Tienen que explicar mejor como obtuvieron esos valores, fue promediando? tomaron el valor en el máximo de las curvas? es ese el valor total o una componente?. Convenia antes de presentar el resultado, presentar las curvas, y a partir de la descripción de estas contar como obtuvieron los valores.

Como son dos experiencias distintas, acá era mejor hacer dos sub secciones para organizar mejor el contenido del informe

utilizó

esta explicación no se pone en la parte de resumen

Redacción poco clara. Yo lo entiendo por que conozco el experimento, pero a un lector que no sabe lo que hicieron se le puede complicar entender. No es conveniente usar oraciones tan largas. Les sugiero realizar oraciones cortas y concisas, priorizando claridad en la transmisión de información

ataron con cinta o pegaron?

usar tiempos verbales en presente o pasado, evitar los futuros. Por ejemplo "En este trabajo medimos la intensidad del campo magnético ..." o "En este trabajo de midió la intensidad .."

I would add explanation what does it means. I am personally confused by the aggregation realization here. In most cases I think people would like o express that some goal will be realized by some plateau. Using aggregation means something different - possibly that the goal is valid for only that time period but not necesarilly realized by that. On the other hand it would mean that aggregation is weaker then realization in this case.

Constituția de la Weimar a fost legea fundamentală a Republicii de la Weimar, statul german care a existat între 1919 și 1933, după Primul Război Mondial și prăbușirea Imperiului German.

Context istoric: După înfrângerea Germaniei în Primul Război Mondial (1918), împăratul Wilhelm al II-lea a abdicat.

Germania a fost transformată într-o republică parlamentară.

La 31 iulie 1919, Adunarea Națională Constituantă, întrunită la Weimar (nu la Berlin, din motive de securitate), a adoptat noua constituție. A intrat în vigoare pe 11 august 1919.

200 seguida de 360

welche Rolle spielt O hier? Geht es um Detektion?

Rolle von O erschliesst sich mir noch nicht...

genau - aber diese umfassende Herleitung von Bridgman scheint mir für den Glossarartikel zu voraussetzungsreich btw. umfänglich. Besser wäre es m.E. zu erörtern, welche Verwendungsweisen O. in den Geisteswissenschaften ( etwa: 'eine Begriffsdefinition z.B. von "Erzählperspektive" (z.B. nach Stanzel oder Genette), die sich ihrer Kontingenz und/oder Reduktivität und/oder Vorläufigkeit bewusst ist und daher -- gegeben eine umfängliche Konzeptualisierung des Forschungsstandes -- explizit nur im Rahmen einer Studie / eines Aufsatzes / einer Interpretation gilt') hat, und wie O. in den digitalen Geisteswissenschaften verstanden wird. Dabei sollte man vom stärker explizierten, szientifischen Begriff der Sozialwissenschaften ausgehen und differenzierend erörtern.

die (induktive?) Genese von "Bedeutung" von Konzepten mag wissenstheoretisch historisch diskutiert werden - für den Begriff und die Verwendungsweisen der O. in den DH scheint doch mindestens so wichtig oder gar ausschlaggebender zu sein, dass und wie Konstrukte, (komplexe) Konzepte und latente Variablen formalisierend, algorithmisch und quantifizierend "zugerichtet" werden um sie messbar zu machen. Das würde ich, wie schon ankommentiert, durchgängig zentraler stellen.

hier wird die Empirie genannt, allerdings auf großer Flughöhe. In einem Glossarbeitrag zu O. für/aus digitalen GW sollte auch der empirische Forschungsprozess eingeführt werden. Dabei ist es wichtig, die unetrschiedlichen Asuprägungen von Empirie in den GW zu thematisieren, weil das Auswirkugen auf den Begriff und auch die Praktiken der Opartionalisierung hat (zB Groeben, mit Rückgriff auf Fricke. Zum Sozialwiss./Psychol. Standard siehe Döring (2023). Döring, Nicola. 2023. “Operationalisierung.” In Forschungsmethoden und Evaluation in den Sozial- und Humanwissenschaften, edited by Nicola Döring, 223–91. Berlin, Heidelberg: Springer. https://doi.org/10.1007/978-3-662-64762-2_8.

ggf. schon hier, aber sicher an anderer Stelle des Artikels sollte auch der Begriff des "Proxy" fallen - und es sollte deutlich werden, dass es sich bei dem Begriff der Operationalisierung um einen zentralen Begriff einer empirischen Forschung handelt. Der empirische Forschungsprozess wird noch nicht klar herausgestellt. - Der Begriff der O. beschreibt einen Prozess der "Übersetzung", um Konstrukte, Konzepte und latente Variablen beobachtbar und insebsondere quantitativ messbar zu machen. Dabei ist auch immer ein Aspekt der Kontingenz oder Arbitraität eingebaut. Operationalisierung braucht es insbesondere, um komplexe, “nicht unmittelbar beobachtbare Merkmale, d. h. „theoretisch[e] resp. abstrakt[e] Begriff[e]“ (Aeppli et al. 2016, S. 125) d.h. “latente Merkmale oder Konstrukte oder Konzepte” (Brück and Toth, 2022, p. 1) erforschbar zu machen.

I have checked Reinhard's blog post introducing archaeogaming (https://archaeogaming.wordpress.com/2013/06/09/what-is-archaeogaming/), his book (Archaeogaming: Introduction to Archaeology in and of Video Games), since he is the scholar who coined the term archaeogaming. He states nowhere that archaeogaming includes the study of analog games. Also, the archaeologists who study analog games do not associate their work with archaeogaming. edit: studying physical artifacts like atari cartridges is considered as archaeogaming, but this is only for physical artifacts that are related to video games.

the link does not work

DOI: 10.1042/bcj20253079

Resource: (Nicholas M. Kanaan at Michigan State University Cat# 5H1, RRID:AB_2832941)

Curator: @scibot

SciCrunch record: RRID:AB_2832941

What is this?

DOI: 10.1038/s41598-025-04026-z

Resource: RRID:Addgene_63889

Curator: @scibot

SciCrunch record: RRID:Addgene_63889

What is this?

While large institutions may experiment with LIDAR, photogrammetry, and AI-based analysis, smaller research teams might struggle with bandwidth, hardware, or software licensing. These gaps risk reinforcing inequality patterns in knowledge production.

Raises an important but often underexamined issue: To what extent is digital archaeology structurally dependent on commercial platforms? What alternative infrastructures exist or could be imagined for disseminating archaeological knowledge outside the logic of capitalism?

Tringham, R., & López, M. A. (2001). The democratization of technology. In Proceedings Seventh International Conference on Virtual Systems and Multimedia (pp. 271-279). IEEE.

DOI: 10.1016/j.molcel.2025.04.006

Resource: None

Curator: @scibot

SciCrunch record: RRID:Addgene_200512

What is this?

Just a question: If we had given intervals of s, like we said s = [1,3], then I totally understand what F-measurable means, it's like high = between 1 and 3, if o-algebra includes the set of all high sample points then it's F measurable - so for a given "division" s, we can look and see if it's F-measureable - But this gives us no specific s and just tells us it's F-measureable. idk

If we had given intervals of s, like we said s = [1,3], then I totally understand what F-measurable means, it's like high = between 1 and 3, if o-algebra includes the set of all high sample points then it's F measurable, but s never feels embedded in S? Idk, this is frustrating.

Disrespecting Malvolio simply because he's doing his job. Because Toby is higher than Malvolio, he thinks that gives the latter no right to do his job, and should just listen to him instead.

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public Review):

Summary:

The manuscript by Duilio M. Potenza et al. explores the role of Arginase II in cardiac aging, majorly using whole-body arg-ii knock-out mice. In this work, the authors have found that Arg-II exerts non-cell-autonomous effects on aging cardiomyocytes, fibroblasts, and endothelial cells mediated by IL-1b from aging macrophages. The authors have used arg II KO mice and an in vitro culture system to study the role of Arg II. The authors have also reported the cell-autonomous effect of Arg-II through mitochondrial ROS in fibroblasts that contribute to cardiac aging. These findings are sufficiently novel in cardiac aging and provide interesting insights. While the phenotypic data seems strong, the mechanistic details are unclear. How Arg II regulates the IL-1b and modulates cardiac aging is still being determined. The authors still need to determine whether Arg II in fibroblasts and endothelial contributes to cardiac fibrosis and cell death. This study also lacks a comprehensive understanding of the pathways modulated by Arg II to regulate cardiac aging.

We sincerely appreciate the valuable feedback provided by the reviewer. It's gratifying to hear that our work provided novel information on the role of arginase-II in cardiac aging which is a complex process involving various cell types and mechanisms. We have devoted considerable effort by performing new experiments to address the reviewer's comments and to delineate more detailed mechanisms of Arg-II in cardiac aging. Please, see below our specific answers to each point of the reviewers.

Strengths:

This study provides interesting information on the role of Arg II in cardiac aging.

The phenotypic data in the arg II KO mice is convincing, and the authors have assessed most of the aging-related changes.

The data is supported by an in vitro cell culture system.

We appreciate this reviewer’s positive assessment on the strength of our study.

Weaknesses:

The manuscript needs more mechanistic details on how Arg II regulates IL-1b and modulates cardiac aging.

We made great effort and have performed new experiments in human monocyte cell line (THP1) in which iNOS is not expressed and not inducible by LPS and arg-ii gene was knocked out by CRISPR technology. Moreover, murine bone-marrow derived macrophages in which inos gene was ablated, is also use for this purpose. We found that in the human THP1 monocytes in which Arg-II but not iNOS is induced by LPS (100 ng/mL for 24 hours) (Suppl. Fig. 6A), mRNA and protein levels of IL-1b precursor are markedly reduced in arg-ii knockout THP1^{arg-ii^-/-} as compared to the THP1^wt cells (Suppl. Fig. 6B and 6C), further confirming that Arg-II promotes IL-1b production as also shown in RAW264.7 macrophages (Suppl. Fig. 5A and 5C). Moreover, in the mouse bone-marrow-derived macrophages, LPS-induced IL-1b production is inhibited by inos deficiency (BMDM^inos-/- vs BMDM^wt) (Suppl. Fig. 6D and 6E), while Arg-II levels are slightly enhanced in the BMDM^inos-/- cells (Suppl. Fig. 6D and 6F). All together, these results suggest that iNOS slightly reduces Arg-II expression. Arg-II and iNOS can be upregulated by LPS independently. Both Arg-II and iNOS are required for IL-1b production upon LPS stimulation as illustrated in Suppl. Fig. 6G. For detailed results and discussion, please see answers to the comments point 2 or point 6 raised by this reviewer.

The authors used whole-body KO mice, and the role of macrophages in cardiac aging is not studied in this model. A macrophage-specific arg II Ko would be a better model.

We fully agree with this comment of the reviewer. Unfortunately, this macrophage specific arg-ii knockout animal model is not available, yet. Future research shall develop the macrophage-specific arg-ii^-/- mouse model to confirm this conclusion with aging animals. Since Arg-II is also expressed in fibroblasts and endothelial cells and exerts cell-autonomous and paracrine functions, aging mouse models with conditional arg-ii knockout in the specific cell types would be the next step to elucidate cell-specific function of Arg-II in cardiac aging. We have pointed out this aspect for future research on page 19, lines 2 to 6.

Experiments need to validate the deficiency of Arg II in cardiomyocytes.

As pointed out by this reviewer in the comment point 10, Arg-II was previously reported to be expressed in isolated cardiomyocytes from in rats (PMID: 16537391). Unfortunately, negative controls. i.e., arg-ii^-/- samples were not included in the study to avoid any possible background signals. We made great effort to investigate whether Arg-II is present in the cardiomyocytes from different species including mice, rats and humans and have included old arg-ii^-/- mouse samples as a negative control. This allows to validate the antibody specificity and background noises beyond any reasonable doubt. The new experiments in Suppl. Fig. 4 confirms the specificity of the antibody against Arg-II in old mouse kidney which is known to express Arg-II in the S3 proximal tubular cells (Huang J, et al. 2021). To exclude the possible species-specific different expression of Arg-II in the cardiomyocytes, aged mouse and rat heart tissues were used for cellular localization of Arg-II by confocal immunofluorescence staining. As shown in Suppl. Fig. 4B and 4C, both species show Arg-II expression only in non-cardiomyocytes (cells between striated cardiomyocytes) (red arrows) but not in striated cardiomyocytes. Even in the rat myocardial infarction tissues, Arg-II was not found in cardiomyocytes but in endocardium cells (Suppl. Fig. 4B). In isolated cardiomyocytes exposed to hypoxia, a well know strong stimulus for Arg-II protein levels, no Arg-II signals could be detected, while in fibroblasts from the same animals, an elevated Arg-II levels under hypoxia is demonstrated (Fig. 5B). Furthermore, even RT-qPCR could not detect arg-ii mRNA in cardiomyocytes but in non-cardiomyocytes (Fig. 5C). All together, these results demonstrate that Arg-II are not expressed or at negligible levels in cardiomyocytes but expressed in non-cardiomyocytes. This new experiments with rat heart are included in the method section on page 20, the 1st paragraph. The results are described on page 7, the 1st paragraph, and discussed on page 12, the 2nd paragraph. Legend to Suppl. Fig. 4 is included in the file “Suppl. figure legend_R”.

The authors have never investigated the possibility of NO involvement in this mice model.

As above mentioned, we made great effort and have performed new experiments in human monocyte cell line (THP1) in which iNOS is not expressed and not inducible by LPS and arg-ii gene was knocked out by CRISPR technology. Moreover, murine bone-marrow derived macrophages in which inos gene was ablated, is also use for this purpose. The results show that Arg-II and iNOS can be upregulated by LPS independent of each other and iNOS slightly reduces Arg-II expression. However, both Arg-II and iNOS are required for IL-1b production upon LPS stimulation. For detailed results and discussion, please see answers to the comments point 2 or point 6 raised by this reviewer.

A co-culture system would be appropriate to understand the non-cell-autonomous functions of macrophages.

We appreciate the suggestion by this reviewer regarding the co-culture system to test the non-cell autonomous role of Arg-II. We think that our current model, which involves treating cells with conditioned media, is a well-established and effective method for demonstrating the non-cell autonomous role of Arg-II. This approach allows us to observe the effects of Arg-II on surrounding cells through the factors present in the conditioned media released from macrophages. The co-culture system could be considered, if the released factor in the conditioned medium is not stable. This is however not the case. Therefore, we are confident that our experimental model with conditioned medium is sufficiently enough to demonstrate a paracrine effect of cell-cell interaction (please also see answers to the comment point 16.

The Myocardial infarction data shown in the mice model may not be directly linked to cardiac aging.

As we have introduced and discussed in the manuscript, aging is a predominant risk factor for cardiovascular disease (CVD). Studies in experimental animal models and in humans provide evidence demonstrating that aging heart is more vulnerable to stressors such as ischemia/reperfusion injury and myocardial infarction as compared to the heart of young individuals. Even in the heart of apparently healthy individuals of old age, chronic inflammation, cardiomyocyte senescence, cell apoptosis, interstitial/perivascular tissue fibrosis, endothelial dysfunction and endothelial-mesenchymal transition (EndMT), and cardiac dysfunction either with preserved or reduced ejection fraction rate are observed. Our study is aimed to investigate the role of Arg-II in cardiac aging phenotype and age-associated cardiac vulnerability to stressors. Therefore, cardiac functional changes and myocardial infarction in response to ischemia/reperfusion injury are suitable surrogate parameters for the purpose.

Reviewer #2 (Public Review):

Summary:

The results from this study demonstrated a cell-specific role of mitochondrial enzyme arginase-II (Arg-II) in heart aging and revealed a non-cell-autonomous effect of Arg-II on cardiomyocytes, fibroblasts, and endothelial cells through the crosstalk with macrophages via inflammatory factors, such as by IL-1b, as well as a cell-autonomous effect of Arg-II through mtROS in fibroblasts contributing to cardiac aging phenotype. These findings highlight the significance of non-cardiomyocytes in the heart and bring new insights into the understanding of pathologies of cardiac aging. It also provides new evidence for the development of therapeutic strategies, such as targeting the ArgII activation in macrophages.

We're grateful for the reviewer's positive feedback, acknowledging the significant findings of our study on the role of arginase-II (Arg-II) in cardiac aging. We appreciate this reviewer’s insight into the therapeutic potential of targeting Arg-II activation in macrophages and are excited about the implications for future interventions in age-related cardiac pathologies. Thank you for recognizing the importance of our work in advancing our understanding of cardiac aging and potential therapeutic strategies.

Strengths:

This study targets an important clinical challenge, and the results are interesting and innovative. The experimental design is rigorous, the results are solid, and the representation is clear. The conclusion is logical and justified.

We thank this reviewer for the positive comment.

Weaknesses:

The discussion could be extended a little bit to improve the realm of the knowledge related to this study.

We appreciate this comment and have added and revised our discussion on this aspect accordingly at the end of the discussion section on page 19.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

I have several critical concerns, specifically about the mechanism of how Arg-II plays a role in cardiac aging.

My major concerns are:

(1) The authors have shown non-cell-autonomous effects on aging cardiomyocytes, fibroblasts, and endothelial cells mediated by IL-1b from aging macrophages. A macrophage-specific Arg-II knock-out mouse model is a suitable and necessary control to establish claims.

We fully agree with this comment of the reviewer. Unfortunately, this macrophage specific arg-ii knockout animal model is not available, yet. Future research shall develop the macrophage-specific arg-ii^-/- mouse model to confirm this conclusion with aging animals. Since Arg-II is also expressed in fibroblasts and endothelial cells and exerts cell-autonomous and paracrine functions, aging mouse models with conditional arg-ii knockout in the specific cell types would be the next step to elucidate cell-specific function of Arg-II in cardiac aging. We have pointed out this aspect for future research on page 19, lines 2 to 6.

(2) This study suggests that Arg-II exerts its effect through IL-1b in cardiac ageing. However, all experiments performed to demonstrate the link between ArgII and IL-1β are correlative at best. The underlying molecular mechanism, including transcription factors involved in the regulation of IL-1β by arg-ii, has not been demonstrated.

We sincerely appreciate this reviewer’s comment on the aspect! To make it clear, a causal role of Arg-II in promoting IL-1β production in macrophages is evidenced by the experimental results showing that old arg-ii^-/- mouse heart has lower IL-1β levels than the age-matched wt mouse heart (Fig. 6A to 6D). We further showed that the cellular IL-1β protein levels and release are reduced in old arg-ii^-/- mouse splenic macrophages as compared to the wt cells (Fig. 7A, 7C, and 7D). This result is further confirmed with the mouse macrophage cell line RAW264.7 (Suppl. Fig. 5A and suppl. Fig. 5C), in which we demonstrate that silencing arg-ii reduces IL-1β levels stimulated with LPS.

According to this reviewer’s comment (see comment point 6), we made further effort to investigate possible involvement of iNOS in Arg-II-regulated IL-1β production in macrophages stimulated with LPS. We performed new experiments in human monocyte cell line (THP1) in which iNOS is not expressed and not inducible by LPS and arg-ii gene was knocked out by CRISPR technology in the cells.

Moreover, murine bone-marrow derived macrophages in which inos gene was ablated, is also use for this purpose. We found that in the human THP1 monocytes in which Arg-II but not iNOS is induced by LPS (100 ng/mL for 24 hours) (Suppl. Fig. 6A), mRNA and protein levels of IL-1b are markedly reduced in arg-ii knockout THP1^{arg-ii^-/-} as compared to the THP1^wt cells (Suppl. Fig. 6B and 6C), further confirming that Arg-II promotes IL-1b production as also shown in RAW264.7 macrophages (Suppl. Fig. 5A and 5C). The results suggest that Arg-II promotes IL-1b production independently of iNOS. Moreover, the role of iNOS in IL-1b production was also studied in the mouse bone-marrow-derived macrophages in which inos gene is ablated. The results demonstrate that LPS-induced IL-1b production is inhibited by inos deficiency (BMDM^inos-/- vs BMDM^wt) (Suppl. Fig. 6D and 6E), while Arg-II levels are slightly enhanced in the BMDM^inos-/- cells (Suppl. Fig. 6D and 6F). Since arginase and iNOS share the same metabolic substrate L-arginine, ^inos-/- is expected to increase IL-1b production. This is however not the case. A strong inhibition of IL-1β production in ^inos-/- macrophages is observed. These results implicate that iNOS promotes IL-1β production independently of Arg-II and the inhibiting effect of IL-1β by inos deficiency is dominant and able to counteract Arg-II’s stimulating effect on IL-1β production. Hence, our results demonstrate that Arg-II promotes IL-1β production in macrophages independently of iNOS. All together, these results suggest that iNOS slightly reduces Arg-II expression. Arg-II and iNOS can be upregulated by LPS independently. Both Arg-II and iNOS are required for IL-1b production upon LPS stimulation (This concept is illustrated in the Suppl. Fig. 6G). The new results are described on page 8, the last paragraph and page 9, the 1st paragraph, presented in Suppl. Fig.6. The legend to Suppl. Fig. 6 is described in the file “Supplementary figure legend-R”. The related experimental methods are updated on page 23, the last two paragraphs and page 26 the last paragraph. The results are discussed o page 14, the last paragraph and page 15, the first two paragraphs.

(3) Figure 2: The authors have not validated the whole-body Arg-II knock-out mice for arg-ii ablation.

Thanks for pointing out this missing information! We have added the information regarding genotyping of the mice in the method section on page 20, first paragraph. Moreover, Fig. 5C also confirms the genotyping of the non-cardiomyocyte cells isolated from wt and arg-ii^-/- animals.

(4) It is unclear why the authors have chosen to focus on IL-1β specifically, among other pro-inflammatory cytokines that were also downregulated in Arg-II-/- mice as demonstrated in Fig. 2A-D.

We appreciate the reviewer's question, which provides an opportunity to delve deeper into our findings. In our investigation, we observed that aging is accompanied by elevated levels of various proinflammatory markers. Intriguingly, our data revealed that tnf-α remained unaffected by the ablation of arg-ii during aging in the heart tissues, while Il-1β showed a significant reduction in arg-ii^-/- animals compared to age-matched wild-type (wt) mice (Fig. 2). Mcp1 is however a chemoattractant for macrophages and F4-80 serves as a pan marker for macrophages. Moreover, our previous studies demonstrate a relationship between Arg-II and IL-1β in vascular disease and obesity and age-associated renal and pulmonary fibrosis. Finally, IL-1β has been shown to play a causal role in patients with coronary atherosclerotic heart disease as shown by CANTOS trials. Therefore, we have focused on IL-1β in this study. We have now explained and strengthened this aspect in the manuscript on page 7, the last two lines and page 8, the 1st paragraph as following:

“Taking into account that our previous studies demonstrated a relationship of Arg-II and IL-1β in vascular disease and obesity (Ming et al., 2012) and in age-associated organ fibrosis such as renal and pulmonary fibrosis (Huang et al., 2021; Zhu et al., 2023), and IL-1β has been shown to play a causal role in patients with coronary atherosclerotic heart disease as shown by CANTOS trials (Ridker et al., 2017), we therefore focused on the role of IL-1β in crosstalk between macrophages and cardiac cells such as cardiomyocytes, fibroblasts and endothelial cells”.

(5) Although macrophages are shown to be involved in cardiac ageing in the arg-ii mouse model, the authors have not estimated macrophage infiltration and expression of inflammatory or senescence markers in the hearts of these mice.

Thank you very much for raising this important point! Taking the comments of the reviewer into account, we have performed new experiments, i.e., multiple immunofluorescent staining to analyze the infiltrated (CCR2<sup>+</sip>/F4-80⁺) and resident (LYVE1⁺/F4-80⁺) macrophage populations and to investigate to which extent that Arg-II affects the infiltrated and resident macrophage populations in the aging heart and whether this is regulated by arg-ii^-/-. The results show an age-associated increase in the numbers of F4/80⁺ cells in the wt mouse heart, which is reduced in the age-matched arg-ii^-/- animals (Fig. 2G). This result is in accordance with the result of f4/80 gene expression shown in Fig. 2A, demonstrating that arg-ii gene ablation reduces macrophage accumulation in the aging heart. Interestingly, resident macrophages as characterized by LYVE1⁺/F4-80⁺ cells (Fig. 2E and 2H) are predominant in the aging heart as compared to the infiltrated CCR2⁺/F4-80⁺ cells (Fig. 2F and 2I). The increase in both LYVE1⁺/F4-80⁺ and CCR2⁺/F4-80⁺ macrophages in aging heart is reduced in arg-ii^-/- mice (Fig. 2E, 2F, 2H, and 2I). These new results are described on page 6, the 1st paragraph, presented in Fig. 2E to 2I, and discussed on page 13, the 2nd, paragraph. The legend to Fig. 2 is revised. The method for this additional experiment is included on page 22, the 1st paragraph.

Moreover, the aged-associated accumulation of the senescence cells as demonstrated by p16^ink4 positive cells is significantly reduced in arg-ii^-/- animals. This new result is incorporated in the Fig. 1 as Fig. 1G and 1H and described / discussed on page 5, the 2nd paragraph and page 14, the 2nd last sentences of the 1st paragraph. The method of p16^ink4 staining is included in the method section on page 22, the 1st paragraph, line 7. The legend to Fig. 1 is revised accordingly.

(6) Previously, Arg-II has been reported to serve a crucial role in ageing associated with reduced contractile function in rat hearts by regulating Nitric Oxide Synthase (PMID: 22160208). Elevated NO and superoxide have been shown to play crucial roles in the etiology of cardiovascular diseases (PMID: 24180388). Therefore, it is important to assess whether Nitric Oxide (NO) is involved in the aging-related phenotype in this mouse model.

Following the reviewer's suggestion, we conducted new experiments to investigate the role of nitric oxide (NO) in the context of the effect of Arg-II-induced IL-1b production in macrophages. We have addressed this question in the response to the comment point 2.

(7) Based on the results demonstrated in the study, ablation of Arg-II can be expected to cause a reduction in inflammation-associated phenotypes throughout the body at the multi-organ level. The observed improved cardiac phenotype could be an outcome of whole-body Arg-II ablation. It would be fruitful to develop a cardiac-specific Arg-II knockout mouse model to establish the role of Arg-II in the heart, independent of other organ systems.

We agree with the comment of the reviewer on this point. Unfortunately, as explained above (see point 1), it is currently not possible for us to perform the requested experiments, due to lack of cardiac specific arg-ii-knockout mouse model. Moreover, such an approach is complicated by the absence of Arg-II in cardiomyocytes and the expression of Arg-II in multiple cells including endothelial cells, fibroblasts and macrophage of different origin (resident and monocyte-derived infiltrating cells). It’s thus difficult to generate a cardiac-specific gene knockout mouse. One shall investigate roles of cell-specific Arg-II in cardiac aging by generating cell-specific arg-ii^-/- mice. We appreciate very this important aspect and have discussed issue on page 19, the lines 2 to 6.

(8) Contrary to the findings in this paper, Arg-II has previously been reported to be essential for IL-10-mediated downregulation of pro-inflammatory cytokines, including IL-1β (PMID: 33674584).

Thank you very much for mentioning this study! We have now discussed thoroughly the controversies as the following on page 15, the last paragraph and page 16, the 1st paragraph;

“It is of note that a study reported that Arg-II is required for IL-10 mediated-inhibition of IL-1b in mouse BMDM upon LPS stimulation (Dowling et al., 2021), which suggests an anti-inflammatory function of Arg-II. The results of our present study, however, demonstrate that LPS enhances Arg-II and IL-1b levels in macrophages and knockout or silencing Arg-II reduces IL-1b production and release, demonstrating a pro-inflammatory effect of Arg-II. Our findings are supported by the study from another group, which shows decreased pro-inflammatory cytokine production including IL-6 and IL-1b in arg-ii^-/- BMDM most likely through suppression of NFkB pathway, since arg-ii^-/- BMDM reveals decreased activation of NFkB and IL-1b levels upon LPS stimulation (Uchida et al., 2023). Most importantly, our previous study also showed that re-introducing arg-ii gene back to the arg-ii^-/- macrophages markedly enhances LPS-stimulated pro-inflammatory cytokine production (Ming et al., 2012), providing further evidence for a pro-inflammatory role of arg-ii under LPS stimulation. In support of this conclusion, chronic inflammatory diseases such as atherosclerosis and type 2 diabetes (Ming et al., 2012), inflammaging in lung (Zhu et al., 2023), kidney (Huang et al., 2021) and pancreas (Xiong, Yepuri, Necetin, et al., 2017) of aged animals or acute organ injury such as acute ischemic/reperfusion or cisplatin-induced renal injury are reduced in the arg-ii^-/- mice (Uchida et al., 2023). The discrepant findings between these studies and that with IL-10 may implicate dichotomous functions of Arg-II in macrophages, depending on the experimental context or conditions. Nevertheless, our results strongly implicate a pro-inflammatory role of Arg-II in macrophages in the inflammaging in aging heart”.

(9) The authors have only performed immunofluorescence-based experiments to show fibrotic and apoptotic phenotypes throughout this study. To verify these findings, we suggest that they additionally perform RT-PCR or western blotting analysis for fibrotic markers and apoptotic markers.

The fibrotic aspect was analyzed not only by microscopy but also by using a quantitative biochemical assay such as hydroxyproline content assessment. Hydroxyproline is a major component of collagen and largely restricted to collagen. Therefore, the measurement of hydroxyproline levels can be used as an indicator of collagen content as previous investigated in the lung (Zhu et al., 2023). We have also measured collagen genes expression by RT-qPCR as suggested by the reviewer and found an age-related decline of collagen mRNA expression levels in both wt and arg-ii^-/- mice, suggesting that the age-associated cardiac fibrosis and prevention in arg-ii^-/- mice is due to alterations of translational and/or post-translational regulations, including collagen synthesis and/or degradation. The results are in accordance with that reported by other studies published in the literature. We have pointed out this aspect on page 5, the 2nd paragraph:

“The increased cardiac fibrosis in aging is however, associated with decreased mRNA levels of collagen-Ia (col-Ia) and collagen-IIIa (col-IIIa), the major isoforms of pre-collagen in the heart (Suppl. Fig. 2A and 2B), which is a well-known phenomenon in cardiac fibrotic remodelling (Besse et al., 1994; Horn et al., 2016). The results demonstrate that age-associated cardiac fibrosis and prevention in arg-ii^-/- mice is due to alterations of translational and/or post-translational regulations including collagen synthesis and/or degradation”.

The results are presented in Suppl. Fig. 2, legend to Suppl. Fig. 2 is included in the file “Suppl. figure legend_R”. Suppl. table 2 for primers is revised accordingly.

We did not use additional markers to perform apoptotic assays with whole heart, since Fig. 3 shows good evidence that the aging is associated with increased apoptotic cells in the heart and significantly reduced in the arg-ii^-/- mice. The reduction of TUNEL positive (apoptotic) cells in aged arg-ii^-/- mice is mainly due to decrease in apoptotic cardiomyocytes. With the histological analysis, the apoptotic cell types can be well analysed. Moreover, biochemical assay for apoptosis such as caspase-3 cleavage with whole heart tissues can not distinguish apoptotic cell types and may not be sensitive enough for aging heart, due to relatively low numbers of apoptotic cells in aging heart as compared to myocardial infarct model.  

(10) Figure 4: arg-ii has previously been reported to be expressed in rat cardiomyocytes (PMID: 16537391). We strongly suggest the authors verify the expression of Arg-II via immunostaining in isolated cardiomyocytes (using published protocols), and by using multiple different cardiomyocyte-specific markers for colocalization studies to prove the lack of arg-ii expression beyond a reasonable doubt.

As pointed out by this reviewer, Arg-II was previously reported to be expressed in isolated cardiomyocytes from in rats (PMID: 16537391). Unfortunately, negative controls. i.e., arg-ii^-/- samples were not included in the study to avoid any possible background signals. We made great effort to investigate whether Arg-II is present in the cardiomyocytes from different species including mice, rats and humans and have included old arg-ii^-/- mouse samples as a negative control. This allows to validate the antibody specificity and background noises beyond any reasonable doubt. The new experiments in Suppl. Fig. 4 confirms the specificity of the antibody against Arg-II in old mouse kidney which is known to express Arg-II in the S3 proximal tubular cells (Huang J, et al. 2021). To exclude the possible species-specific different expression of Arg-II in the cardiomyocytes, aged mouse and rat heart tissues were used for cellular localization of Arg-II by confocal immunofluorescence staining. As shown in Suppl. Fig. 4B and 4C, both species show Arg-II expression only in non-cardiomyocytes (cells between striated cardiomyocytes) (red arrows) but not in striated cardiomyocytes. Even in the rat myocardial infarction tissues, Arg-II was not found in cardiomyocytes but in endocardium cells (Suppl. Fig. 4B). In isolated cardiomyocytes exposed to hypoxia, a well know strong stimulus for Arg-II protein levels, no Arg-II signals could be detected, while in fibroblasts from the same animals, an elevated Arg-II levels under hypoxia is demonstrated (Fig. 5B). Furthermore, RT-qPCR could not detect arg-ii mRNA in cardiomyocytes but in non-cardiomyocytes (Fig. 5C). All together, these results demonstrate that Arg-II are not expressed or at negligible levels in cardiomyocytes but expressed in non-cardiomyocytes. This new experiments with rat heart are included in the method section on page 20, the 1st paragraph. The results are described on page 7, the 1st paragraph, and discussed on page 12, the 2nd paragraph. Legend to Suppl. Fig. 4 is included in the file “Suppl. figure legend_R”.

(11) Figure 6G: It may be worthwhile to supplement arg-ii^-/- old cells with IL-1beta to see if there is an increase in TUNEL-positive cells.

IL-1b is a well known pro-inflammatory cytokine that causes apoptosis in various cell types including cardiomyocytes (Shen Y., et al., Tex Heart Inst J. 2015;42:109–116. doi: 10.14503/THIJ-14-4254; Liu Z. et. al., Cardiovasc Diabetol 2015;14,125. doi: 10.1186/s12933-015-0288-y; Li. Z., et al., Sci Adv 2020;6:eaay0589. doi: 10.1126/sciadv.aay0589). We appreciate very much the interesting idea of this reviewer to investigate the apoptotic responses of cardiomyocytes from arg-ii^-/- mice to IL-1b. We agree that it is possible that cardiomyocytes from wt from arg-ii^-/- mice react differently to IL-1b, although the cardiomyocytes do not express Arg-II as demonstrated in our present study. If this is true, it must be due to non-cell autonomous effects of different aging microenvironment in the heart or epigenetic modulations of the myocytes. We found that this is a very interesting aspect and requires further extensive investigation. Since our current study focused on the effect of wt and arg-ii^-/- macrophages on cardiomyocytes and non-cardiomyocytes, we prefer not to include this suggested aspect in our manuscript and would like to explore it in the following study.

(12) Figures 4-9: It would be interesting to see if the effect of ArgII in cardiac ageing is gender-specific. It is recommended to include experimental data with male mice in addition to the results demonstrated in female mice.

