eLife Assessment

The study presents a comprehensive multi-approach and functional investigation of RBMX2 as a host factor involved in Mycobacterium bovis pathogenesis and its potential role in promoting epithelial-mesenchymal transition and lung cancer progression. The findings are valuable since the possible connection between M. bovis and lung cancer and the underlying mechanisms provides a promising direction for future research. The evidence is solid with methods, data, and analyses broadly supporting the claims, albeit with minor weaknesses that, if addressed, will make the evidence stronger. The study remains of great interest to microbiology, oncology, and drug discovery scientists.

Reviewer #1 (Public review):

Summary:

This manuscript presents a compelling study identifying RBMX2 as a novel host factor upregulated during Mycobacterium bovis infection.

The study demonstrates that RBMX2 plays a role in:

(1) Facilitating M. bovis adhesion, invasion, and survival in epithelial cells.

(2) Disrupting tight junctions and promoting EMT.

(3) Contributing to inflammatory responses and possibly predisposing infected tissue to lung cancer development.

By using a combination of CRISPR-Cas9 library screening, multi-omics, coculture models, and bioinformatics, the authors establish a detailed mechanistic link between M. bovis infection and cancer-related EMT through the p65/MMP-9 signaling axis. Identification of RBMX2 as a bridge between TB infection and EMT is novel.

Strengths:

This topic and data are both novel and significant, expanding the understanding of transcriptomic diversity beyond RBM2 in M. bovis responsive functions.

Weaknesses:

(1) The abstract and introduction sometimes suggest RBMX2 has protective anti-TB functions, yet results show it facilitates pathogen adhesion and survival. The authors need to rephrase claims to avoid contradiction.

(2) While p65/MMP-9 is convincingly implicated, the role of MAPK/p38 and JNK is less clearly resolved.

(3) Metabolomics results are interesting but not integrated deeply into the main EMT narrative.

(4) A key finding and starting point of this study is the upregulation of RBMX2 upon M. bovis infection. However, the authors have only assessed RBMX2 expression at the mRNA level following infection with M. bovis and BCG. To strengthen this conclusion, it is essential to validate RBMX2 expression at the protein level through techniques such as Western blotting or immunofluorescence. This would significantly enhance the credibility and impact of the study's foundational observation.

(5) The manuscript would benefit from a more in-depth discussion of the relationship between tuberculosis (TB) and lung cancer. While the study provides experimental evidence suggesting a link via EMT induction, integrating current literature on the epidemiological and mechanistic connections between chronic TB infection and lung tumorigenesis would provide important context and reinforce the translational relevance of the findings.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

This manuscript presents a compelling study identifying RBMX2 as a novel host factor upregulated during Mycobacterium bovis infection.

The study demonstrates that RBMX2 plays a role in:

(1) Facilitating M. bovis adhesion, invasion, and survival in epithelial cells.

(2) Disrupting tight junctions and promoting EMT.

(3) Contributing to inflammatory responses and possibly predisposing infected tissue to lung cancer development.

By using a combination of CRISPR-Cas9 library screening, multi-omics, coculture models, and bioinformatics, the authors establish a detailed mechanistic link between M. bovis infection and cancer-related EMT through the p65/MMP-9 signaling axis. Identification of RBMX2 as a bridge between TB infection and EMT is novel.

Strengths:

This topic and data are both novel and significant, expanding the understanding of transcriptomic diversity beyond RBM2 in M. bovis responsive functions.

Weaknesses:

(1) The abstract and introduction sometimes suggest RBMX2 has protective anti-TB functions, yet results show it facilitates pathogen adhesion and survival. The authors need to rephrase claims to avoid contradiction.

We sincerely appreciate the reviewer's valuable feedback regarding the need to clarify RBMX2's role throughout the manuscript. We have carefully revised the text to ensure consistent messaging about RBMX2's function in promoting M. bovis infection. Below we detail the specific modifications made:

(1) Introduction Revisions:

Changed "The objective of this study was to elucidate the correlation between host genes and the susceptibility of M.bovis infection" to "The objective of this study was to identify host factors that promote susceptibility to M.bovis infection"

Revised "RBMX2 polyclonal and monoclonal cell lines exhibited favorable phenotypes" to "RBMX2 knockout cell lines showed reduced bacterial survival"

Replaced "The immune regulatory mechanism of RBMX2" with "The role of RBMX2 in facilitating M.bovis immune evasion"

(2) Results Revisions:

Modified "RBMX2 fails to affect cell morphology and the ability to proliferate and promotes M.bovis infection" to "RBMX2 does not alter cell viability but significantly enhances M.bovis infection"

Strengthened conclusion in Figure 4: "RBMX2 actively disrupts tight junctions to facilitate bacterial invasion"

(3) Discussion Revisions:

Revised screening description: "We screened host factors affecting M.bovis susceptibility and identified RBMX2 as a key promoter of infection"

Strengthened concluding statement: "In summary, RBMX2 drives TB pathogenesis by compromising epithelial barriers and inducing EMT"

These targeted revisions ensure that:

All sections consistently present RBMX2 as promoting infection; the language aligns with our experimental finding; potential protective interpretations have been eliminated. We believe these modifications have successfully addressed the reviewer's concern while maintaining the manuscript's original structure and scientific content. We appreciate the opportunity to improve our manuscript and thank the reviewer for this constructive suggestion.

(2) >While p65/MMP-9 is convincingly implicated, the role of MAPK/p38 and JNK is less clearly resolved.

We sincerely appreciate the reviewer's insightful comment regarding the roles of MAPK/p38 and JNK in our study. Our experimental data clearly demonstrated that RBMX2 knockout significantly reduced phosphorylation levels of p65, p38, and JNK (Fig. 5A), indicating potential involvement of all three pathways in RBMX2-mediated regulation.

Through systematic functional validation, we obtained several important findings:

In pathway inhibition experiments, p65 activation (PMA treatment) showed the most dramatic effects on both tight junction disruption (ZO-1, OCLN reduction) and EMT marker regulation (E-cadherin downregulation, N-cadherin upregulation);

p38 activation (ML141 treatment) exhibited moderate effects on these processes;

JNK activation (Anisomycin treatment) displayed minimal impact.

Most conclusively, siRNA-mediated silencing of p65 alone was sufficient to:

Restore epithelial barrier function

Reverse EMT marker expression

Reduce bacterial adhesion and invasion

These results establish a clear hierarchy in pathway importance: p65 serves as the primary mediator of RBMX2's effects, while p38 plays a secondary role and JNK appears non-essential under our experimental conditions. We have now clarified this relationship in the revised Discussion section to strengthen this conclusion.

This refined understanding of pathway hierarchy provides important mechanistic insights while maintaining consistency with all our experimental data. We thank the reviewer for this valuable suggestion that helped improve our manuscript.

(3) Metabolomics results are interesting but not integrated deeply into the main EMT narrative.

Thank you for this constructive suggestion. In this article, we detected the metabolome of RBMX2 knockout and wild-type cells after Mycobacterium bovis infection, which mainly served as supporting evidence for our EMT model. However, we did not conduct an in-depth discussion of these findings. We have now added a detailed discussion of this section to further support our EMT model.

ADD:Meanwhile, metabolic pathways enriched after RBMX2 deletion, such as nucleotide metabolism, nucleotide sugar synthesis, and pentose interconversion, primarily support cell proliferation and migration during EMT by providing energy precursors, regulating glycosylation modifications, and maintaining redox balance; cofactor synthesis and amino sugar metabolism participate in EMT regulation through influencing metabolic remodeling and extracellular matrix interactions; chemokine and cGMP-PKG signaling pathways may further mediate inflammatory responses and cytoskeletal rearrangements, collectively promoting the EMT process.

(4) A key finding and starting point of this study is the upregulation of RBMX2 upon M. bovis infection. However, the authors have only assessed RBMX2 expression at the mRNA level following infection with M. bovis and BCG. To strengthen this conclusion, it is essential to validate RBMX2 expression at the protein level through techniques such as Western blotting or immunofluorescence. This would significantly enhance the credibility and impact of the study's foundational observation.

Thank you for your comment. We have supplemented the experiments in this part and found that Mycobacterium bovis infection can significantly enhance the expression level of RBMX2 protein.

(5) The manuscript would benefit from a more in-depth discussion of the relationship between tuberculosis (TB) and lung cancer. While the study provides experimental evidence suggesting a link via EMT induction, integrating current literature on the epidemiological and mechanistic connections between chronic TB infection and lung tumorigenesis would provide important context and reinforce the translational relevance of the findings.

We sincerely appreciate the valuable comments from the reviewer. We fully agree with your suggestion to further explore the relationship between tuberculosis (TB) and lung cancer. In the revised manuscript, we will add a new paragraph in the Discussion section to systematically integrate the current literature on the epidemiological and mechanistic links between chronic tuberculosis infection and lung cancer development, including the potential bridging roles of chronic inflammation, tissue damage repair, immune microenvironment remodeling, and the epithelial-mesenchymal transition (EMT) pathway. This addition will help more comprehensively interpret the clinical implications of the observed EMT activation in the context of our study, thereby enhancing the biological plausibility and clinical translational value of our findings.

ADD:There is growing epidemiological evidence suggesting that chronic TB infection represents a potential risk factor for the development of lung cancer. Studies have shown that individuals with a history of TB exhibit a significantly increased risk of lung cancer, particularly in areas of the lung with pre-existing fibrotic scars, indicating that chronic inflammation, tissue repair, and immune microenvironment remodeling may collectively contribute to malignant transformation 74. Moreover, EMT not only endows epithelial cells with mesenchymal features that enhance migratory and invasive capacity but is also associated with the acquisition of cancer stem cell-like properties and therapeutic resistance 75. Therefore, EMT may serve as a crucial molecular link connecting chronic TB infection with the malignant transformation of lung epithelial cells, warranting further investigation in the intersection of infection and tumorigenesis.

Reviewer #2 (Public review):

Summary:

I am not familiar with cancer biology, so my review mainly focuses on the infection part of the manuscript. Wang et al identified an RNA-binding protein RBMX2 that links the Mycobacterium bovis infection to the epithelial-Mesenchymal transition and lung cancer progression. Upon mycobacterium infection, the expression of RBMX2 was moderately increased in multiple bovine and human cell lines, as well as bovine lung and liver tissues. Using global approaches, including RNA-seq and proteomics, the authors identified differential gene expression caused by the RBMX2 knockout during M. bovis infection. Knockout of RBMX2 led to significant upregulations of tight-junction related genes such as CLDN-5, OCLN, ZO-1, whereas M. bovis infection affects the integrity of epithelial cell tight junctions and inflammatory responses. This study establishes that RBMX2 is an important host factor that modulates the infection process of M. bovis.

Strengths:

(1) This study tested multiple types of bovine and human cells, including macrophages, epithelial cells, and clinical tissues at multiple timepoints, and firmly confirmed the induced expression of RBMX2 upon M. bovis infection.

(2) The authors have generated the monoclonal RBMX2 knockout cell lines and comprehensively characterized the RBMX2-dependent gene expression changes using a combination of global omics approaches. The study has validated the impact of RBMX2 knockout on the tight-junction pathway and on the M. bovis infection, establishing RBMX2 as a crucial host factor.

Weaknesses:

(1) The RBMX2 was only moderately induced (less than 2-fold) upon M. bovis infection, arguing its contribution may be small. Its value as a therapeutic target is not justified. How RBMX2 was activated by M. bovis infection was unclear.

Thank you for your valuable and constructive comments. In this study, we primarily utilized the CRISPR whole-genome screening approach to identify key factors involved in bovine tuberculosis infection. Through four rounds of screening using a whole-genome knockout cell line of bovine lung epithelial cells infected with Mycobacterium bovis, we identified RBMX2 as a critical factor.

Although the transcriptional level change of RBMX2 was less than two-fold, following the suggestion of Reviewer 1, we examined its expression at the protein level, where the change was more pronounced, and we have added these results to the manuscript.

Regarding the mechanism by which RBMX2 is activated upon M. bovis infection, we previously screened for interacting proteins using a Mycobacterium tuberculosis secreted and membrane protein library, but unfortunately, we did not identify any direct interacting proteins from M. tuberculosis (https://doi.org/10.1093/nar/gkx1173).

(2) Although multiple time points have been included in the study, most analyses lack temporal resolution. It is difficult to appreciate the impact/consequence of M. bovis infection on the analyzed pathways and processes.

We appreciate the valuable comments from the reviewers. Although our study included multiple time points post-infection, in our experimental design we focused on different biological processes and phenotypes at distinct time points:

During the early phase (e.g., 2 hours post-infection), we focused on barrier phenotypes; during the intermediate phase (e.g., 24 hours post-infection), we concentrated more on pathway activation and EMT phenotypes;

And during the later phase (e.g., 48–72 hours post-infection), we focused more on cell death phenotypes, which were validated in another FII article (https://doi.org/10.3389/fimmu.2024.1431207).

We also examined the impact of varying infection durations on RBMX2 knockout EBL cellular lines via GO analysis. At 0 hpi, genes were primarily related to the pathways of cell junctions, extracellular regions, and cell junction organization. At 24 hpi, genes were mainly associated with pathways of the basement membrane, cell adhesion, integrin binding and cell migration By 48 hpi, genes were annotated into epithelial cell differentiation and were negatively regulated during epithelial cell proliferation. This indicated that RBMX2 can regulate cellular connectivity throughout the stages of M. bovis infection.

For KEGG analysis, genes linked to the MAPK signaling pathway, chemical carcinogen-DNA adducts, and chemical carcinogen-receptor activation were observed at 0 hpi. At 24 hpi, significant enrichment was found in the ECM-receptor interaction, PI3K-Akt signaling pathway, and focal adhesion. Upon enrichment analysis at 48 hpi, significant enrichment was noted in the TGF-beta signaling pathway, transcriptional misregulation in cancer, microRNAs in cancer, small cell lung cancer, and p53 signaling pathway.

Reviewer #3 (Public review):

Summary:

This study investigates the role of the host protein RBMX2 in regulating the response to Mycobacterium bovis infection and its connection to epithelial-mesenchymal transition (EMT), a key pathway in cancer progression. Using bovine and human cell models, the authors have wisely shown that RBMX2 expression is upregulated following M. bovis infection and promotes bacterial adhesion, invasion, and survival by disrupting epithelial tight junctions via the p65/MMP-9 signaling pathway. They also demonstrate that RBMX2 facilitates EMT and is overexpressed in human lung cancers, suggesting a potential link between chronic infection and tumor progression. The study highlights RBMX2 as a novel host factor that could serve as a therapeutic target for both TB pathogenesis and infection-related cancer risk.

Strengths:

The major strengths lie in its multi-omics integration (transcriptomics, proteomics, metabolomics) to map RBMX2's impact on host pathways, combined with rigorous functional assays (knockout/knockdown, adhesion/invasion, barrier tests) that establish causality through the p65/MMP-9 axis. Validation across bovine and human cell models and in clinical tissue samples enhances translational relevance. Finally, identifying RBMX2 as a novel regulator linking mycobacterial infection to EMT and cancer progression opens exciting therapeutic avenues.

Weaknesses:

Although it's a solid study, there are a few weaknesses noted below.

(1) In the transcriptomics analysis, the authors performed (GO/KEGG) to explore biological functions. Did they perform the search locally or globally? If the search was performed with a global reference, then I would recommend doing a local search. That would give more relevant results. What is the logic behind highlighting some of the enriched pathways (in red), and how are they relevant to the current study?

We appreciate the reviewer's thoughtful questions regarding our transcriptomic analysis. In this study, we employed a localized enrichment approach focusing specifically on gene expression profiles from our bovine lung epithelial cell system. This cell-type-specific analysis provides more biologically relevant results than global database searches alone.

Regarding the highlighted pathways, these represent:

(1) Temporally significant pathways showing strongest enrichment at each stage:

• 0h: Cell junction organization (immediate barrier response)

• 24h: ECM-receptor interaction (early EMT initiation)

• 48h: TGF-β signaling (chronic remodeling)

(2) Mechanistically linked to our core findings about RBMX2's role in:

• Epithelial barrier disruption

• Mesenchymal transition

• Chronic infection outcomes

We selected these particular pathways because they:

(1) Showed the most statistically significant changes (FDR <0.001)

(2) Formed a coherent biological narrative across infection stages

(3) Were independently validated in our functional assays

This targeted approach allows us to focus on the most infection-relevant pathways while maintaining statistical rigor.

