, fructose). Then we will monitor intracellular bacterial growth. By comparing bacterial growth of ST double mutant with ECK12-WT-GFP under favorable carbon sources such as glycerol, the results will be definitive to answer whether this phenomenon is unique to Salmonella or not.

Specific Comments:

1. Figure 1H: The effect shown here is not compelling due to inconsistent y-axis scaling. Panels 1B, 1C, and 1D should use a unified axis range with 1H to allow direct visual comparison of growth dynamics.

Thank you, we will change it as suggested.

Figures 1B, 1C, 1G, 1H: The current presentation of individual growth traces makes it difficult to appreciate the population-level trend. A smoothed average line overlaid on these plots could better represent the average dynamics of replicative vs. non-replicative infections. Or alternatively the total fraction of cells that proliferate summarized as a segmented bar plot (possibly binned per time point).

We will plot the results as suggested, the total fraction of infected cells harboring bacteria that proliferate as a segmented bar plot, binned per time point.

Figure 2G: This panel would benefit from including a comparable condition with the SPI-1/SPI-2 double mutant to aid interpretation. Additionally, the authors should explore whether this nutrient-supported replication is seen in non-phagocytic cells such as HeLa or Caco-2, which would help delineate whether the observed phenomenon is macrophage-specific.

The graph asked by Reviewer is Figure S1D. As we are representing ST growth in macrophages supporting Salmonella replication, some of the conditions, such as lactate, cannot be shown in the infection conditions using the double mutant because there are no cells supporting the replication of the double mutant, so there are no cells to plot.

As suggested, we are also going to perform the same experiments in HeLa cells to investigate whether the observed phenomenon is macrophage specific.

Line 117: The sentence stating that the double mutant can undergo "exponential intracellular growth even in the absence of T3SS-dependent secretion" is an overstatement. The data suggest only a modest improvement in growth, restricted to a minority of infected cells. This claim should be revised accordingly, as should similar overstatements in the discussion (e.g., lines 203-204).

We will remove the term 'exponential' and revise the sentence at line 117 and those in the discussion. Line 203-204 will be: 'we demonstrated that providing macrophages with preferred nutrients allows a subpopulation of ST to replicate intracellularly without the need for a functional T3SS'.

Line 162: The authors should clarify that glycerol had the strongest effect in primary macrophages, while multiple alternative carbon sources had notable effects primarily in RAW cells.

We will add this clarification in the text.