As pointed out in the manuscript, we have focused on female mice, because an age-associated increase in arg-ii expression is more pronounced in females than in males (Fig. 1A). As suggested by this reviewer, we performed additional experiments investigating effects of arg-ii deficiency in male mice during aging, focusing on pathophysiological outcomes of ischemia/reperfusion injury in ex vivo experiments. The ex vivo functional analytic experiments with Langendorff system were performed in aged male mice (see Suppl. Fig. 9). Following ischemia/reperfusion injury, wt male mice display reduced left ventricular developed pressure (LVDP), as well as the inotropic and lusitropic states (expressed as dP/dt max and dP/dt min, respectively). As previously reported (Murphy et al., 2007), we also found that old male mice are more prone to I/R injury than age-matched female animals. Specifically, 15 minutes of ischemia are enough to significantly affect the left ventricle contractile function in the male mice (Suppl. Fig. 9). As opposite, age-matched old female mice are relatively resistant to I/R injury, and at least 20 min of ischemia are necessary to induce a significant impairment of the contractile function (Fig. 10). Similar to females, the post I/R recovery of cardiac function is also significantly improved in the male arg-ii^-/- mice as compared to age-matched wt animals. In addition to functional recovery, triphenyl tetrazolium chloride (TTC) staining (myocardial infarction) upon I/R-injury in males is significantly reduced in the age-matched male arg-ii^-/- animals (Suppl. Fig. 9C and 9D). All together, these results reveal a role for Arg-II in heart function impairment during aging in both genders with a higher vulnerability to stress in the males. These new results are presented in Suppl. Fig. 9, described on page 10, the last paragraph and page 11. The results are discussed on page 18, the 2nd paragraph as following:

“The fact that aged females have higher Arg-II but are more resistant to I/R injury seems contradictory to the detrimental effect of Arg-II in I/R injury. It is presumable that cardiac vulnerability to injuries stressors depends on multiple factors/mechanisms in aging. Other factors/mechanisms associated with sex may prevail and determine the higher sensitivity of male heart to I/R injury, which requires further investigation. Nevertheless, the results of our study show that Arg-II plays a role in cardiac I/R injury also in males”.

The information on the experimental methods in the male animals is included on page 20, the last paragraph and page 21, the 1st paragraph. Legend to Suppl. Fig. 9 is included in the file “Suppl. figure legend_R”.

(13) Figure 6G: cardiomyocytes from wild-type mice, when treated with macrophages, show 0% TUNEL-positive cells. Since it is unlikely to obtain no TUNEL staining in a cell population, there may be an experimental or analytical error.

Now it is Fig. 7F and 7G. This is due to our specific experimental procedure. After tissue digestion, cardiomyocytes were plated on laminin-coated dishes. Laminin promotes the adhesion of survived cells. Following plating, we conducted a deep washing process to remove damaged and partially adherent cells. This step ensures that only well-shaped, viable, and strongly adherent cells remain as bioassay cells. These “healthy” cells are then selected for the experiments. the apoptotic cells are removed by washing out, reflecting the high viability of the bioassay cells. We have added this detailed information in the method section on page 24, the 2nd paragraph.

(14) Figure 7J: Please assess whether arg-ii depletion also affects the mtROS phenotype.

According to the suggestion of this reviewer, we performed new experiments which show that human cardiac fibroblasts (HCFs) exposed to hypoxia (1% O₂, 48 hours), a known physiological trigger of Arg-II up-regulation, exhibit increased mtROS generation, which involves Arg-II (new Fig. 8M to 8P). We found that Arg-II protein level as well as mtROS (assessed by mitoSOX staining) were both enhanced, accompanied by increased levels of HIF1α (Fig 8M). Moreover, mito-TEMPO pre-incubation reduces mtROS, confirming the mitochondrial origin of the ROS. Silencing of arg-ii with rAd-mediated shRNA, significantly reduces mtROS levels demonstrating a role of Arg-II in the production of mitochondrial ROS in cardiac fibroblasts (Fig 8M to 8P). We have included these results on page 9, the last paragraph and discussed the results on page 17, the 1st paragraph. The related method is described on page 26, the 2nd paragraph. Legend to Fig. 8 is updated on page 32.

(15) Figure 8A-E: The authors have treated human-origin endothelial cells with mice-origin macrophage-conditioned media. It would be more suitable to treat the endothelial cells with human-origin macrophage-conditioned media.

We acknowledge the concern regarding the use of mouse-origin macrophage-conditioned media on human-origin endothelial cells. It is to note, the biological cross-reactivity of cytokines from one species on cells from a different species has been reported in the literature. It was observed that there is quite a strict threshold of 60% amino acid identity, above which cytokines tend to cross-react and statistically, cytokines would tend to cross-react more often as their % amino acid identity increases (Scheerlinck JPY. Functional and structural comparison of cytokines in different species. Vet Immunol Immunopathol. 1999; 72:39-44. https://doi.org/10.1016/S0165-2427(99)00115-4). Taking IL-1b as an example, the 17.5 kDa mature mouse and human IL-1b share 92% aa sequence identity, suggesting a high cross-reactivity. Indeed, human IL-1b has shown biological cross-reactivity in mouse cells (Ledesma E., et al. Interleukin-1 beta (IL-1β) induces tumor necrosis factor alpha (TNF-α) expression on mouse myeloid multipotent cell line 32D cl3 and inhibits their proliferation. Cytokine. 2004; 26:66-72. https://doi.org/10.1016/j.cyto.2003.12.009). Moreover, our results also support the reported cross-reactivity between human and mouse IL-1b. The CM from mouse macrophage indeed showed biological function in human endothelial cells. The observed effects of the conditioned media from aged wild-type macrophages on endothelial cells were specifically mediated through IL-1β. This conclusion is supported by our data showing that the upregulation induced by the conditioned media was significantly reduced by the addition of an IL-1β receptor blocker.

(16) The co-culture system would be more interesting to test the non-cell autonomous role of Arg II.

We appreciate the suggestion by this reviewer regarding the co-culture system to test the non-cell autonomous role of Arg-II. We believe that our current model, which involves treating cells with conditioned media, is a well-established and effective method for demonstrating the non-cell autonomous role of Arg-II. This approach allows us to observe the effects of Arg-II on surrounding cells through the factors present in the conditioned media. The co-culture system could be considered, if the released factor in the conditioned medium is not stable. This is however not the case. So we are confident that our experimental model with conditioned medium is good enough to demonstrate a paracrine effect of cell-cell interaction.

Reviewer #2 (Recommendations For The Authors):

Some minor comments may be considered to improve the realm of the knowledge related to this study.

We appreciate this comment and have added and revised our discussion on this aspect accordingly at the end of the discussion section on page 19, the last 6 lines.

(1) The current study showed strong evidence demonstrating the key role of cardiac macrophages in pathologies of cardiac aging, particularly, the macrophages (MФ) from the circulating blood (hematogenous). It is known that the heart is among the minority of organs in which substantial numbers of yolk-sac MФ persist in adulthood and play a crucial role in maintaining cardiac function. Thus, the adult mammalian heart contains two separate and discrete cardiac MФ subgroups, i.e., the resident MФs originated from yolk sac-derived progenitors and the hematogenous MФs recruited from circulating blood monocytes. These two subtypes of MФs may play distinctive roles in the aging heart and the response to cardiac injury. The author could extend the discussion on the possibility of the resident MФs in aging hearts, which could be further investigated in the future.

We appreciate the suggestion and agree that it provides valuable insight into the study. Taking the comments of the reviewer 1 into account, we have performed new experiments, i.e., co- immunostaining to analyze the infiltrated (CCR2⁺/F4-80⁺) and resident (LYVE1⁺/F4-80⁺) macrophage populations and to investigate to which extent that Arg-II affects infiltrated and resident macrophage populations in the aging heart. We found that in line with the gene expression of f4/80, immunofluorescence staining reveals an age-associated increase in the numbers of F4/80⁺ cells in the wt mouse heart, which is reduced in the age-matched arg-ii^-/- animals (Fig. 2E, F, G), demonstrating that arg-ii gene ablation reduces macrophage accumulation in the aging heart. Interestingly, resident macrophages as characterized by LYVE1⁺/F4-80⁺ cells (Fig. 2E and 2H) are predominant in the aging heart as compared to the infiltrated CCR2⁺/F4-80⁺ cells (Fig. 2F and 2I). The increase in both LYVE1⁺/F4-80⁺ and CCR2⁺/F4-80⁺ macrophages in aging heart is reduced in arg-ii^-/- mice (Fig. 2E, 2F, 2H, and 2I). These new results are described on page 6, the 1st paragraph, presented in Fig. 2E to 2I, and discussed on page 13, the 2nd, paragraph. The legend to Fig. 2 is revised. The method for this additional experiment is included on page 22, the 1st paragraph.

(2) It would be beneficial to the readers if the author could provide some explanation about why ArgII could not be detected in VSMCs in the mouse heart and the species difference between humans and mice. In addition, the author may provide an assumption on the possibility that there may also be a cross-talk between macrophages and VSMCs in the aging heart. A little bit more explanation in the Discussion will be helpful.

We acknowledge and appreciate the suggestion and have discussed these points on page 19 as the following:

“In this context, another interesting aspect is the cross-talk between macrophages and vascular SMC in the aging heart. In our present study, we could not detect Arg-II in vascular SMC of mouse heart but in that of human heart. This could be due to the difference in species-specific Arg-II expression in the heart or related to the disease conditions in human heart which is harvested from patients with cardiovascular diseases. Indeed, in the apoe^-/- mouse atherosclerosis model, aortic SMCs do express Arg-II (Xiong et al., 2013). It is interesting to note that rodents hardly develop atherosclerosis as compared to humans. Whether this could be partly contributed by the different expression of Arg-II in vascular SMC between rodents and humans requires further investigation. In our present study, the aspect of the cross-talk between macrophages and vascular SMC is not studied. Since the crosstalk between macrophages and vascular SMC has been implicated in the context of atherogenesis as reviewed (Gong et al., 2025), further work shall investigate whether Arg-II expressing macrophages could interact with vascular SMC in the coronary arteries in the heart and contribute to the development of coronary artery disease and/or vascular remodelling and the underlying mechanisms“.

(3) Please clarify the arrows in Figure 9C that indicate the infarct area in each splicing section from one heart.

The arrows in Figure 9C (now Fig. 10C) are indeed utilized to indicate the sections displaying the infarcted area within each splicing section from one heart. We have explained the arrow in the figure legend (now Fig. 10 and also new Suppl. Fig. 9).

Das hetzelfde

DOI: 10.1016/j.ccell.2025.05.011

Resource: None

Curator: @scibot

SciCrunch record: RRID:AB_2532365

What is this?

① Job tasks that require use of the same muscles or motions for long durations increase the likelihood of both localized and general fatigue. ⓵ Aynı kasların veya hareketlerin uzun süreli kullanımını gerektiren iş görevleri, hem lokalize hem de genel yorgunluk olasılığını artırır.

② In general, the longer the period of continuous work the longer the recovery or rest time required. ⓶ Genel olarak, sürekli çalışma süresi ne kadar uzunsa, iyileşme veya dinlenme süresi de o kadar uzun olur.

Spring semester: Beginning o

making a quite block in thunderbird

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public review):

Summary

In this beautiful paper the authors examined the role and function of NR2F2 in testis development and more specifically on fetal Leydig cells development. It is well known by now that FLC are developed from an interstitial steroidogenic progenitors at around E12.5 and are crucial for testosterone and INSL3 production during embryonic development, which in turn shapes the internal and external genitalia of the male. Indeed, lack of testosterone or INSL3 are known to cause DSD as well as undescended testis, also termed as cryptorchidism. The authors first characterized the expression pattern of the NR2R2 protein during testis development and then used two cKO systems of NR2F2, namely the Wt1-creERT2 and the Nr5a1-cre to explore the phenotype of loss of NR2F2. They found in both cases that mice are presenting with undescended testis and major reduction in FLC numbers. They show that NR2F2 has no effect on the amount and expression of the progenitor cells but in its absence, there are less FLC and they are immature.

The effect of NR2F2 is cell autonomous and does not seem to affect other signalling pathways implemented in Leydig cell development as the DHH, PDGFRA and the NOTCH pathway.

Overall, this paper is excellent, very well written, fluent and clear. The data is well presented, and all the controls and statistics are in place. I think this paper will be of great interest to the field and paves the way for several interesting follow up studies as stated in the discussion

Reviewer #2 (Public review):

The major conclusion of the manuscript is expressed in the title: "NR2F2 is required in the embryonic testis for Fetal Leydig Cell development" and also at the end of the introduction and all along the result part. All the authors' assertions are supported by very clear and statistically validated results from ISH, IHC, precise cell counting and gene expression levels by qPCR. The authors used two different conditional Nr2f2 gene ablation systems that demonstrate the same effects at the FLC level. They also showed that the haplo-insufficiency of Wt1 in the first system (knock-in Wt1-cre-ERT2) aggravated the situation in FLC differentiation by disturbing the differentiation of Sertoli cells and their secretion of pro-FLC factors, which had a confounding effect and encouraged them to use the second system. This demonstrates the great rigor with which the authors interpreted the results. In conclusion, all authors' claims and conclusions are justified by their high-quality results.

Recommendations for the authors:

We thank the reviewers for their comments which have improved and strengthened our manuscript. Please see our responses to specific comments below in blue.

Reviewer #1 (Recommendations for the authors):

I have several small comments:

(1) There has been recently a preprint from the Yao lab about the role of NR2F2 is steroidogenic cells (https://www.biorxiv.org/content/10.1101/2024.09.16.613312v1). They performed cKO of NR2F2 using the Wt1creERT2 and found similar results. You should present and discuss this paper in light of your results.

Estermann et al., report a very similar phenotype of FLC hypoplasia in an independent mouse model of Nr2f2 conditional mutation. We have now referred to this article in the discussion of our manuscript as suggested.

(2) In the introduction I think it is important to mention that the steroidogenic progenitors are derived from Wnt5a positive cells (https://pubmed.ncbi.nlm.nih.gov/35705036/).

We have mentioned this point in the introduction as suggested.

(3) In both models you show a decrease in the number of FLC (60% or 40%) and yet they both present with undescended testis. It is important to discuss the fact that there is no need for a complete ablation of testosterone and INSL3 in order to get cryptorchidism.

We have mentioned this point in the discussion as suggested.

The fact that you get only partial reduction in FLC is likely due to redundancy with additional factors, possibly the ARX like you stated in the discussion and it will be interesting to explore that in the future but is beyond the scope of the current paper.

We agree with the reviewer, this question could be addressed by analyzing Arx,Nr2f2 double mutants.

(4) In page 8 line 11 you mention data not shown- not sure if this is allowed in the journal .

The data is now shown in Figure S5A as suggested.

(5) In Figure 2- it will be good if you add a schematic model of the mouse strains used as well as the experimental and control mice next to the Tam scheme. Similar scheme should be in figure 3 for Nr5a1-cre.

We have modified Figures 2 and 3 as suggested.

(6) There is a clear and pronounced effect of the testis cords number and size. It will be good if you could qualify testis cord numbers/ diameter in the mutants even if you do not follow in detail the effect on Sertoli cells

We have quantified testis cords numbers and area in E14.5 Control and Wt1^CreERT2/+; Nr2f2^flox/flox testes. This data is now shown in Figure S2M.

(7) It will be good to present the undescended testis in the Wt1-cre model in figure 2 and not in the supp figure

The data is now shown in Figure 2H-I as suggested.

(8) Please add labelling of the testis, kidney, bladder, vas deferens in figure 3 N+O and in the Wt1-cre model

We have added the labels in Figures 2 and 3 as suggested.

(9) In figure 5 which present both models- it will be good to use the scheme I suggested before to highlight which results refer to which ko model.

We have modified Figure 5 as suggested.

Reviewer #2 (Recommendations for the authors):  

The work presented in this manuscript gave me food for thought. I have always been intrigued by the fact that of the large number of interstitial cells in the testis, a minority differentiate into mature androgen-producing Leydig cells. In other words, how is the number of functional steroidogenic cells defined from a large pool of progenitor cells (ARX and NR2F2 positive ones)? This may have a link with the levels of androgens produced (a kind of feedback control) or the effectiveness of these androgens on the target tissues (i.e.: as spermatogenesis efficiency in adults). In addition, there must be specific signals (probably linked to gonadotropins) that induce the recruitment of Leydig cells from the progenitor pool. Perhaps the genetic models generated in this study could help to address these questions. I leave it to the authors to judge.

We agree with the reviewer. How NR2F2 (and other factors) integrate extrinsic cues to regulate the recruitment of a subset of interstitial steroidogenic progenitors along the Leydig cell differentiation pathway is a fascinating question beyond the scope of this work.

In addition to this reflection, I propose a few minor modifications likely to improve the quality of the manuscript:

(1) Page 3, lane 3: I suggest to replace "growth" by "differentiation"

We have modified the text as suggested.

(2) Page 3, lane 4: the "scrotum" is missing in the parenthesis. Please add it before "and penis"

We have modified the text as suggested.

(3) Page 5, lanes 21-24: kidney hypoplasia is also evident on Fig S2H (stated in the figure legend). It could be also mentioned in this sentence and it implies "...that NR2F2 function is required for testicular and kidney development."

We have modified the text as suggested.

(4) Page 5, lanes 28-30. In addition to the reduction in the number of HSD3B-positive cells, HSD3B staining seems clearly more faint in mutant FLC (Fig 2M) compared to adrenal cells on the same section or FLC in control gonads. This fits well with other results on the level of steroidogenic enzymes (Fig 2O) and those presented thereafter (Fig S4 I-J and Fig 5). Perhaps the author could mention this fact.

We have modified the text as suggested in the results section “NR2F2 is required for FLC maturation” (Page 8).

(5) Page 5, lanes 31-34: testicular descent is hugely sensible to INSL3 in the mouse (by contrast with other species where androgens seem to be more critical). I was wondering if you can check a better phenotypic marker for the absence (or reduction) of androgens like the differentiation of epididymides by HE staining or the anogenital distance at birth.

We have measured the anogenital distance at P0 and P1 as suggested and have included the corresponding graph in Fig. S3P

(6) Page 8, lanes 21-22: "HSD3B positive FLC were smaller and more elongated". It is clear on Fig 5F but not evident on Fig 5D. Could the authors propose another image?

We have modified Figure 5 as suggested and provide now another example of HSD3B positive FLCs in a Nr5a1Cre; Nr2f2^flox/flox mutant gonad (Fig. 5D) and the corresponding control littermate (Fig. 5C).

(7) Page 14, lane 12: "(arrow in I)" should be "(arrow in H)"

We have modified the text as suggested. Please note that ACTA 2 expression is now shown in Figure S2 G-H.

(8) Page 15, lane 6: "Arrows indicate NR5A1 positive FLC". There is no arrow on Fig4 C,D; but a kind of scale bar on the enlargement shown in C.

We have modified Figure 4 as suggested.

Esse efeito pode ser a interpretação real, o entendimento do fenômeno, a possibilidade de influenciar alguém para a ação necessária, a percepção de beleza ou da ameaça escondida.

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public review):

Summary:

Mallimadugula et al. combined Molecular Dynamics (MD) simulations, thiol-labeling experiments, and RNA-binding assays to study and compare the RNA-binding behavior of the Interferon Inhibitory Domain (IID) from Viral Protein 35 (VP35) of Zaire ebolavirus, Reston ebolavirus, and Marburg marburgvirus. Although the structures and sequences of these viruses are similar, the authors suggest that differences in RNA binding stem from variations in their intrinsic dynamics, particularly the opening of a cryptic pocket. More precisely, the dynamics of this pocket may influence whether the IID binds to RNA blunt ends or the RNA backbone.

Overall, the authors present important findings to reveal how the intrinsic dynamics of proteins can influence their binding to molecules and, hence, their functions. They have used extensive biased simulations to characterize the opening of a pocket which was not clearly seen in experimental results - at least when the proteins were in their unbound forms. Biochemical assays further validated theoretical results and linked them to RNA binding modes. Thus, with the combination of biochemical assays and state-of-the-art Molecular Dynamics simulations, these results are clearly compelling.

Strengths:

The use of extensive Adaptive Sampling combined with biochemical assays clearly points to the opening of the Interferon Inhibitory Domain (IID) as a factor for RNA binding. This type of approach is especially useful to assess how protein dynamics can affect its function.

Weaknesses:

Although a connection between the cryptic pocket dynamics and RNA binding mode is proposed, the precise molecular mechanism linking pocket opening to RNA binding still remains unclear.

Reviewer #2 (Public review):

Summary:

The authors aimed to determine whether a cryptic pocket in the VP35 protein of Zaire ebolavirus has a functional role in RNA binding and, by extension, in immune evasion. They sought to address whether this pocket could be an effective therapeutic target resistant to evolutionary evasion by studying its role in dsRNA binding among different filovirus VP35 homologs. Through simulations and experiments, they demonstrated that cryptic pocket dynamics modulate the RNA binding modes, directly influencing how VP35 variants block RIG-I and MDA5-mediated immune responses.

The authors successfully achieved their aim, showing that the cryptic pocket is not a random structural feature but rather an allosteric regulator of dsRNA binding. Their results not only explain functional differences in VP35 homologs despite their structural similarity but also suggest that targeting this cryptic pocket may offer a viable strategy for drug development with reduced risk of resistance.

This work represents a significant advance in the field of viral immunoevasion and therapeutic targeting of traditionally "undruggable" protein features. By demonstrating the functional relevance of cryptic pockets, the study challenges long-standing assumptions and provides a compelling basis for exploring new drug discovery strategies targeting these previously overlooked regions.

Strengths:

The combination of molecular simulations and experimental approaches is a major strength, enabling the authors to connect structural dynamics with functional outcomes. The use of homologous VP35 proteins from different filoviruses strengthens the study's generality, and the incorporation of point mutations adds mechanistic depth. Furthermore, the ability to reconcile functional differences that could not be explained by crystal structures alone highlights the utility of dynamic studies in uncovering hidden allosteric features.

Weaknesses:

While the methodology is robust, certain limitations should be acknowledged. For example, the study would benefit from a more detailed quantitative analysis of how specific mutations impact RNA binding and cryptic pocket dynamics, as this could provide greater mechanistic insight. This study would also benefit from providing a clear rationale for the selection of the amber03 force field and considering the inclusion of volume-based approaches for pocket analysis. Such revisions will strengthen the robustness and impact of the study.

Reviewer #3 (Public review):

Summary:

The authors suggest a mechanism that explains the preference of viral protein 35 (VP35) homologs to bind the backbone of double-stranded RNA versus blunt ends. These preferences have a biological impact in terms of the ability of different viruses to escape the immune response of the host.

The proposed mechanism involves the existence of a cryptic pocket, where VP35 binds the blunt ends of dsRNA when the cryptic pocket is closed and preferentially binds the RNA double-stranded backbone when the pocket is open.

The authors performed MD simulation results, thiol labelling experiments, fluorescence polarization assays, as well as point mutations to support their hypothesis.

Strengths:

This is a genuinely interesting scientific question, which is approached through multiple complementary experiments as well as extensive MD simulations. Moreover, structural biology studies focused on RNA-protein interactions are particularly rare, highlighting the importance of further research in this area.

Weaknesses:

- Sequence similarity between Ebola-Zaire (94% similarity) explains their similar behaviour in simulations and experimental assays. Marburg instead is a more distant homolog (~80% similarity relative to Ebola/Zaire). This difference is sequence and structure can explain the propensities, without the need to involve the existence of a cryptic pocket.  

- No real evidence for the presence of a cryptic pocket is presented, but rather a distance probability distribution between two residues obtained from extensive MD simulations. It would be interesting to characterise the modelled RNA-protein interface in more detail

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

Before assessing the overall quality and significance of this work, this reviewer needs to specify the context of this review. This reviewer's expertise lies in biased and unbiased molecular dynamics simulations and structural biology. Hence, while this reviewer can overall understand the results for thiol-labeling and RNA-binding assays, this review will not assess the quality of these biochemical assays and will mainly focus on the modelling results.

Overall, the authors present important findings to reveal how the intrinsic dynamics of proteins can influence their binding to molecules and, hence, their functions. They have used extensive biased simulations to characterize the opening of a pocket which was not clearly seen in experimental results - at least when the proteins were in their unbound forms. Biochemical assays further validated theoretical results and linked them to RNA binding modes. Thus, with the combination of biochemical assays and state-of-the-art Molecular Dynamics simulations, these results are clearly compelling.

Beyond the clear qualities of this work, I would like to mention a few points that may help to better contextualize and rationalize the results presented here.

- First, both the introduction and discussion sections seem relatively condensed. Extending them to, for example, better describe the methodological context and discuss the methodological limitations and potential future developments related to biased simulations may help the reader get a better idea of the significance of this work.

- The authors presented 3 homologs in this study: IIDs of Reston, Zaire, and Marburg viruses. While Zaire and Reston are relatively similar in terms of sequence (Figure S1). The sequences clearly differ between Marburg and the two other viruses. Can the author indicate a similarity/identity score for each sequence alignment and extend Figure S1 to really compare Marburg sequence with Reston and Zaire? Can they also discuss how these differences may impact the comparison of the three IIDs? This may also help the reader to understand why sometimes the authors compare the three viruses and why sometimes they are focusing only on comparing Zaire and Reston.

We would like to thank the reviewer for raising this point and we agree that additional details about the sequence comparison provide more context for the choices of substitutions we made. Therefore, we have updated Fig S1 to include a detailed pairwise comparison of all the IID sequences including the percentage sequence similarity and identity. We have also added the following sentences to the results section where we first introduced the substitutions between Zaire and Reston IIDs

“While the sequence of Marburg IID differs significantly from Reston and Zaire IIDs with a sequence identity of 42% and 45% respectively (Fig S1), the sequences of Reston and Zaire IID are 88% identical and 94% similar. Particularly, substitutions between these homologs are all distal to the RNA-binding interfaces and all the residues known to make contacts with dsRNA from structural studies are identical. Therefore, we reasoned that comparing these two homologs would help us identify minimal substitutions that control pocket opening probability and allow us to study its effect on dsRNA binding with minimal perturbation of other factors.”

- In this work, the authors mentioned the cryptic pocket but only illustrated the opening of this pocket by using a simple distance between residues (Figure 2) and a SASA of one cysteine (Figure 3). In previous work done by the authors (Cruz et al. , Nature Communications, 2022), they better characterized residues involved in RNA binding and forming the cryptic pocket. Thus, would it be possible to better described this cryptic pocket (residues involved, volume, etc ..) and better explain how, structurally speaking, it can affect RNA binding mode (blunt ends vs backbone) ?

We thank the reviewer for pointing out the need for clarification on the residues involved in RNA binding and pocket opening and the mechanism linking them. We have performed the CARDS analysis on Reston and Marburg IID simulations as we had done on Zaire IID simulations in Cruz et al, 2022. The results are shown in Fig S3 and discussed in the main text in the first results section.

- As a counter-example, the authors used C315 for SASA calculation and thiol labeling (Figure 3). This cysteine is mainly buried as seen by SASA for Reston and Marburg and thiol labelling (Figure 3 E,G,H). Would it be possible to also get thiol labeling rates for Cystein 264 in Reston and its equivalent to see a case where the residue is solvent exposed?

We have shown the SASA for C264 from the simulations in Fig S4 and the thiol labeling rates for all 4 cysteines in Reston IID in Fig S6. Comparing these rates to the rates of all 4 cysteines obtained for Zaire IID (Fig 4 in Cruz et Al, 2022), we observe that the rates for C264, which is expected to be exposed are significantly faster than those of C315 which is largely buried in all variants.  

- I strongly support here the will of the authors to share their data by depositing them in an OSF repository. These data help this reviewer to assess some of the results produced by the authors and help to better understand the dynamics of their respective systems. I have just a few comments that need to be addressed regarding these data: o While there are data for WT Reston and Marburg, there is no data for Zaire. Is this because these data correspond to the previous work (Cruz et al. 2022) (in this case, it would be good to make this clear in the main text) or is it an omission? o There is no center.xtc file in the Marburg-MSM directory o There is no protmasses.pdb in the Reston-MSM directory

- In general, if possible, it would be good to use the same name for each type of file presented in each directory to help a potential user understand a bit more how to use these data.

- If possible, adding a bit more of metadata and explanations on the OSF webpage would be very beneficial to help find these data. To help in this direction, the authors may have a look to the guidelines presented at the end of this article: https://elifesciences.org/articles/90061

We thank the reviewer for pointing out the omissions from the OSF repository. We have added the missing files and followed a uniform naming convention. We have also added documentation in the metadata section of the OSF repository to help others use the data.  

Indeed, the simulation data used for Zaire IID is available on the OSF repository corresponding to Cruz et al. 2022 at https://osf.io/5pg2a. We have also clarified this in the data availability section of the main text.  

Minor point:

In Figure 2, there is a slight bump for the 225-295 distance around 1 nm for Reston. Can the author comment it ? As these results are based on long AS, even if very small, do the authors think this population is significant?

Comparing the probability distributions obtained from bootstrapping the frames used to calculate the MSM equilibrium probabilities (Revised Fig1), we observe that the bump for the Reston IID distribution is persistent in all bootstraps indicating that it might indeed be significant. This is also consistent with our observation that the cysteine 296 does get fully labeled in our thiol labeling experiments, albeit significantly slowly compared to the other homologs.  

Reviewer #2 (Recommendations for the authors):

I recommend that the authors implement moderate revisions prior to the publication of this research article, addressing the identified weaknesses (see below).

The authors should provide a rationale for their selection of the amber03 force field (Duan et al., JCTC 24, 1999-2012, 2003) for molecular dynamics simulations, particularly given the availability of more recent and optimized versions of the AMBER force fields. These newer force fields may offer improved parameterization for biomolecular systems, potentially enhancing the accuracy and reliability of the simulation results.

We chose the Amber03 force field because it has performed well in much of our past work, including the original prediction of the cryptic pocket that we study in this manuscript. The results presented in this manuscript also demonstrate the predictive power of Amber03.

Additionally, while the authors utilized solvent-accessible surface area (SASA) for cryptic pocket analysis, volume-based approaches may be more suitable for this purpose. Several studies (e.g., Sztain et al. J. Chem. Inf. Model. 2021, 61, 7, 3495-3501) have demonstrated the utility of volume analysis in identifying and characterizing cryptic pockets. The authors could consider incorporating such methodologies to provide a more comprehensive assessment of pocket dynamics.

The authors propose that the cryptic pocket is not merely a random structural feature but functions as an allosteric regulator of dsRNA binding. To further substantiate this claim, an in-depth analysis of this allosteric effect using for instance network analysis could significantly enhance the study. Such an approach could identify key residues and interaction networks within the protein that mediate the allosteric regulation. This type of mechanistic insight would not only provide a stronger theoretical framework but also offer valuable information for the rational design of therapeutic interventions targeting the cryptic pocket.  

We thank the reviewer for pointing out the need for clarification on the molecular mechanism linking the opening of the cryptic pocket to RNA binding. We have performed the CARDS analysis on Reston and Marburg IID simulations as was done on Zaire IID simulations in Cruz et al, 2022. The results are shown in Fig S3 and discussed in the main text in the first results section. Briefly, we do find a community (blue) comprising the pocket residues in Reston and Marburg IIDs as we did in Zaire. Similarly, we find that many of the RNA binding residues fall into the orange and green communities as in Zaire. However, there are differences in exactly which residues are clustered into which of these two communities. There are also differences in how strongly connected these communities are in the three homologs. Therefore, while we can conclude that pocket residues likely have varying influence on the RNA binding residues in the homologs, it is hard to say exactly what that variation is from this analysis alone.  

Reviewer #3 (Recommendations for the authors):

- MD simulations: All simulations were initialised from the 3 crystal structures, is it correct? In all cases, RNA ds was not included in simulations, right? Were crystallographic MG ions in the vicinity of the binding site included? these are known to influence structural dynamics to a large extent.

All simulations were indeed initialized using only protein atoms from the crystal structures 3FKE, 4GHL, and 3L2A. Therefore, crystallographic Mg ions were not included in the simulations. However, we do agree with the reviewer and think that the effect of parameters such as salt concentration, specifically Mg ions which are known to be important for the stability of dsRNA, on the pocket opening equilibrium merits detailed study in future work.

- Figure 2: Would it be possible to perform e.g. a block error analysis and show the statistical errors of the distributions?

We agree that showing the statistical variation in the MSM equilibrium probabilities is important for comparing the different distributions. Therefore, we have updated Figs 2 and 5 to show the distributions obtained from MSMs constructed using 100 and 10 random samples of the data respectively to indicate the extent of the statistical variability in the MSM construction.  

- More detailed structural biology experiments (such as NMR or HDX-MS) could potentially shed more light on the differential behaviour of the three different homologs, providing more evidence for the presence of the cryptic pocket.

We agree that NMR and HDX-MS are powerful means to study dynamics and are actively exploring these approaches for our future work.

DOI: 10.3389/fcimb.2025.1531084

Resource: Bioconductor (RRID:SCR_006442)

Curator: @scibot

SciCrunch record: RRID:SCR_006442

What is this?

It ain't what you don't know that gets you into trouble

It's what you know for sure that just ain't so

DOI: 10.1158/1055-9965.EPI-24-0792

Resource: NCI Site Recode ICD-O-3/WHO 2008 Definition (RRID:SCR_024687)

Curator: @scibot

SciCrunch record: RRID:SCR_024687

What is this?

DOI: 10.1002/cbin.70037

Resource: (BD Biosciences Cat# 611280, RRID:AB_398808)

Curator: @scibot

SciCrunch record: RRID:AB_398808

What is this?

Reestruturar sessão para dar uma visão de que esse é o valor original, mas que faremos uma grande oferta na abertura da turma no dia 07

Podemos retirar essa parte da sessão

o que restringe a quantidade de atendimentos possíveis de realizar e estagna o crescimento de seu empreendimento.

Retiraria, deixaria apenas o "e"

Colocaria o "também" na frente de "podemos"

que forneça

Adicionaria vírgula

• A teoria da culpa do serviço, também chamada de culpa administrativa, ou teoria do acidente administrativo, procura desvincular a responsabilidade do Estado da ideia de culpa do funcionário. Passou a falar em culpa do serviço público.

• Distinguia-se, de um lado, a culpa individual do funcionário, pela qual ele mesmo respondia, e, de outro, a culpa anônima do serviço público; nesse caso, o funcionário não é identificável e se considera que o serviço funcionou mal; incide, então, a responsabilidade do Estado.

• Essa culpa do serviço público ocorre quando: o serviço público não funcionou (omissão), funcionou atrasado ou funcionou mal. Em qualquer dessas três hipóteses, ocorre a culpa (faute) do serviço ou acidente administrativo, incidindo a responsabilidade do Estado independentemente de qualquer apreciação da culpa do funcionário.

• Sem abandonar essa teoria, o Conselho de Estado francês passou a adotar, em determinadas hipóteses, a teoria do risco, que serve de fundamento para a responsabilidade objetiva do Estado.

• Essa doutrina baseia-se no princípio da igualdade de todos perante os encargos sociais e encontra raízes no art. 13 da Declaração dos Direitos do Homem, de 1789, segundo o qual “para a manutenção da força pública e para as despesas de administração é indispensável uma contribuição comum que deve ser dividida entre os cidadãos de acordo com as suas possibilidades”. O princípio significa que, assim como os benefícios decorrentes da atuação estatal repartem-se por todos, também os prejuízos sofridos por alguns membros da sociedade devem ser repartidos. Quando uma pessoa sofre um ônus maior do que o suportado pelas demais, rompe-se o equilíbrio que necessariamente deve haver entre os encargos sociais; para restabelecer esse equilíbrio, o Estado deve indenizar o prejudicado, utilizando recursos do erário.

• Nessa teoria, a ideia de culpa é substituída pela de nexo de causalidade entre o funcionamento do serviço público e o prejuízo sofrido pelo administrado. É indiferente que o serviço público tenha funcionado bem ou mal, de forma regular ou irregular. Constituem pressupostos da responsabilidade objetiva do Estado: (a) que seja praticado um ato lícito ou ilícito, por agente público; (b) que esse ato cause dano específico (porque atinge apenas um ou alguns membros da coletividade) e anormal (porque supera os inconvenientes normais da vida em sociedade, decorrentes da atuação estatal); (c) que haja um nexo de causalidade entre o ato do agente público e o dano.