(2) While the authors show that RBMX2 expression correlates with EMT-related gene expression and barrier dysfunction, the evidence for direct association remains limited in this study. How does RBMX2 activate p65? Does it bind directly to p65 or modulate any upstream kinases? Could ChIP-seq or CLIP-seq provide further evidence for direct RNA or DNA targets of RBMX2 that drive EMT or NF-κB signaling?

We sincerely appreciate the reviewer's in-depth questions regarding the mechanisms by which RBMX2 activates p65 and its association with EMT. Although the molecular mechanism remains to be fully elucidated, our study has provided experimental evidence supporting a direct regulatory relationship between RBMX2 and the p65 subunit of the NF-κB pathway. Specifically, we investigated whether the transcription factor p65 could directly bind to the promoter region of RBMX2 using CHIP experiments. The results demonstrated that the transcription factor p65 can physically bind to the RBMX2 region.

Furthermore, dual-luciferase reporter assays were conducted, showing that p65 significantly enhances the transcriptional activity of the RBMX2 promoter, indicating a direct regulatory effect of RBMX2 on p65 expression.

These findings support our hypothesis that RBMX2 activates the NF-κB signaling pathway through direct interaction with the p65 protein, thereby participating in the regulation of EMT progression and barrier function.

In our subsequent work papers, we will also employ experiments such as CLIP to further investigate the specific mechanisms through which RBMX2 exerts its regulatory functions.

(3) The manuscript suggests that RBMX2 enhances adhesion/invasion of several bacterial species (e.g., E. coli, Salmonella), not just M. bovis. This raises questions about the specificity of RBMX2's role in Mycobacterium-specific pathogenesis. Is RBMX2 a general epithelial barrier regulator or does it exhibit preferential effects in mycobacterial infection contexts? How does this generality affect its potential as a TB-specific therapeutic target?

Thank you for your valuable comments. When we initially designed this experiment, we were interested in whether the RBMX2 knockout cell line could confer effective resistance not only against Mycobacterium bovis but also against Gram-negative and Gram-positive bacteria. Surprisingly, we indeed observed resistance to the invasion of these pathogens, albeit weaker compared to that against Mycobacterium bovis.

Nevertheless, we believe these findings merit publication in eLife. Moreover, RBMX2 knockout does not affect the phenotype of epithelial barrier disruption under normal conditions; its significant regulatory effect on barrier function is only evident upon infection with Mycobacterium bovis.

Importantly, during our genome-wide knockout library screening, RBMX2 was not identified in the screening models for Salmonella or Escherichia coli, but was consistently detected across multiple rounds of screening in the Mycobacterium bovis model.

(4) The quality of the figures is very poor. High-resolution images should be provided.

Thank you for your feedback; we provided higher-resolution images.

(5) The methods are not very descriptive, particularly the omics section.

Thank you for your comments; we have revised the description of the sequencing section.

(6) The manuscript is too dense, with extensive multi-omics data (transcriptomics, proteomics, metabolomics) but relatively little mechanistic integration. The authors should have focused on the key mechanistic pathways in the figures. Improving the narratives in the Results and Discussion section could help readers follow the logic of the experimental design and conclusions.

Thank you for your valuable comments. We have streamlined the figures and revised the description of the results section accordingly.

Reviewer #2 (Public review):

Summary:

I am not familiar with cancer biology, so my review mainly focuses on the infection part of the manuscript. Wang et al identified an RNA-binding protein RBMX2 that links the Mycobacterium bovis infection to the epithelial-Mesenchymal transition and lung cancer progression. Upon mycobacterium infection, the expression of RBMX2 was moderately increased in multiple bovine and human cell lines, as well as bovine lung and liver tissues. Using global approaches, including RNA-seq and proteomics, the authors identified differential gene expression caused by the RBMX2 knockout during M. bovis infection. Knockout of RBMX2 led to significant upregulations of tight-junction related genes such as CLDN-5, OCLN, ZO-1, whereas M. bovis infection affects the integrity of epithelial cell tight junctions and inflammatory responses. This study establishes that RBMX2 is an important host factor that modulates the infection process of M. bovis.

Strengths:

(1) This study tested multiple types of bovine and human cells, including macrophages, epithelial cells, and clinical tissues at multiple timepoints, and firmly confirmed the induced expression of RBMX2 upon M. bovis infection.

(2) The authors have generated the monoclonal RBMX2 knockout cell lines and comprehensively characterized the RBMX2-dependent gene expression changes using a combination of global omics approaches. The study has validated the impact of RBMX2 knockout on the tight-junction pathway and on the M. bovis infection, establishing RBMX2 as a crucial host factor.

Weaknesses:

(1) The RBMX2 was only moderately induced (less than 2-fold) upon M. bovis infection, arguing its contribution may be small. Its value as a therapeutic target is not justified. How RBMX2 was activated by M. bovis infection was unclear.

(2) Although multiple time points have been included in the study, most analyses lack temporal resolution. It is difficult to appreciate the impact/consequence of M. bovis infection on the analyzed pathways and processes.

Reviewer #3 (Public review):

Summary:

This study investigates the role of the host protein RBMX2 in regulating the response to Mycobacterium bovis infection and its connection to epithelial-mesenchymal transition (EMT), a key pathway in cancer progression. Using bovine and human cell models, the authors have wisely shown that RBMX2 expression is upregulated following M. bovis infection and promotes bacterial adhesion, invasion, and survival by disrupting epithelial tight junctions via the p65/MMP-9 signaling pathway. They also demonstrate that RBMX2 facilitates EMT and is overexpressed in human lung cancers, suggesting a potential link between chronic infection and tumor progression. The study highlights RBMX2 as a novel host factor that could serve as a therapeutic target for both TB pathogenesis and infection-related cancer risk.

Strengths:

The major strengths lie in its multi-omics integration (transcriptomics, proteomics, metabolomics) to map RBMX2's impact on host pathways, combined with rigorous functional assays (knockout/knockdown, adhesion/invasion, barrier tests) that establish causality through the p65/MMP-9 axis. Validation across bovine and human cell models and in clinical tissue samples enhances translational relevance. Finally, identifying RBMX2 as a novel regulator linking mycobacterial infection to EMT and cancer progression opens exciting therapeutic avenues.

Weaknesses:

Although it's a solid study, there are a few weaknesses noted below.

(1) In the transcriptomics analysis, the authors performed (GO/KEGG) to explore biological functions. Did they perform the search locally or globally? If the search was performed with a global reference, then I would recommend doing a local search. That would give more relevant results. What is the logic behind highlighting some of the enriched pathways (in red), and how are they relevant to the current study?

(2) While the authors show that RBMX2 expression correlates with EMT-related gene expression and barrier dysfunction, the evidence for direct association remains limited in this study. How does RBMX2 activate p65? Does it bind directly to p65 or modulate any upstream kinases? Could ChIP-seq or CLIP-seq provide further evidence for direct RNA or DNA targets of RBMX2 that drive EMT or NF-κB signaling?

(3) The manuscript suggests that RBMX2 enhances adhesion/invasion of several bacterial species (e.g., E. coli, Salmonella), not just M. bovis. This raises questions about the specificity of RBMX2's role in Mycobacterium-specific pathogenesis. Is RBMX2 a general epithelial barrier regulator or does it exhibit preferential effects in mycobacterial infection contexts? How does this generality affect its potential as a TB-specific therapeutic target?

(4) The quality of the figures is very poor. High-resolution images should be provided.

(5) The methods are not very descriptive, particularly the omics section.

(6) The manuscript is too dense, with extensive multi-omics data (transcriptomics, proteomics, metabolomics) but relatively little mechanistic integration. The authors should have focused on the key mechanistic pathways in the figures. Improving the narratives in the Results and Discussion section could help readers follow the logic of the experimental design and conclusions.

Exposé Détaillé : Les États Hybrides Veille-Sommeil et les Fonctions Cognitives du Sommeil

Ce document de synthèse présente les thèmes principaux, les idées essentielles et les faits marquants issus des extraits de la conférence de Stanislas Dehaene et de la présentation d'Isabelle Arnulf intitulée "Les états hybrides veille sommeil une fenêtre sur les fonctions cognitives du sommeil".

1. Introduction : L'accès au Rêve et la Notion d'États Hybrides

Isabelle Arnulf, Professeur de neurologie et chef de service des pathologies du sommeil à l'hôpital de la Pitié-Salpêtrière, souligne la difficulté majeure d'étudier les fonctions cognitives du sommeil : l'accès au rêve. Elle définit le rêve non seulement comme les "grands scénarios immersifs" mais aussi comme "tout ce qui est pensée, émotion, sensation, ressenti pendant le sommeil". Le défi réside dans le fait que le contenu cognitif est vécu pendant le sommeil, et l'approche traditionnelle de réveil du dormeur pour recueillir des souvenirs présente des biais significatifs :

Oubli massif : "plus de 99 % de nos rêves soient oubliés à partir du moment où on se réveille". Distorsion du récit : La transformation du rêve en récit peut être inexacte en raison de la reconstruction difficile et de l'auto-censure. Manque de temporalité : L'incapacité de situer précisément le rêve par rapport au moment du réveil. Pour pallier ces difficultés, la recherche s'est orientée vers l'obtention de "marqueurs du rêve au moment où il se passe pendant le sommeil", notamment grâce aux "états hybrides" entre veille et sommeil. Ces états sont des zones frontalières où le cerveau n'est ni totalement éveillé ni totalement endormi, donnant lieu à des comportements ou des expériences particulières.

2. Les États Hybrides Veille-Sommeil comme Fenêtre sur le Rêve

Isabelle Arnulf identifie plusieurs états hybrides, qu'elle classe selon qu'ils se produisent entre :

Veille et sommeil lent profond : somnambulisme, terreurs nocturnes, parler dans le sommeil (somniloquie), éveils confusionnels. Veille et sommeil paradoxal : cataplexie, troubles comportementaux en sommeil paradoxal (TCSP), parler en sommeil paradoxal, rêve lucide, hypnagogies (entre veille et sommeil lent léger). La présentation se focalise sur les TCSP, le somnambulisme, les terreurs nocturnes, la somniloquie et le rêve lucide, car ils offrent des pistes directes pour l'étude des rêves.

2.1. Les Troubles Comportementaux en Sommeil Paradoxal (TCSP)

Définition : Rêves et cauchemars agités, survenant généralement chez les personnes de plus de 50 ans. Le dormeur "va souvent donner des coups de poing, des coups de pied, parler, insulter dans son sommeil". Congruence avec le rêve : Le contenu mental rapporté au réveil est "congruent et isomorphe au comportement qu'on a vu avant", ce qui en fait un "outil assez extraordinaire pour voir les rêves en comportement". Physiologie : Normalement, le sommeil paradoxal est caractérisé par une atonie musculaire (paralysie). Chez les patients TCSP, le "verrou qui nous paralyse dans la phase paradoxale n'est pas complètement effectif", permettant l'expression physique des rêves. Conséquences : Les comportements violents peuvent entraîner des blessures pour le dormeur ou son conjoint. La paralysie pendant le sommeil paradoxal est donc cruciale pour la sécurité. Variété des comportements : Au-delà des violences, la perte de l'atonie musculaire peut révéler des comportements non-violents, mais toujours associés à des souvenirs de rêve congruents, comme chanter ou mimer des actions. Analogie animale : Michel Jouvet a pu déclencher des comportements oniriques chez les chats en ciblant la zone du tronc cérébral responsable de l'atonie, confirmant l'existence d'une dégénérescence similaire chez les patients TCSP. Marqueur précoce de maladies neurodégénératives : Le TCSP est un "très haut risque" de développer dans les années qui suivent une maladie de Parkinson ou des corps de Lewy. La dégénérescence d'une région focale du cerveau (locus coeruleus et subcoeruleus) est associée à ce trouble. Cela en fait un sujet de recherche majeur pour le prodrome de ces maladies.

2.2. Les Parasomnies de Sommeil Lent Profond (Somnambulisme, Terreurs Nocturnes)

Définition : Comportements anormaux (marche, fuite, cris) observés à la lisière de l'éveil et du sommeil lent profond. Cerveau hybride : Pendant ces épisodes, le "la partie postérieure du cerveau est réveillé la partie antérieure est encore dans du sommeil profond". Contenu onirique : Contrairement à l'idée reçue, les somnambules peuvent rêver. "76 % des adultes avec ces phénomènes là [...] ont occasionnellement le rappel d'un contenu de rêve qui est associé à l'épisode et qui une fois de plus est isomorphe au comportement qu'on a vu avant". Ces rêves sont souvent plus brefs et liés à des "catastrophes imminentes". Perception de l'environnement : À la différence des TCSP où le dormeur est dans un autre monde, les somnambules et sujets aux terreurs nocturnes ont une "perception de la chambre et de leur contenu de rêve qui apparaît comme une sorte d'hologramme dans la chambre". Origine des rêves : L'accélération cardiaque et l'activation des régions postérieures du cerveau jusqu'à 20 secondes avant le début du comportement suggèrent que l'expérience mentale commence avant le réveil moteur, et n'est pas simplement une création au moment de l'éveil partiel.

3. Les Fonctions Cognitives du Sommeil et du Rêve

La recherche utilise ces états hybrides pour "mieux décrire le contenu mental" et "tester des hypothèses sur les fonctions cognitives du sommeil et du rêve".

3.1. La Simulation de la Menace (Théorie de Revonsuo) Hypothèse : La théorie de Revonsuo (2000) suggère que le rêve permet de "s'entraîner à faire face à la menace" et aurait une fonction darwinienne.

Corrélation avec les TCSP et le somnambulisme : "60 à 70 % se rappelle que c'était un scénario de menace" dans les rêves des patients TCSP et des somnambules. Somnambules (sommeil lent profond) : Majoritairement des catastrophes (inondation, murs qui s'écroulent) avec une réponse de fuite ("100 % [...] fuit hors du lit"). TCSP (sommeil paradoxal) : Plus d'agressions par des personnes et des animaux avec une réponse de contre-attaque ("75 % [...] contre-attaque dans le lit"). Rêves des étudiants en médecine avant le concours : Une étude auprès de 2324 étudiants a montré que 171 sur 188 qui se souvenaient d'un rêve la veille de l'examen avaient "rêvé de l'examen" et l'avaient "raté de toutes les manières possibles". Le fait de rêver du concours, même négativement, "donne un demi-point de plus" à la note, suggérant un "gain cognitif" et corroborant la théorie de Revonsuo sur l'entraînement face à des situations stressantes.

3.2. Le Langage pendant le Sommeil (Somniloquie) Méthodologie : Enregistrement et transcription de 883 vocalisations (dont 703 phrases complètes) sur environ 200 personnes.

Caractéristiques du langage nocturne :Négativité : Le mot le plus fréquent est "non", et les tournures négatives représentent plus de 9 % des énoncés, bien plus que dans la conversation éveillée. Vulgarité : "les mots vulgaires sont beaucoup plus fréquents que dans la conversation d'éveil". Ils sont plus fréquents en sommeil lent qu'en sommeil paradoxal (où ce sont plutôt des insultes). Structure : Le langage est "adressé à une personne", respecte la grammaire, le tour de parole, et peut être complexe. Les gestes co-verbaux sont également "parfaitement conservés". Différences avec l'éveil : Plus de marmonnements, une parole "plus négative, plus répétée" et "plus vulgaire", suggérant une expression moins censurée ou plus primitive du contenu mental.

3.3. La Consolidation de la Mémoire et la Réexécution des Apprentissages

• Hypothèse : Le sommeil consolide la mémoire. La question est de savoir si les apprentissages récents sont réexécutés dans les rêves.
• Expérience : Des patients somnambules et TCSP ont appris une chorégraphie ou un texte avant de dormir.
• Résultats : La mémoire procédurale (chorégraphie) et verbale (texte) était bien consolidée. Cependant, la réexécution des comportements appris pendant le sommeil était "une grande déception" et "assez rare", suggérant que la fenêtre d'observation est trop étroite.
• Cas anecdotique : Un patient TCSP a reproduit des mouvements de "taping" (examen neurologique) pendant son sommeil paradoxal, montrant une certaine transparence de l'apprentissage dans le rêve.