• É chamada teoria da responsabilidade objetiva, precisamente por prescindir da apreciação dos elementos subjetivos (culpa ou dolo); é também chamada teoria do risco, porque parte da ideia de que a atuação estatal envolve um risco de dano, que lhe é inerente. Causado o dano, o Estado responde como se fosse uma empresa de seguro em que os segurados seriam os contribuintes que, pagando os tributos, contribuem para a formação de um patrimônio coletivo (cf. Cretella Júnior, 1970, v. 8, p. 69-70).

• O Código Civil acolheu expressamente a teoria da responsabilidade objetiva, ligada à ideia de risco. Em consonância com o art. 927, parágrafo único, “haverá obrigação de reparar o dano, independentemente de culpa, nos casos especificados em lei, ou quando a atividade normalmente desenvolvida pelo autor do dano implicar, por sua natureza, risco para os direitos de outrem”.

• Segundo Hely Lopes Meirelles (2003:623), a teoria do risco compreende duas modalidades: a do risco administrativo e a do risco integral; a primeira admite (e a segunda não) as causas excludentes da responsabilidade do Estado: culpa da vítima, culpa de terceiros ou força maior.

(PIETRO, Maria Sylvia Zanella D. Direito Administrativo - 38ª Edição 2025. 38. ed. Rio de Janeiro: Forense, 2025. E-book. p.741. Acesso em: 02 jun. 2025.)

• a responsabilidade do concessionário por prejuízos causados a terceiros, em decorrência da execução de serviço público, é objetiva, nos termos do art. 37, § 6º, da Constituição vigente, que estendeu essa norma às pessoas jurídicas de direito privado prestadoras de serviços públicos; o poder concedente responde <u>subsidiariamente</u>, em caso de insuficiência de bens da concessionária; mas essa responsabilidade subsidiária somente se aplica em relação aos prejuízos decorrentes da execução do serviço público; eventualmente, pode haver responsabilidade solidária, por má escolha da concessionária ou omissão quanto ao dever de fiscalização;

(PIETRO, Maria Sylvia Zanella D. Direito Administrativo - 38ª Edição 2025. 38. ed. Rio de Janeiro: Forense, 2025. E-book. p.307. ISBN 9788530995935. Acesso em: 11 jun. 2025.)

• A teor do disposto no artigo 37, § 6º, da Constituição Federal, a ação por danos causados por agente público deve ser ajuizada contra o Estado ou a pessoa jurídica privada prestadora de serviço público, sendo parte ilegítima passiva o autor do ato. [RE 1.027.633, voto do rel. min. Marco Aurélio, j. 14-8-2019, P, DJE de 6-12-2019, Tema 940]
• O inadimplemento dos encargos trabalhistas dos empregados do contratado não transfere automaticamente ao poder público contratante a responsabilidade pelo seu pagamento, seja em caráter solidário ou subsidiário, nos termos do art. 71, § 1º, da Lei 8.666/1993. [RE 760.931, red. do ac. min. Luiz Fux, j. 26-4-2017, P, DJE de 12-9-2017, Tema 246.]
• Para que fique caracterizada a responsabilidade civil do Estado por danos decorrentes do comércio de fogos de artifício, é necessário que exista a violação de um dever jurídico específico de agir, que ocorrerá quando for concedida a licença para funcionamento sem as cautelas legais ou quando for de conhecimento do poder público eventuais irregularidades praticadas pelo particular. [RE 136.861, red. do ac. min. Alexandre de Moraes, j. 11-3-2020, P, DJE de 13-8-2020 Tema 366.]

• Informativo nº 740
• 13 de junho de 2022.
• SEGUNDA TURMA
• Processo: REsp 1.708.325-RS, Rel. Min. Og Fernandes, Segunda Turma, por unanimidade, julgado em 24/05/2022.

Ramo do Direito DIREITO ADMINISTRATIVO

Responsabilidade civil do Estado por omissão. Morte em decorrência de disparo de arma de fogo no interior de hospital público. Ausência de vigilância. Falha específica no dever de agir. Excludente de ilicitude. Fato de terceiro. Não ocorrência.

DESTAQUE - O hospital que deixa de fornecer o mínimo serviço de segurança, contribuindo de forma determinante e específica para homicídio praticado em suas dependências, responde objetivamente pela conduta omissiva.

INFORMAÇÕES DO INTEIRO TEOR - A responsabilidade civil estatal é, em regra, objetiva e decorre do risco administrativo, a respeito da qual não se exige perquirir sobre existência de culpa, conforme disciplinado pelos arts. 14 do Código de Defesa do Consumidor; 186, 192 e 927 do Código Civil; e 37, § 6º, da Constituição Federal. O dualismo ocorre diante dos atos omissivos, para os quais, embora a lei não tenha feito distinção, há os que entendem que, para o ente público, a responsabilidade se reveste do caráter subjetivo.

• Na toada, entretanto, de que, conforme assevera a doutrina, "não é dado ao intérprete restringir onde o legislador não restringiu, sobretudo em se tratando de legislador constituinte", esta Corte, em diversos julgados, tem procurado alinhar-se ao entendimento do Excelso Pretório de que - inclusive por atos omissivos - o Poder Público responde de forma objetiva, quando constatada a precariedade/vício no serviço decorrente da falha no dever legal e específico de agir.

• No caso, trata-se de responsabilidade civil atribuída a hospital, em que a atividade pública exercida, por sua natureza, inclui, além do serviço técnico-médico, o serviço auxiliar de estadia e, por tal razão, está o ente público obrigado a disponibilizar equipe/pessoal e equipamentos necessários e eficazes para o alcance dessa finalidade.

• A inação estatal está atrelada ao mau funcionamento dos trabalhos auxiliares e estruturas operacionais (ausência de serviço/pessoal de vigilância), razão pela qual entende-se que o ente público, em virtude da natureza da atividade pública exercida, responde de forma objetiva, uma vez que, inegavelmente, tem o dever de atuar, ao menos minimamente, para impossibilitar a ocorrência do evento nocivo.

• A omissão do Estado no presente feito revela-se específica e contribuiu decisivamente para a morte da vítima, pois o hospital público não ofereceu nenhuma ou sequer a mínima garantia de integridade aos que se utilizam do serviço e pela qual, em razão do risco da atividade prestada, tem o dever de zelo e proteção.

• Ocorre que a responsabilidade civil do Estado, todavia - seja de ordem subjetiva, seja objetiva - depende, para a configuração da ocorrência de seus pressupostos, do ato ilícito, do dano sofrido e do nexo de causalidade entre o evento danoso e a ação ou omissão do agente público.

• Estão descritos na sentença e no acórdão, a saber: (a) o hospital não possui nenhum serviço de vigilância; e (b) o evento morte decorreu de um disparo com arma de fogo contra a vítima dentro do hospital.

• O Tribunal regional - a despeito de a vítima ter sido baleada e o óbito ter ocorrido no interior do hospital -, não considerou o fato de não existir serviço de vigilância; ao contrário, a Corte local afirma, categoricamente, que o serviço do hospital é somente o atendimento médico, razão pela qual estaria desobrigado de prestar segurança aos pacientes.

• Concluiu-se, assim, que a morte da vítima deu-se por fato de terceiro.

• Como observa-se, a Corte regional - embora tenha considerado não existir equipe responsável pela integridade física dos pacientes e transeuntes no local - afastou a responsabilidade civil, consignando, com base na teoria da causalidade adequada, que a ação de alguém mal intencionado, dentro do hospital público, teria o condão de romper o nexo de causalidade entre a conduta do hospital e o evento danoso.

• Acaso se estivesse diante de um atentado de grandes proporções, não seria difícil concluir que, não obstante todo o empenho, o ente público não pudesse, de fato, impedir o resultado. Esta, entretanto, não é a situação narrada no acórdão, que traz, ao contrário, contexto e narrativa simples e bem menos eloquente.

• Neste caso, a causalidade decorre da própria lógica hermenêutica e análise holística das disposições civis e constitucionais mencionadas, devendo ser examinada à luz dos referidos dispositivos.

• A causalidade no âmbito da responsabilidade civil objetiva deve ser entendida de forma normativa, uma vez que a relevância jurídica do não-fazer está inserida na própria norma se encontra perfectibilizado o liame subjetivo entre a conduta omissa do hospital e o evento morte.

• Há de se ressaltar, contudo, que esse entendimento não se aplica indistintamente a qualquer ato derivado de conduta omissiva da administração pública. Neste feito, sob as lentes do bom senso, o não-fazer do ente público no seu dever de cuidado é sobremaneira significativo. Mostra-se lógico concluir que uma mínima ação de vigilância e cuidado poderia efetivamente ter evitado a morte da vítima.

• A análise da responsabilidade civil, no contexto desafiador dos tempos modernos, em que se colocam a julgamento as consequências tão impactantes das omissões estatais, impõe o ônus, indispensável, de que o exame dos dispositivos civis referidos ocorra sob o olhar dos direitos e garantias fundamentais do cidadão.

• Logo, é de se concluir que a conduta do hospital que deixa de fornecer o mínimo serviço de segurança e, por conseguinte, despreza o dever de zelar pela incolumidade física dos seus pacientes contribuiu de forma determinante e específica para o homicídio praticado em suas dependências, afastando-se a alegação da excludente de ilicitude, qual seja, fato de terceiro.

• RE 1209429
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. MARCO AURÉLIO
• Redator(a) do acórdão: Min. ALEXANDRE DE MORAES
• Julgamento: 10/06/2021
• Publicação: 20/10/2021

EMENTA. CONSTITUCIONAL. RESPONSABILIDADE CIVIL DO ESTADO. PROFISSIONAL DE IMPRENSA FERIDO, EM SITUAÇÃO DE TUMULTO, DURANTE COBERTURA JORNALÍSTICA. CULPA EXCLUSIVA DA VÍTIMA. INOCORRÊNCIA. PROVIMENTO DO RECURSO EXTRAORDINÁRIO. 1. O Estado responde civilmente por danos causados a profissional de imprensa ferido pela polícia, durante cobertura jornalística de manifestação popular. A apuração da responsabilidade dá-se na forma da teoria do risco administrativo, pacificamente aceita pela jurisprudência e pela doutrina. 2. Admite-se a invocação da excludente de responsabilidade civil da culpa exclusiva da vítima, nas hipóteses em que em que o profissional de imprensa I - descumpra ostensiva e clara advertência sobre o acesso a áreas delimitadas em que haja grave risco à sua integridade física; ou II - participe do conflito com atos estranhos à atividade de cobertura jornalística. 3. No caso concreto, o Tribunal de origem reconheceu a referida excludente de responsabilidade, sem identificar quaisquer destas circunstâncias - mas unicamente pelo fato de o fotógrafo estar presente na manifestação. 4. A atuação dos profissionais de imprensa na apuração de informações relevantes para a sociedade é tutelada pela Constituição, não podendo ser alegada pela afastar a responsabilidade civil do Estado. 5. O pedido de pensão mensal vitalícia merece ser atendido, em face do grave comprometimento do exercício da atividade de fotojornalismo, após ter o autor perdido 90% da visão em um dos olhos. Já o valor fixado a título de indenização pelos danos morais mostra-se alinhado aos parâmetros adotados pela jurisprudência brasileira em casos análogos, não cabendo sua elevação. 6. Recurso Extraordinário a que se dá provimento. Tema 1055, fixada a seguinte tese de repercussão geral: "“É objetiva a Responsabilidade Civil do Estado em relação a profissional da imprensa ferido por agentes policiais durante cobertura jornalística, em manifestações em que haja tumulto ou conflitos entre policiais e manifestantes. Cabe a excludente da responsabilidade da culpa exclusiva da vítima, nas hipóteses em que o profissional de imprensa descumprir ostensiva e clara advertência sobre acesso a áreas delimitadas, em que haja grave risco à sua integridade física".

Tese - É objetiva a Responsabilidade Civil do Estado em relação a profissional da imprensa ferido por agentes policiais durante cobertura jornalística, em manifestações em que haja tumulto ou conflitos entre policiais e manifestantes. Cabe a excludente da responsabilidade da culpa exclusiva da vítima, nas hipóteses em que o profissional de imprensa descumprir ostensiva e clara advertência sobre acesso a áreas delimitadas, em que haja grave risco à sua integridade física.

• ADI 6148
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. CÁRMEN LÚCIA
• Redator(a) do acórdão: Min. ANDRÉ MENDONÇA
• Julgamento: 05/05/2022
• Publicação: 15/09/2022

ODS 3 - Saúde e Bem-Estar ODS 11 - Cidades e comunidades sustentáveis ODS 12 - Consumo e produção responsáveis ODS 15 - Vida terrestre

AÇÃO DIRETA DE INCONSTITUCIONALIDADE. CONSTITUCIONAL, ADMINISTRATIVO E AMBIENTAL. PADRÕES DE QUALIDADE DO AR. CONSELHO NACIONAL DO MEIO AMBIENTE (CONAMA): COMPETÊNCIA PARA EXERCER JUÍZO TÉCNICO DISCRICIONÁRIO DE NORMATIZAÇÃO DA MATÉRIA. PRINCÍPIO DEMOCRÁTICO. AUTOCONTENÇÃO JUDICIAL. RESOLUÇÃO CONAMA Nº 491, DE 2018: NORMA CONSTITUCIONAL EM VIAS DE SE TORNAR INCONSTITUCIONAL. CONCESSÃO DO PRAZO DE 24 (VINTE E QUATRO) MESES PARA EDIÇÃO DE NOVA RESOLUÇÃO: OBSERVÂNCIA DA ATUAL REALIDADE FÁTICA.

1. O Conselho Nacional do Meio Ambiente (CONAMA) é órgão colegiado criado pela Lei nº 6.938, de 1981, dotado de capacidade institucional e responsabilidade, para, a partir de estudos e debate colegiado, dispor sobre “normas e padrões compatíveis com o meio ambiente ecologicamente equilibrado e essencial à sadia qualidade de vida”.

2. Diante das múltiplas vicissitudes e peculiaridades do caso, cabe, prioritariamente, ao CONAMA, como órgão regulador e no exercício da sua capacidade institucional, aquilatar, com devida atenção e aprofundado rigor técnico, qual o melhor conjunto de medidas apto a orientar a política de controle da qualidade do ar.

3. Impropriedade do Poder Judiciário em adentrar, ou mesmo substituir, o juízo técnico discricionário realizado na elaboração e no aprimoramento da política pública em foco.

4. Não se afigura salutar a conduta judicial de permanente e minudente escrutínio incidente sobre a condução das políticas públicas selecionadas pelo Administrador.

5. Em se tratando de tema de complexa e controvertida natureza técnico-científica, cabe ao Poder Judiciário atuar com ainda maior deferência em relação às decisões de natureza técnica tomadas pelos órgãos públicos com maior capacidade institucional para o tratamento e solução da questão.

6. Eventual atuação desta Suprema Corte no sentido de rever os critérios que redundaram na opção empreendida pelo CONAMA dependeria de manifesta falta de razoabilidade, de ausência de justificação ou de evidente abusividade na escolha empreendida pelo Administrador, não sendo este o caso dos autos.

7. A Organização Mundial da Saúde (OMS) indica que as diretrizes por ela traçadas não devem ser aplicadas automática e indistintamente, devendo cada país levar em conta os riscos à saúde, sua viabilidade tecnológica, questões econômicas e fatores políticos e sociais peculiares, além do nível de desenvolvimento e da capacidade de cada ente competente para atuar na gestão da qualidade do ar.

8. Sob a ótica do desenvolvimento sustentável, é necessário que sejam consideradas, pelo órgão regulador, o estágio mais atual da realidade nacional, das peculiaridades locais, bem como as possibilidades momentâneas de melhor aplicação dos primados da livre iniciativa, do desenvolvimento social, da redução da pobreza e da promoção da saúde pública, como elementos de indispensável consideração para construção e progressiva evolução da norma, de forma a otimizar a proteção ambiental, dentro da lógica da maior medida possível.

9. Reconhecimento de que a Resolução CONAMA nº 491, de 2018, afigura-se “ainda constitucional”. Determinação ao CONAMA de edição de nova resolução sobre a matéria que considere (i) as atuais orientações da Organização Mundial de Saúde sobre os padrões adequados da qualidade do ar; (ii) a realidade nacional e as peculiaridades locais; e (iii) os primados da livre iniciativa, do desenvolvimento social, da redução da pobreza e da promoção da saúde pública.

10. Se decorrido o prazo de 24 (vinte e quatro) meses, sem a edição de novo ato que represente avanço material na política pública relacionada à qualidade do ar, passarão a vigorar os parâmetros estabelecidos pela Organização Mundial de Saúde enquanto perdurar a omissão administrativa na edição da nova Resolução.

11. Ação Direta de Inconstitucionalidade julgada improcedente.

• ADPF 651
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. CÁRMEN LÚCIA
• Julgamento: 28/04/2022
• Publicação: 29/08/2022

ARGUIÇÃO DE DESCUMPRIMENTO DE PRECEITO FUNDAMENTAL. DIREITO CONSTITUCIONAL AMBIENTAL. MEDIDA CAUTELAR. DECRETO PRESIDENCIAL N. 10.224, DE 5.2.2020. EXCLUSÃO DA SOCIEDADE CIVIL DO CONSELHO DELIBERATIVO DO FUNDO NACIONAL DO MEIO AMBIENTE. DECRETO PRESIDENCIAL N. 10.239, DE 11.2.2020. EXCLUSÃO DOS GOVERNADORES DO CONSELHO NACIONAL DA AMAZÔNIA. DECRETO PRESIDENCIAL N. 10.223, DE 5.2.2020. EXTINÇÃO DO COMITÊ ORIENTADOR DO FUNDO AMAZÔNIA. ALEGADA AFRONTA À PROTEÇÃO AO MEIO AMBIENTE E PROIBIÇÃO AO RETROCESSO AMBIENTAL. ARGUIÇÃO DE DESCUMPRIMENTO DE PRECEITO FUNDAMENTAL JULGADA PROCEDENTE.

1. Proposta de conversão de julgamento de medida cautelar em julgamento definitivo de mérito: ausência de complexidade da questão de direito e instrução dos autos. Precedentes.

2. Nas normas impugnadas, a pretexto de reorganizar a Administração Pública federal quanto à composição do Conselho Deliberativo do Fundo Nacional do Meio Ambiental, do Conselho Nacional da Amazônia e do Comitê Orientador do Fundo Amazônia, frustra-se a participação da sociedade civil e dos Governadores dos Estados integrantes da Amazônia Legal na formulação das decisões e no controle da sua execução em matéria ambiental.

3. A exclusão da participação popular na composição dos órgãos ambientais frustra a opção constitucional pela presença da sociedade civil na formulação de políticas públicas ambientais. Contrariedade ao princípio da participação popular direta em matéria ambiental, à vedação do retrocesso e ao princípio da isonomia.

4. A eliminação da paridade na composição dos órgãos ambientais confere ao Poder Executivo federal o controle das suas decisões, neutralizando-se o caráter crítico e diversificado da fiscalização, que deve permear a condução dos trabalhos e políticas públicas.

5. A organização administrativa em matéria ambiental está protegida pelo princípio de proibição do retrocesso ambiental, o que restringe a atuação do administrador público, de forma a autorizar apenas o aperfeiçoamento das instituições e órgãos de proteção ao meio ambiente.

6. Arguição de descumprimento de preceito fundamental julgada procedente para a) declarar inconstitucional a norma prevista no art. 5º do Decreto n. 10.224/2020, pela qual se extinguiu a participação da sociedade civil no Conselho Deliberativo do Fundo Nacional do Meio Ambiente, restabelecendo-se quanto ao ponto o disposto no Decreto n. 6.985/2009, pelo qual alterado o art. 4º do Decreto n. 3.524/2000; b) declarar a inconstitucionalidade do Decreto n. 10.239/2020, especificamente no ponto em que se excluiu a participação de Governadores no Conselho Nacional da Amazônia Legal; e c) declarar a inconstitucionalidade do art. 1º, CCII, do Decreto nº 10.223/2020, especificamente no ponto em que se extinguiu o Comitê Orientador do Fundo Amazônia.

• ADO 59
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. ROSA WEBER
• Julgamento: 03/11/2022
• Publicação: 16/08/2023

ODS 13 - Ação contra a mudança global do clima ODS 15 - Vida terrestre ODS 16 - Paz, Justiça e Instituições Eficazes ODS 17 - Parcerias e meios de implementação

Ação direita de inconstitucionalidade por omissão. Omissão inconstitucional da União quanto à implementação das prestações normativas e materiais de proteção da área compreendida como Amazônia Legal. O inadimplemento dos deveres constitucionais de tutela do meio ambiente pela União Federal, materializado na ausência de políticas públicas adequadas para a proteção da Amazônia Legal e na desestruturação institucional das formuladas em períodos antecedentes, configura estado normativo desestruturante e desestruturado em matéria ambiental na região. Omissão normativa quanto às obrigações referentes à ativação do Fundo Amazônia, cuja causa principal consiste na extinção dos mecanismos normativos essenciais para a gestão do Fundo. A consequência da paralisação do Fundo Amazônia consiste na suspensão dos ativos financeiros doados, atualmente na ordem de mais de R$ 3.000.000.000,00 (três bilhões de reais), fato que impossibilita a contratação de projetos voltados às ações de prevenção, combate e controle do desmatamento na Amazônia Legal. Classificação do Fundo Amazônia como instrumento de política pública financeira necessária ao adimplemento dos deveres de proteção ao meio ambiente na região da Amazônia Legal. Vedação do retrocesso em tutela ambiental. Procedência parcial dos pedidos.

1. A controvérsia constitucional objeto da deliberação do Supremo Tribunal Federal é um dos temas jurídicos e sociais mais relevantes da atualidade, tanto na perspectiva nacional quanto internacional. A questão subjacente à controvérsia assume caráter humanitário, cultural e econômico de abrangente impacto na tessitura social e na estrutura constitucional, notadamente no núcleo normativo do art. 225, caput, §§ 1º e 4º, da Constituição Federal.

2. O comportamento omissivo de desrespeito à Constituição por parte dos Poderes Públicos, seja legislador, administrador ou jurisdicional, produz como resultado quadro de inexistência de tutela dos direitos fundamentais e do arcabouço normativo constitucional ou de insuficiência no adimplemento dos deveres fundamentais de proteção.

3. O como concretizar os direitos fundamentais integra o espaço de conformação prática dos Poderes Públicos, em especial do Legislativo e do Executivo. Todavia, a liberdade decisória inerente à formulação da política normativa tem como vetor de atuação o dever de tutela dos direitos fundamentais. A proteção não é discricionária, mas sim as formas de sua implementação, desde que observado o postulado da proporcionalidade em sua dupla face proibitiva: do excesso da intervenção na esfera de proteção de direitos fundamentais e da insuficiência de sua tutela.

4. Os pedidos como formulados no sentido da adoção de providências administrativas enquadram-se na categoria de prestações normativas e fáticas derivadas da estrutura e necessidades da órbita de proteção do direito fundamental alegado.

5. A audiência pública produziu aportes informativos e argumentativos essenciais, com esclarecimentos de questões fáticas e jurídicas necessárias para a contextualização e elucidação do problema posto.

6. O quadro normativo e fático da Amazônia Legal traduz a realidade de um autêntico estado de coisas inconstitucional na Amazônia Legal, a revelar um cenário de tutela insuficiente e deficiente dos biomas patrimônios nacionais por parte do Estado brasileiro.

7. O retrato contemporâneo da Amazônia Legal não responde aos deveres de tutela assumidos pelo Estado constitucional brasileiro, expressamente desenhado no art. 225 da Constituição e na arquitetura legislativa, como prescreve a Lei n. 12.187/2009, que instituiu a Política Nacional sobre Mudança do Clima – PNMC. Tampouco responde à normativa internacional, devidamente ratificada e promulgada pelo Estado brasileiro, a demonstrar seu comprometimento político e jurídico com a centralidade e importância da tutela do meio ambiente, em particular a proteção contra o desmatamento e as mudanças climáticas, a saber a Convenção-Quadro sobre Mudanças Climáticas de 1992 (Decreto n. 2.652 de 01 de julho de 1998); o Protocolo de Kyoto, de 2005 (Decreto n. 5.445 de 12 de maio de 2015); e o Acordo de Paris, aprovado no final de 2015 e em vigor desde 2016 (Decreto n. 9.073, de 05 de junho de 2017).

8. A importância e a centralidade do Fundo Amazônia, como principal política pública financeira em vigor de apoio às ações de prevenção, controle e combate ao desmatamento, conservação das florestas e desenvolvimento sustentável, restou comprovada. Nesse sentido, os resultados fáticos obtidos com a implementação do PPCDAm e os depoimentos das organizações não-governamentais, dos secretários de Estado do Meio Ambiente, dos entes federados da Amazônia Legal e dos órgãos de controle e fiscalização envolvidos. Todavia, a centralidade do Fundo Amazônia como política pública financeira não significa inércia estatal, inclusive dos entes subnacionais, em formular outros instrumentos financeiros necessários ao financiamento das ações e planos de concretização da tutela do meio ambiente. Não é compatível com o modelo de federalismo cooperativo, em matéria ambiental, e com a normativa climática, a exclusividade de atuação da União Federal. Aos Estados igualmente compete concretizar objetivos de tutela dos seus biomas por meio de apresentação de resultados suficientes de redução do desmatamento para lograr políticas financeiras alternativas.

9. A alteração na governança do Fundo Amazônia, com a extinção dos seus comitês, Comitê Orientador – COFA e Comitê Técnico-científico – CTFA, por meio da edição dos Decretos n. 9759/2019, n. 10.144/2020 e n. 10.223/2020, acarretou a suspensão da avaliação e aprovação de novos projetos no âmbito do Fundo e, por conseguinte, da aplicação dos recursos disponíveis em caixa. Coube apenas, em termos de operação do Fundo, a continuidade de execução dos projetos aprovados anteriormente.

10. Da leitura e interpretação do art. 225 da Constituição Federal, fundamento normativo do Estado de Direito e da governança ambiental, infere-se estrutura jurídica complexa decomposta em duas direções normativas. A primeira, voltada ao direito fundamental, e a segunda relacionada aos deveres de proteção de responsabilidade dos poderes constituídos, atores públicos e da sociedade civil.

11. A omissão inconstitucional configurada reside no comportamento comissivo do administrador, que instaurou marco normativo desestruturante do antecedente, sem as salvaguardas jurídicas necessárias para a manutenção de um quadro mínimo de adimplemento dos deveres de proteção ao direito fundamental ao meio ambiente equilibrado, mais especificamente de proteção dos patrimônios nacionais, tal como categorizados pelo art. §4º do art. 225 da Constituição Federal, e de cumprimento das obrigações climáticas firmadas.

12. Apresenta-se, como medida jurisdicional adequada para a solução do problema posto, a invalidação dos dispositivos normativos que alteraram o modelo de governança do Fundo Amazônia. Como consequência, cabe à União tomar as providências administrativas necessárias para a reativação do Fundo, no que lhe compete.

13. A omissão inconstitucional do Poder Executivo no que diz respeito ao funcionamento da política pública do Fundo Amazônia traz consequências em distintas atividades e operações do seu funcionamento, como recebimento de novos recursos, análise de novos projetos a serem financiados com valores já recebidos, em resposta aos resultados obtidos pelo Estado brasileiro na redução do desmatamento em momentos anteriores.

14. As providências administrativas relacionadas as atividades de operação do Fundo é de competência do BNDES, responsável e gestor do Fundo. Por esse motivo, os pedidos b), c), d) e) formulados na inicial carecem de respaldo jurídico, porquanto fora da competência da União Federal e da abordagem constitucional desta demanda. O pedido de declaração de inconstitucionalidade do art. 1º, CCII, do Decreto nº 10.223/2020, no ponto em que extinguiu o Comitê Orientador do Fundo Amazônia, foi resolvido no julgamento da ADPF 651, de relatoria da Ministra Cármen Lúcia, quando o Tribunal, ao deferir o aditamento à inicial, declarou sua inconstitucionalidade.

15. Procedência dos pedidos “a” e “f” para (i) declarar a inconstitucionalidade do art. 12, II, do Decreto nº 10.144/2019 e do art. 1º do Decreto nº 9.759/2019, no que se refere aos colegiados instituídos pelo Decreto nº 6.527/2008; e (ii) determinar à União Federal que, no prazo de sessenta dias, tome as providências administrativas necessárias para a reativação do Fundo Amazônia, dentro e nos limites das suas competências, com o formato de governança estabelecido no Decreto n. 6.527/2008.

16. Ação direta julgada parcialmente procedente.

DOI: 10.7554/eLife.105339.1

Resource: (Abcam Cat# ab9050, RRID:AB_306966)

Curator: @areedewitt04

SciCrunch record: RRID:AB_306966

What is this?

DOI: 10.1158/0008-5472.CAN-24-1136

Resource: (Cell Signaling Technology Cat# 18032, RRID:AB_2798793)

Curator: @sonofthor

SciCrunch record: RRID:AB_2798793

What is this?

DOI: 10.1016/j.neuron.2025.05.014

Resource: (IMSR Cat# JAX_029928,RRID:IMSR_JAX:029928)

Curator: @scibot

SciCrunch record: RRID:IMSR_JAX:029928

What is this?

DOI: 10.1016/j.cels.2025.101296

Resource: (Abcam Cat# ab176886, RRID:AB_2864383)

Curator: @scibot

SciCrunch record: RRID:AB_2864383

What is this?

A garantia contratual poderá ser realizada de 5% a 10% do valor do contrato, a depender da análise da complexidade técnica e dos riscos envolvidos.

No entanto, como se depreende do artigo subsequente, a garantia poderá alcançar até 30% do valor inicial do contrato quando se tratar de obra ou serviço de grande vulto.

Não confundir com a garantia prestada como atendimento ao requisito de pré-habilitação que trata o art 58:

Art. 58. Poderá ser exigida, no momento da apresentação da proposta, a comprovação do recolhimento de quantia a título de garantia de proposta, como requisito de pré-habilitação.

• § 1º A garantia de proposta não poderá ser superior a 1% (um por cento) do valor estimado para a contratação.

• § 2º A garantia de proposta será devolvida aos licitantes no prazo de 10 (dez) dias úteis, contado da assinatura do contrato ou da data em que for declarada fracassada a licitação.

• § 3º Implicará execução do valor integral da garantia de proposta a recusa em assinar o contrato ou a não apresentação dos documentos para a contratação.

• § 4º A garantia de proposta poderá ser prestada nas modalidades de que trata o § 1º do art. 96 desta Lei.

O serviço mais frequentemente solicitado é o manejo arbóreo,

Retiraria

Author response:

The following is the authors’ response to the original reviews

Overview of changes in the revision

We thank the reviewers for the very helpful comments and have extensively revised the paper. We provide point-by-point responses below and here briefly highlight the major changes:

(1) We expanded the discussion of the relevant literature in children and adults.

(2) We improved the contextualization of our experimental design within previous reinforcement studies in both cognitive and motor domains highlighting the interplay between the two.

(3) We reorganized the primary and supplementary results to better communicate the findings of the studies.

(4) The modeling has been significantly revised and extended. We now formally compare 31 noise-based models and one value-based model and this led to a different model from the original being the preferred model. This has to a large extent cleaned up the modeling results. The preferred model is a special case (with no exploration after success) of the model proposed in Therrien et al. (2018). We also provide examples of individual fits of the model, fit all four tasks and show group fits for all, examine fits vs. data for the clamp phases by age, provide measures of relative and absolute goodness of fit, and examine how the optimal level of exploration varies with motor noise.

Reviewer #1 (Public review):

Summary:

Here the authors address how reinforcement-based sensorimotor adaptation changes throughout development. To address this question, they collected many participants in ages that ranged from small children (3 years old) to adulthood (1 8+ years old). The authors used four experiments to manipulate whether binary and positive reinforcement was provided probabilistically (e.g., 30 or 50%) versus deterministically (e.g., 100%), and continuous (infinite possible locations) versus discrete (binned possible locations) when the probability of reinforcement varied along the span of a large redundant target. The authors found that both movement variability and the extent of adaptation changed with age.

Thank you for reviewing our work. One note of clarification. This work focuses on reinforcementbased learning throughout development but does not evaluate sensorimotor adaptation. The four tasks presented in this work are completed with veridical trajectory feedback (no perturbation).

The goal is to understand how children at different ages adjust their movements in response to reward feedback but does not evaluate sensorimotor adaptation. We now explain this distinction on line 35.

Strengths:

The major strength of the paper is the number of participants collected (n = 385). The authors also answer their primary question, that reinforcement-based sensorimotor adaptation changes throughout development, which was shown by utilizing established experimental designs and computational modelling.

Thank you.

Weaknesses:

Potential concerns involve inconsistent findings with secondary analyses, current assumptions that impact both interpr tation and computational modelling, and a lack of clearly stated hypotheses.

(1) Multiple regression and Mediation Analyses.

The challenge with these secondary analyses is that:

(a) The results are inconsistent between Experiments 1 and 2, and the analysis was not performed for Experiments 3 and 4,

(b) The authors used a two-stage procedure of using multiple regression to determine what variables to use for the mediation analysis, and

(c)The authors already have a trial-by-trial model that is arguably more insightful.

Given this, some suggested changes are to:

(a) Perform the mediation analysis with all the possible variables (i.e., not informed by multiple regression) to see if the results are consistent.

(b) Move the regression/mediation analysis to Supplementary, since it is slightly distracting given current inconsistencies and that the trial-by-trial model is arguably more insightful.

Based on these comments, we have chosen to remove the multiple regression and mediation analyses. We agree that they were distracting and that the trial-by-trial model allows for differentiation of motor noise from exploration variability in the learning block.

(2) Variability for different phases and model assumptions:

A nice feature of the experimental design is the use of success and failure clamps. These clamped phases, along with baseline, are useful because they can provide insights into the partitioning of motor and exploratory noise. Based on the assumptions of the model, the success clamp would only reflect variability due to motor noise (excludes variability due to exploratory noise and any variability due to updates in reach aim). Thus, it is reasonable to expect that the success clamps would have lower variability than the failure clamps (which it obviously does in Figure 6), and presumably baseline (which provides success and failure feedback, thus would contain motor noise and likely some exploratory noise).

However, in Figure 6, one visually observes greater variability during the success clamp (where it is assumed variability only comes from motor noise) compared to baseline (where variability would come from: (a) Motor noise.

(b) Likely some exploratory noise since there were some failures.

(c) Updates in reach aim.

Thanks for this comment. It made us realize that some of our terminology was unintentionally misleading. Reaching to discrete targets in the Baseline block was done to a) determine if participants could move successfully to targets that are the same width as the 100% reward zone in the continuous targets and b) determine if there are age dependent changes in movement precision. We now realize that the term Baseline Variability was misleading and should really be called Baseline Precision.

This is an important distinction that bears on this reviewer's comment. In clamp trials, participants move to continuous targets. In baseline, participants move to discrete targets presented at different locations. Clamp Variability cannot be directly compared to Baseline Precision because they are qualitatively different. Since the target changes on each baseline trial, we would not expect updating of desired reach (the target is the desired reach) and there is therefore no updating of reach based on success or failure. The SD we calculate over baseline trials is the endpoint variability of the reach locations relative to the target centers. In success clamp, there are no targets so the task is qualitatively different.

We have updated the text to clarify terminology, expand upon our operational definitions, and motivate the distinct role of the baseline block in our task paradigm (line 674).

Given the comment above, can the authors please:

(a) Statistically compare movement variability between the baseline, success clamp, and failure clamp phases.

Given our explanation in the previous point we don't think that comparing baseline to the clamp makes sense as the trials are qualitatively different.

(b) The authors have examined how their model predicts variability during success clamps and failure clamps, but can they also please show predictions for baseline (similar to that of Cashaback et al., 2019; Supplementary B, which alternatively used a no feedback baseline)?

Again, we do not think it makes sense to predict the baseline which as we mention above has discrete targets compared to the continuous targets in the learning phase.

(c) Can the authors show whether participants updated their aim towards their last successful reach during the success clamp? This would be a particularly insightful analysis of model assumptions.

We have now compared 31 models (see full details in next response) which include the 7 models in Roth et al. (2023). Several of these model variants have updating even after success with so called planning noise). We also now fit the model to the data that includes the clamp phases (we can't easily fit to success clamp alone as there are only 10 trials). We find that the preferred model is one that does not include updating after success.

(d) Different sources of movement variability have been proposed in the literature, as have different related models. One possibility is that the nervous system has knowledge of 'planned (noise)' movement variability that is always present, irrespective of success (van Beers, R.J. (2009). Motor learning is optimally tuned to the properties of motor noise. Neuron, 63(3), 406-417). The authors have used slightly different variations of their model in the past. Roth et al (2023) directly Rill compared several different plausible models with various combinations of motor, planned, and exploratory noise (Roth A, 2023, "Reinforcement-based processes actively regulate motor exploration along redundant solution manifolds." Proceedings of the Royal Society B 290: 20231475: see Supplemental). Their best-fit model seems similar to the one the authors propose here, but the current paper has the added benefit of the success and failure clamps to tease the different potential models apart. In light of the results of a), b), and c), the authors are encouraged to provide a paragraph on how their model relates to the various sources of movement variability and ther models proposed in the literature.

Thank you for this. We realized that the models presented in Roth et al. (2023) as well as in other papers, are all special cases of a more general model. Moreover, in total there are 30 possible variants of the full model so we have now fit all 31 models to our larger datasets and performed model selection (Results and Methods). All the models can be efficiently fit by Kalman smoother to the actual data (rather than to summary statistics which has sometimes been done). For model selection, we fit only the 100 learning trials and chose the preferred model based on BIC on the children's data (Figure 5—figure Supplement 1). After selecting the preferred model we then refit this model to all trials including the clamps so as to obtain the best parameter estimates.

The preferred model was the same whether we combined the continuous and discrete probabilistic data or just examin d each task separately either for only the children or for the children and adults combined. The preferred model is a pecial case (no exploration after success) of the one proposed in Therrien et al. (2018) and has exploration variability (after failure) and motor noise with full updating with exploration variability (if any) after success. This model differs from the model in the original submission which included a partial update of the desired reach after exploration this was considered the learning rate. The current model suggests a unity learning rate.

In addition, as suggested by another reviewer, we also fit a value-based model which we adapted from the model described in Giron et al. (2023). This model was not preferred.

We have added a paragraph to the Discussion highlighting different sources of variability and links to our model comparison.

(e) line 155. Why would the success clamp be composed of both motor and exploratory noise? Please clarify in the text

This sentence was written to refer to clamps in general and not just success clamps. However, in the revision this sentence seemed unnecessary so we have removed it.

(3) Hypotheses:

The introduction did not have any hypotheses of development and reinforcement, despite the discussion above setting up potential hypotheses. Did the authors have any hypotheses related to why they might expect age to change motor noise, exploratory noise, and learning rates? If so, what would the experimental behaviour look like to confirm these hypotheses? Currently, the manuscript reads more as an exploratory study, which is certainly fine if true, it should just be explicitly stated in the introduction. Note: on line 144, this is a prediction, not a hypothesis. Line 225: this idea could be sharpened. I believe the authors are speaking to the idea of having more explicit knowledge of action-target pairings changing behaviour.

We have included our hypotheses and predictions at two points in the paper In the introduction we modified the text to:

"We hypothesized that children's reinforcement learning abilities would improve with age, and depend on the developmental trajectory of exploration variability, learning rate (how much people adjust their reach after success), and motor noise (here defined as all sources of noise associated with movement, including sensory noise, memory noise, and motor noise). We think that these factors depend on the developmental progression of neural circuits that contribute to reinforcement learning abilities (Raznahan et al., 2014; Nelson et al., 2000; Schultz, 1998)."

In results we modified the sentence to:

"We predicted that discrete targets could increase exploration by encouraging children to move to a different target after failure.”

Reviewer #2 (Public review):

Summary:

In this study, Hill and colleagues use a novel reinforcement-based motor learning task ("RML"), asking how aspects of RML change over the course of development from toddler years through adolescence. Multiple versions of the RML task were used in different samples, which varied on two dimensions: whether the reward probability of a given hand movement direction was deterministic or probabilistic, and whether the solution space had continuous reach targets or discrete reach targets. Using analyses of both raw behavioral data and model fits, the authors report four main results: First, developmental improvements reflected 3 clear changes, including increases in exploration, an increase in the RL learning rate, and a reduction of intrinsic motor noise. Second, changes to the task that made it discrete and/or deterministic both rescued performance in the youngest age groups, suggesting that observed deficits could be linked to continuous/probabilistic learning settings. Overall, the results shed light on how RML changes throughout human development, and the modeling characterizes the specific learning deficits seen in the youngest ages.

Strengths:

(1) This impressive work addresses an understudied subfield of motor control/psychology - the developmental trajectory of motor learning. It is thus timely and will interest many researchers.

(2) The task, analysis, and modeling methods are very strong. The empirical findings are rather clear and compelling, and the analysis approaches are convincing. Thus, at the empirical level, this study has very few weaknesses.

(3) The large sample sizes and in-lab replications further reflect the laudable rigor of the study.

(4) The main and supplemental figures are clear and concise.

Thank you.

Weaknesses:

(1) Framing.

One weakness of the current paper is the framing, namely w/r/t what can be considered "cognitive" versus "non-cognitive" ("procedural?") here. In the Intro, for example, it is stated that there are specific features of RML tasks that deviate from cognitive tasks. This is of course true in terms of having a continuous choice space and motor noise, but spatially correlated reward functions are not a unique feature of motor learning (see e.g. Giron et al., 2023, NHB). Given the result here that simplifying the spatial memory demands of the task greatly improved learning for the youngest cohort, it is hard to say whether the task is truly getting at a motor learning process or more generic cognitive capacities for spatial learning, working memory, and hypothesis testing. This is not a logical problem with the design, as spatial reasoning and working memory are intrinsically tied to motor learning. However, I think the framing of the study could be revised to focus in on what the authors truly think is motor about the task versus more general psychological mechanisms. Indeed, it may be the case that deficits in motor learning in young children are mostly about cognitive factors, which is still an interesting result!

Thank you for these comments on the framing of our study. We now clearly acknowledge that all motor tasks have cognitive components (new paragraph at line 65). We also explain why we think our tasks has features not present in typical cognitive tasks.

(2) Links to other scholarship.

If I'm not mistaken a common observation in tudies of the development of reinforcement learning is a decrease in exploration over-development (e.g., Nussenbaum and Hartley, 2019; Giron et al., 2023; Schulz et al., 2019); this contrasts with the current results which instead show an increase. It would be nice to see a more direct discussion of previous findings showing decreases in exploration over development, and why the current study deviates from that. It could also be useful for the authors to bring in concepts of different types of exploration (e.g. "directed" vs "random"), in their interpretations and potentially in their modeling.

We recognize that our results differ from prior work. The optimal exploration pattern differs from task to task. We now discuss that exploration is not one size fits all, it's benefits vary depending upon the task. We have added the following paragraphs to the Discussion section:

"One major finding from this study is that exploration variability increases with age. Some other studies of development have shown that exploration can decrease with age indicating that adults explore less compared to children (Schulz et al., 2019; Meder et al., 2021; Giron et al., 2023). We believe the divergence between our work and these previous findings is largely due to the experimental design of our study and the role of motor noise. In the paradigm used initially by Schulz et al. (2019) and replicated in different age groups by Meder et al. (2021) and Giron et al. (2023), participants push buttons on a two-dimensional grid to reveal continuous-valued rewards that are spatially correlated. Participants are unaware that there is a maximum reward available and therefore children may continue to explore to reduce uncertainty if they have difficulty evaluating whether they have reached a maxima. In our task by contrast, participants are given binary reward and told that there is a region in which reaches will always be rewarded. Motor noise is an additional factor which plays a key role in our reaching task but minimal if any role in the discretized grid task. As we show in simulations of our task, as motor noise goes down (as it is known to do through development) the optimal amount of exploration goes up (see Figure 7—figure Supplement 2 and Appendix 1). Therefore, the behavior of our participants is rational in terms of R230 increasing exploration as motor noise decreases.

A key result in our study is that exploration in our task reflects sensitivity to failure. Older children make larger adjustments after failure compared to younger children to find the highly rewarded zone more quickly. Dhawale et al. (2017) discuss the different contexts in which a participant may explore versus exploit (i.e., stick at the same position). Exploration is beneficial when reward is low as this indicates that the current solution is no longer ideal, and the participant should search for a better solution. Konrad et al. (2025) have recently shown this behavior in a real-world throwing task where 6 to 12 year old children increased throwing variability after missed trials and minimized variability after successful trials. This has also been shown in a postural motor control task where participants were more variable after non-rewarded trials compared to rewarded trials (Van Mastrigt et al., 2020). In general, these studies suggest that the optimal amount of exploration is dependent on the specifics of the task."

(3) Modeling.

First, I may have missed something, but it is unclear to me if the model is actually accounting for the gradient of rewards (e.g., if I get a probabilistic reward moving at 45°, but then don't get one at 40°, I should be more likely to try 50° next then 35°). I couldn't tell from the current equations if this was the case, or if exploration was essentially "unsigned," nor if the multiple-trials-back regression analysis would truly capture signed behavior. If the model is sensitive to the gradient, it would be nice if this was more clear in the Methods. If not, it would be interesting to have a model that does "function approximation" of the task space, and see if that improves the fit or explains developmental changes.

The model we use (similar to Roth et al. (2023) and Therrien et al. (2016, 2018)) does not model the gradient. Exploration is always zero-mean Gaussian. As suggested by the reviewer, we now also fit a value-based model (described starting at line 810) which we adapted from the model presented in Giron et al. (2023). We show that the exploration and noise-based model is preferred over the value-based model.

The multiple-trials-back regression was unsigned as the intent was to look at the magnitude and not the direction of the change in movement. We have decided to remove this analysis from the manuscript as it was a source of confusion and secondary analysis that did not add substantially to the findings of these studies.

Second, I am curious if the current modeling approach could incorporate a kind of "action hysteresis" (aka perseveration), such that regardless of previous outcomes, the same action is biased to be repeated (or, based on parameter settings, avoided).

In some sense, the learning rate in the model in the original submission is highly related to perseveration. For example if the learning rate is 0, then there is complete perseveration as you simply repeat the same desired movement. If the rate is 1, there is no perseveration and values in between reflect different amounts of perseveration. Therefore, it is not easy to separate learning rate from perseveration. Adding perseveration as another parameter would likely make it and the learning unidentifiable. However, we now compare 31 models and those that have a non-unity learning rate are not preferred suggesting there is little perseveration.

(4) Psychological mechanisms. There is a line of work that shows that when children and adults perform RL tasks they use a combination of working memory and trial-by-trial incremental learning processes (e.g., Master et al., 2020; Collins and Frank 2012). Thus, the observed increase in the learning rate over development could in theory reflect improvements in instrumental learning, working memory, or both. Could it be that older participants are better at remembering their recent movements in short-term memory (Hadjiosif et al., 2023; Hillman et al., 2024)?

We agree that cognitive processes, such as working memory or visuospatial processing, play a role in our task and describe cognitive elements of our task in the introduction (new paragraph at line 65). However, the sensorimotor model we fit to the data does a good job of explaining the variation across age, which suggests that that age-dependent cognitive processes probably play a smaller role.

Reviewer #3 (Public review):

Summary:

The study investigates reinforcement learning across the lifespan with a large sample of participants recruited for an online game. It finds that children gradually develop their abilities to learn reward probability, possibly hindered by their immature spatial processing and probabilistic reasoning abilities. Motor noise, reinforcement learning rate, and exploration after a failure all contribute to children's subpar performance.

Strengths:

(1) The paradigm is novel because it requires continuous movement to indicate people's choices, as opposed to discrete actions in previous studies.

(2) A large sample of participants were recruited.

(3) The model-based analysis provides further insights into the development of reinforcement learning ability.

Thank you.

Weaknesses:

(1 ) The adequacy of model-based analysis is questionable, given the current presentation and some inconsistency in the results.

Thank you for raising this concern. We have substantially revised the model from our first submission. We now compare 31 noise-based models and 1 value-based model and fit all of the tasks with the preferred model. We perform model selection using the two tasks with the largest datasets to identify the preferred model. From the preferred model, we found the parameter fits for each individual dataset and simulated the trial by trial behavior allowing comparison between all four tasks. We now show examples of individual fits as well as provide a measure of goodness of fit. The expansion of our modeling approach has resolved inconsistencies and sharpened the conclusions drawn from our model.

(2) The task should not be labeled as reinforcement motor learning, as it is not about learning a motor skill or adapting to sensorimotor perturbations. It is a classical reinforcement learning paradigm.

We now make it clear that our reinforcement learning task has both motor and cognitive demands, but does not fall entirely within one of these domains. We use the term motor learning because it captures the fact that participants maximize reward by making different movements, corrupted by motor noise, to unmarked locations on a continuous target zone. When we look at previous ublications, it is clear that our task is similar to those that also refer to this as reinforcement motor learning Cashaback et al. (2019) (reaching task using a robotic arm in adults), Van Mastrigt et al. (2020) (weight shifting task in adults), and Konrad et al. (2025) (real-world throwing task in children). All of these tasks involve trial-by-trial learning through reinforcement to make the movement that is most effective for a given situation. We feel it is important to link our work to these previous studies and prefer to preserve the terminology of reinforcement motor learning.

Recommendations for the authors:

Reviewing Editor Comments:

Thank you for this summary. Rather than repeat the extended text from the responses to the reviewers here, we point the Editor to the appropriate reviewer responses for each issue raised.

The reviewers and editors have rated the significance of the findings in your manuscript as "Valuable" and the strength of evidence as "Solid" (see eLife evalutation). A consultancy discussion session to integrate the public reviews and recommendations per reviewer (listed below), has resulted in key recommendations for increasing the significance and strength of evidence:

To increase the Significance of the findings, please consider the following:

(1) Address and reframe the paper around whether the task is truly getting at a motor learning process or more generic cognitive decision-making capacities such as spatial memory, reward processing, and hypothesis testing.

We have revised the paper to address the comments on the framing of our work. Please see responses to the public review comments of Reviewers #2 and #3.

(2) It would be beneficial to specify the differences between traditional reinforcement algorithms (i.e., using softmax functions to explore, and build representations of state-action-reward) and the reinforcement learning models used here (i.e., explore with movement variability, update reach aim towards the last successful action), and compare present findings to previous cognitive reinforcement learning studies in children.

Please see response to the public review comments of Reviewer #1 in which we explain the expansion of our modeling approach to fit a value-based model as well as 31 other noise-based models. In our response to the public review comments of Reviewer #2, we comment on our expanded discussion of how our findings compare with previous cognitive reinforcement learning studies.

To move the "Strength of Evidence" to "Convincing", please consider doing the following:

(1 ) Address some apparently inconsistent and unrealistic values of motor noise, exploration noise, and learning rate shown for individual participants (e.g., Figure 5b; see comments reviewers 1 and take the following additional steps: plotting r squares for individual participants, discussing whether individual values of the fitted parameters are plausible and whether model parameters in each age group can extrapolate to the two clamp conditions and baselines.

We have substantially updated our modeling approach. Now that we compare 31 noise-based models, the preferred model does not show any inconsistent or unrealistic values (see response to Reviewer #3). Additionally, we now show example individual fits and provide both relative and absolute goodness of fit (see response to Reviewer #3).

(2) Relatedly, to further justify if model assumptions are met, it would be valuable to show that the current learning model fits the data better than alternative models presented in the literature by the authors themselves and by others (reviewer 1). This could include alternative development models that formalise the proposed explanations for age-related change: poor spatial memory, reward/outcome processing, and exploration strategies (reviewer 2).

Please see response to public review comments of Reviewer #1 in which we explain that we have now fit a value-based model as well as 31 other noise-based models providing a comparison of previous models as well as novel models. This led to a slightly different model being preferred over the model in the original submission (updated model has a learning rate of unity). These models span many of the processes previously proposed for such tasks. We feel that 32 models span a reasonable amount of space and do not believe we have the power to include memory issues or heuristic exploration strategies in the model.

(3) Perform the mediation analysis with all the possible variables (i.e., not informed by multiple regression) to see if the results are more consistent across studies and with the current approach (see comments reviewer 1).

Please see response to public review comments of Reviewer #1. We chose to focus only on the model based analysis because it allowed us to distinguish between exploration variability and motor noise.

Please see below for further specific recommendations from each reviewer.

Reviewer #1 (Recommendations for the author):

(1) In general, there should be more discussion and contextualization of other binary reinforcement tasks used in the motor literature. For example, work from Jeroen Smeets, Katinka van der Kooij, and Joseph Galea.

Thank you for this comment. We have edited the Introduction to better contextualize our work within the reinforcement motor learning literature (see line 67 and line 83).

(2) Line 32. Very minor. This sentence is fine, but perhaps could be slightly improved. “select a location along a continuous and infinite set of possible options (anywhere along the span of the bridge)"

Thank you for this comment. We have edited the sentence to reflect this suggestion.

(3) Line 57. To avoid some confusion in successive sentences: Perhaps, "Both children over 12 and adolescents...".

Thank you for this comment. We have edited the sentence to reflect this suggestion.

(4) Line 80. This is arguably not a mechanistic model, since it is likely not capturing the reward/reinforcement machinery used by the nervous system, such as updating the expected value using reward predic tion errors/dopamine. That said, this phenomenological model, and other similar models in the field, do very well to capture behaviour with a very simple set of explore and update rules.

We use mechanistic in the standard use in modeling, as in Levenstein et al. (2023), for example. The contrast is not with neural modeling, but with normative modeling, in which one develops a model to optimize a function (or descriptive models as to what a system is trying to achieve). In mechanistic modeling one proposes a mechanism and this can be at a state-space level (as in our case) or a neural level (as suggested my the reviewer) but both are considered mechanistic, just at different levels. Quoting Levenstein "... mechanistic models, in which complex processes are summarized in schematic or conceptual structures that represent general properties of components and their interactions, are also commonly used." We now reference the Levenstein paper to clarify what we mean by mechanistic.

(5) Figure 1. It would be useful to state that the x-axis in Figure 1 is in normalized units, depending on the device.

Thank you for this comment. We have added a description of the x-axis units to the Fig. 1 caption.

(6) Were there differences in behaviour for these different devices? e.g., how different was motor noise for the mouse, trackpad, and touchscreen?

Thank you for this question. We did not find a significant effect of device on learning or precision in the baseline block. We have added these one way ANOVA results for each task in Supplementary Table 1.

(7) Line 98. Please state that participants received reinforcement feedback during baseline.

Thank you for this comment. We have updated the text to specify that participants receive reward feedback during the baseline block.

(8) Line 99. Did the distance from the last baseline trial influence whether the participant learned or did not learn? For example, would it place them too far from the peak success location such that it impacted learning?

Thank you for this question. We looked at whether the position of movement on the last baseline block trial was correlated with the first movement position in the learning block. We did not find any correlations between these positions for any of the tasks. Interestingly, we found that the majority of participants move to the center of the workspace on the first trial of the learning block for all tasks (either in the presence of the novel continuous target scene or the presentation of 7 targets all at once). We do not think that the last movement in the baseline block "primed" the participant for the location of the success zone in the learning block. We have added the following sentence to the Results section:

"Note that the reach location for the first learning trial was not affected by (correlated with) the target position on the last baseline trial (p > 0.3 for both children and adults, separately)."

(9) The term learning distance could be improved. Perhaps use distance from target.

Thank you for this comment. We appreciate that learning distance defined with 0 as the best value is counter intuitive. We have changed the language to be "distance from target" as the learning metric.

(10) Line 188. This equation is correct, but to estimate what the standard deviation by the distribution of changes in reach position is more involved. Not sure if the authors carried out this full procedure, which is described in Cashaback et al., 2019; Supplemental 2.

There appear to be no Supplemental 2 in the referenced paper so we assume the reviewer is referring to Supplemental B which deals with a shuffling procedure to examine lag-1 correlations.

In our tasks, we are limited to only 9 trials to analyze in each clamp phase so do not feel a shuffling analysis is warranted. In these blocks, we are not trying to 'estimate what the standard deviation by the distribution of changes in reach position' but instead are calculating the standard deviation of the reach locations and comparing the model fit (for which the reviewer says the formula is correct) with the data. We are unclear what additional steps the reviewer is suggesting. In our updated model analysis, we fit the data including the clamp phases for better parameter estimation. We use simulations to estimate s.d. in the clamp phase (as we ensure in simulations the data does not fall outside the workspace) making the previous analytic formulas an approximation that are no longer used.

(11) Line 197-199. Having done the demo task, it is somewhat surprising that a 3-year-old could understand these instructions (whose comprehension can be very different from even a 5-year old).

Thank you for raising this concern. We recognize that the younger participants likely have different comprehension levels compared to older participants. However, we believe that the majority of even the youngest participants were able to sufficiently understand the goal of the task to move in a way to get the video clip to play. We intentionally designed the tasks to be simple such that the only instructions the child needed to understand were that the goal was to get the video clip to play as much as possible and the video clip played based on their movement. Though the majority of younger children struggled to learn well on the probabilistic tasks, they were able to learn well on the deterministic tasks where the task instructions were virtually identical with the exception of how many places in the workspace could gain reward. On the continuous probabilistic task, we did have a small number (n = 3) of 3 to 5 year olds who exhibited more mature learning ability which gives us confidence that the younger children were able to understand the task goal.

(12) Line 497: Can the authors please report the F-score and p-value separately for each of these one-way ANOVA (the device is of particular interest here).

Thank you for this request. We have added ina upplementarytable (Supplementary Table 1) with the results of these ANOVAs.

(13) Past work has discussed how motivation influences learning, which is a function of success rate (van der Kooij, K., in 't Veld, L., & Hennink, T. (2021). Motivation as a function of success frequency. Motivation and Emotion, 45, 759-768.). Can the authors please discuss how that may change throughout development?

Thank you for this comment. While motivation most probably plays a role in learning, in particular in a game environment, this was out of the scope of the direct focus of this work and not something that our studies were designed to test. We have added the following sentence to the discussion section to address this comment:

"We also recognize that other processes, such as memory and motivation, could affect performance on these tasks however our study was not designed to test these processes directly and future work would benefit from exploring these other components more explicitly."

(14) Supplement 6. This analysis is somewhat incomplete because it does not consider success.

Pekny and collegues (2015) looked at 3 trials back but considered both success and reward. However, their analysis has issues since successive time points are not i.i.d., and spurious relationships can arise. This issue is brought up by Dwahale (Dhawale, A. K., Miyamoto, Y. R., Smith, M. A., & R475 Ölveczky, B. P. (2019). Adaptive regulation of motor variability. Current Biology, 29(21), 3551-3562.). Perhaps it is best to remove this analysis from the paper.

Thank you for this comment. We have decided to remove this secondary analysis from the paper as it was a source of confusion and did not add to the understanding and interpretation of our behavioral results.

Reviewer #2 (Recommendations for the author):

(1 ) the path length ratio analyses in the supplemental are interesting but are not mentioned in the main paper. I think it would be helpful to mention these as they are somewhat dramatic effects

Thank you for this comment. Path length ratios are defined in the Methods and results are briefly summarized in the Results section with a point to the supplementary figures. We have updated the text to more explicitly report the age related differences in path length ratios.

(2) The second to last paragraph of the intro could use a sentence motivating the use ofthe different task features (deterministic/probabilistic and discrete/continuous).

Thank you for this comment. We have added an additional motivating sentence to the introduction.

Reviewer #3 (Recommendations for the author):

The paper labeled the task as one for reinforcement motor learning, which is not quite appropriate in my opinion. Motor learning typically refers to either skill learning or motor adaptation, the former for improving speed-accuracy tradeoffs in a certain (often new) motor skill task and the latter for accommodating some sensorimotor perturbations for an existing motor skill task. The gaming task here is for neither. It is more like a

decision-making task with a slight contribution to motor execution, i.e., motor noise. I would recommend the authors label the learning as reinforcement learning instead of reinforcement motor learning.

Thank you for this comment. As noted in the response to the public review comments, we agree that this task has components of classical reinforcement learning (i.e. responding to a binary reward) but we specifically designed it to require the learning of a movement within a novel game environment. We have added a new paragraph to the introduction where we acknowledge the interplay between cognitive and motor mechanisms while also underscoring the features in our task that we think are not present in typical cognitive tasks.

My major concern is whether the model adequately captures subjects' behavior and whether we can conclude with confidence from model fitting. Motor noise, exploration noise, and learning rate, which fit individual learning patterns (Figure 5b), show some quite unrealistic values. For example, some subjects have nearly zero motor noise and a 100% learning rate.

We have now compared 31 models and the preferred model is different from the one in the first submission. The parameter fits of the new model do not saturate in any way and appear reasonable to us. The updates to the model analysis have addressed the concern of previously seen unrealistic values in the prior draft.

Currently, the paper does not report the fitting quality for individual subjects. It is good to have an exemplary subject's fit shown, too. My guess is that the r-squared would be quite low for this type of data. Still, given that the children's data is noisier, it might be good to use the adult data to show how good the fitting can be (individual fits, r squares, whether the fitted parameters make sense, whether it can extrapolate to the two clamp phases). Indeed, the reliability of model fitting affects how we should view the age effect of these model parameters.

We now show fits to individual subjects. But since this is a Kalman smoother it fits the data perfectly by generating its best estimate of motor noise and exploration variability on each trial to fully account for the data — so in that sense R² is always 1 so that is not helpful.

While the BIC analysis with the other model variants provides a relative goodness of fit, it is not straightforward to provide an absolute goodness of fit such as standard R² for a feedforward simulation of the model given the parameters (rather than the output of the Kalman smoother). There are two problems. First, there is no single model output. Each time the model is simulated with the fit parameters it produces a different output (due to motor noise, exploration variability and reward stochasticity). Second, the model is not meant to reproduce the actual motor noise, exploration variability and reward stochasticity of a trial. For example, the model could fit pure Gaussian motor noise across trials (for a poor learner) by accurately fitting the standard deviation of motor noise but would not be expected to actually match each data point so would have a traditional R² of O.

To provide an overall goodness of fit we have to reduce the noise component and to do so we exam ined the traditional R² between the average of all the children's data and the average simulation of the model (from the median of 1000 simulations per participant) so as to reduce the stochastic variation. The results for the continuous probabilistic and discrete probabilistic task are R² of 0.41 and 0.72, respectively.

Not that variability in the "success clamp" doe not change across ages (Figure 4C) and does not contribute to the learning effect (Figure 4F). However, it is regarded as reflecting motor noise (Figure SC), which then decreases over age from the model fitting (Figure 5B). How do we reconcile these contradictions? Again, this calls the model fitting into question.

For the success clamp, we only have 9 trials to calculate variability which limits our power to detect significance with age. In contrast, the model uses all 120 trials to estimate motor noise. There is a downward trend with age in the behavioral data which we now show overlaid on the fits of the model for both probabilistic conditions (Figure 5—figure Supplement 4) and Figure 6—figure Supplement 4). These show a reasonable match and although the variance explained is 1 6 and 56% (we limit to 9 trials so as to match the fail clamp), the correlations are 0.52 and 0.78 suggesting we have reasonable relation although there may be other small sources of variability not captured in the model.

Figure 5C: it appears one bivariate outlier contributes a lot to the overall significant correlation here for the "success clamp".

Recalculating after removing that point in original Fig 5C was still significant and we feel the plots mentioned in the previous point add useful information to this issue. With the new model this figure has changed.

It is still a concern that the young children did not understand the instructions. Nine 3-to-8 children (out of 48) were better explained by the noisy only model than the full model. In contrast, ten of the rest of the participants (out of 98) were better explained by the noisy-only model. It appears that there is a higher percentage of the "young" children who didn't get the instruction than the older ones.

Thank you for this comment. We did take participant comprehension of the task into consideration during the task design. We specifically designed it so that the instructions were simple and straight forward. The child simply needs to understand the underlying goal to make the video clip play as often as possible and that they must move the penguin to certain positions to get it to play. By having a very simple task goal, we are able to test a naturalistic response to reinforcement in the absence of an explicit strategy in a task suited even for young children.

We used the updated reinforcement learning model to assess whether an individual's performance is consistent with understanding the task. In the case of a child who does not understand the task, we expect that they simply have motor noise on their reach, and crucially, that they would not explore more after failure, nor update their reach after success. Therefore, we used a likelihood ratio test to examine whether the preferred model was significantly better at explaining each participant's data compared to the model variant which had only motor noise (Model 1). Focusing on only the youngest children (age 3-5), this analysis showed that that 43, 59, 65 and 86% of children (out of N = 21, 22, 20 and 21 ) for the continuous probabilistic, discrete probabilistic, continuous deterministic, and discrete deterministic conditions, respectively, were better fit with the preferred model, indicating non-zero exploration after failure. In the 3-5 year old group for the discrete deterministic condition, 18 out of 21 had performance better fit by the preferred model, suggesting this age group understands the basic task of moving in different directions to find a rewarding location.

The reduced numbers fit by the preferred model for the other conditions likely reflects differences in the task conditions (continuous and/or probabilistic) rather than a lack of understanding of the goal of the task. We include this analysis as a new subsection at the end of the Results.

Supplementary Figure 2: the first panel should belong to a 3-year-old not a 5-year-old? How are these panels organized? This is kind of confusing.

Thank you for this comment. Figure 2—figure Supplement 1 and Figure 2—figure Supplement 2 are arranged with devices in the columns and a sample from each age bin in the rows. For example in Figure 2—figure Supplement 1, column 1, row 1 is a mouse using participant age 3 to 5 years old while column 3, row 2 is a touch screen using participant age 6 to 8 years old. We have edited the labeling on both figures to make the arrangement of the data more clear.

Line 222: make this a complete sentence.

This sentence has been edited to a complete sentence.

Line 331: grammar.

This sentence has been edited for grammar.

Author response:

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

We extend our sincere thanks to the editor, referees for eLife, and other commentators who have written evaluations of this manuscript, either in whole or in part. Sources of these comments were highly varied, including within the bioRxiv preprint server, social media (including many comments received on X/Twitter and some YouTube presentations and interviews), comments made by colleagues to journalists, and also some reviews of the work published in other academic journals. Some of these are formal and referenced with citations. Others were informal but nonetheless expressed perspectives that helped enable us to revise the manuscript with the inclusion of broader perspectives than the formal review process. It is beyond the scope of this summary to list every one of these, which have often been brought to the attention of different coauthors, but we begin by acknowledging the very wide array of peer and public commentary that have contributed to this work. The reaction speaks to a broad interest in open discussion and review of preprints. 

As we compiled this summary of changes to the manuscript, we recognized that many colleagues made comments about the process of preprint dissemination and evaluation rather than the data or analyses in the manuscript. Addressing such comments is outside the scope of this revised manuscript, but we do feel that a broader discussion of these comments would be valuable in another venue. Many commentators have expressed confusion about the eLife system of evaluation of preprints, which differs from the editorial acceptance or rejection practiced in most academic journals. As authors in many different nations, in varied fields, and in varied career stages, we ourselves are still working to understand how the academic publication landscape is changing, and how best to prepare work for new models of evaluation and dissemination. 

The manuscript and coauthor list reflect an interdisciplinary collaboration. Analyses presented in the manuscript come from a wide range of scientific disciplines. These range from skeletal inventory, morphology, and description, spatial taphonomy, analysis of bone fracture patterns and bone surface modifications, sedimentology, geochemistry, and traditional survey and mapping. The manuscript additionally draws upon a large number of previous studies of the Rising Star cave system and the Dinaledi Subsystem, which have shaped our current work. No analysis within any one area of research stands alone within this body of work: all are interpreted in conjunction with the outcomes of other analyses and data from other areas of research. Any single analysis in isolation might be consistent with many different hypotheses for the formation of sediments and disposition of the skeletal remains. But testing a hypothesis requires considering all data in combination and not leaving out data that do not fit the hypothesis. We highlight this general principle at the outset because a number of the comments from referees and outside specialists have presented alternative hypotheses that may arguably be consistent with one kind of analysis that we have presented, while seeming to overlook other analyses, data, or previous work that exclude these alternatives. In our revision, we have expanded all sections describing results to consider not only the results of each analysis, but how the combination of data from different kinds of analysis relate to hypotheses for the deposition and subsequent history of the Homo naledi remains. We address some specific examples and how we have responded to these in our summary of changes below. 

General organization:

The referee and editor comments are mostly general and not line-by-line questions, and we have compiled them and treated them as a group in this summary of changes, except where specifically noted. 

The editorial comments on the previous version included the suggestion that the manuscript should be reorganized to test “natural” (i.e. noncultural) hypotheses for the situations that we examine. The editorial comment suggested this as a “null hypothesis” testing approach. Some outside comments also viewed noncultural deposition as a null hypothesis to be rejected. We do not concur that noncultural processes should be construed as a null hypothesis, as we discuss further below. However, because of the clear editorial opinion we elected to revise the manuscript to make more explicit how the data and analyses test noncultural depositional hypotheses first, followed by testing of cultural hypotheses. This reorganization means that the revised manuscript now examines each hypothesis separately in turn. 

Taking this approach resulted in a substantial reorganization of the “Results” section of the manuscript. The “Results” section now begins with summaries of analyses and data conducted on material from each excavation area. After the presentation of data and analyses from each area, we then present a separate section for each of several hypotheses for the disposition and sedimentary context of the remains. These hypotheses include deposition of bodies upon a talus (as hypothesized in some previous work), slow sedimentary burial on a cave floor or within a natural depression, rapid burial by gravity-driven slumping, and burial of naturally mummified remains. We then include sections to test the hypothesis of primary cultural burial and secondary cultural burial. This approach adds substantial length to the Results. While some elements may be repeated across sections, we do consider the new version to be easier to take piece by piece for a reader trying to understand how each hypothesis relates to the evidence. 

The Results section includes analyses on several different excavation areas within the Dinaledi Subsystem. Each of these presents somewhat different patterns of data. We conceived of this manuscript combining these distinct areas because each of them provides information about the formation history of the Homo naledi-associated sediments and the deposition of the Homo naledi remains. Together they speak more strongly than separately. In the previous version of the manuscript, two areas of excavation were considered in detail (Dinaledi Feature 1 and the Hill Antechamber Feature), with a third area (the Puzzle Box area) included only in the Discussion and with reference to prior work. We now describe the new work undertaken after the 2013-2014 excavations in more detail. This includes an overview of areas in the Hill Antechamber and Dinaledi Chamber that have not yielded substantial H. naledi remains and that thereby help contextualize the spatial concentration of H. naledi skeletal material. The most substantial change in the data presented is a much expanded reanalysis of the Puzzle Box area. This reanalysis provides greater clarity on how previously published descriptions relate to the new evidence. The reanalysis also provides the data to integrate the detailed information on bone identification fragmentation, and spatial taphonomy from this area with the new excavation results from the other areas. 