3.4. La Régulation des Émotions

• Fonction connue : Le sommeil aide à "digérer les émotions négatives", comme le montre le fait qu'une nuit de sommeil permet de répondre de manière plus rationnelle à un e-mail agressif.
• Expression faciale des émotions pendant le sommeil :Sourires : Les adultes, y compris les personnes normales, sourient pendant leur sommeil (sommeil lent léger et paradoxal). 52 % de ces sourires sont des "sourires de Duchène", associés à de "vraies émotions positives".
• Émotions négatives : Les froncements de sourcils (corrugator) sont fréquents dans tous les sujets. Les expressions complètes de mécontentement, peur ou colère sont observées "uniquement les troubles comportementaux en sommeil paradoxal".
• Lien avec les mouvements oculaires rapides (MOR) : Les émotions négatives sont "très fortement associées [...] aux bouffées de mouvement oculaire" en sommeil paradoxal. Cela rappelle la technique de l'EMDR (Eye Movement Desensitization and Reprocessing) utilisée pour digérer les traumatismes, suggérant que les MOR rapides pourraient aider à traiter les émotions négatives pendant le sommeil paradoxal.
• Dynamique des émotions pendant le sommeil paradoxal : Les émotions négatives apparaissent plus tôt (pic autour de 10 minutes) et sont plus présentes au début de la phase. Le cerveau semble traiter les émotions "à très haute vitesse", passant rapidement du positif au négatif, ce qui pourrait "éviter trop d'émotions négatives si elle se suivait sans arrêt".

3.5. La Créativité et les Hypnagogies

• Anectodes de chercheurs et artistes : Einstein et Mendeleïev auraient eu des intuitions créatives en rêve.
• Méthode d'Edison : Thomas Edison utilisait une méthode pour se réveiller au début de l'endormissement (hypnagogies) pour capturer des "bonnes idées". Kekulé aurait découvert la structure du benzène lors d'une rêverie hypnagogique.
• Expérience sur la résolution de problèmes : Des participants ont tenté de résoudre une tâche de réduction de nombres (avec une règle cachée) avant de dormir, puis se sont reposés avec une bouteille à la main (méthode d'Edison).
• Résultats : Ceux qui se sont réveillés après 30 à 60 secondes de sommeil lent léger (stade N1) grâce à la chute de la bouteille ont eu un taux de réussite de 83 % pour trouver la solution cachée. Ceux qui sont restés éveillés ou qui sont passés en sommeil N2 n'ont pas trouvé la solution.
• Explication : La résolution du problème semble nécessiter un "cocktail EEG favorable", un mélange de "réflexion de l'éveil et de déjà l'imagination la pensée un peu métaphorique en image du sommeil". Les hypnagogies, images et petits scénarios qui apparaissent à l'endormissement, sont clés pour cette créativité.

4. Conclusion : Le Sommeil comme "Boîte Noire" du Contenu Mental

Isabelle Arnulf conclut que "il y a une activité mentale dans tous les stades de sommeil" avec des fonctions potentielles de "mémoriser de simuler les menaces de réguler les émotions peut-être de développer la créativité peut-être peut-être aussi l'empathie". Les parasomnies (états hybrides) sont des "fenêtres directes étroites [...] mais riches sur ces conditions et ses émotions pendant le sommeil". Le sommeil est une "boîte noire" que ces "fragmentations" permettent d'explorer, révélant le "cristal qui est à l'intérieur qui est ce contenu mental".

Enfin, elle mentionne l'importance clinique de cette recherche, notamment la capacité de modifier les cauchemars récurrents par la "répétition d'image mentale" (Image Rehearsal Therapy), soulignant comment la compréhension du traitement cognitif pendant le sommeil peut être utilisée pour des interventions thérapeutiques.

① “Impacted” means that a tooth either has not come in (“erupted”) when expected or a tooth that cannot erupt because it does not have room or may be coming in the wrong direction or position. ① “Gömülü” terimi, bir dişin beklenen zamanda çıkmamış (“sürmemiş”) olması veya yeterli alan olmadığı için ya da yanlış yönde veya pozisyonda çıkmaya çalıştığı için çıkamaması anlamına gelir.

② What causes an impacted tooth may not be known – for some people, it just happens. ② Bir dişin gömülü olmasının nedenleri bilinmeyebilir – bazı kişilerde bu sadece olur.

③ Genetics can play a role, so if a parent had an impacted tooth, their child may experience the same problem. ③ Genetik faktörler etkili olabilir, bu yüzden bir ebeveynin gömülü dişi varsa, çocuğunda da aynı problem görülebilir.

① Tooth rotation is defined as mesiolingual or distolingual intra-alveolar displacement of the tooth around its longitudinal axis. ① Diş rotasyonu, dişin uzunlamasına ekseni etrafında mesiolingual veya distolingual intra-alveoler yer değiştirmesi olarak tanımlanır.

② A number of factors are involved in the rotation of teeth, such as space inadequacy, abnormal tooth eruption sequence, and undesirable forces exerted by the tongue and lips or any combination of these. ② Diş rotasyonunda; yer yetersizliği, anormal diş sürme dizisi ve dil ile dudakların uyguladığı istenmeyen kuvvetler ya da bunların herhangi bir kombinasyonu gibi birçok faktör rol oynar.

③ Biomechanical principles involve the application of a single or couple of forces for correction of rotation. ③ Biyomekanik prensipler, rotasyonun düzeltilmesi için tek veya birkaç kuvvet uygulanmasını içerir.

④ Rotated teeth can be corrected by removable, semifixed or fixed appliances depending upon the severity of the rotation. ④ Dönen dişler, rotasyonun şiddetine bağlı olarak hareketli, yarı sabit veya sabit apareylerle düzeltilebilir.

① The most common clinical symptoms in dystopic teeth are infection and pain. ① Distopik dişlerde en yaygın klinik belirtiler enfeksiyon ve ağrıdır.

② The etiology of this type of developmental anomalies is not known exactly. ② Bu tür gelişimsel anomalilerin etiyolojisi tam olarak bilinmemektedir.

③ - Trauma to the oral area ③ - Ağız bölgesine travma

④ - Dermoid cysts ④ - Dermoid kistler

⑤ - The importance of the presence of extraodontogenic epithelium is pointed out. ⑤ - Ekstraodontojenik epitelyumun varlığının önemi vurgulanmaktadır.

① Transmigration is a phenomenon of movement of an unerupted tooth in the bone across the midline. ① Transmigration, sürmemiş bir dişin kemikte orta hatta doğru hareket etmesi olgusudur.

② This anomaly is not often found. ② Bu anomali sık rastlanmaz.

③ Transmigration is more prevalent in females than in males, and more often encountered in the mandible than maxilla, it affects mostly canines. ③ Transmigration kadınlarda erkeklere göre daha yaygındır ve üst çeneye göre alt çenede daha sık görülür; en çok kaninleri etkiler.

⑥ The reason for the inversion of teeth as documented is unusual proliferation of odontogenic epithelium before the development of the tooth germ. ⑥ Dişlerin inversiyonunun sebebi olarak, diş tomurcuğu gelişiminden önce odontojenik epitelin alışılmadık çoğalması gösterilmiştir.

① Inversion has been defined as "the malposition of a tooth in which the tooth has reversed and is positioned upside down." ① İnversiyon, dişin ters döndüğü ve baş aşağı pozisyonda bulunduğu malpozisyon olarak tanımlanmıştır.

① Ectopic eruption is a disturbance in which the tooth does not follow its usual course. ① Ektopik sürme, dişin normal yolunu takip etmediği bir bozukluktur.

① Tooth transposition; is a disturbance of tooth eruption and is defined as change in the position of two adjacent teeth within the same quadrant. ① Diş transpozisyonu; diş sürmesinde bir bozukluktur ve aynı çeyrekteki bitişik iki dişin pozisyonlarının yer değiştirmesi olarak tanımlanır.

④ Dystopia is the abnormal location of the tooth unit outside the arc. ④ Distopi, diş biriminin diş arkının dışında anormal konumda olmasıdır.

⑤ Pathology is expressed when the tooth erupts in the wrong place or position. ⑤ Diş yanlış yerde veya pozisyonda sürerse patoloji ortaya çıkar.

⑥ It can grow outside the dentition, be tilted or rotated around its own axis. ⑥ Diş, diş dizisinin dışında büyüyebilir, eğilebilir veya kendi ekseni etrafında dönebilir.

⑦ In practice, most often there is a dystopia of wisdom teeth. ⑦ Pratikte en sık görülen distopi, yirmi yaş dişlerindedir.

⑧ A popular reason is the problematic growth of 3rd molar. ⑧ Yaygın bir neden, 3. büyük azı dişinin problemli büyümesidir.

⑨ When there is not enough space in the dentition, they begin to squeeze the “neighbors”. ⑨ Diş dizisinde yeterli alan olmadığında, komşu dişlere baskı yapmaya başlarlar.

⑩ This makes the 2nd molar move. ⑩ Bu, 2. büyük azı dişinin yer değiştirmesine neden olur.

⑪ Also genetic is a main factor. ⑪ Ayrıca genetik önemli bir faktördür.

⑫ A child can inherit from its parents with too narrow a jaw or wide crowns. ⑫ Çocuk, ebeveynlerinden çok dar bir çene veya geniş kronlar kalıtabilir.

① Dentinal dysplasia (DD) should not be confused with dentinogenesis (or sometimes called odontogenesis) imperfecta. ① Dentin displazisi (DD), dentinogenez (ya da bazen odontogenez olarak da adlandırılan) imperfecta ile karıştırılmamalıdır.

② The first differential diagnosis is that DD does not affect the entire dentition. ② İlk ayırıcı tanı olarak DD tüm dişleri etkilemez.

③ In DD the roots are short and conical, hence the name rootless teeth, and the pulp chambers are obliterated by multiple nodules of poorly organized dentine. ③ DD’de kökler kısa ve konik şekildedir, bu nedenle “kökü olmayan dişler” olarak adlandırılır ve pulpa odaları düzensiz dentin nodülleriyle tamamen tıkanmıştır.

④ Usually patients lose these teeth early after eruption. ④ Genellikle hastalar dişlerin sürmesinden sonra erken dönemde bu dişleri kaybederler.

⑤ There are two types of DD: ⑤ İki tip DD vardır:

⑥ - Type 1 involves radicular dentin (rootless teeth) and ⑥ - Tip 1, radiküler dentini (kökü olmayan dişler) içerir ve

⑦ - Type 2 involves coronal dentin and is easily mistaken for dentinogenesis imperfecta. ⑦ - Tip 2, koronal dentini içerir ve kolaylıkla dentinogenez imperfecta ile karıştırılır.

⑧ DD type 2 teeth are similar to dentinogenesis imperfecta teeth, but the coronal pulp chambers contain denticles accompanied by thistle tube appearance of the pulps in the permanent teeth. ⑧ DD tip 2 dişleri dentinogenez imperfecta dişlerine benzer, ancak koronal pulpa odaları dentiküller ve kalıcı dişlerde “thistle tube” (deve dikeni tüpü) görünümü ile karakterizedir.

⑨ However the pulps will be totally obliterated in deciduous teeth, which clinically resemble dentinogenesis imperfecta (amber colored and translucent). ⑨ Ancak, süt dişlerinde pulpa odaları tamamen tıkanır ve klinik olarak dentinogenez imperfecta gibi (kehribar renkli ve şeffaf) görünür.

④ The essential features are: ④ Temel özellikler şunlardır:

⑤ Bone fragility. ⑤ Kemik kırılganlığı.

⑥ Blue sclera. ⑥ Mavi sklera (göz akında mavi renk).

⑦ Progressive hearing loss. ⑦ İlerleyici işitme kaybı.

⑧ Dentinal changes. ⑧ Dentin değişiklikleri.

① Amber, grey to purple-bluish discoloration or opalescence. ① Amber, gri ila morumsu-mavi renkte renk değişikliği veya opalesans.

② Pulpal obliteration. ② Pulpa odasının tıkanması.

③ Relatively bulbous crowns. ③ Görece soğanımsı taç yapısı.

④ Short, narrow roots. ④ Kısa ve dar kökler.

⑤ Enamel may be lost after tooth eruption, exposing the soft dentine, which rapidly wears. This is probably due to inherent weakness in the dentine rather than because of an enamel defect or abnormality at the dentinoenamel junction. ⑤ Diş sürmesinden sonra mine kaybolabilir ve yumuşak dentin açığa çıkar, bu da hızlı aşınmaya neden olur. Bu muhtemelen mine defekti veya dentino-mine birleşimindeki anormallikten ziyade dentinin doğuştan zayıf olmasındandır.

⑥ Mantle dentine appears normal. ⑥ Mantle dentin normal görünür.

⑦ Circumpulpal dentine has poorly formed dentine with abnormal direction of tubules. Small soft-tissue inclusions represent remnants of pulpal tissue. ⑦ Pulpa çevresi dentinde kötü gelişmiş dentin ve tubüllerin anormal yönü bulunur. Küçük yumuşak doku parçaları pulpal doku kalıntılarını temsil eder.

① What is specific to dentinogenesis imperfecta is that all teeth in both dentitions are affected. ① Dentinogenezis imperfektaya özgü olan, hem süt hem de kalıcı dişlerde tüm dişlerin etkilenmesidir.

② That is the greatest differential with the dentinal dysplasia types. ② Bu, dentin displazi tiplerinden en büyük farkıdır.

③ Since the dentin is affected, the adherence between enamel and dentin is compromised and patients will suffer from chipping enamel and hence sensitive teeth, which subsequently wear down quickly too. ③ Dentin etkilendiği için, mine ve dentin arasındaki tutunma bozulur ve hastalar mine çatlaması ve buna bağlı hassasiyet yaşar; bu da dişlerin hızlı aşınmasına yol açar.

④ The teeth have a blue to brown color when they erupt. ④ Dişler sürerken mavi ila kahverengi arasında bir renge sahiptir.

⑤ Pulp chambers may get obliterated, but sometimes they don’t and seem to be expanded, which renders the radiographic appearance typical and is called “shell teeth.” ⑤ Pulpa odaları tıkanabilir, ancak bazen tıkalı olmaz ve genişlemiş gibi görünür; bu da radyografide tipik bir görünüm verir ve buna “kabuk dişler” denir.

Type 2 is isolated opalescent teeth. ① Tip 2, izole (yalnızca) opalesan (sedefimsi) dişlerdir.

② DI type II affects both the primary and the permanent dentition, but the former is usually more severely affected. ② Dentinogenezis imperfekta Tip II, hem süt dişlerini hem de kalıcı dişleri etkiler; ancak genellikle süt dişleri daha ağır etkilenir.

③ The teeth usually have an opalescent hue, ranging from amber brown to grayish-blue. ③ Dişler genellikle kehribar kahverenginden grimsi maviye kadar değişen opalesan bir renge sahiptir.

① Type 1 is associated with osteogenesis imperfecta and teeth appear opalescent. ① Tip 1, osteogenezis imperfekta ile ilişkilidir ve dişler opalesan (sedefimsi) görünür.

② DI type 1 is often not diagnosed accurately as it has a great variety in appearance and expression and therefore osteogenesis imperfecta might be underdiagnosed as well, ② Dentinogenezis imperfekta Tip 1 sıklıkla doğru teşhis edilemez çünkü görünüş ve ifade açısından büyük çeşitlilik gösterir; bu nedenle osteogenezis imperfekta da yeterince tanınmayabilir.

③ as this condition also shows a myriad of expressions (bruising, prolonged bleeding, spraining, fractures, hearing impairment, joint hypermobility, and blue sclerae) ③ Çünkü bu durum aynı zamanda morarma, uzun süren kanama, burkulma, kemik kırıkları, işitme kaybı, eklem aşırı hareketliliği ve mavi sklera gibi çeşitli belirtiler gösterir.

① Hypocalcified type (type 3, in which the enamel is normal in quantity but poorly calcified, which gives it an opaque appearance and makes it weak). ① Hipokalsifiye tipi (tip 3), burada mine miktar olarak normaldir fakat kötü kalsifiye olmuştur; bu durum mineye opak bir görünüm verir ve mineyi zayıf yapar.

② In hypocalcification AI, the enamel is extremely soft, but has the normal thickness, as opposed to the hypoplastic form. ② Hipokalsifikasyon amelogenez imperfectada, mine çok yumuşaktır, ancak kalınlığı normaldir; bu, hipoplazi formunun aksine bir durumdur.

③ However, due to its hypocalcified nature, the enamel is quickly worn and lost after eruption, leaving the remaining enamel to be rough and discolored. ③ Ancak, hipokalsifiye doğası nedeniyle, mine diş sürmesinden sonra hızla aşınır ve kaybolur; kalan mine ise pürüzlü ve renk değiştirmiş olur.

① Hypomaturation type (type 2, in which the form appears normal, but the enamel is soft and vulnerable, and brownish-yellow in color); ① Hipomaturasyon tipi (tip 2), burada mine şekil olarak normal görünür, ancak mine yumuşak ve hassas olup kahverengi-sarımsı renktedir;

② In hypomaturation AI, the enamel appears brown or yellowish due to a defective mechanism during maturation of the enamel matrix, though the enamel has the normal thickness. ② Hipomaturasyon amelogenez imperfectada, mine matriksinin olgunlaşması sırasında mekanizmadaki bozukluk nedeniyle mine kahverengi veya sarımsı görünür, ancak mine kalınlığı normaldir.

③ This type also leads to quick wear and chipping off of the enamel. ③ Bu tip, mine yüzeyinde hızlı aşınma ve çatlama (dökülme) ile sonuçlanır.

① Hypoplastic type (type 1 in which the enamel matrix is inadequately formed, causing pitted, grooved, and thin enamel). ① Hipoplastik tip (tip 1), burada mine matriksi yetersiz oluşur ve bunun sonucu olarak çukurlu, oluklu ve ince mine görülür.

② In hypoplastic AI, the enamel is thin and the interproximal contacts are often missing (spacing between teeth) and the patients also have a tendency to develop an anterior open bite. ② Hipoplastik amelogenez imperfectada, mine incedir ve dişler arası temaslar çoğunlukla yoktur (dişler arasında boşluklar olur) ve hastalarda ön açık kapanış (anterior open bite) gelişme eğilimi de vardır.

① Phenotypes range from markedly hypoplastic (thin) enamel (either uniformly with spacing between adjacent teeth or irregularly giving rise to pits or grooves) to varying degrees of hypomineralization (poorly formed enamel) with altered colour and translucency. ① Fenotipler belirgin hipoplastik (ince) mine yapısından (ya komşu dişler arasında düzgün boşluklarla ya da düzensiz, çukurlar veya oluklar oluşturarak) değişen derecelerde hipomineralizasyona (kötü oluşmuş mine) kadar değişir; renk ve saydamlıkta değişiklikler olur.

② In many cases both hypoplasia and hypomineralization are seen together. ② Birçok vakada hem hipoplazi hem de hipomineralizasyon birlikte görülür.

③ The colour of the teeth is presumed to reflect the degree of hypomineralization of the enamel – the darker the colour the more severe the degree of hypomineralization. ③ Dişlerin rengi, mine hipomineralizasyon derecesini yansıttığı varsayılır – renk ne kadar koyuysa, hipomineralizasyon o kadar şiddetlidir.

Hypoplasia (Hipoplazi) Hypoplasia refers to a defect in the quantity of enamel formed.

Hipoplazi, oluşan mine miktarındaki eksiklik anlamına gelir.

It is caused by a failure or reduced deposition of enamel matrix during tooth development.

Diş gelişimi sırasında mine matriksinin yetersiz veya eksik birikmesi sonucu oluşur.

Clinically, hypoplasia appears as pits, grooves, or a thin enamel layer, often leading to surface contour defects.

Klinikte, hipoplazi çukurlar, oluklar veya ince mine tabakası olarak görülür, diş yüzeyinde şekil bozuklukları oluşturabilir.

Hypomineralization (Hipomineralizasyon) Hypomineralization refers to a defect in the quality of enamel mineralization.

Hipomineralizasyon, mine mineralizasyon kalitesindeki bozukluktur.

The amount of enamel may be normal, but the mineral content and structure are defective, leading to softer, more porous enamel.

Mine miktarı normal olabilir, ancak mineral içeriği ve yapısı bozuk olduğu için mine daha yumuşak ve gözeneklidir.

Clinically, it appears as opacities or discolorations without loss of enamel thickness.

Klinikte, mine kalınlığında azalma olmadan opasiteler (saydamlığın azalması) veya renk değişiklikleri olarak görünür.

① Based on a combination of the mode of inheritance and clinical and radiographic appearances ① Kalıtım biçimi ile klinik ve radyografik görünümün birleşimine dayanarak

① The term amelogenesis imperfecta is usually applied to inherited defects of the enamel of both primary and permanent teeth. ① "Amelogenesis imperfecta" terimi, hem süt hem de daimi dişlerin minelerinde görülen kalıtsal bozukluklara genellikle uygulanır.

② It presents as a change in enamel quality, ranging from localized opacity through opacity with discoloration and obvious poor quality to enamel loss, probably due to post-eruptive breakdown. ② Mine kalitesinde bir değişim şeklinde ortaya çıkar; bu değişim, lokalize opaklıktan renk değişikliğiyle birlikte opaklığa, belirgin düşük kaliteye ve muhtemelen sürme sonrası kırılmaya bağlı mine kaybına kadar değişebilir.

① If Turner's hypoplasia is found on a canine or a premolar, the most likely cause is an infection that was present when the primary (baby) tooth was still in the mouth. ① Turner hipoplazisi bir kanin veya premolar dişte bulunursa, en olası nedeni süt dişi hâlâ ağızdayken mevcut olan bir enfeksiyondur.

② Most likely, the primary tooth was heavily decayed and an area of inflamed tissues around the root of the tooth (called a periapical inflammation), affecting the development of the permanent tooth. ② Büyük olasılıkla, süt dişi ciddi şekilde çürümüştür ve diş kökü etrafındaki dokularda bir iltihaplanma (periapikal inflamasyon) oluşmuş, bu da daimi dişin gelişimini etkilemiştir.

③ The appearance of the abnormality will depend on the severity and longevity of the infection. ③ Anomalinin görünümü, enfeksiyonun şiddetine ve süresine bağlı olarak değişecektir.

① Turner's hypoplasia is an abnormality found in teeth. ① Turner hipoplazisi, dişlerde görülen bir anormalliktir.

② Its appearance is variable, though usually is manifested as a portion of missing or diminished enamel on permanent teeth. ② Görünümü değişkenlik gösterse de genellikle daimi dişlerde mine kaybı veya azalması şeklinde kendini gösterir.

③ Unlike other abnormalities which affect multiple teeth, Turner's hypoplasia usually affects only one tooth in the mouth and, it is referred to as a Turner's tooth. ③ Birden fazla dişi etkileyen diğer anomalilerin aksine, Turner hipoplazisi genellikle ağızda sadece bir dişi etkiler ve bu dişe "Turner dişi" denir.

① A defect in quantity that causes a defect of contour in the surface of the enamel. ① Mine yüzeyinde şekil bozukluğuna neden olan niceliksel bir bozukluktur.

② This is usually caused by initial failure of the deposition of enamel protein, but the same clinical effect could also result if there is a mineralization defect which leads to loss of enamel substance after eruption. ② Bu genellikle mine proteininin ilk birikiminin başarısızlığıyla oluşur, ancak benzer klinik etki, mine süresince meydana gelen bir mineralizasyon bozukluğundan kaynaklı madde kaybı durumunda da oluşabilir.

③ In the former case the enamel is often hard and glassy, in the latter it will usually pit on probing. ③ İlk durumda mine genellikle sert ve cam gibi olurken, ikinci durumda sond ile muayenede çukurlaşma gözlenir.

① Extrinsic stains can be removed with abrasives. ① Dışsal lekeler aşındırıcı maddelerle giderilebilir.

② Mild discoloration may be improved using professionally or home-applied peroxide based bleaching agents. ② Hafif renklenmeler, profesyonelce ya da evde uygulanan peroksit bazlı beyazlatıcı ajanlarla iyileştirilebilir.

③ Intrinsic stains, if superficial, may be removed with microabrasion techniques. ③ İçsel lekeler, yüzeyel olmaları durumunda mikroabrazyon teknikleriyle giderilebilir.

① In its mildest forms, fluorosis is manifest as hypomineralization of the enamel, leading to opacities. ① Florozisin en hafif formlarında, mine hipomineralizasyonu olarak ortaya çıkar ve opasiteler oluşur.

② These can range from tiny white flecks to confluent opacities throughout the enamel, making the crown totally lacking in translucency. ② Bunlar, minede küçük beyaz lekelerden tüm yüzeye yayılmış birleşik opaklıklara kadar değişebilir ve bu durum, kronun tamamen saydamlığını kaybetmesine neden olur.

③ Hypoplasia occurs at higher concentrations of fluoride. ③ Daha yüksek florür konsantrasyonlarında hipoplazi oluşur.

④ When the tooth first erupts, the surface of even the most severely affected enamel may be intact; however, with wear, areas of enamel are lost and stains are taken up into the porosities. ④ Diş ilk sürdüğünde, en ağır şekilde etkilenmiş mine bile yüzey olarak sağlam olabilir; ancak aşınmayla birlikte mine alanları kaybolur ve gözenekli alanlara lekeler yerleşir.

⑤ At 1 ppm of fluoride in public water supplies, up to 10% of the population will show very mild opacities attributable to fluorosis (though this may depend on individual water consumption); ⑤ Halk su kaynaklarında florür seviyesi 1 ppm olduğunda, nüfusun %10’una kadarında florozise bağlı çok hafif opaklıklar görülebilir (bu bireysel su tüketimine bağlı olabilir);

⑥ interestingly, this seems to be a minimum value and the proportion of opacities increases as fluoride levels either fall below 1 ppm or rise above 2 ppm. ⑥ İlginçtir ki bu oran minimum gibi görünür ve florür seviyesi 1 ppm’in altına düştüğünde ya da 2 ppm’in üzerine çıktığında opaklık oranı artar.

⑦ Severely affected cases may require microabrasion or restoration with composite resin, either in a localized or a more generalized manner, or porcelain veneers. ⑦ Ağır şekilde etkilenen olgular, lokal ya da daha yaygın bir şekilde mikroabrazyon, kompozit rezin restorasyonu veya porselen veneer uygulaması gerektirebilir

① Gray or brown in color, these deep, dark stains either cover the entire tooth or appear as a pattern of horizontal stripes. ① Gri veya kahverengi renkte olan bu derin ve koyu lekeler ya tüm dişi kaplar ya da yatay çizgiler şeklinde bir desen olarak ortaya çıkar.

④ Many adults also currently suffer from tetracycline stains on their teeth. ④ Birçok yetişkin de günümüzde dişlerinde tetrasilin lekeleriyle yaşamaktadır.

⑤ Tetracycline teeth stains develop on permanent teeth while they are still forming under the gum line. ⑤ Tetrasilin lekeleri, kalıcı dişler henüz diş eti altında oluşurken gelişir.

⑥ During development, the drug becomes calcified in the tooth, generating tetracycline tooth stains. ⑥ Gelişim sırasında ilaç diş içinde kalsifiye olur ve tetrasilin lekelerinin oluşmasına neden olur.

⑦ Children are susceptible to tetracycline tooth stains from the time they are in utero until the age of 8. ⑦ Çocuklar, rahimde bulundukları zamandan 8 yaşına kadar tetrasilin lekelerine karşı hassastır.

⑧ Since teeth start to develop before we're even born, pregnant women should not take tetracycline to prevent the possibility of the drug affecting the unborn baby's oral health. ⑧ Dişler doğmadan önce gelişmeye başladığı için, hamile kadınlar, ilacın doğmamış bebeğin ağız sağlığını etkileme ihtimaline karşı tetrasilin almamalıdır.

① Tooth discoloration may be extrinsic or intrinsic in nature. ① Diş renginde değişiklik, dışsal veya içsel nitelikte olabilir.

② Extrinsic staining is superficial and occurs after tooth eruption. ② Dışsal lekelenme yüzeyseldir ve diş sürmesinden sonra meydana gelir.

③ Intrinsic discoloration may result from a developmental defect of enamel or internal staining of the tooth. ③ İçsel renk değişikliği, minenin gelişimsel bir defektinden veya dişin iç kısmındaki lekelenmeden kaynaklanabilir.

④ Although such internal staining is manifest as a change in tooth color, the intrinsic defect may affect the dentine primarily or exclusively. ④ Bu iç lekelenme diş renginde değişiklik olarak ortaya çıkmasına rağmen, içsel defekt esas olarak ya da sadece dentini etkileyebilir.

① Opacities result from a defect in the quality of the enamel, affecting the lucency of the tissue. ① Opasiteler, minenin kalitesindeki bir bozukluktan kaynaklanır ve dokunun saydamlığını etkiler.

② Hypomineralization results in a change in the porosity of the enamel, causing opacity. ② Hipomineralizasyon, minenin gözenekliliğinde değişikliğe yol açar ve bu da opasiteye neden olur.

③ This may be located below the enamel surface, which otherwise remains intact. ③ Bu, genellikle minenin yüzeyinin altında yer alır; yüzey ise sağlam kalır.

❻ An enamel pearl is a condition of teeth where enamel is found in locations where enamel is not supposed to be, such as on a root surface. ❻ Mine incisi, minenin bulunmaması gereken yerlerde, örneğin kök yüzeyinde, mine bulunması durumudur.

❼ They are usually found in the area between roots, which is called a furcation, of molars. ❼ Genellikle molar dişlerin kökler arasındaki furkasyon bölgesinde bulunurlar.

❽ Enamel pearls are not common in teeth with a single root. ❽ Tek köklü dişlerde mine incileri yaygın değildir.

❾ The most common location of enamel pearls is the furcation areas of the maxillary (75%) and mandibular third molar roots. ❾ Mine incilerinin en sık bulunduğu yer, maksiller (%75) ve mandibular üçüncü molar köklerinin furkasyon bölgesidir.

❿ Enamel pearls are formed from the Hertwig's Epithelial root sheath. ❿ Mine incileri, Hertwig’in Epitel Kök Kılıfından oluşur.

❸ X-linked vitamin D-resistant rickets (hypophosphataemic rickets). ❸ X'e bağlı vitamin D’ye dirençli raşitizm (hipofosfatemik raşitizm).

❹ Vitamin D-dependent rickets. ❹ Vitamin D’ye bağımlı raşitizm.

❺ Hypophosphatasia. ❺ Hipofosfatazi.

❻ Dentinogenesis imperfecta (some cases). ❻ Dentinogenezis imperfekta (bazı vakalar).

❼ Regional odontodysplasia. ❼ Bölgesel odontodisplazi.

❽ Klinefelter’s syndrome. ❽ Klinefelter sendromu.

❾ Shell teeth. ❾ Kabuk dişler (shell teeth).

❸ The tooth, therefore, has a long body and short roots, with a tendency towards a single root or apical displacement of the furcation. ❸ Bu nedenle dişin uzun bir gövdesi ve kısa kökleri olur; tek kök olma ya da bifurkasyonun apikal yönde yer değiştirme eğilimi vardır.

❶ Taurodontism is an enlargement of a multiradicular tooth’s body and pulp chamber with consequent apical displacement of the pulpal floor and bifurcation of the roots of the tooth (molar teeth with a pulp chamber that is vertically enlarged at the expense of the roots). ❶ Taurodontizm, çok köklü bir dişin gövdesi ve pulpa odasının büyümesi olup, bunun sonucunda pulpa tabanının apikal yönde yer değiştirmesi ve diş köklerinin bifurkasyonu oluşur (köklerin pahasına pulpa odasının dikey olarak genişlemesiyle molar dişlerde görülür).

❸ This tapering or screwdriver-like appearance is important in the differential diagnosis as there are other causes of non-syphilitic notching of the incisal edge (e.g. trauma). ❸ Bu daralma ya da tornavida benzeri görünüm ayırıcı tanıda önemlidir çünkü kesici uçta frengi dışı çentiklenmeye neden olan başka durumlar da vardır (örneğin travma).