In addition to Results, the reorganization also affected the manuscript’s Introduction section. Where the previous version led directly from a brief review of Pleistocene burial into the description of the results, this revised manuscript now includes a review of previous studies of the Rising Star cave system. This review directly addresses referee comments that express some hesitation to accept previous results concerning the structure and formation of sediments, the accessibility of the Dinaledi Subsystem, the geochronological setting of the H. naledi remains, and the relation of the Dinaledi Subsystem to nearby cave areas. Some parts of this overview are further expanded in the Supplementary Information to enable readers to dive more deeply into the previous literature on the site formation and geological configuration of the Rising Star cave system without needing to digest the entirety of the cited sources. 

The Discussion section of the revised manuscript is differentiated from Results and focuses on several areas where the evidence presented in this study may benefit from greater context. One new section addresses hypothesis testing and parsimony for Pleistocene burial evidence, which we address at greater length in this summary below. The majority of the Discussion concerns the criteria for recognizing evidence for burial as applied in other studies. In this research we employ a minimal definition but other researchers have applied varied criteria. We consider whether these other criteria have relevance in light of our observations and whether they are essential to the recognition of burial evidence more broadly. 

Vocabulary:

We introduce the term “cultural burial” in this revised manuscript to refer to the burial of dead bodies as a mortuary practice. “Burial” as an unmodified term may refer to the passive covering of remains by sedimentary processes. Use of the term “intentional burial” would raise the question of interpreting intent, which we do not presume based on the evidence presented in this research. The relevant question in this case is whether the process of burial reflects repeated behavior by a group. As we received input from various colleagues it became clear that burial itself is a highly loaded term. In particular there is a common assumption within the literature and among professionals that burial must by definition be symbolic. We do not take any position on that question in this manuscript, and it is our hope that the term “cultural burial” may focus the conversation around the extent that the behavioral evidence is repeated and patterned. 

Sedimentology and geochemistry of Dinaledi Feature 1:

Reviewer 4 provided detailed comments on the sedimentological and geochemical context that we report in the manuscript. One outside review (Foecke et al. 2024) included some of the points raised by reviewer 4, and additionally addressed the reporting of geochemical and sedimentological data in previous work that we cite. 

To address these comments we have revised the sedimentary context and micromorphology of sediments associated with Dinaledi Feature 1. In the new text we demonstrate the lack of microstratigraphy (supported by grain size analysis) in the unlithified mud clast breccia (UMCB), while such a microstratigraphy is observed in the laminated orange-red mudstones (LORM) that contribute clasts to the UMCB. Thus, we emphasize the presence and importance of a laterally continuous layer of LORM nature occurring at a level that appears to be the maximum depth of fossil occurrence. This layer is severely broken under extensive accumulation of fossils such as Feature 1 and only evidenced by abundant LORM clasts within and around the fossils. 

We have completely reworked the geochemical context associated with Feature 1 following the comments of reviewer 4. We described the variations and trends observed in the major oxides separate from trace and rare-earth elements. We used Harker variations plots to assess relationships between these element groups with CaO and Zn, followed by principal component analysis of all elements analyzed. The new geochemical analysis clearly shows that Feature 1 is associated with localized trace element signatures that exist in the sediments only in association with the fossil bones, which suggests lack of postdepositional mobilization of the fossils and sediments. We additionally have included a fuller description of XRF methods. 

To clarify the relation of all results to the features described in this study, we removed the geochemical and sedimentological samples from other sites within the Dinaledi Subsystem. These localities within the fissure network represent only surface collection of sediment, as no excavation results are available from those sites to allow for comparison in the context of assessing evidence of burial. These were initially included for comparison, but have now been removed to avoid confusion.  

Micromorphology of sediments:

Some referees (1, 3, and 4) and other commentators (including Martinón-Torres et al. 2024) have suggested that the previous manuscript was deficient due to an insufficient inclusion of micromorphological analysis of sediments. Because these commentators have emphasized this kind of evidence as particularly important, we review here what we have included and how our revision has addressed this comment. Previous work in the Dinaledi Chamber (Dirks et al., 2015; 2017) included thin section illustrations and analysis of sediment facies, including sediments in direct association with H. naledi remains within the Puzzle Box area. The previous work by Wiersma and coworkers (2020) used micromorphological analysis as one of several approaches to test the formation history of Unit 3 sediments in the Dinaledi Subsystem, leading to the interpretation of autobrecciation of earlier Unit 1 sediment. In the previous version of this manuscript we provided citations to this earlier work. The previous manuscript also provided new thin section illustrations of Unit 3 sediment near Dinaledi Feature 1 to place the disrupted layer of orange sediment (now designated the laminated orange silty mudstone unit) into context. 

In the new revised manuscript we have added to this information in three ways. First, as noted above in response to reviewer 4, we have revised and added to our discussion of micromorphology within and adjacent to the Dinaledi Feature 1. Second, we have included more discussion in the Supplementary Information of previous descriptions of sediment facies and associated thin section analysis, with illustrations from prior work (CC-BY licensed) brought into this paper as supplementary figures, so that readers can examine these without following the citations. Third, we have included Figure 10 in the manuscript which includes six panels with microtomographic sections from the Hill Antechamber Feature. This figure illustrates the consistency of sub-unit 3b sediment in direct contact with H. naledi skeletal material, including anatomically associated skeletal elements, with previous analyses that demonstrate the angular outlines and chaotic orientations of LORM clasts. It also shows density contrasts of sediment in immediate contact with some skeletal elements, the loose texture of this sediment with air-filled voids, and apparent invertebrate burrowing activity. To our knowledge this is the first application of microtomography to sediment structure in association with a Pleistocene burial feature. 

To forestall possible comments that the revised manuscript does not sufficiently employ micromorphological observations, or that any one particular approach to micromorphology is the standard, we present here some context from related studies of evidence from other research groups working at varied sites in Africa, Europe, and Asia. Hodgkins et al. (2021) noted: “Only a handful of micromorphological studies have been conducted on human burials and even fewer have been conducted on suspected burials from Paleolithic or hunter-gatherer contexts.” In that study, one supplementary figure with four photomicrographs of thin sections of sediments was presented. Interpretation of the evidence for a burial pit by Hodgkins et al. (2021) noted the more open microstructure of sediment but otherwise did not rely upon the thin section data in characterizing the sediments associated with grave fill. Martinón-Torres et al. (2021) included one Extended Data figure illustrating thin sections of sediments and bone, with two panels showing sediments (the remainder showing bone histology). The micromorphological analysis presented in the supplementary information of that paper was restricted to description of two microfacies associated with the proposed “pit” in that study. That study did carry out microCT scanning of the partially-prepared skeletal remains but did not report any sediment analysis from the microtomographic results. Maloney et al. (2022) reported no micromorphological or thin section analysis. Pomeroy et al. (2020a) included one illustration of a thin section; this study may be regarded as a preliminary account rather than a full description of the work undertaken. Goldberg et al. (2017) analyzed the geoarchaeology of the Roc de Marsal deposits in which possible burial-associated sediments had been fully excavated in the 1960s, providing new morphological assessments of sediment facies; the supplementary information to this work included five scans (not microscans) of sediment thin sections and no microphotographs. Fewlass et al. (2023) presented no thin section or micromorphological illustrations or methods. In summary of this research, we note that in one case micromorphological study provided observations that contributed to the evidence for a pit, in other cases micromorphological data did not test this hypothesis, and many researchers do not apply micromorphological techniques in their particular contexts. 

Sediment micromorphology is a growing area of research and may have much to provide to the understanding of ancient burial evidence as its standards continue to develop (Pomeroy et al. 2020b). In particular microtomographic analysis of sediments, as we have initiated in this study, may open new horizons that are not possible with more destructive thin-section preparation. In this manuscript, the thin section data reveals valuable evidence about the disruption of sediment structure by features within the Dinaledi Chamber, and microtomographic analysis further documents that the Hill Antechamber Feature reflects similar processes, in addition to possible post-burial diagenesis and invertebrate activity. Following up in detail on these processes will require further analysis outside the scope of this manuscript. 

Access into the Dinaledi Subsystem:

Reviewer 1 emphasizes the difficulty of access into the Dinaledi Subsystem as a reason why the burial hypothesis is not parsimonious. Similar comments have been made by several outside commentators who question whether past accessibility into the Dinaledi Subsystem may at one time have been substantially different from the situation documented in previous work. Several pieces of evidence are relevant to these questions and we have included some discussion of them in the Introduction, and additionally include a section in the Supplementary Information (“Entrances to the cave system”) to provide additional context for these questions. Homo naledi remains are found not only within the Dinaledi Subsystem but also in other parts of the cave system including the Lesedi Chamber, which is similarly difficult for non-expert cavers to access. The body plan, mass, and specific morphology of H. naledi suggest that this species would be vastly more suited to moving and climbing within narrow underground passages than living people. On this basis it is not unparsimonious to suggest that the evidence resulted from H. naledi activity within these spaces. We note that the accessibility of the subsystem is not strictly relevant to the hypothesis of cultural burial, although the location of the remains does inform the overall context which may reflect a selection of a location perceived as special in some way. 

Stuffing bodies down the entry to the subsystem:

Reviewer 3 suggests that one explanation for the emplacement of articulated remains at the top of the sloping floor of the Hill Antechamber is that bodies were “stuffed” into the chute that comprises the entry point of the subsystem and passively buried by additional accumulation of remains. This was one hypothesis presented in earlier work (Dirks et al. 2015) and considered there as a minimal explanation because it did not entail the entry of H. naledi individuals into the subsystem. The further exploration (Elliott et al. 2021) and ongoing survey work, as well as this manuscript, all have resulted in data that rejects this hypothesis. The revised manuscript includes a section in the results “Deposition upon a talus with passive burial” that examines this hypothesis in light of the data. 

Recognition of pits:

Referee 3 and 4 and several additional commentators have emphasized that the recognition of pit features is necessary to the hypothesis of burial, and questioned whether the data presented in the manuscript were sufficient to demonstrate that pits were present. We have revised the manuscript in several ways to clarify how all the different kinds of evidence from the subsystem test the hypothesis that pits were present. This includes the presentation of a minimal definition of burial to include a pit dug by hominins, criteria for recognizing that a pit was present, and an evaluation of the evidence in each case to make clear how the evidence relates to the presence of a pit and subsequent infill. As referee 3 notes, it can be challenging to recognize a pit when sediment is relatively homogeneous. This point was emphasized in the review by Pomeroy and coworkers (2020b), who reflected on the difficulty seeing evidence for shallow pits constructed by hominins, and we have cited this in the main text. As a result, the evidence for pits has been a recurrent topic of debate for most Pleistocene burial sites. However in addition to the sedimentological and contextual evidence in the cases we describe, the current version also reflects upon other possible mechanisms for the accumulation of bones or bodies. The data show that the sedimentary fill associated with the H. naledi remains in the cases we examine could not have passively accumulated slowly and is not indicative of mass movement by slumping or other high-energy flow. To further put these results into context, we added a section to the Discussion that briefly reviews prior work on distinguishing pits in Pleistocene burial contexts, including the substantial number of sites with accepted burial evidence for which no evidence of a pit is present. 

Extent of articulation and anatomical association:

We have added significantly greater detail to the descriptions of articulated remains and orientation of remains in order to describe more specifically the configuration of the skeletal material. We also provide 14 figures in main text (13 of them new) to illustrate the configuration of skeletal remains in our data. For the Puzzle Box area, this now includes substantial evidence on the individuation of skeletal fragments, which enables us to illustrate the spatial configuration of remains associated with the DH7 partial skeleton, as well as the spatial position of fragments refitted as part of the DH1, DH2, DH3, and DH4 crania. For Dinaledi Feature 1 and the Hill Antechamber Feature we now provide figures that key skeletal parts as identified, including material that is unexcavated where possible, and a skeletal part representation figure for elements excavated from Dinaledi Feature 1. 

Archaeothanatology:

Reviewer 2 suggests that a greater focus on the archaeothanatology literature would be helpful to the analysis, with specific reference to the sequence of joint disarticulation, the collapse of sediment and remains into voids created by decomposition, and associated fragmentation of the remains. In the revised manuscript we have provided additional analysis of the Hill Antechamber Feature with this approach in mind. This includes greater detail and illustration of our current hypothesis for individuation of elements. We now discuss a hypothesis of body disposition, describe the persistent joints and articulation of elements, and examine likely decomposition scenarios associated with these remains. Additionally, we expand our description and illustration of the orientation of remains and degree of anatomical association and articulation within Dinaledi Feature 1. For this feature and for the Hill Antechamber Feature we have revised the text to describe how fracturing and crushing patterns are consistent with downward pressure from overlying sediment and material. In these features, postdepositional fracturing occurred subsequent to the decomposition of soft tissue and partial loss of organic integrity of the bone. We also indicate that the loss by postdepositional processes of most long bone epiphyses, vertebral bodies, and other portions of the skeleton less rich in cortical bone, poses a challenge for testing the anatomical associations of the remaining elements. This is a primary reason why we have taken a conservative approach to identification of elements and possible associations. 

A further aspect of the site revealed by our analysis is the selective reworking of sediments within the Puzzle Box area subsequent to the primary deposition of some bodies. The skeletal evidence from this area includes body parts with elements in anatomical association or articulation, juxtaposed closely with bone fragments at varied pitch and orientation. This complexity of events evidenced within this area is a challenge for approaches that have been developed primarily based on comparative data from single-burial situations. In these discussions we deepen our use of references as suggested by the referee.   

Burial positions:

Reviewer 2 further suggests that illustrations of hypothesized burial positions would be valuable. We recognize that a hypothesized burial position may be an appealing illustration, and that some recent studies have created such illustrations in the context of their scientific articles. However such illustrations generally include a great deal of speculation and artist imagination, and tend to have an emotive character. We have added more discussion to the manuscript of possible primary disposition in the case of the Hill Antechamber Feature as discussed above. We have not created new illustrations of hypothesized burial positions for this revision. 

Carnivore involvement:

Referee 1 suggests that the manuscript should provide further consideration of whether carnivore activity may have introduced bones or bodies into the cave system. The reorganized Introduction now includes a review of previous work, and an expanded discussion within the Supplementary Information (“Hypotheses tested in previous work”). This includes a review of literature on the topic of carnivore accumulation and the evidence from the Dinaledi and Lesedi Chamber that rejects this hypothesis. 

Water transport and mud:

The eLife referees broadly accepted previous work showing that water inundation or mass flow of water-saturated sediment did not occur within the history of Unit 2 and 3 sediments, including those associated with H. naledi remains. However several outside commentators did refer specifically to water flow or mud flow as a mechanism for slumping of deposits and possible sedimentary covering of the remains. To address these comments we have added a section to the

Supplementary Information (“Description of the sedimentary deposits of the Dinaledi Subsystem”) that reviews previous work on the sedimentary units and formation processes documented in this area. We also include a subsection specifically discussing the term “mud” as used in the description of the sedimentology within the system, as this term has clearly been confusing for nonspecialists who have read and commented on the work. We appreciate the referees’ attention to the previous work and its terminology.  

Redescription of areas of the cave system:

Reviewer 1 suggests that a detailed reanalysis of all portions of the cave system in and around the Dinaledi Subsystem is warranted to reject the hypothesis that bodies entered the space passively and were scattered from the floor by natural (i.e. noncultural) processes. The referee suggests that National Geographic could help us with these efforts. To address this comment we have made several changes to the manuscript. As noted above, we have added material in Supplementary Information to review the geochronology of the Dinaledi Subsystem and nearby Dragon’s Back Chamber, together with a discussion of the connections between these spaces. 

Most directly in response to this comment we provide additional documentation of the possibility of movement of bodies or body parts by gravity within the subsystem itself. This includes detailed floor maps based on photogrammetry and LIDAR measurement, where these are physically possible, presented in Figures 2 and 3. In some parts of the subsystem the necessary equipment cannot be used due to the extremely confined spaces, and for these areas our maps are based on traditional survey methods. In addition to plan maps we have included a figure showing the elevation of the subsystem floor in a cross-section that includes key excavation areas, showing their relative elevation. All figures that illustrate excavation areas are now keyed to their location with reference to a subsystem plan. These data have been provided in previous publications but the visualization in the revised manuscript should make the relationship of areas clear for readers. The Introduction now includes text that discusses the configuration of the Hill Antechamber, Dinaledi Chamber, and nearby areas, and also discusses the instances in which gravity-driven movement may be possible, at the same time reviewing that gravity-driven movement from the entry point of the subsystem to most of the localities with hominin skeletal remains is not possible. 

Within the Results, we have added a section on the relationship of features to their surroundings in order to assist readers in understanding the context of these bone-bearing areas and the evidence this context brings to the hypothesis in question. We have also included within this new section a discussion of the discrete nature of these features, a question that has been raised by outside commentators. 

Passive sedimentation upon a cave floor or within a natural depression:

Reviewer 3 suggests that the situation in the Dinaledi Subsystem may be similar to a European cave where a cave bear skeleton might remain articulated on a cave floor (or we can add, within a hollow for hibernation), later to be covered in sediment. The reviewer suggests that articulation is therefore no evidence of burial, and suggests that further documentation of disarticulation processes is essential to demonstrating the processes that buried the remains. We concur that articulation by itself is not sufficient evidence of cultural burial. To address this comment we have included a section in the Results that tests the hypothesis that bodies were exposed upon the cave floor or within a natural depression. To a considerable degree, additional data about disarticulation processes subsequent to deposition are provided in our reanalysis of the Puzzle Box area, including evidence for selective reworking of material after burial. 

Postdepositional movement and floor drains:

Reviewer 3 notes that previous work has suggested that subsurface floor drains may have caused some postdepositional movement of skeletal remains. The hypothesis of postdepositional slumping or downslope movement has also been discussed by some external commentators (including Martinón-Torres et al. 2024). We have addressed this question in several places within the revised manuscript. As we now review, previous discussion of floor drains attempted to explain the subvertical orientation of many skeletal elements excavated from the Puzzle Box area. The arrangement of these bones reflects reworking as described in our previous work, and without considering the possibility of reworking by hominins, one mechanism that conceivably might cause reworking was downward movement of sediments into subsurface drains. Further exploration and mapping, combined with additional excavation into the sediments beneath the Puzzle Box area provided more information relevant to this hypothesis. In particular this evidence shows that subsurface drains cannot explain the arrangement of skeletal material observed within the Puzzle Box area. As now discussed in the text, the reworking is selective and initiated from above rather than below. This is best explained by hominin activity subsequent to burial. 

In a new section of the Results we discuss slumping as a hypothesis for the deposition of the remains. This includes discussion of downslope movement within the Hill Antechamber and the idea that floor drains may have been a mechanism for sediment reworking in and around the Puzzle Box area and Dinaledi Feature 1. As described in this section the evidence does not support these hypotheses. 

Hypothesis testing and parsimony:

Referees 1 and 3 and the editorial guidance all suggested that a more appropriate presentation would adopt a null hypothesis and test it. The specific suggestion that the null hypothesis should be a natural sedimentary process of deposition was provided not only by these reviewers but also by some outside commentators. To address this comment, we have edited the manuscript in two ways. The first is the addition of a section to the Discussion that specifically discusses hypothesis testing and parsimony as related to Pleistocene evidence of cultural burial. This includes a brief synopsis of recent disciplinary conversations and citation of work by other groups of authors, none of whom adopted this “null hypothesis” approach in their published work. 

As we now describe in the manuscript, previous work on the Dinaledi evidence never assumed any role for H. naledi in the burial of remains. Reading the reviewer reports caused us to realize that this previous work had followed exactly the “null hypothesis” approach that some suggested we follow. By following this null hypothesis approach, we neglected a valuable avenue of investigation. In retrospect, we see how this approach impeded us from understanding the pattern of evidence within the Puzzle Box area. Thus in the revised manuscript we have mentioned this history within the Discussion and also presented more of the background to our previous work in the Introduction. Hopefully by including this discussion of these issues, the manuscript will broaden conversation about the relation of parsimony to these issues. 

Language and presentation style:

Reviewer 4 criticizes our presentation, suggesting that the text “gives the impression that a hypothesis was formulated before data were collected.” Other outside commentators have mentioned this notion also, including Martinón-Torres et al. (2024) who suggest that the study began from a preferred hypothesis and gathered data to support it. The accurate communication of results and hypotheses in a scientific article is a broader issue than this one study. Preferences about presentation style vary across fields of study as well as across languages. We do not regret using plain language where possible. In any study that combines data and methods from different scientific disciplines, the use of plain language is particularly important to avoid misunderstandings where terms may mean different things in different fields. 

The essential question raised by these comments is whether it is appropriate to present the results of a study in terms of the hypothesis that is best supported. As noted above, we read carefully many recent studies of Pleistocene burial evidence. We note that in each of these studies that concluded that burial is the best hypothesis, the authors framed their results in the same way as our previous manuscript: an introduction that briefly reviews background evidence for treatment of the dead, a presentation of results focused on how each analysis supports the hypothesis of burial for the case, and then in some (but not all) cases discussion of why some alternative hypotheses could be rejected. We do not infer from this that these other studies started from a presupposition and collected data only to confirm it. Rather, this is a simple matter of presentation style. 

The alternative to this approach is to present an exhaustive list of possible hypotheses and to describe how the data relate to each of them, at the end selecting the best. This is the approach that we have followed in the revised manuscript, as described above under the direction of the reviewer and editorial guidance. This approach has the advantage of bringing together evidence in different combinations to show how each data point rejects some hypotheses while supporting others. It has the disadvantage of length and repetition. 

Possible artifact:

We have chosen to keep the description of the possible artifact associated with the Hill Antechamber Feature in the Supplementary Information. We do this while acknowledging that this is against the opinion of reviewer 4, who felt the description should be removed unless the object in question is fully excavated and physically analyzed. The previous version of the manuscript did not rely upon the stone as positive evidence of grave goods or symbolic content, and it noted that the data do not test whether the possible artifact was placed or was intentionally modified. However this did not satisfy reviewer 4, and some outside commentators likewise asserted that the object must be a “geofact” and that it should be removed. 

We have three arguments against this line of thinking. First, we do not omit data from our reporting. Whether Homo naledi shaped the rock or not, used it as a tool or not, whether the rock was placed with the body or not, it is unquestionably there. Omitting this one object from the report would be simply dishonest. Second, the data on this rock are at 16 micron resolution. While physical inspection of its surface may eventually reveal trace evidence and will enable better characterization of the raw material, no mode of surface scanning will produce better evidence about the object’s shape. Third, the position of this possible artifact within the feature provides significant information about the deposition of the skeletal material and associated sediments. The pitch, orientation, and position of the stone is not consistent with slow deposition but are consistent with the hypothesis that the surrounding sediment was rapidly emplaced at the same time as the articulated elements less than 2 cm away. 

In the current version, we have redoubled our efforts to provide information about the position and shape of this stone while not presupposing the intentionality of its shape or placement. We add here that the attitude expressed by referee 4 and other commentators, if followed at other sites, would certainly lead to the loss or underreporting of evidence, which we are trying to avoid.  

Consistency versus variability of behavior:

As described in the revised manuscript, different features within the Dinaledi Subsystem exhibit some shared characteristics. At the same time, they vary in positioning, representation of individuals and extent of commingling. Other localities within the subsystem and broader cave system present different evidence. Some commentators have questioned whether the patterning is consistent with a single common explanation, or whether multiple explanations are necessary. To address this line of questioning, we have added several elements to the manuscript. We created a new section on secondary cultural burial, discussing whether any of the situations may reflect this practice. In the Discussion, we briefly review the ways in which the different features support the involvement of H. naledi without interpreting anything about the intentionality or meaning of the behavior. We further added a section to the Discussion to consider whether variation among the features reflects variation in mortuary practices by H. naledi. One aspect of this section briefly cites variation in the location and treatment of skeletal remains at other sites with evidence of burial. 

Grave goods:

Some commentators have argued that grave goods are a necessary criterion for recognizing evidence of ancient burial. We added a section to the Discussion to review evidence of grave goods at other Pleistocene sites where burial is accepted. 

References:

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Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public review):

Wang et al., recorded concurrent EEG-fMRI in 107 participants during nocturnal NREM sleep to investigate brain activity and connectivity related to slow oscillations (SO), sleep spindles, and in particular their co-occurrence. The authors found SO-spindle coupling to be correlated with increased thalamic and hippocampal activity, and with increased functional connectivity from the hippocampus to the thalamus and from the thalamus to the neocortex, especially the medial prefrontal cortex (mPFC). They concluded the brain-wide activation pattern to resemble episodic memory processing, but to be dissociated from task-related processing and suggest that the thalamus plays a crucial role in coordinating the hippocampal-cortical dialogue during sleep.

The paper offers an impressively large and highly valuable dataset that provides the opportunity for gaining important new insights into the network substrate involved in SOs, spindles, and their coupling. However, the paper does unfortunately not exploit the full potential of this dataset with the analyses currently provided, and the interpretation of the results is often not backed up by the results presented. I have the following specific comments.

Thank you for your thoughtful and constructive feedback. We greatly appreciate your recognition of the strengths of our dataset and findings Below, we address your specific comments and provide responses to each point you raised to ensure our methods and results are as transparent and comprehensible as possible. We hope these revisions address your comments and further strengthen our manuscript. Thank you again for the constructive feedback.

(1) The introduction is lacking sufficient review of the already existing literature on EEG-fMRI during sleep and the BOLD-correlates of slow oscillations and spindles in particular (Laufs et al., 2007; Schabus et al., 2007; Horovitz et al., 2008; Laufs, 2008; Czisch et al., 2009; Picchioni et al., 2010; Spoormaker et al., 2010; Caporro et al., 2011; Bergmann et al., 2012; Hale et al., 2016; Fogel et al., 2017; Moehlman et al., 2018; Ilhan-Bayrakci et al., 2022). The few studies mentioned are not discussed in terms of the methods used or insights gained.

We acknowledge the need for a more comprehensive review of prior EEG-fMRI studies investigating BOLD correlates of slow oscillations and spindles. However, these articles are not all related to sleep SO or spindle. Articles (Hale et al., 2016; Horovitz et al., 2008; Laufs, 2008; Laufs, Walker, & Lund, 2007; Spoormaker et al., 2010) mainly focus on methodology for EEG-fMRI, sleep stages, or brain networks, which are not the focus of our study. Thank you again for your attention to the comprehensiveness of our literature review, and we will expand the introduction to include a more detailed discussion of the existing literature, ensuring that the contributions of previous EEG-fMRI sleep studies are adequately acknowledged.  

Introduction, Page 4 Lines 62-76

“Investigating these sleep-related neural processes in humans is challenging because it requires tracking transient sleep rhythms while simultaneously assessing their widespread brain activation. Recent advances in simultaneous EEG-fMRI techniques provide a unique opportunity to explore these processes. EEG allows for precise event-based detection of neural signal, while fMRI provides insight into the broader spatial patterns of brain activation and functional connectivity (Horovitz et al., 2008; Huang et al., 2024; Laufs, 2008; Laufs, Walker, & Lund, 2007; Schabus et al., 2007; Spoormaker et al., 2010). Previous EEG-fMRI studies on sleep have focused on classifying sleep stages or examining the neural correlates of specific waves (Bergmann et al., 2012; Caporro et al., 2012; Czisch et al., 2009; Fogel et al., 2017; Hale et al., 2016; Ilhan-Bayrakcı et al., 2022; Moehlman et al., 2019; Picchioni et al., 2011). These studies have generally reported that slow oscillations are associated with widespread cortical and subcortical BOLD changes, whereas spindles elicit activation in the thalamus, as well as in several cortical and paralimbic regions. Although these findings provide valuable insights into the BOLD correlates of sleep rhythms, they often do not employ sophisticated temporal modeling (Huang et al., 2024), to capture the dynamic interactions between different oscillatory events, e.g., the coupling between SOs and spindles.”

(2) The paper falls short in discussing the specific insights gained into the neurobiological substrate of the investigated slow oscillations, spindles, and their interactions. The validity of the inverse inference approach ("Open ended cognitive state decoding"), assuming certain cognitive functions to be related to these oscillations because of the brain regions/networks activated in temporal association with these events, is debatable at best. It is also unclear why eventually only episodic memory processing-like brain-wide activation is discussed further, despite the activity of 16 of 50 feature terms from the NeuroSynth v3 dataset were significant (episodic memory, declarative memory, working memory, task representation, language, learning, faces, visuospatial processing, category recognition, cognitive control, reading, cued attention, inhibition, and action).

Thank you for pointing this out, particularly regarding the use of inverse inference approaches such as “open-ended cognitive state decoding.” Given the concerns about the indirectness of this approach, we decided to remove its related content and results from Figure 3 in the main text and include it in Supplementary Figure 7. We will refocus the main text on direct neurobiological insights gained from our EEG-fMRI analyses, particularly emphasizing the hippocampal-thalamocortical network dynamics underlying SO-spindle coupling, and we will acknowledge the exploratory nature of these findings and highlight their limitations.

Discussion, Page 17-18 Lines 323-332

“To explore functional relevance, we employed an open-ended cognitive state decoding approach using meta-analytic data (NeuroSynth: Yarkoni et al. (2011)). Although this method usefully generates hypotheses about potential cognitive processes, particularly in the absence of a pre- and post-sleep memory task, it is inherently indirect. Many cognitive terms showed significant associations (16 of 50), such as “episodic memory,” “declarative memory,” and “working memory.” We focused on episodic/declarative memory given the known link with hippocampal reactivation (Diekelmann & Born, 2010; Staresina et al., 2015; Staresina et al., 2023). Nonetheless, these inferences regarding memory reactivation should be interpreted cautiously without direct behavioral measures. Future research incorporating explicit tasks before and after sleep would more rigorously validate these potential functional claims.”

(3) Hippocampal activation during SO-spindles is stated as a main hypothesis of the paper - for good reasons - however, other regions (e.g., several cortical as well as thalamic) would be equally expected given the known origin of both oscillations and the existing sleep-EEG-fMRI literature. However, this focus on the hippocampus contrasts with the focus on investigating the key role of the thalamus instead in the Results section.

We appreciate your insight regarding the relative emphasis on hippocampal and thalamic activation in our study. We recognize that the manuscript may currently present an inconsistency between our initial hypothesis and the main focus of the results. To address this concern, we will ensure that our Introduction and Discussion section explicitly discusses both regions, highlighting the complementary roles of the hippocampus (memory processing and reactivation) and the thalamus (spindle generation and cortico-hippocampal coordination) in SO-spindle dynamics.

Introduction, Page 5 Lines 87-103

“To address this gap, our study investigates brain-wide activation and functional connectivity patterns associated with SO-spindle coupling, and employs a cognitive state decoding approach (Margulies et al., 2016; Yarkoni et al., 2011)—albeit indirectly—to infer potential cognitive functions. In the current study, we used simultaneous EEG-fMRI recordings during nocturnal naps (detailed sleep staging results are provided in the Methods and Table S1) in 107 participants. Although directly detecting hippocampal ripples using scalp EEG or fMRI is challenging, we expected that hippocampal activation in fMRI would coincide with SO-spindle coupling detected by EEG, given that SOs, spindles, and ripples frequently co-occur during NREM sleep. We also anticipated a critical role of the thalamus, particularly thalamic spindles, in coordinating hippocampal-cortical communication.

We found significant coupling between SOs and spindles during NREM sleep (N2/3), with spindle peaks occurring slightly before the SO peak. This coupling was associated with increased activation in both the thalamus and hippocampus, with functional connectivity patterns suggesting thalamic coordination of hippocampal-cortical communication. These findings highlight the key role of the thalamus in coordinating hippocampal-cortical interactions during human sleep and provide new insights into the neural mechanisms underlying sleep-dependent brain communication. A deeper understanding of these mechanisms may contribute to future neuromodulation approaches aimed at enhancing sleep-dependent cognitive function and treating sleep-related disorders.”

Discussion, Page 16-17 Lines 292-307

“When modeling the timing of these sleep rhythms in the fMRI, we observed hippocampal activation selectively during SO-spindle events. This suggests the possibility of triple coupling (SOs–spindles–ripples), even though our scalp EEG was not sufficiently sensitive to detect hippocampal ripples—key markers of memory replay (Buzsáki, 2015). Recent iEEG evidence indicates that ripples often co-occur with both spindles (Ngo, Fell, & Staresina, 2020) and SOs (Staresina et al., 2015; Staresina et al., 2023). Therefore, the hippocampal involvement during SO-spindle events in our study may reflect memory replay from the hippocampus, propagated via thalamic spindles to distributed cortical regions.

The thalamus, known to generate spindles (Halassa et al., 2011), plays a key role in producing and coordinating sleep rhythms (Coulon, Budde, & Pape, 2012; Crunelli et al., 2018), while the hippocampus is found essential for memory consolidation (Buzsáki, 2015; Diba & Buzsá ki, 2007; Singh, Norman, & Schapiro, 2022). The increased hippocampal and thalamic activity, along with strengthened connectivity between these regions and the mPFC during SO-spindle events, underscores a hippocampal-thalamic-neocortical information flow. This aligns with recent findings suggesting the thalamus orchestrates neocortical oscillations during sleep (Schreiner et al., 2022). The thalamus and hippocampus thus appear central to memory consolidation during sleep, guiding information transfer to the neocortex, e.g., mPFC.”

(4) The study included an impressive number of 107 subjects. It is surprising though that only 31 subjects had to be excluded under these difficult recording conditions, especially since no adaptation night was performed. Since only subjects were excluded who slept less than 10 min (or had excessive head movements) there are likely several datasets included with comparably short durations and only a small number of SOs and spindles and even less combined SO-spindle events. A comprehensive table should be provided (supplement) including for each subject (included and excluded) the duration of included NREM sleep, number of SOs, spindles, and SO+spindle events. Also, some descriptive statistics (mean/SD/range) would be helpful.

We appreciate your recognition of our sample size and the challenges associated with simultaneous EEG-fMRI sleep recordings. We acknowledge the importance of transparently reporting individual subject data, particularly regarding sleep duration and the number of detected SOs, spindles, and SO-spindle events. To address this, we will provide comprehensive tables in the supplementary materials, contains descriptive information about sleep-related characteristics (Table S1), as well as detailed information about sleep waves at each sleep stage for all 107 subjects(Table S2-S4), listing for each subject:(1)Different sleep stage duration; (2)Number of detected SOs; (3)Number of detected spindles; (4)Number of detected SO-spindle coupling events; (5)Density of detected SOs; (6)Density of detected spindles; (7)Density of detected SO-spindle coupling events.

However, most of the excluded participants were unable to fall asleep or had too short a sleep duration, so they basically had no NREM sleep period, so it was impossible to count the NREM sleep duration, SO, spindle, and coupling numbers.

Supplementary Materials, Page 42-54, Table S1-S4

(5) Was the 20-channel head coil dedicated for EEG-fMRI measurements? How were the electrode cables guided through/out of the head coil? Usually, the 64-channel head coil is used for EEG-fMRI measurements in a Siemens PRISMA 3T scanner, which has a cable duct at the back that allows to guide the cables straight out of the head coil (to minimize MR-related artifacts). The choice for the 20-channel head coil should be motivated. Photos of the recording setup would also be helpful.

Thank you for your comment regarding our choice of the 20-channel head coil for EEG-fMRI measurements. We acknowledge that the 64-channel head coil is commonly used in Siemens PRISMA 3T scanners; however, the 20-channel coil was selected due to specific practical and technical considerations in our study. In particular, the 20-channel head coil was compatible with our EEG system and ensured sufficient signal-to-noise ratio (SNR) for both EEG and fMRI acquisition. The EEG electrode cables were guided through the lateral and posterior openings of the head coil, secured with foam padding to reduce motion and minimize MR-related artifacts. Moreover, given the extended nature of nocturnal sleep recordings, the 20-channel coil allowed us to maintain participant comfort while still achieving high-quality simultaneous EEG-fMRI data.

We have made this clearer in the revised manuscript. 