❶ Although now very rare in most parts of the world, congenital syphilis presents with several important diagnostic dental manifestations. ❶ Dünyanın çoğu yerinde artık çok nadir görülse de, konjenital frengi birkaç önemli tanısal diş bulgusu ile ortaya çıkar.

❷ Both primary and permanent incisors have tapering crowns and central notching of the incisal edge. ❷ Hem süt hem de kalıcı kesici dişlerin kronları daralan şekildedir ve kesici uçta merkezi bir çentik bulunur.

eLife Assessment

This work describes an inference technique for extracting information about relative contributions of excitatory and inhibitory synaptic drive onto single neurons in neural networks. The electrophysiological techniques and results are of high quality, and the analytical work is novel and potentially powerful, yet with several untested assumptions underlying the approach. This is nevertheless solid work that will be valuable to neuroscience labs interested in exploring alternative approaches to studies of integrated synaptic connectivity.

Reviewer #2 (Public review):

Summary:

By measuring intracellular changes in membrane voltage from a single neuron of the medulla the authors attempted to develop a method for determining the balance of excitatory and inhibitory synaptic drive onto a single neuron.

Strengths:

This data-driven approach to explore neural circuits is described well in this study and could be valuable in identifying microcircuits that generate rhythms. Importantly, perhaps, this inference method could enable microcircuits to be studied without the need for time-consuming anatomical tracing or other more involved electrophysiological techniques. Therefore, I can see the value in developing an approach of this type.

Weaknesses:

The implications of several assumptions associated with this inference technique have been considered by the authors.

Most importantly, it is my understanding that this approach assumes a linear I-V when extracting information about the excitatory and inhibitory synaptic conductances (see equations 6 and 7). In Figure 6, the authors explore the impact of varying the reversal potential for the extraction of information about synaptic drive, but this still assumes that the underlying conductance is linear. However, open rectification will be a feature of any conductance generated by asymmetric distributions of ions (see the GHK current equation) and will therefore be a particular issue for the inhibition resulting from asymmetrical Cl- ion gradients across GABA-A receptors as well as the K+ conductance indirectly activated by GABA-B receptor activation. The mixed cation conductance that underlies most synaptic excitation will also generate a non-linear I-V relationship due to the inward rectification associated with polyamine block of AMPA receptors. The authors present evidence that the I-V relationship is linear over most of the voltage range examined, and this is a helpful addition. The authors have discussed the absence of active conductances contributing to the I-V, but I still wonder how the extraction of information concerning the excitatory and inhibitory conductances relies on the assumption of a linear I-V for these conductances.

This approach has similarities to earlier studies undertaken in the visual cortex that estimated the excitatory and inhibitory synaptic conductance changes that contributed to membrane voltage changes during receptive field stimulation. However, these approaches also involved the recording of transmembrane current changes during visual stimulation that were undertaken in voltage-clamp at various command voltages to estimate the underlying conductance changes. Molkov et al have attempted to essentially deconvolve the underlying conductance changes without this information and I am concerned that this simply may not be possible. However, I appreciate the efforts taken by the authors to address this issue.

The current balance equation (1) cited in this study is based upon the parallel conductance model developed by Hodgkin & Huxley. One key element of the HH equations is the inclusion of an estimate of the capacitive current generated due to the change in voltage across the membrane capacitance. While the present study considers the impact of membrane capacitance, a deeper discussion on how variations in capacitance across different neuron types might affect inference accuracy would be useful. Differences in capacitance could introduce variability in inferred conductances, potentially influencing model predictions.

Studies using acute slicing preparations to examine circuit effects have often been limited to the study of small microcircuits, especially feedforward and feedback interneuron circuits. It is widely accepted that any information gained from this approach will always be compromised by the absence of patterned afferent input from outside the brain region being studied. In this study, descending control from the Pons and the neocortex will not be contributing much to the synaptic drive and ascending information from respiratory muscles will also be absent completely. This may not have been such a major concern if this study had been limited to demonstrating the feasibility of a methodological approach. However, this limitation does need to be considered when using an approach of this type to speculate on the prevalence of specific circuit motifs within the medulla (Figure 4). Therefore, I would argue that some discussion of this limitation should be included in this manuscript.

Author response:

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

Reviewer #1 (Public Review):

Comments on revisions)

The authors have done a good job at revising the manuscript to put this work into the context of earlier work on brainstem central pattern generators.

Thank you.

I still believe the case for the method is not as convincing as it would have been if the method had been validated first on oscillations produced by a known CPG model. Why would the inference of synaptic types from the model CPG voltage oscillations be predetermined? Such inverse problems are quite complicated and their solution is often not unique or sufficiently constrained. Recovering synaptic weights (or CPG parameters) from limited observations of a highly nonlinear system is not warranted (Gutenkunst et al., Universally sloppy parameter sensitivities in systems biology models, PLoS Comp. Biol. 2007; www.doi.org/10.1371/journal.pcbi.0030189) especially when using surrogate biological models like Hodgkin-Huxley models.

The model of the CPG is irrelevant for such a test of validity because what we reconstruct are postsynaptic conductances of an individual neuron. The network creates a periodic input to this neuron and thus forms a periodic pattern of excitatory and inhibitory conductances. The nature of this input, whether autonomously generated or created artificially (say by periodic optogenetic stimulation), is generally not important. To illustrate this, we used a one-compartment conductance-based (Hodgkin-Huxley style) model neuron incorporating a certain common set of channels (fast sodium (I_NaF), potassium delayed rectifier (I_Kdr), persistent sodium (I_NaP), calcium-dependent potassium (I_KCa), and cationic non-specific current (I_CAN)), as well as excitatory and inhibitory synaptic channels whose conductances were implemented as predefined periodic functions. The test suggested by the reviewer would be to implement a current-step protocol similar to the experiments and apply our technique to see if the reconstructed conductance profiles match those predefined functions. Below we show the reconstruction steps for the following arbitrarily chosen pattern:

𝑔_𝐸𝑋𝐶(𝑡) /𝑔_{𝐿𝐸𝐴𝐾} = 0.1(1 + sin(π𝑡)) and 𝑔_𝐼𝑁𝐻(𝑡)/𝑔_{𝐿𝐸𝐴𝐾} = 0.1 (1 + cos(π𝑡)). Author response image 1 below shows the baseline activity of this model neuron in the absence of the injected current.

Author response image 1.

Then we applied a current-step protocol with four steps producing different levels of hyperpolarization and applied our method by calculating the total conductance using linear regression (see the current-voltage plots below) and then decomposing it into the excitatory and inhibitory components.

Author response image 2.

As one can see, the reconstructed conductances in Author response image 3 below are nearly identical to their theoretical profiles. This is not surprising because all voltage-dependent currents in the model neuron were inactive in the range of voltages matching our experimental conditions. Therefore, the model could be reduced to just the leak current, synaptic currents and the injected current, which matches precisely the model we used in our manuscript.

Author response image 3.

In p.2, the edited section refers to the interspike interval being much smaller than the period of the network. More important is to mention the relationship between the decay time of inhibitory synapses and the period of the network.

This interpretation misunderstands the focus of our method. The edited sections (including in the theory section of Results) highlight the conditions under which the capacitive current becomes negligible, emphasizing that the membrane time constant must be much smaller than the network oscillation period. This separation of time scales ensures that the membrane potential adjusts quickly to changes in postsynaptic conductance, rendering the capacitive current insignificant over the network’s rhythm. In contrast, the synaptic decay time governs how presynaptic inputs are transduced into postsynaptic conductances—a process relevant to understanding synaptic dynamics but not directly tied to our method’s core objective. Our approach reconstructs postsynaptic conductances from intracellular recordings, not presynaptic spike trains. While interpreting these conductance profiles in terms of specific synaptic connections would indeed involve synaptic decay dynamics, such an analysis exceeds the scope of our paper. Thus, the condition emphasized in the edited sections—concerning the membrane time constant and network period—is the critical one for our method’s applicability, and the synaptic decay time, while relevant to broader synaptic modeling, does not undermine our conclusions.

We have added the requirement for a much smaller membrane time constant in the Introduction on page 2. The Results theory section already incorporates an extensive discussion of this requirement.

Comments from the editors:

We apologize for the delay in coming to this decision, but there was quite a bit of post-review discussion that needed to be resolved. There are two issues that the reviewers agree should be addressed. They remain unconvinced that the simplifying assumptions of the approach are valid. 1) The main issue with the phase argument is that the biological synaptic conductance depends on time and not on the phase of the respiratory cycle as mentioned in the first round of reviews. The approximation g(t)=g(phase) seems to be far too simple to be biologically realistic.

As we elaborate below, time and phase are fundamentally and mathematically equivalent representations of the same underlying dynamics in a periodic system, and thus, a phase-based representation—where conductances are expressed as functions of the cycle’s phase—is a justified and effective approach for capturing their behavior. We have added this explanation to the theory section of Results. Below are the bases for our assertion.

In a periodic system, such as the respiratory CPG, the system’s behavior repeats at regular intervals, defined by a period T. For the respiratory cycle in our experimental preparation, this period is approximately 3–4 seconds, encompassing phases like inspiration, post-inspiration, and expiration. In such systems:

Time (t) is a continuous variable that progresses linearly.

Phase (φ) represents the position within one cycle, typically normalized between 0 and 1 (or 0 to 2π in some contexts). It can be mathematically related to time via: φ(t) = (t mod T)/T, where (t mod T) is the time elapsed within the current cycle.

Because the system is periodic, any variable that repeats with period T—such as synaptic conductance in a rhythmically active network—can be expressed as a function of either time or phase. Specifically, if g(t) is periodic with period T, then g(t) = g(t+T). This periodicity allows us to redefine g(t) in terms of phase: g(t) = g(φ(t)), where φ(t) maps time onto a repeating cycle. Thus, in a periodic system, time and phase are fundamentally equivalent representations of the same underlying dynamics. Saying that synaptic conductance depends on phase is mathematically equivalent to saying it depends on time in a periodic manner.

In a rhythmically active network like the respiratory central pattern generator (CPG), the synaptic conductances, regardless of the specific mechanisms by which they are formed, exhibit periodicity that matches the network’s oscillatory cycle. This occurs because the conductances are driven by the repetitive activity of presynaptic neurons, which are synchronized to the network’s overall rhythm. As a result, the synaptic conductances vary with the same period as the network, making a phase-based representation—where conductances are expressed as functions of the cycle’s phase—a justified and effective approach for capturing their behavior. In our study, we utilized the in situ arterially perfused brainstem-spinal cord preparation from mature rats, which is known to produce a highly periodic respiratory rhythm. To ensure the consistency of this periodicity, we carefully selected recordings where the coefficient of variation of the respiratory cycle period was less than 10%, as outlined in our methods. This strict selection criterion confirms the stability and regularity of the rhythm, supporting the validity of using a phase representation to analyze the synaptic conductances.

(2) Figure S1 is problematic. First, the currents injected appear to be infinitesimally small.

There was a typo in the current units, which should be nA and not pA, as evident from the injected current–membrane potential plots in Figure 1B. Figure S1 has been corrected.

Second, the input resistance is completely independent of voltage, as though there was little or no contribution from hyperpolarization activated currents, which would be surprising.

While hyperpolarization-activated currents are indeed present in many neuronal types and could theoretically affect input resistance, our data consistently show linear I-V relationships across the voltage range tested (-60 to -100 mV) for the neurons analyzed (see Figure S1 and Author response image 4-9 below). This linearity suggests that, under our experimental conditions, the contribution of voltage-dependent currents, such as h-currents, is negligible within this range.

Additionally, we now indicate in the manuscript in the theory section of Results how the presence of significant hyperpolarization-activated h-currents would impact our synaptic conductance reconstruction method. In current-clamp recordings, non-linearity from h-currents could introduce voltage-dependent changes in total conductance unrelated to synaptic inputs, potentially skewing the reconstruction. However, this concern does not apply to voltage-clamp recordings, where the membrane potential is held constant, eliminating contributions from voltage-dependent intrinsic currents. As strong evidence of the minimal influence of h-currents, we directly compared synaptic conductance reconstructions using both current-clamp and voltage-clamp protocols in a subset of neurons. The results from these two approaches were highly consistent, indicating that h-currents do not significantly affect our findings. This robustness across experimental methods reinforces the reliability of our conclusions.

Together, the linear I-V relationships and the agreement between current- and voltage-clamp reconstructions provide compelling evidence that our method accurately captures synaptic conductances without interference from h-currents.

Typical examples of I-V relationships for each respiratory neuron firing phenotype:

Author response image 4.

ramp-I

Author response image 5.

pre-I/I

Author response image 6.

post-I

Author response image 7.

aug-E

Author response image 8.

early-I

Author response image 9.

late-I

ここから始まる一文が長くて読みづらい？

感じずに、出した

（修飾関係が混乱するので）

PyREPLが説明無く使われている

質問: この「私」は誰？

結局、が、英語っぽい？この流れでは日本語ではあまり使わない気がします。 あと、「新しいREPL」は何に対して新しいのか？と思いました。

代案 見つけたIssue #127960 は、Python 3.13 で強化されたREPLでは、

Bouchra Interessée il ma dit d 'envoyer un mail / je l'ai deja envoyer

preguntas al profe @JuliethDG

Metabolism

Anaerobic Exercise

El titulo no expresa que en este trabajo también se midió el campo magnético terrestre

mas allá de que esta bastante desprolija esta forma de presentar el resultado. Falto un poco de discusión acerca de este último resultado

la numeración de la bibliografía debe ser única, no puede haber dos [1]

esta forma de presentar los resultados es un poco desprolija. Mejor en una tabla o dentro del mismo párrafo

usar el símbolo ASCII ±

la referencia bibliografica no se pone al lado de una ecuación. En este caso, había que ponerla en "...esta dada por [2], " y luego va la ecuación

proteins and exercising

Vegetarian lysine sources

PDCAAS Formula

Second Component of Protein Quality

First Component of Protein Quality

Complete and Incomplete Protein Sources

USDA Recommendation

Animal Based Foods

Sources of Protein

Nitrogen balance

doel: voorkomen van toekomstig herhaald slachtofferschap. handvaten worden gegeven voor hoe om te gaan met specifieke situaties.

doel: 1) kind kan gevoelens en gedachten delen over trauma, 2) ouders reageren hierop helpend, begripvol en positief, en 3) therapeut draagt stokje terug over naar ouders; ouders zijn weer primaire agents of change.

doel: kind leert, door blootstelling aan triggers, dat hij/zij veilig is en dat hij/zij niet bang hoeft te zijn voor bepaalde dingen die hij/zij vermijdt (overcoming maladaptive emotional reactions). daarnaast is het doel dat het kind alle geleerde vaardigheden toepast en leert dat hij/zij de triggers 'aan kan' (mastery).

doel: een tijdlijn maken van alle gebeurtenissen die te maken hebben met/van belang zijn voor het trauma. Door het narratief vervolgens te verwerken kunnen dysfunctionele cognities worden herkend en omgebogen naar helpende cognities. Het verschil met de fase hiervoor (cognitieve coping skills) is dat het hier gaat om cognities rondom het trauma: hoe iemand terugdenkt aan het trauma en denkpatronen die voortkomen uit het trauma.

doel: kinderen leren hoe zij denken over alledaagse dingen/gebeurtenissen en of die gedachten helpend zijn. Vervolgens leren ze niet-helpende gedachten om te buigen naar helpende gedachten. belangrijk is dat het niet gaat over gedachten met betrekking tot het trauma, dit gebeurt pas in de fase daarna. Ouders leren hoe ze denken over het trauma van hun kind en of deze helpend zijn. Vervolgens wordt ouders geleerd hoe ze niet-helpende gedachten kunnen ombuigen.

doel: kinderen leren hun eigen gevoel te begrijpen en herkennen en hoe ze hun gevoelens kunnen uiten op een gezonde manier. ouders leren hoe ze hun eigen gevoelens op een gezonde manier kunnen uiten zodat ze een voorbeeld kunnen zijn voor hun kind.

doel: kind leert dat hij/zij zichzelf kan kalmeren wanneer hij/zij wordt getriggerd. ouders leren hoe ze hun kind daarbij kunnen ondersteunen

doel: ouders leren hoe ze moeten reageren op en omgaan met het trauma van hun kind. Hierdoor helpen ze hun kind, ervaren ze zelf minder stress, verbetert relatie tussen kind en ouders.

doel: kind en ouders informeren

la componente vertical no se mide respecto del norte o sur magnético, no me queda claro que fue lo que quisieron. Deberían haber aclarado mejor en la descripción de la experiencia como fue que ""giraron" la sonda. En estos casos lo mejor es definir un sistema de coordenadas y referir la descripción a ese sistema, sin ambigüedades

Esta un poco largo el resumen

hay que formalizar un poco más el lenguaje. Hay que decir si el ajuste es bueno o malo y por que

Esta discusión es un poco trivial, les recomiendo que vayan directamente al grano. "Dado que en nuestro arreglo experimental tenemos dos solenoides en serie, utilizamos para el ajuste el modelo dado por al ecuación (2)"

veo un error de concepto acá. El norte magnético es diferente al norte geográfico. La brújula informa el norte magnético, por eso les da lo mismo

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public review):

Summary:

The study aims to create a comprehensive repository about the changes in protein abundance and their modification during oocyte maturation in Xenopus laevis.

Strengths:

The results contribute meaningfully to the field.

Weaknesses:

The manuscript could have benefitted from more comprehensive analyses and clearer writing. Nonetheless, the key findings are robust and offer a valuable resource for the scientific community.

We would like to thank the reviewer for his/her positive feedback on our article. The public review points out that "The manuscript could have benefitted from more comprehensive analyses and clearer writing." We have rewritten several sections and provided more detailed explanations of the analysis and interpretation of some data (see below for details). We have also followed all of the reviewer's recommendations, some of which specifically highlighted areas lacking clarity. We would also like to thank the reviewer for pointing out some errors, for which we apologize, and which have now been corrected. We sincerely appreciate the reviewer's thorough work, as it has greatly enhanced the clarity and precision of the manuscript.

Reviewer #2 (Public review):

Summary:

The authors analyzed Xenopus oocytes at different stages of meiosis using quantitative phosphoproteomics. Their advanced methods and analyses revealed changes in protein abundances and phosphorylation states to an unprecedented depth and quantitative detail. In the manuscript they provide an excellent interpretation of these findings putting them in the context of past literature in Xenopus as well as in other model systems.

Strengths:

High quality data, careful and detailed analysis, outstanding interpretation in the context of the large body of the literature.

Weaknesses:

Merely a resource, none of the findings are tested in functional experiments.

I am very impressed by the quality of the data and the careful and detailed interpretation of the findings. In this form the manuscript will be an excellent resource to the cell division community in general, and it presents a very large number of hypotheses that can be tested in future experiments. Xenopus has been and still is a popular and powerful model system that led to critical discoveries around countless cellular processes, including the spindle, nuclear envelope, translational regulation, just to name a few. This also includes a huge body of literature on the cell cycle describing its phosphoregulation. It is indeed somewhat frustrating to see that these earlier studies using phosphomutants and phospho-antibodies were just scratching the surface. The phosphoproteomics analysis presented here reveals much more extensive and much more dynamic changes in phosphorylation states. Thereby, in my opinion, this manuscript opens a completely new chapter in this line of research, setting the stage for more systematic future studies.

We thank the reviewer for his/her extremely positive comments. The public review points out that "none of the findings are tested in functional experiments." This is entirely accurate. We focused our work on obtaining the highest quality proteomic and phosphoproteomic data possible, and then sought to highlight these data by connecting them with existing functional data from the literature. This approach has opened up research avenues with enormous, previously unforeseen potential, in a wide range of biological fields (cell cycle, meiosis, oogenesis, embryonic development, cell biology, cellular physiology, signaling, evolution, etc.). We chose not to delay publication by experimentally investigating the narrow area in which we are specialists (meiotic maturation), while our data offer a vast array of research opportunities across various fields. Our goal was, therefore, to present this extensive dataset as a resource for different scientific communities, who can explore their specific biological questions using our data. This is why we submitted our article to the "Repository" section of eLife. Nevertheless, in the context of the comparative analysis of the mouse and Xenopus phosphoproteomes performed at the reviewer’s request, we felt it was important to complement this new section with functional experiments that not only validate the proteomic data but also provide new insights into certain proteins and their regulation by Cdk1 (new paragraph lines 824-860 and new Figure 9).

We are also grateful to the reviewer for the recommendation to improve the manuscript by including more comparisons between our Xenopus data and those from other systems. We have followed this suggestion (see below), which has significantly enriched the article (new paragraph lines 824-860 and new Figure 9).

Reviewer #3 (Public review):

Summary:

The authors performed time-resolved proteomics and phospho-proteomics in Xenopus oocytes from prophase I through the MII arrest of the unfertilized egg. The data contains protein abundance and phosphorylation sites of a large number set of proteins at different stages of oocyte maturation. The large sets of the data are of high quality. In addition, the authors discussed several key pathways critical for the maturation. The data is very useful for the researchers not only researchers in Xenopus oocytes but also those in oocyte biology in other organisms.

Strengths:

The data of proteomics and phospho-proteomics in Xenopus oocyte maturation is very useful for future studies to understand molecular networks in oocyte maturation.

Weaknesses:

Although the authors offered molecular pathways of the phosphorylation in the translation, protein degradation, cell cycle regulation, and chromosome segregation. The author did not check the validity of the molecular pathways based on their proteomic data by the experimentation.

We thank the reviewer for his/her positive comments. The public review points out that "The author did not check the validity of the molecular pathways based on their proteomic data by the experimentation." This is entirely accurate. We focused our work on obtaining the highest quality proteomic and phosphoproteomic data possible, and then sought to highlight these data by connecting them with existing functional data from the literature. This approach has opened up research avenues with enormous, previously unforeseen potential, in a wide range of biological fields (cell cycle, meiosis, oogenesis, embryonic development, cell biology, cellular physiology, signaling, evolution, etc.). We chose not to delay publication by experimentally investigating the very narrow area in which we are specialists (meiotic maturation), while our data offer a vast array of research opportunities across various fields. Our goal was, therefore, to present this extensive dataset as a resource for different scientific communities, who can explore their specific biological questions using our data. This is why we submitted our article to the "Repository" section of eLife. Nevertheless, in the context of the comparative analysis of the mouse and Xenopus phosphoproteomes performed at the reviewer’s request, we felt it was important to complement this new section with functional experiments that not only validate the proteomic data but also provide new insights into certain proteins and their regulation by Cdk1 (new paragraph lines 824-860 and new Figure 9).

We have also followed all of the reviewer's recommendations and thank him/her, as the suggestions have significantly enhanced the manuscript.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

(1) Fig. 1 -> In the Figure legend "mPRβ" is called "mPRb". In the Figure, it is indicated that PKA substrates are always activated by the phosphorylation. As the relevant substrates and the mode-of-action of the Arpp19 phosphorylation are not clear at the moment, this seems to be preliminary. It could for example also be conceivable that PKA phosphorylation inhibits a translation activator. In addition, the PG-dependent translation of RINGO/Speedy should be included in the model.

We fully agree with the reviewer. PKA substrates can either be activators of the Cdk1 activation pathway, which are inhibited by phosphorylation by PKA, or repressors of the same pathway, which are activated by phosphorylation by PKA. This is now illustrated in the new Fig. 1. In addition, we have also included RINGO/Speedy in the model and in the text (lines 78-79) and corrected "mPRb" in the legend.

(2) Lane 51-52 -> it is questionable if the meiotic divisions can be called "embryonic processes"

We agree with the reviewer comment, and we have removed the word “embryonic”.

(3) Lane 53 and lane 106-107 -> recent data have indicated that transcription already starts during cell cycle 12 and 13 in most cells (e.g. Blitz and Cho: Control of zygotic genome activation in Xenopus (2021))

We apologize for this mistake. The text has been corrected and the reference added (lines 53 and 107).

(4) Lane 61-62 -> "MI" and "MII" are given as abbreviation for "first and second meiotic spindle"

The text has been clarified to explain that MI is referred to metaphase I and MII stands for metaphase II (lines 61-64).

(%) Lane 131-132 -> "single-cell" is mentioned redundantly in this sentence.

The sentence has been corrected (lines 131-132).

(6) Fig. 2B -> it is not explained what is plotted as "Average levels" on the x-Axis. Is it the average of expression over all samples or at a given time point? Are the values given as a concentration or are the values normalized? If so, how were they normalized?

We agree with the reviewer comment that “Average levels” may have been unclear. In the new Fig. 2B, we have re-plotted the graph using the average protein concentration during meiosis, measured as described in the Methods section.

(7) In Fig. 2-supplement 3E -> from the descriptions it is not entirely clear to me what the difference to the data in Fig. 2B is?

We thank the reviewer for his/her question regarding the relationship between the data in Fig. 2B and Fig. 2-supplement 3E. We confirm that the raw data visualized in Fig. 2-supplement 3E are the same as those in Fig. 2B. However, in Fig. 2-supplement 3E, the data are color-coded differently to highlight the number of proteins whose concentrations change during meiotic divisions, based on the threshold adopted. The legend of Fig. 2-supplement 3E has been modified to clarify this point.

(8) Lane 225-226 -> Kifc1 is a minus-end directed motor

This mistake has been corrected (lines 232-233).

(9) Lane 271 -> Serbp1, here mentioned to be involved in stabilization of mRNAs, has also been implicated in the regulation of ribosomes (e.g. Leesch et al. 2023). Regarding the overall topic of this manuscript, this could be mentioned as well.

We agree with the referee that the important role of Serbp1 in the control of ribosome hibernation needs to be mentioned. We have included this point in the revised manuscript together with the reference (lines 277-279).

(10) Lane 360-363 -> it is mentioned that APPL1 and Akt2 act "to induce meiosis". Furthermore, in the Nader et al. 2020 paper, Akt2 phosphorylation is reported to happen within 30min after PG treatment. In the present work, they only seem to get phosphorylated when Cdk1 is activated. Is there an explanation for this discrepancy?

Indeed, Nader et al. (2020) indicate that Akt2 is phosphorylated on Ser473 (actually, they should have mentioned Ser474, which is the phosphorylated residue on Akt2; Ser473 corresponds to the numbering of Akt1) between 5 and 30 minutes post-Pg, which supports their hypothesis of an early role for this kinase. However, these conclusions should be taken with caution, considering that their functional experiment using antisense against Akt2 depletes only 25% of the protein, the antibody used to visualize Akt2 phosphorylation also recognizes phosphorylated Akt1 and Akt3, and they did not analyze phosphorylation of the protein after 30 minutes. Therefore, we cannot determine whether the level observed at 30 minutes represents a maximum or if it is just the onset of the phosphorylation that peaks later, possibly after activation of Cdk1, for example.

Regarding our measurements: we clearly observe phosphorylation of Akt2 following Cdk1 activation on Ser131. We did not detect Akt2 phosphorylation on Ser474, but since our measurements started 1 hour post-Pg, this protein may have returned to a dephosphorylated state on Ser474.

Therefore, the observations of Nader et al. and ours involve different residues and different phosphorylation kinetics, Nader et al. limiting their analysis to the first 30 minutes, whereas we started at 1 hour.

We have revised the manuscript text to make these aspects clearer (lines 387-392).

(11) Fig. 3B -> it could be made clearer in the Figure that all these sites belong to class I

A title “Class I proteins” has been added in Fig. 3B to clarify it.

(12) Lane 433-434 -> the authors write that the proteomic data of this study confirm that PATL1 is accumulating during meiotic maturation. However, in Fig. 2B PATL1 is not among the significantly enriched proteins.

We apologize for this error. Indeed, PATL1 protein is not significantly enriched. The text has been corrected (lines 461-465).

(13) Fig. 4B -> Zar2 is color-coded to increase in abundance. This is clearly different to published results and what is shown in Fig. 2B of this manuscript.

Indeed, our dataset shows that the quantity of Zar2 decreases. This does not appear anymore in Figure 2B since Zar2 average concentration cannot be estimated. We made an error in the color coding, which has now been corrected in Figure 4B.

(14) Lane 442-444 -> it might be worth mentioning that the interaction between CPEB1 and Maskin, and thus probably its role in regulation of translation, could not be reproduced in other studies (Minshall et al.: CPEB interacts with an ovary-specific eIF4E and 4E-T in early Xenopus oocytes (2007) or Duran-Arque et al.: Comparative analyses of vertebrate CPEB proteins define two subfamilies with coordinated yet distinct functions in post-transcriptional gene regulation (2022)).

This clarification is now mentioned in the text, supported by the two references that have been added (lines 471-477).

(15) Lane 483-485 -> The meaning of these sentences is not entirely clear to me. What exactly is the similarity with the function of Emi1? What does "...binding of Cyclin B1..." mean (binding to which other protein?). What is the similarity between Emi1 and CPEB1/BTG4, both of which are regulators of mRNA stability/polyadenylation?

We apologize if these sentences were unclear. Our intention was to emphasize the central role of ubiquitin ligases in regulating multiple events during meiotic divisions. We used SCF^βTrCP, a wellstudied ubiquitin ligase in Xenopus and mouse oocytes during meiosis, as an example. SCF^βTrCP regulates the degradation of several substrates, including Emi1, Emi2, CPEB1, and Btg4, whose degradation or stabilization is essential for the proper progression of meiosis. Lastly, we highlighted that these regulatory processes, mediated by protein degradation, may be conserved in mitosis, as for example the destruction of Emi1. We have rewritten this paragraph for clarity (lines 513-518).

(16) Lane 521-522 and 572-573 -> the authors write that Myt1 was not detected in their proteome. However, in Fig. 6A they list "pkmyt1" as a class II protein. On Xenbase, "pkmyt1" is the Cdk1 kinase, "Myt1" is a transcription factor, so the authors might have been looking for the wrong protein.

We thank the reviewer for this accurate observation. We have modified the text to correct this error (lines 554 and 607).

(17) Lane 564-565 -> The authors state that Cdk1 activity can be measured by analyzing Cdc27 S428 phosphorylation. However, in vivo the net phosphorylation of a site is always depending on the relevant kinase and phosphatase activities. As S428 is a Cdk1 site, it is not unlikely that it is dephosphorylated by PP2A-B55, which by itself is under the control of Cdk1. Do the authors have direct evidence that the change in phosphorylation of S428 can only be attributed to the changes in Cdk1 activity?

There is evidence in the literature that Cdc27 is dephosphorylated by PP2A (Torres et al., 2010). In Xenopus oocytes, PP2A activity is high during prophase (Lemonnier et al., 2021) and decreases at the time of Cdk1 activation, mediated by the Greatwall-ENSA/Arpp19 system, remaining low until MII (Labbé et al., 2021). Therefore, the period where fluctuations in Cdk1 activity are difficult to assess, from NEBD to MII, corresponds to a phase of inhibited PP2A activity. As a result, the phosphorylation level of Cdc27 reflects primarily the activity of Cdk1. We have added this clarification in the text (lines 597-600).

(18) Fig. 7C and 7D -> in 7C, for Nup35/Nup53 there is a phospho-peptide GIMEVRS(60)PPLHSGG. In Fig. 7D phosphorylation of GVMEMRS(59)PLFSGG is analyzed. Is this the same phosphosite/region of Nup35/Nup53? How can there be a slightly different version of the same peptide in one protein? Are these the L- and S-version of Nup35/Nup53? It is also very surprising that the two phosphosites belong to different classes, class III and class II, respectively.

We thank the reviewer for this observation. The peptides GIMEVRS(60)PPLHSGG and GVMEMRS(59)PLFSGG correspond to the same phosphorylation site in the L and S versions of Xenopus laevis Nup35, respectively. The L version peptide was classified as Class III, while the S version was not assigned to any class due to its high phosphorylation level in prophase, which prevented it from meeting the log₂ fold-change threshold of 1 required by our analysis to detect significant differences.

(19) Table 1 -> second last column is headed "Whur, 2014"

The typo has been corrected.

(20) Fig. 8 -> Why are all the traces starting at t=1h after PG?

The labeling of the graphs in Fig. 8 has been corrected, and the traces now begin at t0.

(21) Lane 754 -> Although a minority, there are also some minus-end directed kinesins, e.g. Kifc1

We agree with the reviewer. We should have mentioned that, in addition to dyneins, some kinesins are minus-end directed motors, especially since one of them, Kifc1, is regulated at the level of its accumulation. We have rephrased the relevant sentences to incorporate this observation (lines 790-793).