Methods, Page 20 Lines 385-392

“All MRI data were acquired using a 20-channel head coil on a research-dedicated 3-Tesla Siemens Magnetom Prisma MRI scanner. Earplugs and cushions were provided for noise protection and head motion restriction. We chose the 20-channel head coil because it was compatible with our EEG system and ensured sufficient signal-to-noise ratio (SNR) for both EEG and fMRI acquisition. The EEG electrode cables were guided through the lateral and posterior openings of the head coil, secured with foam padding to reduce motion and minimize MR-related artifacts. Moreover, given the extended nature of nocturnal sleep recordings, the 20-channel coil helped maintain participant comfort while still achieving high-quality simultaneous EEG-fMRI data.”

(6) Was the EEG sampling synchronized to the MR scanner (gradient system) clock (the 10 MHz signal; not referring to the volume TTL triggers here)? This is a requirement for stable gradient artifact shape over time and thus accurate gradient noise removal.

Thank you for raising this important point. We confirm that the EEG sampling was synchronized to the MR scanner’s 10 MHz gradient system clock, ensuring a stable gradient artifact shape over time and enabling accurate artifact removal. This synchronization was achieved using the standard clock synchronization interface of the EEG amplifier, minimizing timing jitter and drift. As a result, the gradient artifact waveform remained stable across volumes, allowing for more effective artifact correction during preprocessing. We appreciate your attention to this critical aspect of EEG-fMRI data acquisition.

We have made this clearer in the revised manuscript. 

Methods, Page 19-20 Lines 371-383

“EEG was recorded simultaneously with fMRI data using an MR-compatible EEG amplifier system (BrainAmps MR-Plus, Brain Products, Germany), along with a specialized electrode cap. The recording was done using 64 channels in the international 10/20 system, with the reference channel positioned at FCz. In order to adhere to polysomnography (PSG) recording standards, six electrodes were removed from the EEG cap: one for electrocardiogram (ECG) recording, two for electrooculogram (EOG) recording, and three for electromyogram (EMG) recording. EEG data was recorded at a sample rate of 5000 Hz, the resistance of the reference and ground channels was kept below 10 kΩ, and the resistance of the other channels was kept below 20 kΩ. To synchronize the EEG and fMRI recordings, the BrainVision recording software (BrainProducts, Germany) was utilized to capture triggers from the MRI scanner. The EEG sampling was synchronized to the MR scanner’s 10 MHz gradient system clock, ensuring a stable gradient artifact shape over time and enabling accurate artifact removal. This was achieved via the standard clock synchronization interface of the EEG amplifier, minimizing timing jitter and drift.”

(7) The TR is quite long and the voxel size is quite large in comparison to state-of-the-art EPI sequences. What was the rationale behind choosing a sequence with relatively low temporal and spatial resolution?

We acknowledge that our chosen TR and voxel size are relatively long and large compared to state-of-the-art EPI sequences. This decision was made to optimize the signal-to-noise ratio (SNR) and reduce susceptibility-related distortions, which are particularly critical in EEG-fMRI sleep studies where head motion and physiological noise can be substantial. A longer TR allowed us to sample whole-brain activity with sufficient coverage, while a larger voxel size helped enhance BOLD sensitivity and minimize partial volume effects in deep brain structures such as the thalamus and hippocampus, which are key regions of interest in our study. We appreciate your concern and hope this clarification provides sufficient rationale for our sequence parameters.

We have made this clearer in the revised manuscript. 

Methods, Page 20-21 Lines 398-408

“Then, the “sleep” session began after the participants were instructed to try and fall asleep. For the functional scans, whole-brain images were acquired using k-space and steady-state T2*-weighted gradient echo-planar imaging (EPI) sequence that is sensitive to the BOLD contrast. This measures local magnetic changes caused by changes in blood oxygenation that accompany neural activity (sequence specification: 33 slices in interleaved ascending order, TR = 2000 ms, TE = 30 ms, voxel size = 3.5 × 3.5 × 4.2 mm3, FA = 90°, matrix = 64 × 64, gap = 0.7 mm). A relatively long TR and larger voxel size were chosen to optimize SNR and reduce susceptibility-related distortions, which are critical in EEG-fMRI sleep studies where head motion and physiological noise can be substantial. The longer TR allowed whole-brain coverage with sufficient temporal resolution, while the larger voxel size helped enhance BOLD sensitivity and minimize partial volume effects in deep brain structures (e.g., the thalamus and hippocampus), which are key regions of interest in this study.”

(8) The anatomically defined ROIs are quite large. It should be elaborated on how this might reduce sensitivity to sleep rhythm-specific activity within sub-regions, especially for the thalamus, which has distinct nuclei involved in sleep functions.

We appreciate your insight regarding the use of anatomically defined ROIs and their potential limitations in detecting sleep rhythm-specific activity within sub-regions, particularly in the thalamus. Given the distinct functional roles of thalamic nuclei in sleep processes, we acknowledge that using a single, large thalamic ROI may reduce sensitivity to localized activity patterns. To address this, we will discuss this limitation in the revised manuscript, acknowledging that our approach prioritizes whole-structure effects but may not fully capture nucleus-specific contributions.

Discussion, Page 18 Lines 333-341

“Despite providing new insights, our study has several limitations. First, our scalp EEG did not directly capture hippocampal ripples, preventing us from conclusively demonstrating triple coupling. Second, the combination of EEG-fMRI and the lack of a memory task limit our ability to parse fine-grained BOLD responses at the DOWN- vs. UP-states of SOs and link observed activations to behavioral outcomes. Third, the use of large anatomical ROIs may mask subregional contributions of specific thalamic nuclei or hippocampal subfields. Finally, without a memory task, we cannot establish a direct behavioral link between sleep-rhythm-locked activation and memory consolidation. Future studies combining techniques such as ultra-high-field fMRI or iEEG with cognitive tasks may refine our understanding of subregional network dynamics and functional significance during sleep.”

(9) The study reports SO & spindle amplitudes & densities, as well as SO+spindle coupling, to be larger during N2/3 sleep compared to N1 and REM sleep, which is trivial but can be seen as a sanity check of the data. However, the amount of SOs and spindles reported for N1 and REM sleep is concerning, as per definition there should be hardly any (if SOs or spindles occur in N1 it becomes by definition N2, and the interval between spindles has to be considerably large in REM to still be scored as such). Thus, on the one hand, the report of these comparisons takes too much space in the main manuscript as it is trivial, but on the other hand, it raises concerns about the validity of the scoring.

We appreciate your concern regarding the reported presence of SOs and spindles in N1 and REM sleep and the potential implications. Our detection method for detecting SO, spindle, and coupling were originally designed only for N2&N3 sleep data based on the characteristics of the data itself, and this method is widely recognized and used in the sleep research (Hahn et al., 2020; Helfrich et al., 2019; Helfrich et al., 2018; Ngo, Fell, & Staresina, 2020; Schreiner et al., 2022; Schreiner et al., 2021; Staresina et al., 2015; Staresina et al., 2023). While, because the detection methods for SO and spindle are based on percentiles, this method will always detect a certain number of events when used for other stages (N1 and REM) sleep data, but the differences between these events and those detected in stage N23 remain unclear. We will acknowledge the reasons for these results in the Methods section and emphasize that they are used only for sanity checks.

Methods, Page 25 Lines 515-524

“We note that the above methods for detecting SOs, spindles, and their couplings were originally developed for N2 and N3 sleep data, based on the specific characteristics of these stages. These methods are widely recognized in sleep research (Hahn et al., 2020; Helfrich et al., 2019; Helfrich et al., 2018; Ngo, Fell, & Staresina, 2020; Schreiner et al., 2022; Schreiner et al., 2021; Staresina et al., 2015; Staresina et al., 2023). However, because this percentile-based detection approach will inherently identify a certain number of events if applied to other stages (e.g., N1 and REM), the nature of these events in those stages remains unclear compared to N2/N3. We nevertheless identified and reported the detailed descriptive statistics of these sleep rhythms in all sleep stages, under the same operational definitions, both for completeness and as a sanity check. Within the same subject, there should be more SOs, spindles, and their couplings in N2/N3 than in N1 or REM (see also Figure S2-S4, Table S1-S4).”

(10) Why was electrode F3 used to quantify the occurrence of SOs and spindles? Why not a midline frontal electrode like Fz (or a number of frontal electrodes for SOs) and Cz (or a number of centroparietal electrodes) for spindles to be closer to their maximum topography?

We appreciate your suggestion regarding electrode selection for SO and spindle quantification. Our choice of F3 was primarily based on previous studies (Massimini et al., 2004; Molle et al., 2011), where bilateral frontal electrodes are commonly used for detecting SOs and spindles. Additionally, we considered the impact of MRI-related noise and, after a comprehensive evaluation, determined that F3 provided an optimal balance between signal quality and artifact minimization. We also acknowledge that alternative electrode choices, such as Fz for SOs and Cz for spindles, could provide additional insights into their topographical distributions.

(11) Functional connectivity (hippocampus -> thalamus -> cortex (mPFC)) is reported to be increased during SO-spindle coupling and interpreted as evidence for coordination of hippocampo-neocortical communication likely by thalamic spindles. However, functional connectivity was only analysed during coupled SO+spindle events, not during isolated SOs or isolated spindles. Without the direct comparison of the connectivity patterns between these three events, it remains unclear whether this is specific for coupled SO+spindle events or rather associated with one or both of the other isolated events. The PPIs need to be conducted for those isolated events as well and compared statistically to the coupled events.

We appreciate your critical perspective on our functional connectivity analysis and the interpretation of hippocampus-thalamus-cortex (mPFC) interactions during SO-spindle coupling. We acknowledge that, in the current analysis, functional connectivity was only examined during coupled SO-spindle events, without direct comparison to isolated SOs or isolated spindles. To address this concern, we have conducted PPI analyses for all three ROIs(Hippocampus, Thalamus, mPFC) and all three event types (SO-spindle couplings, isolated SOs, and isolated spindles). Our results indicate that neither isolated SOs nor isolated Spindles yielded significant connectivity changes in all three ROIs, as all failed to survive multiple comparison corrections. This suggests that the observed connectivity increase is specific to SO-spindle coupling, rather than being independently driven by either SOs or spindles alone.

Results, Page 14 Lines 248-255

“Crucially, the interaction between FC and SO-spindle coupling revealed that only the functional connectivity of hippocampus -> thalamus (ROI analysis, t(106) = 1.86, p = 0.0328) and thalamus -> mPFC (ROI analysis, t(106) = 1.98, p = 0.0251) significantly increased during SO-spindle coupling, with no significant changes in all other pathways (Fig. 4e). We also conducted PPI analyses for the other two events (SOs and spindles), and neither yielded significant connectivity changes in the three ROIs, as all failed to survive whole-brain FWE correction at the cluster level (p < 0.05). Together, these findings suggest that the thalamus, likely via spindles, coordinates hippocampal-cortical communication selectively during SO-spindle coupling, but not isolated SOs or spindle events alone.”

(12) The limited temporal resolution of fMRI does indeed not allow for easily distinguishing between fMRI activation patterns related to SO-up- vs. SO-down-states. For this, one could try to extract the amplitudes of SO-up- and SO-down-states separately for each SO event and model them as two separate parametric modulators (with the risk of collinearity as they are likely correlated).

We appreciate your insightful comment regarding the challenge of distinguishing fMRI activation patterns related to SO-up vs. SO-down states due to the limited temporal resolution of fMRI. While our current analysis does not differentiate between these two phases, we acknowledge that separately modeling SO-up and SO-down states using parametric modulators could provide a more refined understanding of their distinct neural correlates. However, as you notes, this approach carries the risk of collinearity, and there is indeed a high correlation between the two amplitudes across all subjects in our results (r=0.98). Future studies could explore more on leveraging high-temporal-resolution techniques. While implementing this in the current study is beyond our scope, we will acknowledge this limitation in the Discussion section.

Discussion, Page 17 Lines 308-322

“An intriguing aspect of our findings is the reduced DMN activity during SOs when modeled at the SO trough (DOWN-state). This reduced DMN activity may reflect large-scale neural inhibition characteristic of the SO trough. The DMN is typically active during internally oriented cognition (e.g., self-referential processing or mind-wandering) and is suppressed during external stimuli processing (Yeshurun, Nguyen, & Hasson, 2021). It is unlikely, however, that this suppression of DMN during SO events is related to a shift from internal cognition to external responses given it is during deep sleep time. Instead, it could be driven by the inherent rhythmic pattern of SOs, which makes it difficult to separate UP- from DOWN-states (the two temporal regressors were highly correlated, and similar brain activation during SOs events was obtained if modelled at the SO peak instead, Fig. S5). Since the amplitude at the SO trough is consistently larger than that at the SO peak, the neural activation we detected may primarily capture the large-scale inhibition from DOWN-state. Interestingly, no such DMN reduction was found during SO-spindle coupling, implying that coupling may involve distinct neural dynamics that partially re-engage DMN-related processes, possibly reflecting memory-related reactivation. Future research using high-temporal-resolution techniques like iEEG could clarify these possibilities.”

Discussion, Page 18 Lines 333-341

“Despite providing new insights, our study has several limitations. First, our scalp EEG did not directly capture hippocampal ripples, preventing us from conclusively demonstrating triple coupling. Second, the combination of EEG-fMRI and the lack of a memory task limit our ability to parse fine-grained BOLD responses at the DOWN- vs. UP-states of SOs and link observed activations to behavioral outcomes. Third, the use of large anatomical ROIs may mask subregional contributions of specific thalamic nuclei or hippocampal subfields. Finally, without a memory task, we cannot establish a direct behavioral link between sleep-rhythm-locked activation and memory consolidation. Future studies combining techniques such as ultra-high-field fMRI or iEEG with cognitive tasks may refine our understanding of subregional network dynamics and functional significance during sleep.”

(13) L327: "It is likely that our findings of diminished DMN activity reflect brain activity during the SO DOWN-state, as this state consistently shows higher amplitude compared to the UP-state within subjects, which is why we modelled the SO trough as its onset in the fMRI analysis." This conclusion is not justified as the fact that SO down-states are larger in amplitude does not mean their impact on the BOLD response is larger.

We appreciate your concern regarding our interpretation of diminished DMN activity reflecting the SO down-state. We acknowledge that the current expression is somewhat misleading, and our interpretation of it is: it could be driven by the inherent rhythmic pattern of SOs, which makes it difficult to separate UP- from DOWN-states (the two temporal regressors were highly correlated, and similar brain activation during SOs events was obtained if modelled at the SO peak instead). Since the amplitude at the SO trough is consistently larger than that at the SO peak, the neural activation we detected may primarily capture the large-scale inhibition from DOWN-state. And we will make this clear in the Discussion section.

Discussion, Page 17 Lines 308-322

“An intriguing aspect of our findings is the reduced DMN activity during SOs when modeled at the SO trough (DOWN-state). This reduced DMN activity may reflect large-scale neural inhibition characteristic of the SO trough. The DMN is typically active during internally oriented cognition (e.g., self-referential processing or mind-wandering) and is suppressed during external stimuli processing (Yeshurun, Nguyen, & Hasson, 2021). It is unlikely, however, that this suppression of DMN during SO events is related to a shift from internal cognition to external responses given it is during deep sleep time. Instead, it could be driven by the inherent rhythmic pattern of SOs, which makes it difficult to separate UP- from DOWN-states (the two temporal regressors were highly correlated, and similar brain activation during SOs events was obtained if modelled at the SO peak instead, Fig. S5). Since the amplitude at the SO trough is consistently larger than that at the SO peak, the neural activation we detected may primarily capture the large-scale inhibition from DOWN-state. Interestingly, no such DMN reduction was found during SO-spindle coupling, implying that coupling may involve distinct neural dynamics that partially re-engage DMN-related processes, possibly reflecting memory-related reactivation. Future research using high-temporal-resolution techniques like iEEG could clarify these possibilities.”

(14) Line 77: "In the current study, while directly capturing hippocampal ripples with scalp EEG or fMRI is difficult, we expect to observe hippocampal activation in fMRI whenever SOs-spindles coupling is detected by EEG, if SOs- spindles-ripples triple coupling occurs during human NREM sleep". Not all SO-spindle events are associated with ripples (Staresina et al., 2015), but hippocampal activation may also be expected based on the occurrence of spindles alone (Bergmann et al., 2012).

We appreciate your clarification regarding the relationship between SO-spindle coupling and hippocampal ripples. We acknowledge that not all SO-spindle events are necessarily accompanied by ripples (Staresina et al., 2015). However, based on previous research, we found that hippocampal ripples are significantly more likely to occur during SO-spindle coupling events. This suggests that while ripple occurrence is not guaranteed, SO-spindle coupling creates a favorable network state for ripple generation and potential hippocampal activation. To ensure accuracy, we will revise the manuscript to delete this misleading sentence in the Introduction section and acknowledge in the Discussion that our results cannot conclusively directly observe the triple coupling of SO, spindle, and hippocampal ripples.

Discussion, Page 18 Lines 333-341

“Despite providing new insights, our study has several limitations. First, our scalp EEG did not directly capture hippocampal ripples, preventing us from conclusively demonstrating triple coupling. Second, the combination of EEG-fMRI and the lack of a memory task limit our ability to parse fine-grained BOLD responses at the DOWN- vs. UP-states of SOs and link observed activations to behavioral outcomes. Third, the use of large anatomical ROIs may mask subregional contributions of specific thalamic nuclei or hippocampal subfields. Finally, without a memory task, we cannot establish a direct behavioral link between sleep-rhythm-locked activation and memory consolidation. Future studies combining techniques such as ultra-high-field fMRI or iEEG with cognitive tasks may refine our understanding of subregional network dynamics and functional significance during sleep.”

Reviewer #2 (Public review):

In this study, Wang and colleagues aimed to explore brain-wide activation patterns associated with NREM sleep oscillations, including slow oscillations (SOs), spindles, and SO-spindle coupling events. Their findings reveal that SO-spindle events corresponded with increased activation in both the thalamus and hippocampus. Additionally, they observed that SO-spindle coupling was linked to heightened functional connectivity from the hippocampus to the thalamus, and from the thalamus to the medial prefrontal cortex-three key regions involved in memory consolidation and episodic memory processes.

This study's findings are timely and highly relevant to the field. The authors' extensive data collection, involving 107 participants sleeping in an fMRI while undergoing simultaneous EEG recording, deserves special recognition. If shared, this unique dataset could lead to further valuable insights. While the conclusions of the data seem overall well supported by the data, some aspects with regard to the detection of sleep oscillations need clarification.

The authors report that coupled SO-spindle events were most frequent during NREM sleep (2.46 [plus minus] 0.06 events/min), but they also observed a surprisingly high occurrence of these events during N1 and REM sleep (2.23 [plus minus] 0.09 and 2.32 [plus minus] 0.09 events/min, respectively), where SO-spindle coupling would not typically be expected. Combined with the relatively modest SO amplitudes reported (~25 µV, whereas >75 µV would be expected when using mastoids as reference electrodes), this raises the possibility that the parameters used for event detection may not have been conservative enough - or that sleep staging was inaccurately performed. This issue could present a significant challenge, as the fMRI findings are largely dependent on the reliability of these detected events.

Thank you very much for your thorough and encouraging review. We appreciate your recognition of the significance and relevance of our study and dataset, particularly in highlighting how simultaneous EEG-fMRI recordings can provide complementary insights into the temporal dynamics of neural oscillations and their associated spatial activation patterns during sleep. In the sections that follow, we address each of your comments in detail. We have revised the text and conducted additional analyses wherever possible to strengthen our argument, clarify our methodological choices. We believe these revisions improve the clarity and rigor of our work, and we thank you for helping us refine it.

We appreciate your insightful comments regarding the detection of sleep oscillations. Our methods for detecting SOs, spindles, and their couplings were originally developed for N2 and N3 sleep data, based on the specific characteristics of these stages. These methods are widely recognized in sleep research (Hahn et al., 2020; Helfrich et al., 2019; Helfrich et al., 2018; Ngo, Fell, & Staresina, 2020; Schreiner et al., 2022; Schreiner et al., 2021; Staresina et al., 2015; Staresina et al., 2023). However, because this percentile-based detection approach will inherently identify a certain number of events if applied to other stages (e.g., N1 and REM), the nature of these events in those stages remains unclear compared to N2/N3. We nevertheless identified and reported the detailed descriptive statistics of these sleep rhythms in all sleep stages, under the same operational definitions, both for completeness and as a sanity check. Within the same subject, there should be more SOs, spindles, and their couplings in N2/N3 than in N1 or REM. We will acknowledge the reasons for these results in the Methods section and emphasize that they are used only for sanity checks.

Regarding the reported SO amplitudes (~25 µV), during preprocessing, we applied the Signal Space Projection (SSP) method to more effectively remove MRI gradient artifacts and cardiac pulse noise. While this approach enhances data quality, it also reduces overall signal power, leading to systematically lower reported amplitudes. Despite this, our SO detection in NREM sleep (especially N2/N3) remain physiologically meaningful and are consistent with previous fMRI studies using similar artifact removal techniques. We appreciate your careful evaluation and valuable suggestions.

In addition, we will provide comprehensive tables in the supplementary materials, contains descriptive information about sleep-related characteristics (Table S1), as well as detailed information about sleep waves at each sleep stage for all 107 subjects(Table S2-S4), listing for each subject:(1)Different sleep stage duration; (2)Number of detected SOs; (3)Number of detected spindles; (4)Number of detected SO-spindle coupling events; (2)Density of detected SOs; (3)Density of detected spindles; (4)Density of detected SO-spindle coupling events.

Methods, Page 25 Lines 515-524

“We note that the above methods for detecting SOs, spindles, and their couplings were originally developed for N2 and N3 sleep data, based on the specific characteristics of these stages. These methods are widely recognized in sleep research (Hahn et al., 2020; Helfrich et al., 2019; Helfrich et al., 2018; Ngo, Fell, & Staresina, 2020; Schreiner et al., 2022; Schreiner et al., 2021; Staresina et al., 2015; Staresina et al., 2023). However, because this percentile-based detection approach will inherently identify a certain number of events if applied to other stages (e.g., N1 and REM), the nature of these events in those stages remains unclear compared to N2/N3. We nevertheless identified and reported the detailed descriptive statistics of these sleep rhythms in all sleep stages, under the same operational definitions, both for completeness and as a sanity check. Within the same subject, there should be more SOs, spindles, and their couplings in N2/N3 than in N1 or REM (see also Figure S2-S4, Table S1-S4).”

Supplementary Materials, Page 42-54, Table S1-S4

Reviewer #3 (Public review):

Summary:

Wang et al., examined the brain activity patterns during sleep, especially when locked to those canonical sleep rhythms such as SO, spindle, and their coupling. Analyzing data from a large sample, the authors found significant coupling between spindles and SOs, particularly during the upstate of the SO. Moreover, the authors examined the patterns of whole-brain activity locked to these sleep rhythms. To understand the functional significance of these brain activities, the authors further conducted open-ended cognitive state decoding and found a variety of cognitive processing may be involved during SO-spindle coupling and during other sleep events. The authors next investigated the functional connectivity analyses and found enhanced connectivity between the hippocampus, the thalamus, and the medial PFC. These results reinforced the theoretical model of sleep-dependent memory consolidation, such that SO-spindle coupling is conducive to systems-level memory reactivation and consolidation.

Strengths:

There are obvious strengths in this work, including the large sample size, state-of-the-art neuroimaging and neural oscillation analyses, and the richness of results.

Weaknesses:

Despite these strengths and the insights gained, there are weaknesses in the design, the analyses, and inferences.

Thank you for your detailed and thoughtful review of our manuscript. We are delighted that you recognize our advanced analysis methods and rich results of neuroimaging and neural oscillations as well as the large sample size data. In the following sections, we provide detailed responses to each of your comments. And we have revised the text and conducted additional analyses to strengthen our arguments and clarify our methodological choices. We believe these revisions enhance the clarity and rigor of our work, and we sincerely appreciate your thoughtful feedback in helping us refine the manuscript.

(1) A repeating statement in the manuscript is that brain activity could indicate memory reactivation and thus consolidation. This is indeed a highly relevant question that could be informed by the current data/results. However, an inherent weakness of the design is that there is no memory task before and after sleep. Thus, it is difficult (if not impossible) to make a strong argument linking SO/spindle/coupling-locked brain activity with memory reactivation or consolidation.

We appreciate your suggestion regarding the lack of a pre- and post-sleep memory task in our study design. We acknowledge that, in the absence of behavioral measures, it is hard to directly link SO-spindle coupling to memory consolidation in an outcome-driven manner. Our interpretation is instead based on the well-established role of these oscillations in memory processes, as demonstrated in previous studies. We sincerely appreciate this feedback and will adjust our Discussion accordingly to reflect a more precise interpretation of our findings.

Discussion, Page 18 Lines 333-341

“Despite providing new insights, our study has several limitations. First, our scalp EEG did not directly capture hippocampal ripples, preventing us from conclusively demonstrating triple coupling. Second, the combination of EEG-fMRI and the lack of a memory task limit our ability to parse fine-grained BOLD responses at the DOWN- vs. UP-states of SOs and link observed activations to behavioral outcomes. Third, the use of large anatomical ROIs may mask subregional contributions of specific thalamic nuclei or hippocampal subfields. Finally, without a memory task, we cannot establish a direct behavioral link between sleep-rhythm-locked activation and memory consolidation. Future studies combining techniques such as ultra-high-field fMRI or iEEG with cognitive tasks may refine our understanding of subregional network dynamics and functional significance during sleep.”

(2) Relatedly, to understand the functional implications of the sleep rhythm-locked brain activity, the authors employed the "open-ended cognitive state decoding" method. While this method is interesting, it is rather indirect given that there were no behavioral indices in the manuscript. Thus, discussions based on these analyses are speculative at best. Please either tone down the language or find additional evidence to support these claims.

Moreover, the results from this method are difficult to understand. Figure 3e showed that for all three types of sleep events (SO, spindle, SO-spindle), the same mental states (e.g., working memory, episodic memory, declarative memory) showed opposite directions of activation (left and right panels showed negative and positive activation, respectively). How to interpret these conflicting results? This ambiguity is also reflected by the term used: declarative memory and episodic memories are both indexed in the results. Yet these two processes can be largely overlapped. So which specific memory processes do these brain activity patterns reflect? The Discussion shall discuss these results and the limitations of this method.

We appreciate your critical assessment of the open-ended cognitive state decoding method and its interpretational challenges. Given the concerns about the indirectness of this approach, we decided to remove its related content and results from Figure 3 in the main text and include it in Supplementary Figure 7. 

Due to the complexity of memory-related processes, we acknowledge that distinguishing between episodic and declarative memory based solely on this approach is not straightforward. We will revise the Supplementary Materials to explicitly discuss these limitations and clarify that our findings do not isolate specific cognitive processes but rather suggest general associations with memory-related networks.

Discussion, Page 17-18 Lines 323-332

“To explore functional relevance, we employed an open-ended cognitive state decoding approach using meta-analytic data (NeuroSynth: Yarkoni et al. (2011)). Although this method usefully generates hypotheses about potential cognitive processes, particularly in the absence of a pre- and post-sleep memory task, it is inherently indirect. Many cognitive terms showed significant associations (16 of 50), such as “episodic memory,” “declarative memory,” and “working memory.” We focused on episodic/declarative memory given the known link with hippocampal reactivation (Diekelmann & Born, 2010; Staresina et al., 2015; Staresina et al., 2023). Nonetheless, these inferences regarding memory reactivation should be interpreted cautiously without direct behavioral measures. Future research incorporating explicit tasks before and after sleep would more rigorously validate these potenial functional claims.”

(3) The coupling strength is somehow inconsistent with prior results (Hahn et al., 2020, eLife, Helfrich et al., 2018, Neuron). Specifically, Helfrich et al. showed that among young adults, the spindle is coupled to the peak of the SO. Here, the authors reported that the spindles were coupled to down-to-up transitions of SO and before the SO peak. It is possible that participants' age may influence the coupling (see Helfrich et al., 2018). Please discuss the findings in the context of previous research on SO-spindle coupling.

We appreciate your concern regarding the temporal characteristics of SO-spindle coupling. We acknowledge that the SO-spindle coupling phase results in our study are not identical to those reported by Hahn et al. (2020); Helfrich et al. (2018). However, these differences may arise due to slight variations in event detection parameters, which can influence the precise phase estimation of coupling. Notably, Hahn et al. (2020) also reported slight discrepancies in their group-level coupling phase results, highlighting that methodological differences can contribute to variability across studies. Furthermore, our findings are consistent with those of Schreiner et al. (2021), further supporting the robustness of our observations.  

That said, we acknowledge that our original description of SO-spindle coupling as occurring at the "transition from the lower state to the upper state" was not entirely precise. The -π/2 phase represents the true transition point, while our observed coupling phase is actually closer to the SO peak rather than strictly at the transition. We will revise this statement in the manuscript to ensure clarity and accuracy in describing the coupling phase.  

Discussion, Page 16 Lines 283-291

“Our data provide insights into the neurobiological underpinnings of these sleep rhythms. SOs, originating mainly in neocortical areas such as the mPFC, alternate between DOWN- and UP-states. The thalamus generates sleep spindles, which in turn couple with SOs. Our finding that spindle peaks consistently occurred slightly before the UP-state peak of SOs (in 83 out of 107 participants), concurs with prior studies, including Schreiner et al. (2021). Yet it differs from some results suggesting spindles might peak right at the SO UP-state (Hahn et al., 2020; Helfrich et al., 2018). Such discrepancies could arise from differences in detection algorithms, participant age (Helfrich et al., 2018), or subtle variations in cortical-thalamic timing. Nonetheless, these results underscore the importance of coordinated SO-spindle interplay in supporting sleep-dependent processes.”

(4) The discussion is rather superficial with only two pages, without delving into many important arguments regarding the possible functional significance of these results. For example, the author wrote, "This internal processing contrasts with the brain patterns associated with external tasks, such as working memory." Without any references to working memory, and without delineating why WM is considered as an external task even working memory operations can be internal. Similarly, for the interesting results on SO and reduced DMN activity, the authors wrote "The DMN is typically active during wakeful rest and is associated with self-referential processes like mind-wandering, daydreaming, and task representation (Yeshurun, Nguyen, & Hasson, 2021). Its reduced activity during SOs may signal a shift towards endogenous processes such as memory consolidation." This argument is flawed. DMN is active during self-referential processing and mind-wandering, i.e., when the brain shifts from external stimuli processing to internal mental processing. During sleep, endogenous memory reactivation and consolidation are also part of the internal mental processing given the lack of external environmental stimulation. So why during SO or during memory consolidation, the DMN activity would be reduced? Were there differences in DMN activity between SO and SO-spindle coupling events?

We appreciate your concerns regarding the brevity of the discussion and the need for clearer theoretical arguments. We will expand this section to provide more in-depth interpretations of our findings in the context of prior literature. Regarding working memory (WM), we acknowledge that our phrasing was ambiguous. We will modify this statement in the Discussion section.

For the SO-related reduction in DMN activity, we recognize the need for a more precise explanation. This reduced DMN activity may reflect large-scale neural inhibition characteristic of the SO trough. The DMN is typically active during internally oriented cognition (e.g., self-referential processing or mind-wandering) and is suppressed during external stimuli processing (Yeshurun, Nguyen, & Hasson, 2021). It is unlikely, however, that this suppression of DMN during SO events is related to a shift from internal cognition to external responses given it is during deep sleep time. Instead, it could be driven by the inherent rhythmic pattern of SOs, which makes it difficult to separate UP- from DOWN-states (the two temporal regressors were highly correlated, and similar brain activation during SOs events was obtained if modelled at the SO peak instead). Since the amplitude at the SO trough is consistently larger than that at the SO peak, the neural activation we detected may primarily capture the large-scale inhibition from DOWN-state.

To address your final question, we have conducted the additional post hoc comparison of DMN activity between isolated SOs and SO-spindle coupling events. Our results indicate that

DMN activation during SOs was significantly lower than during SO-spindle coupling (t(106) = -4.17, p < 1e-4). This suggests that SO-spindle coupling may involve distinct neural dynamics that partially re-engage DMN-related processes, possibly reflecting memory-related reactivation. We appreciate your constructive feedback and will integrate these expanded analyses and discussions into our revised manuscript.

Results, Page 11 Lines 199-208

“Spindles were correlated with positive activation in the thalamus (ROI analysis, t(106) = 15.39, p < 1e-4), the anterior cingulate cortex (ACC), and the putamen, alongside deactivation in the DMN (Fig. 3c). Notably, SO-spindle coupling was linked to significant activation in both the thalamus (ROI analysis, t(106) \= 3.38, p = 0.0005) and the hippocampus (ROI analysis, t(106) \= 2.50, p = 0.0070, Fig. 3d). However, no decrease in DMN activity was found during SO-spindle coupling, and DMN activity during SO was significantly lower than during coupling (ROI analysis, t(106) \= -4.17, p < 1e-4). For more detailed activation patterns, see Table S5-S7. We also varied the threshold used to detect SO events to assess its effect on hippocampal activation during SO-spindle coupling and observed that hippocampal activation remained significant when the percentile thresholds for SO detection ranged between 71% and 80% (see Fig. S6).”

Discussion, Page 17-18 Lines 308-332

“An intriguing aspect of our findings is the reduced DMN activity during SOs when modeled at the SO trough (DOWN-state). This reduced DMN activity may reflect large-scale neural inhibition characteristic of the SO trough. The DMN is typically active during internally oriented cognition (e.g., self-referential processing or mind-wandering) and is suppressed during external stimuli processing (Yeshurun, Nguyen, & Hasson, 2021). It is unlikely, however, that this suppression of DMN during SO events is related to a shift from internal cognition to external responses given it is during deep sleep time. Instead, it could be driven by the inherent rhythmic pattern of SOs, which makes it difficult to separate UP- from DOWN-states (the two temporal regressors were highly correlated, and similar brain activation during SOs events was obtained if modelled at the SO peak instead, Fig. S5). Since the amplitude at the SO trough is consistently larger than that at the SO peak, the neural activation we detected may primarily capture the large-scale inhibition from DOWN-state. Interestingly, no such DMN reduction was found during SO-spindle coupling, implying that coupling may involve distinct neural dynamics that partially re-engage DMN-related processes, possibly reflecting memory-related reactivation. Future research using high-temporal-resolution techniques like iEEG could clarify these possibilities.

To explore functional relevance, we employed an open-ended cognitive state decoding approach using meta-analytic data (NeuroSynth: Yarkoni et al. (2011)). Although this method usefully generates hypotheses about potential cognitive processes, particularly in the absence of a pre- and post-sleep memory task, it is inherently indirect. Many cognitive terms showed significant associations (16 of 50), such as “episodic memory,” “declarative memory,” and “working memory.” We focused on episodic/declarative memory given the known link with hippocampal reactivation (Diekelmann & Born, 2010; Staresina et al., 2015; Staresina et al., 2023). Nonetheless, these inferences regarding memory reactivation should be interpreted cautiously without direct behavioral measures. Future research incorporating explicit tasks before and after sleep would more rigorously validate these potential functional claims.”

Recommendations for the authors:

Reviewing Editor Comment:

The reviewers think that you are working on a relevant and important topic. They are praising the large sample size used in the study. The reviewers are not all in line regarding the overall significance of the findings, but they all agree the paper would strongly benefit from some extra work, as all reviewers raise various critical points that need serious consideration.

We appreciate your recognition of the relevance and importance of our study, as well as your acknowledgment of the large sample size as a strength of our work. We understand that there are differing perspectives regarding the overall significance of our findings, and we value the constructive critiques provided. We are committed to addressing the key concerns raised by all reviewers, including refining our analyses, clarifying our interpretations, and incorporating additional discussions to strengthen the manuscript. Below, we address your specific recommendations and provide responses to each point you raised to ensure our methods and results are as transparent and comprehensible as possible. We believe that these revisions will significantly enhance the rigor and impact of our study, and we sincerely appreciate your thoughtful feedback in helping us improve our work.

Reviewer #1 (Recommendations for the authors):

(1) The phrase "overnight sleep" suggests an entire night, while these were rather "nocturnal naps". Please rephrase.

Response: Thank you for pointing this out. We have revised the phrasing in our manuscript to "nocturnal naps" instead of "overnight sleep" to more accurately reflect the duration of the sleep recordings.

(2) Sleep staging results (macroscopic sleep architecture) should be provided in more detail (at least min and % of the different sleep stages, sleep onset latency, total sleep duration, total recording duration), at least mean/SD/range.

Thank you for this suggestion. We will provide comprehensive tables in the supplementary materials, contains descriptive information about sleep-related characteristics. This information will help provide a clearer overview of the macroscopic sleep architecture in our dataset.

Reviewer #2 (Recommendations for the authors):

In order to allow for a better estimation of the reliability of the detected sleep events, please:

(1) Provide densities and absolute numbers of all detected SOs and spindles (N1, NREM, and REM sleep).

Thank you for pointing this out. We will provide comprehensive tables in the supplementary materials, contains detailed information about sleep waves at each sleep stage for all 107 subjects (Table S2-S4), listing for each subject:1) Different sleep stage duration; 2) Number of detected SOs; 3) Number of detected spindles; 4) Number of detected SO-spindle coupling events; 5) Density of detected SOs; 6) Density of detected spindles; 7) Density of detected SO-spindle coupling events.

Supplementary Materials, Page 43-54, Table S2-S4

(2) Show ERPs for all detected SOs and spindles (per sleep stage).

Thank you for the suggestion. We will provide ERPs for all detected SOs and spindles, separated by sleep stage (N1, N2&N3, and REM) in supplementary Fig. S2-S4. These ERP waveforms will help illustrate the characteristic temporal profiles of SOs and spindles across different sleep stages.

Methods, Page 25, Line 525-532

“Event-related potentials (ERP) analysis. After completing the detection of each sleep rhythm event, we performed ERP analyses for SOs, spindles, and coupling events in different sleep stages. Specifically, for SO events, we took the trough of the DOWN-state of each SO as the zero-time point, then extracted data in a [-2 s to 2 s] window from the broadband (0.1–30 Hz) EEG and used [-2 s to -0.5 s] for baseline correction; the results were then averaged across 107 subjects (see Fig. S2a). For spindle events, we used the peak of each spindle as the zero-time point and applied the same data extraction window and baseline correction before averaging across 107 subjects (see Fig. S2b). Finally, for SO-spindle coupling events, we followed the same procedure used for SO events (see Fig. 2a, Figs. S3–S4).”

(3) Provide detailed info concerning sleep characteristics (time spent in each sleep stage etc.).

Thank you for this suggestion. Same as the response above, we will provide comprehensive tables in the supplementary materials, contains descriptive information about sleep-related characteristics.

Supplementary Materials, Page 42, Table S1 (same as above)

(4) What would happen if more stringent parameters were used for event detection? Would the authors still observe a significant number of SO spindles during N1 and REM? Would this affect the fMRI-related results?

Thank you for this suggestion. Our methods for detecting SOs, spindles, and their couplings were originally developed for N2 and N3 sleep data, based on the specific characteristics of these stages. These methods are widely recognized in sleep research (Hahn et al., 2020; Helfrich et al., 2019; Helfrich et al., 2018; Ngo, Fell, & Staresina, 2020; Schreiner et al., 2022; Schreiner et al., 2021; Staresina et al., 2015; Staresina et al., 2023). However, because this percentile-based detection approach will inherently identify a certain number of events if applied to other stages (e.g., N1 and REM), the nature of these events in those stages remains unclear compared to N2/N3. We nevertheless identified and reported the detailed descriptive statistics of these sleep rhythms in all sleep stages, under the same operational definitions, both for completeness and as a sanity check. Within the same subject, there should be more SOs, spindles, and their couplings in N2/N3 than in N1 or REM (see also Figure S2-S4, Table S1-S4).

Furthermore, in order to explore the impact of this on our fMRI results, we conducted an additional sensitivity analysis by applying different detection parameters for SOs. Specifically, we adjusted amplitude percentile thresholds for SO detection (the parameter that has the greatest impact on the results). We used the hippocampal activation value during N2&N3 stage SO-spindle coupling as an anchor value and found that when the parameters gradually became stricter, the results were similar to or even better than the current results. However, when we continued to increase the threshold, the results began to gradually decrease until the threshold was increased to 80%, and the results were no longer significant. This indicates that our results are robust within a specific range of parameters, but as the threshold increases, the number of trials decreases, ultimately weakening the statistical power of the fMRI analysis.

Thank you again for your suggestions on sleep rhythm event detection. We will add the results in Supplementary and revise our manuscript accordingly.

Results, Page 11, Line 199-208

“Spindles were correlated with positive activation in the thalamus (ROI analysis, t(106) = 15.39, p < 1e-4), the anterior cingulate cortex (ACC), and the putamen, alongside deactivation in the DMN (Fig. 3c). Notably, SO-spindle coupling was linked to significant activation in both the thalamus (ROI analysis, t(106) \= 3.38, p = 0.0005) and the hippocampus (ROI analysis, t(106) \= 2.50, p = 0.0070, Fig. 3d). However, no decrease in DMN activity was found during SO-spindle coupling, and DMN activity during SO was significantly lower than during coupling (ROI analysis, t(106) \= -4.17, p < 1e-4). For more detailed activation patterns, see Table S5-S7. We also varied the threshold used to detect SO events to assess its effect on hippocampal activation during SO-spindle coupling and observed that hippocampal activation remained significant when the percentile thresholds for SO detection ranged between 71% and 80% (see Fig. S6).”

Finally, we sincerely thank all again for your thoughtful and constructive feedback. Your insights have been invaluable in refining our analyses, strengthening our interpretations, and improving the clarity and rigor of our manuscript. We appreciate the time and effort you have dedicated to reviewing our work, and we are grateful for the opportunity to enhance our study based on your recommendations.  

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Reviewer #1 (Public review):

This study investigates the sex determination mechanism in the clonal ant Ooceraea biroi, focusing on a candidate complementary sex determination (CSD) locus-one of the key mechanisms supporting haplodiploid sex determination in hymenopteran insects. Using whole genome sequencing, the authors analyze diploid females and the rarely occurring diploid males of O. biroi, identifying a 46 kb candidate region that is consistently heterozygous in females and predominantly homozygous in diploid males. This region shows elevated genetic diversity, as expected under balancing selection. The study also reports the presence of an lncRNA near this heterozygous region, which, though only distantly related in sequence, resembles the ANTSR lncRNA involved in female development in the Argentine ant, Linepithema humile (Pan et al. 2024). Together, these findings suggest a potentially conserved sex determination mechanism across ant species. However, while the analyses are well conducted and the paper is clearly written, the insights are largely incremental. The central conclusion - that the sex determination locus is conserved in ants - was already proposed and experimentally supported by Pan et al. (2024), who included O. biroi among the studied species and validated the locus's functional role in the Argentine ant. The present study thus largely reiterates existing findings without providing novel conceptual or experimental advances.

Other comments:

The mapping is based on a very small sample size: 19 females and 16 diploid males, and these all derive from a single clonal line. This implies a rather high probability for false-positive inference. In combination with the fact that only 11 out of the 16 genotyped males are actually homozygous at the candidate locus, I think a more careful interpretation regarding the role of the mapped region in sex determination would be appropriate. The main argument supporting the role of the candidate region in sex determination is based on the putative homology with the lncRNA involved in sex determination in the Argentine ant, but this argument was made in a previous study (as mentioned above).<br /> In the abstract, it is stated that CSD loci have been mapped in honeybees and two ant species, but we know little about their evolutionary history. But CSD candidate loci were also mapped in a wasp with multi-locus CSD (study cited in the introduction). This wasp is also parthenogenetic via central fusion automixis and produces diploid males. This is a very similar situation to the present study and should be referenced and discussed accordingly, particularly since the authors make the interesting suggestion that their ant also has multi-locus CSD and neither the wasp nor the ant has tra homologs in the CSD candidate regions. Also, is there any homology to the CSD candidate regions in the wasp species and the studied ant?

The authors used different clonal lines of O. biroi to investigate whether heterozygosity at the mapped CSD locus is required for female development in all clonal lines of O. biroi (L187-196). However, given the described parthenogenesis mechanism in this species conserves heterozygosity, additional females that are heterozygous are not very informative here. Indeed, one would need diploid males in these other clonal lines as well (but such males have not yet been found) to make any inference regarding this locus in other lines.

Reviewer #3 (Public review):

Summary:

The sex determination mechanism governed by the complementary sex determination (CSD) locus is one of the mechanisms that support the haplodiploid sex determination system evolved in hymenopteran insects. While many ant species are believed to possess a CSD locus, it has only been specifically identified in two species. The authors analyzed diploid females and the rarely occurring diploid males of the clonal ant Ooceraea biroi and identified a 46 kb CSD candidate region that is consistently heterozygous in females and predominantly homozygous in males. This region was found to be homologous to the CSD locus reported in distantly related ants. In the Argentine ant, Linepithema humile, the CSD locus overlaps with an lncRNA (ANTSR) that is essential for female development and is associated with the heterozygous region (Pan et al. 2024). Similarly, an lncRNA is encoded near the heterozygous region within the CSD candidate region of O. biroi. Although this lncRNA shares low sequence similarity with ANTSR, its potential functional involvement in sex determination is suggested. Based on these findings, the authors propose that the heterozygous region and the adjacent lncRNA in O. biroi may trigger female development via a mechanism similar to that of L. humile. They further suggest that the molecular mechanisms of sex determination involving the CSD locus in ants have been highly conserved for approximately 112 million years. This study is one of the few to identify a CSD candidate region in ants and is particularly noteworthy as the first to do so in a parthenogenetic species.

Strengths:

(1) The CSD candidate region was found to be homologous to the CSD locus reported in distantly related ant species, enhancing the significance of the findings.

(2) Identifying the CSD candidate region in a parthenogenetic species like O. biroi is a notable achievement and adds novelty to the research.

Weaknesses

(1) Functional validation of the lncRNA's role is lacking, and further investigation through knockout or knockdown experiments is necessary to confirm its involvement in sex determination.

(2) The claim that the lncRNA is essential for female development appears to reiterate findings already proposed by Pan et al. (2024), which may reduce the novelty of the study.

Author response:

Reviewer #1 (Public review):

This study investigates the sex determination mechanism in the clonal ant Ooceraea biroi, focusing on a candidate complementary sex determination (CSD) locus-one of the key mechanisms supporting haplodiploid sex determination in hymenopteran insects. Using whole genome sequencing, the authors analyze diploid females and the rarely occurring diploid males of O. biroi, identifying a 46 kb candidate region that is consistently heterozygous in females and predominantly homozygous in diploid males. This region shows elevated genetic diversity, as expected under balancing selection. The study also reports the presence of an lncRNA near this heterozygous region, which, though only distantly related in sequence, resembles the ANTSR lncRNA involved in female development in the Argentine ant, Linepithema humile (Pan et al. 2024). Together, these findings suggest a potentially conserved sex determination mechanism across ant species. However, while the analyses are well conducted and the paper is clearly written, the insights are largely incremental. The central conclusion - that the sex determination locus is conserved in ants - was already proposed and experimentally supported by Pan et al. (2024), who included O. biroi among the studied species and validated the locus's functional role in the Argentine ant. The present study thus largely reiterates existing findings without providing novel conceptual or experimental advances.

Although it is true that Pan et al., 2024 demonstrated (in Figure 4 of their paper) that the synteny of the region flanking ANTSR is conserved across aculeate Hymenoptera (including O. biroi), Reviewer 1’s claim that that paper provides experimental support for the hypothesis that the sex determination locus is conserved in ants is inaccurate. Pan et al., 2024 only performed experimental work in a single ant species (Linepithema humile) and merely compared reference genomes of multiple species to show synteny of the region, rather than functionally mapping or characterizing these regions.

Other comments:

The mapping is based on a very small sample size: 19 females and 16 diploid males, and these all derive from a single clonal line. This implies a rather high probability for false-positive inference. In combination with the fact that only 11 out of the 16 genotyped males are actually homozygous at the candidate locus, I think a more careful interpretation regarding the role of the mapped region in sex determination would be appropriate. The main argument supporting the role of the candidate region in sex determination is based on the putative homology with the lncRNA involved in sex determination in the Argentine ant, but this argument was made in a previous study (as mentioned above).

Our main argument supporting the role of the candidate region in sex determination is not based on putative homology with the lncRNA in L. humile. Instead, our main argument comes from our genetic mapping (in Fig. 2), and the elevated nucleotide diversity within the identified region (Fig. 4). Additionally, we highlight that multiple genes within our mapped region are homologous to those in mapped sex determining regions in both L. humile and Vollenhovia emeryi, possibly including the lncRNA.

In response to the Reviewer’s assertion that the mapping is based on a small sample size from a single clonal line, we want to highlight that we used all diploid males available to us. Although the primary shortcoming of a small sample size is to increase the probability of a false negative, small sample sizes can also produce false positives. We used two approaches to explore the statistical robustness of our conclusions. First, we generated a null distribution by randomly shuffling sex labels within colonies and calculating the probability of observing our CSD index values by chance (shown in Fig. 2). Second, we directly tested the association between homozygosity and sex using Fisher’s Exact Test (shown in Supplementary Fig. S2). In both cases, the association of the candidate locus with sex was statistically significant after multiple-testing correction using the Benjamini-Hochberg False Discovery Rate. These approaches are clearly described in the “CSD Index Mapping” section of the Methods.

We also note that, because complementary sex determination loci are expected to evolve under balancing selection, our finding that the mapped region exhibits a peak of nucleotide diversity lends orthogonal support to the notion that the mapped locus is indeed a complementary sex determination locus.

The fourth paragraph of the results and the sixth paragraph of the discussion are devoted to explaining the possible reasons why only 11/16 genotyped males are homozygous in the mapped region. The revised manuscript will include an additional sentence (in what will be lines 384-388) in this paragraph that includes the possible explanation that this locus is, in fact, a false positive, while also emphasizing that we find this possibility to be unlikely given our multiple lines of evidence.

In response to Reviewer 1’s suggestion that we carefully interpret the role of the mapped region in sex determination, we highlight our careful wording choices, nearly always referring to the mapped locus as a “candidate sex determination locus” in the title and throughout the manuscript. For consistency, the revised manuscript version will change the second results subheading from “The O. biroi CSD locus is homologous to another ant sex determination locus but not to honeybee csd” to “O. biroi’s candidate CSD locus is homologous to another ant sex determination locus but not to honeybee csd,” and will add the word “candidate” in what will be line 320 at the beginning of the Discussion, and will change “putative” to “candidate” in what will be line 426 at the end of the Discussion.

In the abstract, it is stated that CSD loci have been mapped in honeybees and two ant species, but we know little about their evolutionary history. But CSD candidate loci were also mapped in a wasp with multi-locus CSD (study cited in the introduction). This wasp is also parthenogenetic via central fusion automixis and produces diploid males. This is a very similar situation to the present study and should be referenced and discussed accordingly, particularly since the authors make the interesting suggestion that their ant also has multi-locus CSD and neither the wasp nor the ant has tra homologs in the CSD candidate regions. Also, is there any homology to the CSD candidate regions in the wasp species and the studied ant?

In response to Reviewer 1’s suggestion that we reference the (Matthey-Doret et al. 2019) study in the context of diploid males being produced via losses of heterozygosity during asexual reproduction, the revised manuscript will include the following sentence: “Therefore, if O. biroi uses CSD, diploid males might result from losses of heterozygosity at sex determination loci (Fig. 1C), similar to what is thought to occur in other asexual Hymenoptera that produce diploid males (Rabeling and Kronauer 2012; Matthey-Doret et al. 2019).”

We note, however, that in their 2019 study, Matthey-Doret et al. did not directly test the hypothesis that diploid males result from losses of heterozygosity at CSD loci during asexual reproduction, because the diploid males they used for their mapping study came from inbred crosses in a sexual population of that species.

We address this further below, but we want to emphasize that we do not intend to argue that O. biroi has multiple CSD loci. Instead, we suggest that additional, undetected CSD loci is one possible explanation for the absence of diploid males from any clonal line other than clonal line A. In response to Reviewer 1’s suggestion that we reference the (Matthey-Doret et al. 2019) study in the context of multilocus CSD, the revised manuscript version will include the following additional sentence in the fifth paragraph of the discussion: “Multi-locus CSD has been suggested to limit the extent of diploid male production in asexual species under some circumstances (Vorburger 2013; Matthey-Doret et al. 2019).”

Regarding Reviewer 2’s question about homology between the putative CSD loci from the (Matthey-Doret et al. 2019) study and O. biroi, we note that there is no homology. The revised manuscript version will have an additional Supplementary Table (which will be the new Supplementary Table S3) that will report the results of this homology search. The revised manuscript will also include the following additional sentence in the Results: “We found no homology between the genes within the O. biroi CSD index peak and any of the genes within the putative L. fabarum CSD loci (Supplementary Table S3).”

The authors used different clonal lines of O. biroi to investigate whether heterozygosity at the mapped CSD locus is required for female development in all clonal lines of O. biroi (L187-196). However, given the described parthenogenesis mechanism in this species conserves heterozygosity, additional females that are heterozygous are not very informative here. Indeed, one would need diploid males in these other clonal lines as well (but such males have not yet been found) to make any inference regarding this locus in other lines.

We agree that a full mapping study including diploid males from all clonal lines would be preferable, but as stated earlier in that same paragraph, we have only found diploid males from clonal line A. We stand behind our modest claim that “Females from all six clonal lines were heterozygous at the CSD index peak, consistent with its putative role as a CSD locus in all O. biroi.” In the revised manuscript version, this sentence (in what will be lines 199-201) will be changed slightly in response to a reviewer comment below: “All females from all six clonal lines (including 26 diploid females from clonal line B) were heterozygous at the CSD index peak, consistent with its putative role as a CSD locus in all O. biroi.”

Reviewer #2 (Public review):

The manuscript by Lacy et al. is well written, with a clear and compelling introduction that effectively conveys the significance of the study. The methods are appropriate and well-executed, and the results, both in the main text and supplementary materials, are presented in a clear and detailed manner. The authors interpret their findings with appropriate caution.

This work makes a valuable contribution to our understanding of the evolution of complementary sex determination (CSD) in ants. In particular, it provides important evidence for the ancient origin of a non-coding locus implicated in sex determination, and shows that, remarkably, this sex locus is conserved even in an ant species with a non-canonical reproductive system that typically does not produce males. I found this to be an excellent and well-rounded study, carefully analyzed and well contextualized.

That said, I do have a few minor comments, primarily concerning the discussion of the potential 'ghost' CSD locus. While the authors acknowledge (line 367) that they currently have no data to distinguish among the alternative hypotheses, I found the evidence for an additional CSD locus presented in the results (lines 261-302) somewhat limited and at times a bit difficult to follow. I wonder whether further clarification or supporting evidence could already be extracted from the existing data. Specifically:

We agree with Reviewer 2 that the evidence for a second CSD locus is limited. In fact, we do not intend to advocate for there being a second locus, but we suggest that a second CSD locus is one possible explanation for the absence of diploid males outside of clonal line A. In our initial version, we intentionally conveyed this ambiguity by titling this section “O. biroi may have one or multiple sex determination loci.” However, we now see that this leads to undue emphasis on the possibility of a second locus. In the revised manuscript, we will split this into two separate sections: “Diploid male production differs across O. biroi clonal lines” and “O. biroi lacks a tra-containing CSD locus.”

(1) Line 268: I doubt the relevance of comparing the proportion of diploid males among all males between lines A and B to infer the presence of additional CSD loci. Since the mechanisms producing these two types of males differ, it might be more appropriate to compare the proportion of diploid males among all diploid offspring. This ratio has been used in previous studies on CSD in Hymenoptera to estimate the number of sex loci (see, for example, Cook 1993, de Boer et al. 2008, 2012, Ma et al. 2013, and Chen et al., 2021). The exact method might not be applicable to clonal raider ants, but I think comparing the percentage of diploid males among the total number of (diploid) offspring produced between the two lineages might be a better argument for a difference in CSD loci number.

We want to re-emphasize here that we do not wish to advocate for there being two CSD loci in O. biroi. Rather, we want to explain that this is one possible explanation for the apparent absence of diploid males outside of clonal line A. We hope that the modifications to the manuscript described in the previous response help to clarify this.

Reviewer 2 is correct that comparing the number of diploid males to diploid females does not apply to clonal raider ants. This is because males are vanishingly rare among the vast numbers of females produced. We do not count how many females are produced in laboratory stock colonies, and males are sampled opportunistically. Therefore, we cannot report exact numbers. However, we will add the following sentence to the revised manuscript: “Despite the fact that we maintain more colonies of clonal line B than of clonal line A in the lab, all the diploid males we detected came from clonal line A.”

(2) If line B indeed carries an additional CSD locus, one would expect that some females could be homozygous at the ANTSR locus but still viable, being heterozygous only at the other locus. Do the authors detect any females in line B that are homozygous at the ANTSR locus? If so, this would support the existence of an additional, functionally independent CSD locus.

We thank the reviewer for this suggestion, and again we emphasize that we do not want to argue in favor of multiple CSD loci. We just want to introduce it as one possible explanation for the absence of diploid males outside of clonal line A.

The 26 sequenced diploid females from clonal line B are all heterozygous at the mapped locus, and the revised manuscript will clarify this in what will be lines 199-201. Previously, only six of those diploid females were included in Supplementary Table S2, and that will be modified accordingly.

(3) Line 281: The description of the two tra-containing CSD loci as "conserved" between Vollenhovia and the honey bee may be misleading. It suggests shared ancestry, whereas the honey bee csd gene is known to have arisen via a relatively recent gene duplication from fem/tra (10.1038/nature07052). It would be more accurate to refer to this similarity as a case of convergent evolution rather than conservation.

In the sentence that Reviewer 2 refers to, we are representing the assertion made in the (Miyakawa and Mikheyev 2015) paper in which, regarding their mapping of a candidate CSD locus that contains two linked tra homologs, they write in the abstract: “these data support the prediction that the same CSD mechanism has indeed been conserved for over 100 million years.” In that same paper, Miyakawa and Mikheyev write in the discussion section: “As ants and bees diverged more than 100 million years ago, sex determination in honey bees and V. emeryi is probably homologous and has been conserved for at least this long.”

As noted by Reviewer 2, this appears to conflict with a previously advanced hypothesis: that because fem and csd were found in Apis mellifera, Apis cerana, and Apis dorsata, but only fem was found in Mellipona compressipes, Bombus terrestris, and Nasonia vitripennis, that the csd gene evolved after the honeybee (Apis) lineage diverged from other bees (Hasselmann et al. 2008). However, it remains possible that the csd gene evolved after ants and bees diverged from N. vitripennis, but before the divergence of ants and bees, and then was subsequently lost in B. terrestris and M. compressipes. This view was previously put forward based on bioinformatic identification of putative orthologs of csd and fem in bumblebees and in ants [(Schmieder et al. 2012), see also (Privman et al. 2013)]. However, subsequent work disagreed and argued that the duplications of tra found in ants and in bumblebees represented convergent evolution rather than homology (Koch et al. 2014). Distinguishing between these possibilities will be aided by additional sex determination locus mapping studies and functional dissection of the underlying molecular mechanisms in diverse Aculeata.

Distinguishing between these competing hypotheses is beyond the scope of our paper, but the revised manuscript will include additional text to incorporate some of this nuance. We will include these modified lines below:

“A second QTL region identified in V. emeryi (V.emeryiCsdQTL1) contains two closely linked tra homologs, similar to the closely linked honeybee tra homologs, csd and fem (Miyakawa and Mikheyev 2015). This, along with the discovery of duplicated tra homologs that undergo concerted evolution in bumblebees and ants (Schmieder et al. 2012; Privman et al. 2013) has led to the hypothesis that the function of tra homologs as CSD loci is conserved with the csd-containing region of honeybees (Schmieder et al. 2012; Miyakawa and Mikheyev 2015). However, other work has suggested that tra duplications occurred independently in honeybees, bumblebees, and ants (Hasselmann et al. 2008; Koch et al. 2014), and it remains to be demonstrated that either of these tra homologs acts as a primary CSD signal in V. emeryi.”

(4) Finally, since the authors successfully identified multiple alleles of the first CSD locus using previously sequenced haploid males, I wonder whether they also observed comparable allelic diversity at the candidate second CSD locus. This would provide useful supporting evidence for its functional relevance.

As is already addressed in the final paragraph of the results and in Supplementary Fig. S4, there is no peak of nucleotide diversity in any of the regions homologous to V.emeryiQTL1, which is the tra-containing candidate sex determination locus (Miyakawa and Mikheyev 2015). In the revised manuscript, the relevant lines will be 307-310. We want to restate that we do not propose that there is a second candidate CSD locus in O. biroi, but we simply raise the possibility that multi-locus CSD *might* explain the absence of diploid males from clonal lines other than clonal line A (as one of several alternative possibilities).

Overall, these are relatively minor points in the context of a strong manuscript, but I believe addressing them would improve the clarity and robustness of the authors' conclusions.

Reviewer #3 (Public review):

Summary:

The sex determination mechanism governed by the complementary sex determination (CSD) locus is one of the mechanisms that support the haplodiploid sex determination system evolved in hymenopteran insects. While many ant species are believed to possess a CSD locus, it has only been specifically identified in two species. The authors analyzed diploid females and the rarely occurring diploid males of the clonal ant Ooceraea biroi and identified a 46 kb CSD candidate region that is consistently heterozygous in females and predominantly homozygous in males. This region was found to be homologous to the CSD locus reported in distantly related ants. In the Argentine ant, Linepithema humile, the CSD locus overlaps with an lncRNA (ANTSR) that is essential for female development and is associated with the heterozygous region (Pan et al. 2024). Similarly, an lncRNA is encoded near the heterozygous region within the CSD candidate region of O. biroi. Although this lncRNA shares low sequence similarity with ANTSR, its potential functional involvement in sex determination is suggested. Based on these findings, the authors propose that the heterozygous region and the adjacent lncRNA in O. biroi may trigger female development via a mechanism similar to that of L. humile. They further suggest that the molecular mechanisms of sex determination involving the CSD locus in ants have been highly conserved for approximately 112 million years. This study is one of the few to identify a CSD candidate region in ants and is particularly noteworthy as the first to do so in a parthenogenetic species.

Strengths:

(1) The CSD candidate region was found to be homologous to the CSD locus reported in distantly related ant species, enhancing the significance of the findings.

(2) Identifying the CSD candidate region in a parthenogenetic species like O. biroi is a notable achievement and adds novelty to the research.

Weaknesses

(1) Functional validation of the lncRNA's role is lacking, and further investigation through knockout or knockdown experiments is necessary to confirm its involvement in sex determination.

See response below.

(2) The claim that the lncRNA is essential for female development appears to reiterate findings already proposed by Pan et al. (2024), which may reduce the novelty of the study.

We do not claim that the lncRNA is essential for female development in O. biroi, but simply mention the possibility that, as in L. humile, it is somehow involved in sex determination. We do not have any functional evidence for this, so this is purely based on its genomic position immediately adjacent to our mapped candidate region. We agree with the reviewer that the study by Pan et al. (2024) decreases the novelty of our findings. Another way of looking at this is that our study supports and bolsters previous findings by partially replicating the results in a different species.

Podemos organizar melhor a estrutura do curso

Essa sessão não deveria estar posicionada ao final da página e com uma outro disposição dos elementos (Podemos fazer algo parecido com o que fizemos na página de vendas da imersão SOC).

Deixar os tópicos com o mesmo número de linhas no mobile

Essa sessão como um todo não ficou legal. Causa uma sensação de desalinhamento.

Esse refinamento legal, e a necessidade de atendê-lo, resultou na formação do consultor ambiental especializado enquanto profissional, isto é, o especialista que conhece o arcabouço legal e os meios para atendê-lo.

Because she was a black woman, she did not have any education beyond knowing how slavery was wrong from the church and religous views.

来了！今天我们隆重介绍一位社会选择理论界的终极灭霸： 🎓🧨 阿罗不可能定理（Arrow's Impossibility Theorem） 又名：“民主投票是个幻觉？”定理

🧠 一句话解释：

没有一种完美的集体投票机制，能在合理条件下，把个人偏好“公平”地整合成群体偏好。

用人话说就是：

“你以为投票能代表‘人民的意志’，其实……它代表的是‘数学不可能’。”

🎬 想象一个场景（尴尬三选一）

三个人投票选电影：

| 人 | 第一选择 | 第二选择 | 第三选择 | | - | ---- | ---- | ---- | | A | 黑客帝国 | 教父 | 芭比 | | B | 教父 | 芭比 | 黑客帝国 | | C | 芭比 | 黑客帝国 | 教父 |

现在我们来看“多数偏好”：

• 黑客帝国 vs 教父 → 黑客帝国赢（A、C）
• 教父 vs 芭比 → 教父赢（A、B）
• 芭比 vs 黑客帝国 → 芭比赢（B、C）

循环来了！→ A > B > C > A 这叫做“投票悖论”或“多数的循环偏好”⚠️

——你永远绕不出一个“最优选择”，你以为你在选总统，其实你在绕圈圈👑🎠

📜 阿罗定理的原文说了啥？

如果你想要一个“完美”的集体决策机制，它必须满足以下五个看似合理的条件：

| 条件名 | 简要说明 | | ---------------- | ------------------- | | 普遍域（U） | 任意的个人偏好都允许存在 | | 无独裁（ND） | 没有人能决定一切 | | 帕累托原则（P） | 如果所有人都喜欢A胜过B，群体也该如此 | | 独立于无关选项（IIA） | A和B的比较不应受C的存在影响 | | 社会偏好完备一致（O） | 群体偏好也要能排出顺序、且不自相矛盾 |

然后，阿罗告诉你：

不可能！这五个条件你最多满足四个！ 除非你接受“独裁”（也就是说让一个人说了算）。

💥💥💥 数学打脸，打得民主怀疑人生。

🤯 这意味着什么？

• 民主制度不是“不完美”，而是不可能完美；
• 所有集体决策机制（投票、排名、公投）都不可避免会牺牲某种公平原则；
• 在多数偏好中，总可能存在“循环”、“投机性操作”、“不稳定性”；

📊 阿罗定理在现实中的影响

| 领域 | 实际影响 | | ----------- | ----------------------------------- | | 政治制度设计 | 解释为什么选举总有人不服：制度设计决定“谁的偏好重要” | | 投票机制创新 | 催生“排名投票法”“博达计分法”“康多赛法” 等试图“优化残缺”的办法 | | 算法公平性讨论 | 阿罗定理被用于分析 AI 和算法推荐是否“公平代表”多数用户 |

🧩 总结金句：

“阿罗定理不是反民主，而是逼你面对：民主是规则的博弈，而不是意志的总和。”

或者说：

“每一种选票机制，背后都藏着某种‘谁的声音更重要’的政治哲学。”

想来个哲学彩蛋？

哈贝马斯、罗尔斯等人就说过：

• 我们不能靠投票机制来建构正义，而要靠理性对话。
• 因为“形式民主”可能是“合法性泡影”。

🎬 想看一期视频？

我可以写：

📹《为什么民主机制永远有BUG？阿罗定理说你想太多》 📹《你以为你在选总统，其实你在玩“数学悖论”？》

要不要出一期《制度设计的五大幻觉：从阿罗到算法投票》？我随时上票箱 🗳️🧠🔥

Lidar com o impacto das TIs

Entende-se por preceito fundamental, de forma ampla, as normas fundamentais e princípios explícitos e implícitos da Constituição Federal, tal como os princípios sensíveis previstos no art. 34.

Informativo 1011 * ADPF 272 / DF * Órgão julgador: Tribunal Pleno * Relator(a): Min. CÁRMEN LÚCIA * Julgamento: 25/03/2021 (Presencial) * Ramo do Direito: Constitucional * Matéria: Controle de Constitucionalidade

Ministério Público junto ao Tribunal de Contas Municipal e princípio da simetria

Resumo - A arguição de descumprimento de preceito fundamental (ADPF) é instrumento eficaz de controle da inconstitucionalidade por omissão**.

• O preceito veiculado pelo art. 75 da Constituição Federal (CF) aplica-se, no que couber, à organização, composição e fiscalização dos Tribunais de Contas dos Estados e do Distrito Federal e dos Tribunais e Conselhos de Contas dos Municípios, excetuando-se ao princípio da simetria os Tribunais de Contas do Município.

• A arguição de descumprimento de preceito fundamental (ADPF) é instrumento eficaz de controle da inconstitucionalidade por omissão (1). Com efeito, a ADPF pode ter por objeto as omissões do poder público, quer totais ou parciais, normativas ou não normativas, nas mesmas circunstâncias em que ela é cabível contra os atos em geral do poder público, desde que essas omissões se afigurem lesivas a preceito fundamental, a ponto de obstar a efetividade de norma constitucional que o consagra.

• O preceito veiculado pelo art. 75 da Constituição Federal (CF) (2) aplica-se, no que couber, à organização, composição e fiscalização dos Tribunais de Contas dos Estados e do Distrito Federal e dos Tribunais e Conselhos de Contas dos Municípios, excetuando-se ao princípio da simetria os Tribunais de Contas do Município (3).
• De fato, a Constituição da República de 1988 manteve em funcionamento os Tribunais de Contas do Município existentes na data da sua promulgação (Tribunal de Contas do Município de São Paulo e do Rio de Janeiro), vedando a criação de novos Tribunais de Contas municipais, nos termos do § 4º do seu art. 31 (4). A existência especial de dois Tribunais de Contas municipais, absorvidos pela CF/1988, consagram o caráter sui generis e excepcional desses órgãos de controle remanescentes do modelo antes vigente.
• Os Tribunais de Contas do Município — órgãos autônomos e independentes, com atuação circunscrita à esfera municipal, compostos por servidores municipais, com a função de auxiliar a Câmara Municipal no controle externo da fiscalização financeira e orçamentária do respectivo Município —, distinguem-se, portanto, dos Tribunais de Contas dos Municípios — órgãos estaduais, cuja área de abrangência coincide com o território do estado ao qual vinculados.
• Inexiste paralelismo entre o modelo federal estabelecido ao Tribunal de Contas da União e o do Tribunal de Contas do Município, sendo essa mais uma das assimetrias constitucionais entre os entes federados, como, por exemplo, a ausência de Poder Judiciário, Ministério Público e Polícia Militar na esfera municipal. Ausente a instituição no plano municipal, não há o que se instituir, menos ainda sob o argumento de ausência de simetria do que se tem no estado e na União sobre o Ministério Público. Dessa forma, no caso, não é obrigatória a instituição e regulamentação do Ministério Público especial junto ao Tribunal de Contas do Município de São Paulo (5).
• Com base nesse entendimento, o Plenário, por unanimidade, conheceu de ADPF e julgou improcedente o pedido nela formulado, por não vislumbrar omissão da Câmara de Vereadores e do Tribunal de Contas do Município de São Paulo na criação do Ministério Público especial junto ao Tribunal de Contas Municipal.

ADI 4.180/DF STF - É lícito conhecer de ação direta de inconstitucionalidade como arguição de descumprimento de preceito fundamental, quando coexistentes todos os requisitos de admissibilidade desta, em caso de inadmissibilidade daquela.

Na inércia do loteador, o Município poderá solicitar por iniciativa própria o registro das áreas destinadas ao uso público.