(22) Section "Assembly of microtubule spindles and microtubule dynamics" -> Although this section clearly has a strong focus on phosphorylation, it might be worth mentioning again that many regulators of the microtubule spindle, e.g. TXP2, are among the upregulated proteins in Fig. 2B/C

We have already discussed that the protein levels of certain key regulators of the mitotic spindle (Tpx2, PRC1, SSX2IP, Kif11/Eg5 among others) are subject to control during meiotic maturation in a previous chapter “Protein accumulation: the machinery of cell division and DNA replication” (lines 230-239). We agree with the reviewer that this important observation can be mentioned again at the beginning of this chapter on phosphorylation control. We have added a sentence regarding this at the start of the paragraph (lines 774-775).

Reviewer #2 (Recommendations for the authors):

While I find the manuscript excellent and detailed already in its current form, I would appreciate including even more comparisons to other systems. In particular, a similar phosphoproteomics experiment has been performed in starfish oocytes undergoing meiosis (Swartz et al, eLife, 2021), and there are several studies on mitosis of diverse mammalian cells. It would be very exciting to see to what extent changes are conserved.

We thank the reviewer for this recommendation, which we have attempted to follow. We have matched our dataset of mass spectrometry using the the phosphor-occupancy_matlab package, available as part of our code repository (https://github.com/elizabeth-van-itallie) previously described in (Van Itallie et al, 2025). Unfortunately, we were unable to match our dataset with the data from Swartz et al. (2021) on starfish oocyte due to the low sequence conservation. However, we have compared our dataset with the dataset from Sun et al. (2024) on mouse oocyte maturation. We identified a total of 408 conserved phosphorylation sites, which mapped to 320 proteins in Xenopus and 277 in mice (refer to a new paragraph: lines 824-860, new Figure 9, Methods: lines 1011-1032 and 1060-1065, and Appendix 7). The phosphorylation patterns during meiosis showed a significant crossspecies correlation (Pearson r = 0.39, p < 0.0001; see new Figure 9A), demonstrating the evolutionary conservation of phosphoproteomic regulation. Important phosphorylation events, including Plk1 at T201, Gwl at S467, and Erk2 at T188, were upregulated in both species, in line with the activation of the Cdk1 and MAPK signaling cascades (Figure 6B, new Figure 9A-B). We validated several of these phosphorylation sites by western blotting and demonstrated their dependency on Cdk1 activation (new Figure 9C). Together, these findings reinforce the notion that fundamental phospho-regulatory pathways are conserved during oocyte maturation in vertebrates.

Reviewer #3 (Recommendations for the authors):

(1) Page 6, the first paragraph of Results section: Please describe the method on how the authors measured and quantified the proteomes in different stages of Xenopus oocyte maturation briefly. Without the experimental design, it is very hard to evaluate the results in the following paragraphs.

As requested by the reviewer, we added a few sentences describing the method of proteomics and phosphoproteomics measurements in oocytes resuming meiosis (lines 151-158).

(2) In the phospho-proteome, it is better to classify the amino acids for the phosphorylation such as Ser, Thr, and Tyr. Particularly how many tyrosine phosphorylations are in the list.

Our phosphosites dataset contains 80% Ser, 19.9% Thr, and 0.01% Tyr. Phospho-Tyr are slightly less abundant than what has been described in the literature (in most cells “roughly 85-90% of protein phosphorylation happens on Ser, ~10% on Thr, and less than 0.05% on Tyr" after Sharma et al., 2014. The same observation was made regarding the distribution of phosphorylated amino acids in mouse oocytes, where phospho-Tyr abundance is relatively diminished in oocytes compared to mouse organs (Sun et al., 2024). These observations are now reported in the manuscript (lines 309-313).

(3) In class II (Figure 3), when Cdk1 (line 326) is a major kinase, how many phosphorylation sites are a target of Cdk1 (with the Cdk1-motif)? Moreover, do the authors find any other consensus sequences for the phosphorylation? Those are either known or unknown. This information would be useful for the readers.

We thank the reviewer for this valuable comment. To address it, we used the kinase prediction server (https://kinase-library.phosphosite.org/kinase-library/score-site) to analyze Class II phosphosites. These new results are mentioned in lines 340-349 and illustrated in a new Figure (Figure 3—figure supplement 1A). We identified 303 sites predicted to be phosphorylated by Cdk1. Of these, 166 were also predicted as Erk1/2 targets, reflecting the similarity between Cdk1 and Erk1/2 consensus motifs.

Cdk1 substrate phosphorylation is governed by more than just the presence of a consensus sequence. In addition to its preference for the (S/T)P×(K/R) motif, Cdk1/cyclin complexes achieve specificity through docking interactions with short linear motifs (SLiMs) recognized by the cyclin subunit (as LxF motifs)(Loog & Morgan, 2005), and via the Cdk-binding subunits Cks1 or Cks2, which interact with phosphorylated threonine residues in primed substrates (Örd et al, 2019). These mechanisms promote processive multisite phosphorylation and allow Cdk1 to target substrates even at non-canonical sites. Our motif-based analysis captures only part of this complexity and may underestimate the number of true Cdk1 targets.

To further explore kinase involvement across phosphosite classes, we extended the analysis to all clusters and identified the most enriched kinase predictions for each (lines 360-365, new Figure 3— figure supplement 1B). In Class II, the most enriched kinases included Cdk1, Erk2, and Plk1, supporting the conclusions derived from the identification of the phosphosites of this Class. But others such as Cdk2, Cdk3, Cdk5, Cdk16, KIS, JNK1, and JNK3 were also identified.

(4) Figure 3B: Why do the authors show this kind of Table only for Class I, not Classes II-V? It would be informative to show candidate proteins in other classes.

We chose to present the candidate proteins from Class I in a table format because the number of phosphosites (136) was too small to allow a meaningful Gene Ontology (GO) enrichment analysis. Therefore, we manually curated the data and highlighted proteins whose Class I phosphosites are associated with specific biological processes. For Classes II–V, the higher number of phosphosites allowed us to perform GO enrichment analyses. Since several of the enriched processes were shared across different classes, and some proteins have phosphosites in multiple classes, we opted to organize the results by biological processes rather than by class. We agree with the reviewer that it is indeed valuable to highlight interesting proteins with Class II–V phosphosites. We have done so in Figures 4 through 8, using graphical representations instead of tables, in order to make the data more accessible and avoid long tables. Additionally, the Supplementary Figures provide detailed phosphorylation trends for many of the proteins discussed in the main figures.

(5) It would be nice if the authors compare this phospho-proteome in Xenopus oocyte maturation with that in mouse oocyte maturation (Sun et al. 2024) in terms of evolutional conservation of the phospho-proteomes.

We thank the reviewer for this suggestion. As now detailed in the manuscript, we compared our Xenopus phosphoproteome with the dataset from Sun et al. (2024) on mouse oocyte maturation using the the phospho_occupancy_matlab package, available as part of our code repository (https://github.com/elizabeth-van-itallie) previously described in (Van Itallie et al, 2025). We identified 408 conserved phosphorylation sites corresponding to 320 Xenopus and 277 mouse proteins (see new paragraph: lines 824-860, new Figure 9, Methods: lines 1011-1032 and 1060-1065, and Appendix 7). Phosphorylation dynamics across meiosis were significantly correlated between the species (Pearson r = 0.39, p < 0.0001; new Figure 9A), highlighting evolutionary conservation of the phosphoproteomes. Key phosphorylation events such as Plk1 at T201, Gwl at S467, and Erk2 at T188 increased in both species, consistent with activation of the Cdk1 and MAPK pathways (Figure 6B, new Figure 9A–B). We validated experimentally several of these phosphorylation sites by western blot (Erk2, Plk1, Fak1 and Akts1) and demonstrated their dependency on Cdk1 activation (new Figure 9C). Together, these new findings support the conservation of key phospho-regulatory mechanisms across vertebrate oocyte maturation.

Minor points:

(1) Reference lists: Please add Sun et al (2024) shown in line 115.

This important reference has been added (lines 115, 134, 313 and 826).

(2) Figure 1, red arrows for the inhibition: This should be "T" shape for a better understanding of these complicated pathways.

We agree with the reviewer’s remark, and we have modified Figure 1.

(3) Line 236-238: The authors referred to the absence of Cdc6 in oocyte maturation in Xenopus. However, Figure 2C shows that Cdc6 belongs to a list of accumulating proteins with Orc1 and Ocr2 etc. and the authors did not discuss this discrepancy in the text. Please clarity the claim.

We apologize for the unclear wording in our text. The section of the manuscript regarding the pre-RC components may have been misleading. The text has been revised to clarify that Cdc6 was not detected in prophase-arrested oocytes by western blot and that it accumulates during meiotic maturation after MI, enabling oocytes to replicate DNA (lines 243-250).

(4) Line 306: Please add the link to phosphosite.org.

The link has been added (line 319).

eLife Assessment

This important paper describes a comprehensive quantitative phospho-proteomic analysis of the meiotic progression of Xenopus oocytes. Using time-resolved proteomic analyses, the authors provide insights into changes in protein levels and phosphorylation states to an unprecedented depth, quality, and quantitative detail. The key findings are compelling and offer a helpful resource for the scientific community.

Reviewer #1 (Public review):

In the revised version of the manuscript, the authors have adequately addressed all our concerns. The authors should spell check their manuscript, e.g., correct phosphor-site to phospho-site etc.

Summary:

The study aims to create a comprehensive repository about the changes in protein abundance and their modification during oocyte maturation in Xenopus laevis.

Reviewer #2 (Public review):

Summary:

The authors analyzed Xenopus oocytes at different stages of meiosis using quantitative phosphoproteomics. Their advanced methods and analyses revealed changes in protein abundances and phosphorylation states to an unprecedented depth and quantitative detail. In the manuscript they provide an excellent interpretation of these findings putting them in the context of past literature in Xenopus as well as in other model systems. The clarity of these explanations improved significantly in the revised version of the manuscript, and several minor imprecisions have been corrected as well.

Strengths:

High-quality data, careful and detailed analysis, and outstanding interpretation in the context of the large body of literature.

Weaknesses:

Merely a resource, none of the findings are tested in functional experiments.

I am very impressed by the quality of the data and the careful and detailed interpretation of the findings. In this form, the manuscript will be an excellent resource to the cell division community in general, and it presents a very large number of hypotheses that can be tested in future experiments. Xenopus has been and still is a popular and powerful model system that led to critical discoveries around countless cellular processes, including the spindle, nuclear envelope, and translational regulation, just to name a few. This also includes a huge body of literature on the cell cycle describing its phosphoregulation. It is indeed somewhat frustrating to see that these earlier studies using phospho-mutants and phospho-antibodies were just scratching the surface. The phosphoproteomics analysis presented here reveals much more extensive and much more dynamic changes in phosphorylation states. Thereby, in my opinion, this manuscript opens a completely new chapter in this line of research, setting the stage for more systematic future studies.

Reviewer #3 (Public review):

Summary:

The authors performed time-resolved proteomics and phospho-proteomics in Xenopus oocytes from prophase I through the MII arrest of the unfertilized egg. The data contains protein abundance and phosphorylation sites of a large number set of proteins at different stages of oocyte maturation. The large sets of data are of high quality. In addition, the authors discussed several key pathways critical for the maturation. The data is very useful for researchers, not only researchers in Xenopus oocytes but also those in oocyte biology in other organisms.

Strengths:

The data of proteomics and phospho-proteomics in Xenopus oocyte maturation is very useful for future studies to understand molecular networks in oocyte maturation.

Weaknesses:

Although the authors offered molecular pathways of the phosphorylation in translation, protein degradation, cell cycle regulation, and chromosome segregation. The authors did not check the validity of the molecular pathways based on their proteomic data by experimentation. But this is not essential since this is a resource paper.

Healing Process

Most abundant protein in blood

This should be plot_trees

Step 5 of Digestion

Step 4 of Digestion

Step 3 of Digestion

Step 2 of Digestion

Step 1 of Digestion

Protein Digestion

Amino Acids are like Music

Titin Amino Acid

Essential v. Nonessential Amino Acids

Difference between Proteins and Carbs/Lipids

Functions of a Protein

ეს ფაქტი კარგად აჩვენებს მონაწილეობის შედეგს, ბლოგი ზუსტად ასახავს საჯარო პოლიტიკის განხორციელების პრაქტიკულ მაგალითს მთის რეგიონში.

Semi-Vegetarianism

Bouchra sent email

Neue Notation für Einheitengruppe

Understand Flutter state management using Provider and BLoC. Learn how to build scalable and maintainable apps with efficient state handling strategies.

❶ If diagnosed early, an elective (Cvek) pulpotomy can be performed in an attempt to allow normal root formation. ❶ Erken teşhis edilirse, normal kök oluşumuna izin vermek amacıyla seçmeli (Cvek) pulpotomi uygulanabilir.

❶ An enamel-covered tubercle usually projecting from the occlusal surface of a premolar tooth. ❶ Genellikle bir premolar dişin oklüzal yüzeyinden çıkan, mineyle kaplı bir tüberküldür.

❷ It is a cusp-like elevation of enamel located in the central groove or lingual ridge of the buccal cusp of a premolar or permanent molar. ❷ Bu, bir premolar ya da sürekli molar dişin bukkal cuspunun lingual çıkıntısında ya da merkezi olukta yer alan, cusp benzeri bir mine çıkıntısıdır.

❸ It is usually bilateral and more common in the mandible. ❸ Genellikle bilateraldir ve mandibulada daha sık görülür.

❹ It occurs mostly in mandibular premolars and consists of enamel, dentin, and pulp. ❹ En çok mandibular premolarlarda görülür ve mine, dentin ile pulpadan oluşur.

❺ There is evidence of pulp tissue within the tubercle in 43% of cases. ❺ Vakaların %43’ünde tüberkül içinde pulpa dokusu bulunduğuna dair kanıt vardır.

❻ The latter means that attrition may cause pulpal exposure and hence periapical infection can develop. ❻ Bu durum, atrizyonun pulpa açılmasına neden olabileceği ve dolayısıyla periapikal enfeksiyon gelişebileceği anlamına gelir.

❼ One can also find it in shovel-shaped incisors. ❼ Bu oluşuma kürek şeklindeki kesici dişlerde de rastlanabilir.

❽ Radiographs may show occlusal extension of the pulp chamber. ❽ Radyograflarda, pulpa odasının oklüzal yönde uzandığı görülebilir.

❺ Pulp necrosis often occurs soon after the eruption of the affected tooth and may lead to a canine fossa abscess or cellulitis. ❺ Etkilenen dişin sürmesinden kısa bir süre sonra genellikle pulpa nekrozu meydana gelir ve bu durum kanin fossa apsesine veya selülite yol açabilir.

❻ This anomaly may occur in other teeth such as the maxillary central incisors and canines. ❻ Bu anomali, maksiller santral kesiciler ve kaninler gibi diğer dişlerde de görülebilir.

❶ Dens invaginatus is a deep enamel-lined invagination in the crown and/or root of a tooth. ❶ Dens invaginatus, dişin kron ve/veya kök kısmında derin, mine ile kaplı bir invajinasyondur (içe doğru girinti).

❶ Dilaceration is a disturbance in tooth formation that produces a sharp bend or curve in the tooth anywhere in the crown or the root. ❶ Dilaserasyon, diş oluşumu sırasında meydana gelen ve dişin taç (kron) ya da kök kısmında keskin bir eğrilik ya da bükülme ile karakterize bir bozukluktur.

❷ Although this anomaly is likely developmental in nature, one of the oldest concepts is that dilaceration is the result of mechanical trauma to the calcified portion of a partially formed tooth. ❷ Bu anomali büyük olasılıkla gelişimsel kaynaklı olmasına rağmen, en eski görüşlerden biri dilaserasyonun, kısmen oluşmuş bir dişin kalsifiye olmuş (sertleşmiş) kısmına gelen mekanik travma sonucu oluştuğudur.

❶ Dilaceration is a disturbance in tooth formation that produces a sharp bend or curve in the tooth anywhere in the crown or the root. ❶ Dilaserasyon, diş oluşumu sırasında meydana gelen ve dişin taç (kron) ya da kök kısmında keskin bir eğrilik ya da bükülme ile karakterize bir bozukluktur.

❷ Although this anomaly is likely developmental in nature, one of the oldest concepts is that dilaceration is the result of mechanical trauma to the calcified portion of a partially formed tooth. ❷ Bu anomali büyük olasılıkla gelişimsel kaynaklı olmasına rağmen, en eski görüşlerden biri dilaserasyonun, kısmen oluşmuş bir dişin kalsifiye olmuş (sertleşmiş) kısmına gelen mekanik travma sonucu oluştuğudur.

❶ A paramolar cusp or tubercle is a supernumerary cusp occurring on the buccal or lingual surface of upper or lower premolars and molars. ❶ Paramolar cusp veya tüberkül, üst veya alt premolar ve molarların bukkal ya da lingual yüzeyinde görülen süpernümerer (ek) bir tüberküldür.

❷ A paramolar cusp occurring on the buccal surface of the maxillary molars is specifically termed as a parastyle. ❷ Maksiller molarların bukkal yüzeyinde görülen paramolar cusp, özel olarak "parastyle" olarak adlandırılır.

⓴ Composite resin or (eventually) porcelain veneers to improve shape. ⓴ Şekli düzeltmek için kompozit rezin veya (zamanla) porselen laminalar kullanılabilir.

⓴ The profile of the tooth is narrower at the gingival margin than a normal-sized tooth, and there is therefore a limit to how large the tooth can be made without producing an overhang in the gingival region or an unsightly interdental shadow. ⓴ Dişin profili, normal boyuttaki bir dişe göre diş eti kenarında daha dardır; bu nedenle diş, diş eti bölgesinde taşkınlık veya istenmeyen bir diş aralığı gölgesi oluşturmadan ancak belirli bir büyüklüğe kadar yapılabilir

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Nassim - 18/06 - Pas du tout interéssé

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と思ったけど、私のも目を閉じてるな..

eLife Assessment

The authors quantified intentions and knowledge gaps in scientists' use of sex as a biological variable in their work, and used a workshop intervention to show that while willingness was high, pressure points centered on statistical knowledge and perceived additional monetary costs to research. These important findings demonstrate the difficulty in changing understanding - while interventions can improve knowledge and decrease perceived barriers, the impact was small. The evidence was solid, although the sample size was small for the intervention.

Reviewer #1 (Public review):

Summary:

The authors use the theory of planned behavior to understand whether or not intentions to use sex as a biological variable (SABV), as well as attitude (value), subjective norm (social pressure), and behavioral control (ability to conduct behavior), across scientists at a pharmacological conference. They also used an intervention (workshop) to determine the value of this workshop in changing perceptions and misconceptions. Attempts to understand the knowledge gaps were made.

Strengths:

The use of SABV is limited in terms of researchers using sex in the analysis as a variable of interest in the models (and not a variable to control). To understand how we can improve on the number of researchers examining the data with sex in the analyses, it is vital we understand the pressure points that researchers consider in their work. The authors identify likely culprits in their analyses. The authors also test an intervention (workshop) to address the main bias or impediments for researchers' use of sex in their analyses.

Weaknesses:

There are a number of assumptions the authors make that could be revisited:

(1) that all studies should contain across sex analyses or investigations. It is important to acknowledge that part of the impetus for SABV is to gain more scientific knowledge on females. This will require within sex analyses and dedicated research to uncover how unique characteristics for females can influence physiology and health outcomes. This will only be achieved with the use of female-only studies. The overemphasis on investigations of sex influences limits the work done for women's health, for example, as within-sex analyses are equally important.

(2) It should be acknowledged that although the variability within each sex is not different on a number of characteristics (as indicated by meta-analyses in rats and mice), this was not done on all variables, and behavioral variables were not included. In addition, across-sex variability may very well be different, which, in turn, would result in statistical sex significance. In addition, on some measures, there are sex differences in variability, as human males have more variability in grey matter volume than females. PMID: 33044802.

(3) The authors need to acknowledge that it can be important that the sample size is increased when examining more than one sex. If the sample size is too low for biological research, it will not be possible to determine whether or not a difference exists. Using statistical modelling, researchers have found that depending on the effect size, the sample size does need to increase. It is important to bare this in mind as exploratory analyses with small sample size will be extremely limiting and may also discourage further study in this area (or indeed as seen the literature - an exploratory first study with the use of males and females with limited sample size, only to show there is no "significance" and to justify this as an reason to only use males for the further studies in the work.

Reviewer #2 (Public review):

Summary:

The investigators tested a workshop intervention to improve knowledge and decrease misconceptions about sex inclusive research. There were important findings that demonstrate the difficulty in changing opinions and knowledge about the importance of studying both males and females. While interventions can improve knowledge and decrease perceived barriers, the impact was small.

Strengths:

The investigators included control groups and replicated the study in a second population of scientists. The results appear to be well substantiated. These are valuable findings that have practical implications for fields where sex is included as a biological variable to improve rigor and reproducibility.

Weaknesses:

I found the figures difficult to understand and would have appreciated more explanation of what is depicted, as well as greater space between the bars representing different categories.

Reviewer #3 (Public review):

Summary:

This manuscript aims to determine cultural biases and misconceptions in inclusive sex research and evaluate the efficacy of interventions to improve knowledge and shift perceptions to decrease perceived barriers for including both sexes in basic research.

Overall, this study demonstrates that despite the intention to include both sexes and a general belief in the importance of doing so, relatively few people routinely include both sexes. Further, the perceptions of barriers to doing so are high, including misconceptions surrounding sample size, disaggregation, and variability of females. There was also a substantial number of individuals without the statistical knowledge to appropriately analyze data in studies inclusive of sex. Interventions increased knowledge and decreased perception of barriers.

Strengths:

(1) This manuscript provides evidence for the efficacy of interventions for changing attitudes and perceptions of research.

(2) This manuscript also provides a training manual for expanding this intervention to broader groups of researchers.

Weaknesses:

The major weakness here is that the post-workshop assessment is a single time point, soon after the intervention. As this paper shows, intention for these individuals is already high, so does decreasing perception of barriers and increasing knowledge change behavior, and increase the number of studies that include both sexes?

Similarly, does the intervention start to shift cultural factors? Do these contribute to a change in behavior?

Author response:

Reviewer #1 (Public review):

Summary:

The authors use the theory of planned behavior to understand whether or not intentions to use sex as a biological variable (SABV), as well as attitude (value), subjective norm (social pressure), and behavioral control (ability to conduct behavior), across scientists at a pharmacological conference. They also used an intervention (workshop) to determine the value of this workshop in changing perceptions and misconceptions. Attempts to understand the knowledge gaps were made.

Strengths:

The use of SABV is limited in terms of researchers using sex in the analysis as a variable of interest in the models (and not a variable to control). To understand how we can improve on the number of researchers examining the data with sex in the analyses, it is vital we understand the pressure points that researchers consider in their work. The authors identify likely culprits in their analyses. The authors also test an intervention (workshop) to address the main bias or impediments for researchers' use of sex in their analyses.

Weaknesses:

There are a number of assumptions the authors make that could be revisited:

(1) that all studies should contain across sex analyses or investigations. It is important to acknowledge that part of the impetus for SABV is to gain more scientific knowledge on females. This will require within sex analyses and dedicated research to uncover how unique characteristics for females can influence physiology and health outcomes. This will only be achieved with the use of female-only studies. The overemphasis on investigations of sex influences limits the work done for women's health, for example, as within-sex analyses are equally important.

The Sex and Gender Equity in Research (SAGER) guidelines (1) provide guidance that “Where the subjects of research comprise organisms capable of differentiation by sex, the research should be designed and conducted in a way that can reveal sex-related differences in the results, even if these were not initially expected.”. This is a default position of inclusion where the sex can be determined and analysis assessing for sex related variability in response. This position underpins many of the funding bodies new policies on inclusion.

However, we need to place this in the context of the driver of inclusion. The most common reason for including male and female samples is for those studies that are exploring the effect of a treatment and then the goal of inclusion is to assess the generalisability of the treatment effect (exploratory sex inclusion)(2). The second scenario is where sex is included because sex is one of the variables of interest and this situation will arise because there is a hypothesized sex difference of interest (confirmatory sex inclusion).

We would argue that the SABV concept was introduced to address the systematic bias of only studying one sex when assessing treatment effect to improve the generalisability of the research. Therefore, it isn’t directly to gain more scientific knowledge on females. However, this strategy will highlight when the effect is very different between male and female subjects which will potentially generate sex specific hypotheses.

Where research has a hypothesis that is specific to a sex (e.g. it is related to oestrogen levels) it would be appropriate to study only the sex of interest, in this case females. The recently published Sex Inclusive Research Framework gives some guidance here and allows an exemption for such a scenario classifying such proposals “Single sex study justified” (3).

We plan to add an additional paragraph to the introduction to clarify the objectives behind inclusion and how this assists the research process.

(2) It should be acknowledged that although the variability within each sex is not different on a number of characteristics (as indicated by meta-analyses in rats and mice), this was not done on all variables, and behavioral variables were not included. In addition, across-sex variability may very well be different, which, in turn, would result in statistical sex significance. In addition, on some measures, there are sex differences in variability, as human males have more variability in grey matter volume than females. PMID: 33044802.

The manuscript was highlighting the common argument used to exclude the use of females, which is that females are inherently more variable as an absolute truth. We agree there might be situations, where the variance is higher in one sex or another depending on the biology. We will extend the discussion here to reflect this, and we will also link to the Sex Inclusive Research Framework (3) which highlights that in these situations researchers can utlise this argument provided it is supported with data for the biology of interest.

(3) The authors need to acknowledge that it can be important that the sample size is increased when examining more than one sex. If the sample size is too low for biological research, it will not be possible to determine whether or not a difference exists. Using statistical modelling, researchers have found that depending on the effect size, the sample size does need to increase. It is important to bare this in mind as exploratory analyses with small sample size will be extremely limiting and may also discourage further study in this area (or indeed as seen the literature - an exploratory first study with the use of males and females with limited sample size, only to show there is no "significance" and to justify this as an reason to only use males for the further studies in the work.

The reviewer raises a common problem: where researchers have frequently argued that if they find no sex differences in a pilot then they can proceed to study only one sex. The SAGER guidelines (1), and now funder guidelines (4, 5), challenge that position. Instead, the expectation is for inclusion as the default in all experiments (exploratory inclusion strategy) to allow generalisable results to be obtained. When the results are very different between the male and female samples, then this can be determined. This perspective shift (2) requires a change in mindset and understanding that the driver behind inclusion is of generalisability not exploration of sex differences. This will be added to the introduction as an additional paragraph exploring the drivers behind inclusion.

We agree with the reviewer that if the researcher is interested in sex differences in an effect (confirmatory inclusion strategy, aka sex as a primary variable) then the N will need to be higher. However, in this situation, one, of course, must have male and female samples in the same experiment to allow the simultaneous exploration to assess the dependency on sex.

Reviewer #2 (Public review):

Summary:

The investigators tested a workshop intervention to improve knowledge and decrease misconceptions about sex inclusive research. There were important findings that demonstrate the difficulty in changing opinions and knowledge about the importance of studying both males and females. While interventions can improve knowledge and decrease perceived barriers, the impact was small.

Strengths:

The investigators included control groups and replicated the study in a second population of scientists. The results appear to be well substantiated. These are valuable findings that have practical implications for fields where sex is included as a biological variable to improve rigor and reproducibility.

Thank you for assessment and highlighting these strengths. We appreciate your recognition of the value and practical implications of this work.

Weaknesses:

I found the figures difficult to understand and would have appreciated more explanation of what is depicted, as well as greater space between the bars representing different categories.

We plan to review the figures and figure legends to improve clarity of the data.

Reviewer #3 (Public review):

Summary:

This manuscript aims to determine cultural biases and misconceptions in inclusive sex research and evaluate the efficacy of interventions to improve knowledge and shift perceptions to decrease perceived barriers for including both sexes in basic research.

Overall, this study demonstrates that despite the intention to include both sexes and a general belief in the importance of doing so, relatively few people routinely include both sexes. Further, the perceptions of barriers to doing so are high, including misconceptions surrounding sample size, disaggregation, and variability of females. There was also a substantial number of individuals without the statistical knowledge to appropriately analyze data in studies inclusive of sex. Interventions increased knowledge and decreased perception of barriers. Strengths:

(1) This manuscript provides evidence for the efficacy of interventions for changing attitudes and perceptions of research.

(2) This manuscript also provides a training manual for expanding this intervention to broader groups of researchers.

Thank you for highlighting these strengths. We appreciate your recognition that the intervention was effect in changing attitudes and perception. We deliberately chose to share the material to provide the resources to allow a wider engagement.

Weaknesses:

The major weakness here is that the post-workshop assessment is a single time point, soon after the intervention. As this paper shows, intention for these individuals is already high, so does decreasing perception of barriers and increasing knowledge change behavior, and increase the number of studies that include both sexes? Similarly, does the intervention start to shift cultural factors? Do these contribute to a change in behavior?

Measuring change in behaviour following an intervention is challenging and hence we had implemented an intention score as a proxy for behaviour. We appreciate the benefit of a long-term analysis, but it was beyond the scope of this study and would need a larger dataset size to allow for attrition. We agree that the strategy implemented has weaknesses. We plan to extend the limitation section in the discussion to include these.

References

(1) Heidari S, Babor TF, De Castro P, Tort S, Curno M. Sex and Gender Equity in Research: rationale for the SAGER guidelines and recommended use. Res Integr Peer Rev. 2016;1:2.

(2) Karp NA. Navigating the paradigm shift of sex inclusive preclinical research and lessons learnt. Commun Biol. 2025;8(1):681.

(3) Karp NA, Berdoy M, Gray K, Hunt L, Jennings M, Kerton A, et al. The Sex Inclusive Research Framework to address sex bias in preclinical research proposals. Nat Commun. 2025;16(1):3763.

(4) MRC. Sex in experimental design - Guidance on new requirements https://www.ukri.org/councils/mrc/guidance-for-applicants/policies-and-guidance-for-researchers/sex-in-experimental-design/: UK Research and Innovation; 2022

(5) Clayton JA, Collins FS. Policy: NIH to balance sex in cell and animal studies. Nature. 2014;509(7500):282-3.

Nassim - 18/06 - Il travaille déjà avec une banque d'affaire, ne souhaite pas à en parler plus

Nassim - 18/06 - Ne fais plus parti de la boite

Nassim - 18/06 - Pas intéréssé

Nassim - 18/06 - RDV en visio le 24/06 à 9h30

hello

Nassim - 18/06 - Pas de téléphone, message linkedin envoyé

Nassim - 18/06 - Répond pas au tel, message laissé

DOI: 10.1523/jneurosci.2331-24.2025

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SciCrunch record: RRID:CVCL_0030

What is this?

DOI: 10.1038/s44318-025-00483-4

Resource: (RRID:CVCL_1926)

Curator: @sjvitug

SciCrunch record: RRID:CVCL_1926

What is this?

DOI: 10.1038/s44318-025-00483-4

Resource: RRID:Addgene_45911

Curator: @sjvitug

SciCrunch record: RRID:Addgene_45911

What is this?

DOI: 10.1038/s44318-025-00483-4

Resource: (CCLV Cat# CCLV-RIE 1035, RRID:CVCL_0023)

Curator: @sjvitug

SciCrunch record: RRID:CVCL_0023

What is this?

DOI: 10.1038/s44318-025-00483-4

Resource: (TKG Cat# TKG 0331, RRID:CVCL_0030)

Curator: @sjvitug

SciCrunch record: RRID:CVCL_0030

What is this?

DOI: 10.1016/j.molcel.2025.05.030

Resource: (DSMZ Cat# ACC-569, RRID:CVCL_2124)

Curator: @areedewitt04

SciCrunch record: RRID:CVCL_2124

What is this?

DOI: 10.1016/j.molcel.2025.05.030

Resource: (JCRB Cat# JCRB1188, RRID:CVCL_2993)

Curator: @areedewitt04

SciCrunch record: RRID:CVCL_2993

What is this?

DOI: 10.1016/j.molcel.2025.05.030

Resource: (ATCC Cat# CRL-2961, RRID:CVCL_2207)

Curator: @areedewitt04

SciCrunch record: RRID:CVCL_2207

What is this?