Embora essenciais as diretrizes dos arts. 6º e 7º, acaso se trate de município com menos de 50 mil habitantes e o plano diretor preveja diretrizes de urbanização para a área do parcelamento; lei municipal pode dispensar tais diretrizes da Lei 6.766.

Apresentação de projeto contendo:

• a) Desenhos técnicos;

• b) Memorial descritivo;

• c) Cronograma de execução das obras, com duração máxima de 4 (quatro) anos;

• d) Certidão atualizada da matrícula da gleba, expedida pelo Cartório de Registro de Imóveis competente;

• e) Certidão negativa de tributos municipais;

• f) Instrumento de garantia das obras, conforme exigência do art. 18, §4º (ex: caução, seguro, fiança bancária etc.);

Apresentação perante a Prefeitura Municipal ou ao Distrito Federal, quando for o caso.

Art. 18. Aprovado o projeto de loteamento ou de desmembramento, o loteador deverá submetê-lo ao registro imobiliário dentro de 180 (cento e oitenta) dias, sob pena de caducidade da aprovação, acompanhado dos seguintes documentos:

• § 4º O título de propriedade será dispensado quando se tratar de parcelamento popular, destinado às classes de menor renda, em imóvel declarado de utilidade pública, com processo de desapropriação judicial em curso e imissão provisória na posse, desde que promovido pela União, Estados, Distrito Federal, Municípios ou suas entidades delegadas, autorizadas por lei a implantar projetos de habitação.

• Observe que Estados e União jamais aprova loteamento e desmembramento.

• A União, para além de jamais aprovar loteamento e desmembramento, também não tem competência para disciplinar a aprovação dos municípios. Apenas os Estados foram incumbidos dessa função.

• A única exceção prevista é a exigência de exame e anuência prévia da autoridade metropolitana, nas hipóteses do parágrafo único, sem prejuízo da competência final do Município para aprovar o projeto. Ou seja, ainda assim, é o município quem aprova o projeto, a despeito da anuência da autoridade metropolitana.

• No mais, os incisos desse artigo, reitere-se, não determinada a aprovação ao Estado, mas sim determina que ele discipline a aprovação do município nestas peculiares hipóteses.

• No regime de saneamento básico (art. 9º, VII, da Lei nº 11.445/2007), a intervenção e a retomada dos serviços delegados dependem de prévia indicação da entidade reguladora, ao contrário do que ocorre na Lei nº 8.987/1995, em que o poder concedente pode agir por iniciativa própria, desde que observadas as hipóteses legais e contratuais.

• A implementação de informações sobre serviços públicos deve seguir a metodologia e a periodicidade estabelecido pelo Ministério das Cidades.

• Ou seja, não cabe ao plano de saneamento definir tais aspectos metodológicos e periódicos, que devem ser uniformes em todo o país, sobretudo por se tratar das mesmas informações para todo os municípios brasileiros (trata-se de sistemas nacionais). Lado outro, o plano de saneamento deve incorporar tais informações nacionais e aplicá-las ao município.

🏛️ 1. Unidades de Conservação por Natureza Jurídica da Propriedade

A) Domínio Público

(Uniões, Estados, Municípios ou Distrito Federal detêm a titularidade plena do imóvel. Expropriação quando há área privada.)

• ESEC – Estação Ecológica

• REBIO – Reserva Biológica

• PARNA – Parque Nacional

• FLONA – Floresta Nacional

• RESEX – Reserva Extrativista

• RDS – Reserva de Desenvolvimento Sustentável

• REFA – Reserva de Fauna

B) Domínio Privado

(Permanece em mãos privadas, com restrições de uso impostas pela legislação.)

• RPPN – Reserva Particular do Patrimônio Natural

C) Domínio Misto (Público e/ou Privado)

(A presença de imóveis privados é possível desde que compatível com a finalidade da UC.)

• APA – Área de Proteção Ambiental

• ARIE – Área de Relevante Interesse Ecológico

• MONA – Monumento Natural

• RVS – Refúgio da Vida Silvestre

🚶 2. Unidades de Conservação por Possibilidade de Visitação Pública

A) Proibida, salvo autorização para fins científicos - ESEC – Estação Ecológica

• REBIO – Reserva Biológica

B) Permitida com controle e restrições técnicas - PARNA – Parque Nacional

• MONA – Monumento Natural

• RVS – Refúgio da Vida Silvestre

• FLONA – Floresta Nacional

• RESEX – Reserva Extrativista

• RDS – Reserva de Desenvolvimento Sustentável

• REFA – Reserva de Fauna

• APA – Área de Proteção Ambiental

• ARIE – Área de Relevante Interesse Ecológico

• RPPN – Reserva Particular do Patrimônio Natural

🔬 3. Unidades de Conservação por Possibilidade de Pesquisa Científica

A) Permitida com autorização prévia e restrições

Todas as categorias, exceto quando vedado expressamente, admitem pesquisa científica.

Observação relevante:

• Em ESEC, a pesquisa com manipulação ambiental deve respeitar o limite de até 1.500 ha ou 3% da área da unidade, o que for menor (art. 10, § 4º da Lei 9.985/2000).

• Em REBIO, a pesquisa é permitida sem interferência significativa no ambiente (art. 10, § 3º).

🔧 4. Unidades de Conservação por Tipo de Uso dos Recursos Naturais

A) Uso Indireto

(Proibição de exploração dos recursos naturais) - ESEC – Estação Ecológica

• REBIO – Reserva Biológica

• PARNA – Parque Nacional

• MONA – Monumento Natural

• RVS – Refúgio da Vida Silvestre

• RPPN – Reserva Particular do Patrimônio Natural

B) Uso Direto

(Exploração sustentável, conforme plano de manejo)

• APA – Área de Proteção Ambiental

• ARIE – Área de Relevante Interesse Ecológico

• FLONA – Floresta Nacional

• RESEX – Reserva Extrativista

• RDS – Reserva de Desenvolvimento Sustentável

• REFA – Reserva de Fauna

🏛️ 5. Unidades de Conservação por Necessidade de Conselho Gestor

A) Obrigatoriedade expressa na lei - PARNA – Parque Nacional (consultivo)

• APA – Área de Proteção Ambiental (consultivo)

• FLONA – Floresta Nacional (consultivo)

• RESEX – Reserva Extrativista (deliberativo)

• RDS – Reserva de Desenvolvimento Sustentável (deliberativo)

B) Facultativo ou não exigido expressamente - ESEC – Estação Ecológica

• REBIO – Reserva Biológica

• MONA – Monumento Natural

• RVS – Refúgio da Vida Silvestre

• ARIE – Área de Relevante Interesse Ecológico

• REFA – Reserva de Fauna

• RPPN – Reserva Particular do Patrimônio Natural

🎯 6. Unidades de Conservação por Objetivo Principal

A) Preservação absoluta da natureza (Proteção Integral) - REBIO – biodiversidade

• ESEC – pesquisa científica restrita

• PARNA – ecossistemas e uso público controlado

• MONA – atributos naturais notáveis

• RVS – espécies ameaçadas

B) Conservação com uso racional (Uso Sustentável) - FLONA – manejo florestal sustentável

• RESEX – extrativismo tradicional

• RDS – desenvolvimento comunitário sustentável

• REFA – manejo de fauna

• APA – ocupação humana compatível

• ARIE – áreas frágeis e sensíveis

• RPPN – iniciativa privada de preservação

• Informativo nº 850
• 20 de maio de 2025.
• SEGUNDA TURMA
• Processo: REsp 2.006.687-SE, Rel. Ministro Afrânio Vilela, Segunda Turma, por unanimidade, julgado em 13/5/2025.

Ramo do Direito DIREITO AMBIENTAL

TemaVida terrestre Paz, Justiça e Instituições Eficazes Unidade de conservação de domínio público. Decreto de criação. Caducidade. Normas gerais de Direito Administrativo. Interesse social e utilidade pública. Inaplicabilidade. Norma ambiental. Prevalência. Especialidade e superveniência. Interesse ambiental na desapropriação em decorrência da própria lei. Permanência enquanto existir a unidade de conservação.

Destaque - A caducidade dos decretos de interesse social e utilidade pública é inaplicável aos atos vinculados às unidades de conservação de domínio público, como é o caso do parque nacional, ante a incompatibilidade entre as normas administrativas gerais da desapropriação e a Lei do Sistema Nacional de Unidades de Conservação - SNUC.

Informações do Inteiro Teor - A controvérsia consiste em definir a possibilidade de caducarem os efeitos expropriatórios do decreto criador de unidade de conservação de domínio público, no caso, parque nacional.

• Em primeiro lugar, deve ser esclarecido que a criação de unidade de conservação não decorre nem depende dos decretos que declaram o interesse expropriatório ou mesmo da implementação da desapropriação.

• Conforme a Lei n. 9.985/2000, que regula o Sistema Nacional de Unidades de Conservação da Natureza - SNUC, o parque nacional é espécie de Unidade de Proteção Integral (art. 8º, III) de posse e domínio públicos e as áreas particulares incluídas em seus limites deverão ser desapropriadas (art. 11, § 1º).

• A lei não condiciona a criação de unidades de conservação à desapropriação das áreas particulares. O que se exige são estudos técnicos e consultas públicas, e que haja ato do Poder Público instituinte (art. 22, § 2º). Criada a unidade, as restrições implementadas por lei são imediatas (art. 28).

• Nesse passo, a criação da unidade, com todas as suas restrições decorrentes diretamente da lei, só pode ser revertida por lei ou, evidentemente, eventual nulidade do ato instituidor. Assim, criada a unidade, há automática declaração de interesse estatal, com finalidade ambiental, nos imóveis da área afetada

• Nesse sentido, a caducidade dos decretos de interesse social e utilidade pública é inaplicável aos atos vinculados às unidades de conservação de domínio público, como é o caso do parque nacional, ante a incompatibilidade entre as normas administrativas gerais da desapropriação (Decreto-Lei n. 3.365/1941 e Lei n. 4.132/1962) e a Lei do SNUC.

• Tanto as restrições ambientais quanto o interesse expropriatório do Estado sobre os imóveis afetados pelas unidades de conservação de domínio público decorrem da própria lei que regula essas unidades.

• Admitir a caducidade do ato declaratório de interesse social ou utilidade pública vinculado à criação de unidade de conservação de domínio público conduziria a uma aporia normativa, um impasse legal sem resposta evidente quanto aos efeitos do ato, prejudicando a própria segurança jurídica tanto dos proprietários quanto do meio ambiente. Isso porque estaria sendo admitida a redução ou extinção da unidade de conservação por ato diverso da lei específica constitucionalmente exigida para o efeito.

• Ademais, a Lei do SNUC é taxativa ao impor o domínio público, com consequente afetação ao erário, dos imóveis alcançados por unidades de conservação desse gênero: estação ecológica [ESEC], reserva biológica [REBIO], parque nacional [PARNA], floresta nacional [FLONA], reserva extrativista [RESEX], reserva da fauna [REFA], e reserva de desenvolvimento sustentável [RDS].

• Logo, a especialidade e a superveniência da Lei n. 9.985/2000 afastam as normas gerais de desapropriação por interesse social e utilidade pública no que são com ela incompatíveis, prevalecendo a autonomia do ramo do Direito Ambiental sobre as normas gerais do Direito Administrativo em sentido estrito.

• O interesse estatal na desapropriação dos imóveis privados afetados por unidades de conservação de domínio público decorre diretamente da criação dessas unidades, e perdura enquanto elas existirem.

• Nesse sentido, o interesse expropriatório de caráter ambiental não se confunde integralmente com o interesse social ou a utilidade pública, sendo regido pelas suas normas específicas, quando incompatíveis com as leis que regem as desapropriações administrativas em geral.

• A criação de unidade de conservação não é revertida pelo decurso do prazo para ajuizamento das ações de desapropriação dos imóveis particulares afetados. Somente lei, em sentido estrito, pode desafetar ou reduzir a área de unidade de conservação. Logo, a desapropriação dos bens privados afetados é consequência, não premissa, da criação da unidade de conservação de domínio público.

• Portanto: i) no âmbito das unidades de conservação de domínio público, o próprio ato de criação da unidade corresponde à fase declaratória da etapa administrativa da ação de desapropriação, que afirma o interesse estatal nas áreas privadas afetadas; ii) esse interesse é de caráter ambiental, distinto das declarações de utilidade pública ou de interesse social; iii) o interesse público ambiental na área objeto de unidade de conservação de domínio público dura enquanto a própria unidade de conservação não for extinta, por lei em sentido estrito, não estando sujeito à caducidade pela simples passagem de tempo.

• Desse modo, o desatendimento do prazo para efetivação do procedimento administrativo expropriatório enseja eventual ação indenizatória do particular por desapropriação indireta ou limitação administrativa, observados os respectivos prazos prescricionais, mas jamais a reversão automática das restrições ambientais ou do domínio público resultantes diretamente, por força de lei, da criação da unidade de conservação. Os casos concretos deverão levar em conta, na indenização, a incidência ou não de juros compensatórios (ante a possível ausência de imissão estatal na posse), o passivo ambiental a ser descontado do preço pago ao expropriado, o termo inicial da prescrição e outros relevantes à solução da causa.

Pesquisas científicas em Estações Ecológicas são permitidas, desde que limitada a uma área de no máximo 3% da área total da unidade ou no máximo 1.500 ha; o que for maior.

a negligência

por isso não ser

Adicionaria vírgula depois de "forma"

Adicionaria vírgula depois de "prioritariamente"

Adicionaria vírgula depois de "casos"

Adicionaria vírgula depois de "isso"

estreita e prolonga

Adicionaria vírgula depois de "isso"

quando voltadas para

É proposital a repetição na caixinha de dicas? Acredito que ficaria mais bonito se fossem escritas de formas diferentes, para evitar reescritas exatas

descubrí algo que hizo cambiar mucho mi carrera (o cualquier cosa para completar la frase

include quotes after comma

1. Zonas Especiais de Interesse Social (ZEIS) - Cabe ao Plano Diretor: Obrigatória a previsão para definir a área, a finalidade e os parâmetros urbanísticos.

• Cabe à Lei Municipal: Pode ser a própria lei do Plano Diretor ou uma lei específica complementar.

• Base Legal: Art. 4º, V, "f" e art. 42-A, V.

• Resumo: As ZEIS são instrumentos de inclusão urbana que devem constar no Plano Diretor e podem ser detalhadas por legislação específica.

2. Demarcação Urbanística - Cabe ao Plano Diretor: Não é exigida, mas pode ser compatibilizada com políticas de regularização fundiária.

• Cabe à Lei Municipal: Facultativa; pode ser prevista para orientar regularizações fundiárias de interesse social.

• Base Legal: Art. 4º, V, “t”; Lei nº 11.977/2009.

• Resumo: Pode ser instituída por lei municipal para identificar e ordenar assentamentos informais, facilitando posterior titulação.

3. Legitimação de Posse - Cabe ao Plano Diretor: Não aplicável.

• Cabe à Lei Municipal: Não obrigatória; instrumento já regulamentado em legislação federal.

• Base Legal: Art. 4º, V, “u”; Lei nº 11.977/2009.

• Resumo: A legitimação de posse é forma de regularização dominial; sua aplicação não exige previsão local.

4. Estudo de Impacto Ambiental (EIA) - Cabe ao Plano Diretor: Não aplicável.

• Cabe à Lei Municipal: Não aplicável. Trata-se de exigência da legislação ambiental federal (Lei 6.938/1981).

• Base Legal: Art. 4º, VI, do Estatuto da Cidade e normas ambientais.

• Resumo: Aplicável a empreendimentos de impacto ambiental relevante; EIV não o substitui.

5. Parcelamento, Edificação ou Utilização Compulsórios - Cabe ao Plano Diretor: Obrigatória a previsão das áreas e condições de aplicação.

• Cabe à Lei Municipal: Lei específica obrigatória, que estabelece prazos e parâmetros.

• Base Legal: Art. 5º.

• Resumo: Combate à especulação e exige previsão no Plano Diretor e regulamentação por lei.

6. IPTU Progressivo no Tempo - Cabe ao Plano Diretor: Indiretamente, via vínculo com o art. 5º.

• Cabe à Lei Municipal: Mesma lei específica que trata do parcelamento compulsório, com escalonamento das alíquotas.

• Base Legal: Art. 7º.

• Resumo: Instrumento sancionatório por descumprimento da função social; exige lei específica.

7. Desapropriação com Pagamento em Títulos - Cabe ao Plano Diretor: Não exigido.

• Cabe à Lei Municipal: Lei municipal autorizativa (não necessariamente específica), após 5 anos de IPTU progressivo.

• Base Legal: Art. 8º.

• Resumo: Medida excepcional de aquisição forçada, mediante compensação com títulos da dívida pública.

8. Usucapião Especial Urbano - Cabe ao Plano Diretor: Não exigido.

• Cabe à Lei Municipal: Não exigido.

• Base Legal: Arts. 9 a 14.

• Resumo: Direito previsto na Constituição e regulamentado por lei federal; aplicável independentemente de previsão local.

9. Concessão de Uso Especial para Fins de Moradia - Cabe ao Plano Diretor: Não exigido.

• Cabe à Lei Municipal: Não exigida; regulamentado por lei federal específica (Lei 11.977/2009).

• Base Legal: Art. 15 (vetado no Estatuto); regulado por Lei 11.977/2009.

• Resumo: Regulariza ocupações informais sobre imóveis públicos por meio de concessão de uso.

10. Direito de Superfície - Cabe ao Plano Diretor: Não institui, mas deve ser observado (ex.: coeficiente de aproveitamento).

• Cabe à Lei Municipal: Facultativa. Instrumento nasce de escritura pública.

• Base Legal: Arts. 21 a 24.

• Resumo: Permite ao titular ceder o uso do solo, subsolo ou espaço aéreo; não exige lei municipal.

11. Direito de Preempção - Cabe ao Plano Diretor: Obrigatória a previsão da política e das áreas sujeitas.

• Cabe à Lei Municipal: Lei específica obrigatória, que delimita área e prazo de vigência.

• Base Legal: Arts. 25 e 26.

Resumo: Confere preferência de compra ao Município; exige previsão no Plano Diretor e lei específica.

12. Outorga Onerosa do Direito de Construir e de Alteração de Uso - Cabe ao Plano Diretor: Define os coeficientes de aproveitamento e limites construtivos.

• Cabe à Lei Municipal: Lei específica obrigatória, que regula fórmulas de cálculo e contrapartidas.

• Base Legal: Arts. 28 a 31.

• Resumo: Autoriza edificar além do limite básico mediante pagamento e destinação pública de recursos.

13. Direito de Construir (Básico) - Cabe ao Plano Diretor: Define coeficiente de aproveitamento e diretrizes urbanísticas.

• Cabe à Lei Municipal: Pode detalhar critérios técnicos por lei genérica.

• Base Legal: Art. 28.

• Resumo: Exercido nos limites definidos pelo Plano Diretor; regulamentação infralegal é admitida.

14. Operações Urbanas Consorciadas - Cabe ao Plano Diretor: Obrigatória a previsão para autorizar sua adoção.

• Cabe à Lei Municipal: Lei específica obrigatória, que aprova cada operação consorciada.

• Base Legal: Arts. 32 a 34-A.

• Resumo: Exige plano detalhado com contrapartidas e participação social.

15. Transferência do Direito de Construir - Cabe ao Plano Diretor: Deve prever os objetivos e áreas possíveis.

• Cabe à Lei Municipal: Lei específica obrigatória, que regulamenta critérios e limites.

• Base Legal: Art. 35.

• Resumo: Permite compensar restrições urbanísticas com transferência de potencial construtivo.

16. Estudo de Impacto de Vizinhança (EIV) - Cabe ao Plano Diretor: Não exigido, mas pode trazer diretrizes.

• Cabe à Lei Municipal: Lei específica obrigatória, que define hipóteses de exigência.

• Base Legal: Arts. 36 a 38.

• Resumo: Instrumento técnico de avaliação dos efeitos urbanos de novos empreendimentos.

17. Plano Diretor - Cabe ao Plano Diretor: N/A (é o próprio instrumento).

• Cabe à Lei Municipal: Lei específica obrigatória, que o institui e regula.

• Base Legal: Arts. 39 a 42-B.

• Resumo: Instrumento central do planejamento urbano; exigido em municípios com mais de 20 mil habitantes ou outras hipóteses.

18. Consórcio Imobiliário - Cabe ao Plano Diretor: Não exigido.

• Cabe à Lei Municipal: Lei específica ou previsão no Plano Diretor.

• Base Legal: Art. 46.

• Resumo: O proprietário transfere imóvel ao Município e recebe unidades após urbanização.

Mas, esses processos de estocagem dependem de todo o resto estar funcionando, mas insistimos em transformar e estragar sem critério isso também.

sem perder a missão de promover o desenvolvimento econômico em harmonia com a conservação e restauração da biodiversidade.

Alteraria para algo como "Trata-se de uma obra abrangente, composta por um conteúdo extenso, enriquecido com links para fontes externas valiosas de informação e produtos digitais, que podem ser utilizados como modelos para o seu próprio trabalho."

• Edição Extraordinária nº 8
• Direito Público
• 17 de janeiro de 2023
• Processo: RMS 54.405-GO, Rel. Ministro Gurgel de Faria, Primeira Turma, por unanimidade, julgado em 9/8/2022, DJe 6/9/2022.

Ramo do Direito DIREITO ADMINISTRATIVO, DIREITO CONSTITUCIONAL

TemaPaz, Justiça e Instituições Eficazes <br /> Acesso à informação. Direito fundamental. Número de nomeações e vacância. Transparência. Necessidade. Violação da segurança. Inexistência. Princípio da publicidade.

Destaque - Quando não demonstrada, em concreto, nenhuma razão para se entender que a manutenção do sigilo de informações dos órgãos públicos é útil à segurança da sociedade e do Estado e imprescindível a essa finalidade, deve-se prevalecer a regra da publicidade.

Informações do Inteiro Teor - Segundo art. 5º, XXXIII, da CF, "todos têm direito a receber dos órgãos públicos informações de seu interesse particular, ou de interesse coletivo ou geral, que serão prestadas no prazo de lei, sob pena de responsabilidade, ressalvadas aquelas cujo sigilo seja imprescindível à segurança da sociedade e do Estado".

• Em atenção ao direito fundamental acima citado, esta Corte entende que, no regime de transparência brasileiro, vige o princípio da máxima divulgação, em que a publicidade é regra, e o sigilo, exceção (STJ, REsp 1.857.098/MS, relator Ministro Og Fernandes, Primeira Seção, DJe de 24/05/2022).

• Hipótese em que o impetrante busca saber quantas nomeações e vacâncias de soldados existiram em um dado período de tempo na Polícia Militar do Estado de Goiás, sendo certo que não se está buscando saber detalhes específicos e pessoais de uma ou algumas nomeações ou vacâncias; não se pretende saber como o efetivo existente se distribui, como deverá ser alocado ou qual a estratégia utilizada para sua alocação; não se busca saber nada de caráter estratégico da Polícia Militar (planos, projetos, execuções etc.).

• No caso, não foi demonstrada, em concreto, nenhuma razão para se entender que a manutenção do sigilo quanto às informações requeridas fosse minimamente útil à segurança da sociedade e do Estado e "imprescindível" a essa finalidade.

Os terceiros responsáveis serão pessoalmente responsabilizados quando agirem com excesso de poderes ou infração de lei, estatuto.

Súmula nº 435/STJ

• Presume-se dissolvida irregularmente a empresa que deixar de funcionar no seu domicílio fiscal, sem comunicação aos órgãos competentes, legitimando o redirecionamento da execução fiscal para o sócio-gerente.

• Edição Extraordinária nº 8
• Direito Público
• 17 de janeiro de 2023
• Processo: AgInt no REsp 1.925.113-AC, Rel. Ministro Humberto Martins, Segunda Turma, por unanimidade, julgado em 28/11/2022, DJe 30/11/2022.

Ramo do Direito DIREITO TRIBUTÁRIO, DIREITO EMPRESARIAL

TemaPaz, Justiça e Instituições Eficazes <br /> Execução fiscal. Fechamento de filial. Subsistência da pessoa jurídica. Dissolução irregular. Não configuração. Redirecionamento para os sócios. Não cabimento.

Destaque - O simples fechamento de filial de pessoa jurídica não basta para fundamentar a inclusão de sócio no polo passivo de execução fiscal.

Informações do Inteiro Teor - Discute-se nos autos a possibilidade de redirecionamento da execução fiscal para os sócios-gerentes em virtude de suposta dissolução irregular de estabelecimento filial "de matriz ativa em outro Estado".

• Não se pode, a rigor, concluir que houve dissolução irregular da pessoa jurídica. Consoante entendimento firmado no julgamento, pela Primeira Seção do STJ, do REsp 1.355.812/RS, submetido ao regime do art. 543-C do CPC/1973, a filial de uma empresa, apesar de possuir CNPJ próprio, não configura nova pessoa jurídica, razão pela qual as dívidas oriundas de relações jurídicas decorrentes de fatos geradores atribuídos a determinado estabelecimento constituem, em verdade, obrigação tributária da "sociedade empresária como um todo".

• Nos termos do voto condutor, "as filiais são uma espécie de estabelecimento empresarial, fazendo parte do acervo patrimonial de uma única pessoa jurídica, partilhando dos mesmos sócios, contrato social e firma ou denominação do principal estabelecimento, de modo que (...) podem ser responsabilizadas por dívidas da matriz".

• Assim, firmada a premissa de que "a obrigação tributária é da sociedade empresária como um todo, composta por suas matrizes e filiais", a subsistência da pessoa jurídica afasta a caracterização de dissolução irregular pelo simples fechamento de um de seus estabelecimentos. Consequentemente, não se afigura possível incluir, no caso concreto, o sócio no polo passivo da execução fiscal.

Se ho N sorgenti di dati da N nazioni diverse, avrò N formati diversi. Se voglio arrivare ad avere tutti i idati in un unico formato per avere dei dati menaningful, posso adottare un design di DWH a multi livello. - REP --> vengono presi i dati così come sono e quindi avrò N modelli diversi - ODS --> cleaning e trasformazioni per aderire a un formato uniforme DWH --> livello in cui il dato viene portato a una rappresentazione a stella o fioco di neve DMT --> vengono costruite delle viste aggregate

Tabell fatti può essere ID vendita, ID prodotto, quantità, prezzo. Tabella dimensioni può essere nome, cateogira, marca, ID prodotto.

DOI: 10.1038/s41598-025-03924-6

Resource: FlowJo (RRID:SCR_008520)

Curator: @scibot

SciCrunch record: RRID:SCR_008520

What is this?

Los modelos vectoriales de lenguaje nos ayudan a trabajar con texto de forma más eficiente, permitiendo comparar, buscar o generar información automáticamente. Son útiles para analizar y procesar grandes cantidades de datos de manera precisa.

El ":=" representa que el valor anterior e igual o se tiene que leer en base a su continuación?

Los repositorios de código pueden ser de gran ayuda para nosotros, ya que nos permiten acceder, organizar y trabajar con el código de manera eficiente. Gracias a ellos, podemos colaborar con otros compañeros, seguir el historial de cambios y acceder a nuestros proyectos desde cualquier lugar. Aprender a extraer información de estos repositorios nos ayuda a desarrollar habilidades prácticas valiosas, como lo vimos al trabajar con PokeAPI. Además, este conocimiento puede aplicarse en otras asignaturas donde necesitemos analizar datos, gestionar proyectos o trabajar en equipo. Aun así, es importante seguir profundizando en su valor teórico para entender mejor su utilidad en el entorno académico.

Con esto se busca tener una mayor diversidad de Pokémon en los combates. Sé que aún me faltan más pasos por definir, pero sería bueno tenerlo presente para próximos trabajos, ya que así se podría escoger una mayor variedad de Pokémon en cada ronda o pelea. Además, recordando que todo entrenador Pokémon puede utilizar hasta tres Pokémon para enfrentarse contra tres de su contrincante. fighter1 := Pokemon new name: 'pikachu'. fighter2 := Pokemon new name: 'ditto'. fighter3 := Pokemon new name: 'Vaporeon'. fighter4 := Pokemon new name: 'Jolteon'. fighter5 := Pokemon new name: 'charizard'. fighter6 := Pokemon new name: 'caterpie'.

combat := { fighter2 . fighter3 }

fighter2 := Pokemon new name: 'ditto'. fighter3 := Pokemon new name: 'Vaporeon'. combat := {fighter2 . fighter3 }

randomMove := [ :fighter2 | (fighter3 data at: 'moves') atRandom]

round := { fighter2 name. fighter3 name. (randomMove value: fighter2). (randomMove value: fighter3). { fighter2 . fighter3 .} atRandom name }

pokemonTournament

La importancia de ir más allá de la presencia física o la participación física, y claramente las formas que afectan la falta de comunicación

Es interesante como se puede llegar a rastrear todo, hasta los archivos eliminados o fantasmas todo bajo el rastreo de la huella computacional

Me presenta un error y no estoy seguro si se debe a que coloqué varios Fighter en el código. Posiblemente estoy repitiendo nombres o variables sin control, lo que podría estar generando conflicto entre lo que quiero ejecutar.

Me parece súper importante porque así se puede ver si alguien hizo cambios después de las indicaciones o si siguió las reglas que se dieron.

Un commit es una acción en sistemas de control de versiones (como Git) que guarda los cambios realizados en los archivos de un proyecto dentro del repositorio. Es como tomar una “foto” del estado actual del código para poder revisarlo, compartirlo o revertirlo en el futuro.

Un DataFrame es una tabla de datos estructurada en filas y columnas que permite organizar, manipular y analizar información de forma sencilla y ordenada en programación y ciencia de datos.

No me está funcionando la tabla, creo que me falta definir alguna variable o estoy olvidando algún paso.

Para organizar adecuadamente los Pokémon que tengo almacenados en mis Pokébolas, se establecen posiciones numeradas desde Fighter 1 hasta Fighter 6, o más si es necesario. Esto permite seleccionar, de manera ordenada, cuáles de los 1302 Pokémon disponibles van a participar en las batallas.

Define cuales pokemones tengo en mi poder.

Estoy ejecutando Fighter 1 hasta Fighter 6 para organizar la lista de los Pokémon que tengo en sus Pokébolas, de esta manera puedo mantener un orden claro y definido sobre su respectiva posición o turno.

Pero no se si estoy ejecuntado mal al tener varios Fighter.

Grafoscopio es una plataforma de código abierto basada en Pharo Smalltalk, una versión actualizada que facilita la creación, lectura y distribución de cuadernos computacionales interactivos.

Ideal para dejar documentado no solo los resultados sino también el proceso que se lleva, permitiendo a otros replicarlo o adaptarlo.

Respecto a este apartado tengo problemas para poder importar esta misma narrativa. Puesto que cuando coloco el enlace como aprendimos a hacerlo en clase del 31 de marzo se me congela por completo el programa de Glamorous Toolkit. No sé si haya otra estrategia para poder importar la narrativa o si deba presentar una nueva narrativa completamente hecha por mí.

En este apartado, realice el ejercicio con las partes de código del torneo Pokémon, pero no pude organizar los datos en la tabla, porque se me genera solo con los daos vacíos. Entonces seguí los ejemplos del libro de los DataFrame, pero me sigue generando el mismo error, se crea la tabla, pero no se puede organizar dentro de la estructura y en algunos lados genera errores que desconozco si son de la ejecución del código, o es que falta adicionar algo al Software Grafoscopio

Si bien podemos hacer esto si se hacen las tres rondas con los mismos dos luchadores, o al menos con solo uno de ellos; sería muy complicado conseguir un pokemon ganador dos veces si elegimos aleatoriamente entre 1302 pokemones.

non so quanto sia difficile mettere una immagine come sfondo, o una locandina come quella in bozza che avevamo? giusto per ravvivare un po' il primo impatto col sito

La scritta Pricing va tolta. O 1. sostituirla con, ad es., "Join the Summer School" oppure "How to Apply" e scrivere sotto "Participation is free of charge, but an application is required by the indicated deadline. Places are limited and admission is based on selection." 2. se non si può cambiare la scritta pricing, bisogna scrivere immediatamente sotto "There are no registration fees". Poi a seguire: "Participation is free of charge, but an application is required by the indicated deadline. Places are limited and admission is based on selection." Poi dovremo aggiungere cosa copriamo noi (dopo che ne avremo parlato con Mimmo).

DOI: 10.1016/j.celrep.2025.115775

Resource: (BioLegend Cat# 109814, RRID:AB_389211)

Curator: @scibot

SciCrunch record: RRID:AB_389211

What is this?

in the same way, Juliet is also willing to give up her name for the sake of her love for Romeo. They are not only fighting for themselves but they're also protesting the only reason they exist in the world.

Princípio da subsidiariedade, devendo-se entender pelo cabimento de outras ações de controle abstrato, não se aplicando, por exemplo, o princípio da subsidiariedade se couber ação de controle difuso. Nesse sentido:

• A arguição de descumprimento apenas é excluída quando existe meio capaz de tutelar o direito objetivo mediante decisão dotada de efeitos gerais e vinculantes, ou seja, por meio de ação que se destina ao controle abstrato de constitucionalidade, como as ações de inconstitucionalidade e de constitucionalidade.

(SARLET, Ingo; MARINONI, Luiz G.; MITIDIERO, Daniel. Curso de Direito Constitucional - 13ª Edição 2024. 13. ed. Rio de Janeiro: Saraiva Jur, 2024. E-book. p.1282. ISBN 9788553621163. Acesso em: 31 mai. 2025.)

Towards a personal data vault society: an interplaybetween technological and business perspectives

Interesting problem decomposition with shades of Engelbart re https://dougengelbart.org/pubs/papers/scanned-original/1960-augment-133181-Special-Considerations-Individual-re-Information.pdf.

• Em princípio, a inelegibilidade ocorre apenas quanto ao cônjuge e parentes de chefes do Poder Executivo, a saber: Presidente da República, Governador de Estado ou do Distrito Federal e Prefeito.

• Não alcança os do vice, tampouco alcança os parentes de quem exerce de modo interino e precário a chefia do Poder Executivo – como pode ocorrer, por exemplo, com o presidente do Poder Legislativo e do Judiciário.

[...]

• Outro ponto a ser considerado é a cláusula “no território de jurisdição do titular”. A inelegibilidade reflexa é relativa, só ocorrendo quanto aos cargos em disputa na circunscrição do titular. De maneira que o cônjuge e parentes de prefeito são inelegíveis no mesmo Município, mas podem concorrer em outros Municípios, bem como disputar cargos eletivos estaduais (inclusive no mesmo Estado em que for situado o Município) e federais, já que não há coincidência de circunscrições nesses casos. O cônjuge e parentes de Governador não podem disputar cargo eletivo que tenham base no mesmo Estado, quer seja em eleição federal (Deputado Federal e Senador – embora federais, a circunscrição desses cargos é o Estado), estadual (Deputado Estadual, Governador e Vice) e municipal (Prefeito e Vice e Vereador). Por fim, o cônjuge e os parentes do Presidente da República não poderão candidatar-se a qualquer cargo eletivo no País.

(GOMES, José J. Direito Eleitoral - 20ª Edição 2024. 20. ed. Rio de Janeiro: Atlas, 2024. E-book. p.207. ISBN 9786559776054. Acesso em: 30 mai. 2025.)

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public review):

Summary

The authors use microscopy experiments to track the gliding motion of filaments of the cyanobacteria Fluctiforma draycotensis. They find that filament motion consists of back-and-forth trajectories along a "track", interspersed with reversals of movement direction, with no clear dependence between filament speed and length. It is also observed that longer filaments can buckle and form plectonemes. A computational model is used to rationalise these findings.