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DOI: 10.1016/j.isci.2025.113860

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Las semillas inmaduras fueron tratadas con una mezcla de hormonas y sales para romper la dormancia, estimular la germinación y fortalecer el embrión, permitiendo sembrar la siguiente generación en menos de un día.

Es un método usado en mejoramiento genético de cultivos para que las plantas crezcan, florezcan y produzcan semillas mucho más rápido. En lugar de esperar una sola cosecha al año, con la cría rápida se pueden lograr 4 o 5 generaciones de plantas en un mismo año.

La inclusión es el pilar de una estrategia de comunicación efectiva, pues el incluir a todas las partes del personal, nos ayuda a reducir el ruido en la comunicación. Para lograr esto, así como lo expresa el texto es muy acertado el hacer uso de herramientas digitales modernas pues en este tiempo hay que empezar a digitalizarnos, asimismo esto nos puede garantizar que la información llegue a absolutamente todo el personal.

Considero que es esencial el recalcar esto, púes efectivamente cada empresa al tener diferentes giros, debe de efectuar diferentes estrategias, aunque todas deben de guiar hacia el mismo objetivo que es la buena comunicación bidireccional.

En esencia, este párrafo nos muestra como la comunicación interna es el pilar de una cultura empresarial exitosa. Cuando la información fluye de manera clara y constante, los empleados desarrollan un fuerte sentido de pertenencia y confianza en la organización, lo cual es fundamental para el éxito a largo plazo. No se trata solo de hablar, sino de evitar malentendidos; el objetivo es que el mensaje original se entienda perfectamente en todos los niveles, sin que se distorsione en el camino. Y efectivamente nos muestra como el tener un sistema de comunicación efectivo tiene múltiples ventajas.

Función Clave de la Comunicación: Su rol principal es permitir que el plan estratégico se ejecute eficientemente en todos los niveles jerárquicos, optimizando tiempo, recursos (humanos y económicos) y esfuerzo.

Necesidad de Planificación: La comunicación no debe ser un proceso improvisado. El artículo subraya que debe ser estructurada, ordenada y planificada desde la alta dirección, e integrada en el plan estratégico general de la compañía.

Impacto en el Clima Organizacional y la Productividad: Se establece una relación directa entre una comunicación eficiente y un buen clima organizacional. Una comunicación deficiente, incluso en empresas rentables, genera alta rotación de personal, desmotivación y, en última instancia, una disminución de la productividad.

La Comunicación como Pilar Estratégico: El artículo posiciona la comunicación no solo como una herramienta operativa, sino como un recurso estratégico indispensable para la gestión empresarial. Es fundamental para alinear a toda la organización con la visión, misión y objetivos establecidos.

La Comunicación como Pilar Estratégico: El artículo posiciona la comunicación no solo como una herramienta operativa, sino como un recurso estratégico indispensable para la gestión empresarial. Es fundamental para alinear a toda la organización con la visión, misión y objetivos establecidos.

considero que este subtema refleja como una comunicación interna efectiva mejora los procesos operativos al igual que fortalece el sentido de pertenencia y confianza entre los miembros de una organización.

Coincido plenamente con la observación de que la globalización y la competencia obligan a las empresas a diferenciarse mediante estrategias comunicacionales efectivas. Desde mi punto de vista, esta sección destaca un punto clave: la comunicación no solo influye en el marketing o la imagen corporativa, sino también en la eficiencia operativa interna, ya que una información mal transmitida puede generar retrasos o duplicación de esfuerzos. En este sentido, la gestión comunicacional actúa como un sistema nervioso de la empresa, permitiendo la coordinación entre sus distintas áreas.

Las conclusiones resumen con claridad la relevancia de adoptar sistemas de comunicación multidireccionales y aprovechar los canales digitales. Personalmente, creo que este punto conecta con el desafío actual de muchas empresas: mantener la cohesión humana en entornos tecnológicos. Es decir, la digitalización no debe sustituir el contacto personal, sino fortalecerlo a través de la inmediatez y la transparencia. Por tanto, la comunicación empresarial en este siglo XXI debe equilibrar lo tecnológico con lo humano para mantener su efectividad.

El argumento de que una comunicación eficiente mejora el clima laboral y promueve la confianza es totalmente válido. Considero que este punto debería verse también desde la óptica de la inteligencia emocional organizacional: cuando los líderes practican una comunicación empática, abierta y transparente, los equipos se sienten valorados y esto impacta directamente en la productividad. En otras palabras, la comunicación no solo transmite datos, sino que construye relaciones humanas dentro de la empresa.

Me parece muy acertada la relación que los autores establecen entre la deficiente comunicación y el deterioro del clima laboral. Un entorno donde los mensajes no fluyen o son ambiguos tiende a generar desmotivación, frustración y alta rotación de empleados. En mi experiencia, la comunicación organizacional no solo busca informar, sino también dar sentido al trabajo. Cuando el personal entiende el propósito de sus tareas y percibe coherencia entre lo que se dice y lo que se hace, aumenta su compromiso y satisfacción. Esto convierte la comunicación en un pilar de la cultura organizacional.

All states were surrounded by nonstate peoples, but owing to their dispersal, we know precious little about their coming and going, their shifting relationship to states, and their political structures. When a city is burned to the ground, it is often hard to tell whether it was an accidental

fire such as plagued all ancient cities built of combustible ma- terials, a civil war or uprising, or a raid from outside.

1 feb 2023:

Assisi, Fondo Antico presso la Biblioteca del Sacro Convento, ms. 568 https://www.internetculturale.it/it/16/search/detail?id=oai%3Awww.internetculturale.sbn.it%2FTeca%3A20%3ANT0000%3APG0213_ms.568

https://www.internetculturale.it/jmms/iccuviewer/iccu.jsp?id=oai%3Awww.internetculturale.sbn.it%2FTeca%3A20%3ANT0000%3APG0213_ms.568&mode=all&teca=MagTeca+-+ICCU

Heidelberg: https://digi.ub.uni-heidelberg.de/diglit/bav_pal_lat_1326/0335/image,thumbs

BAV: https://digi.vatlib.it/view/MSS_Pal.lat.1326

Archaeology shows that many societies that experimented with freedom, fluid leadership, and non-coercive systems. People were not forced into hierarchy by a law of progress, but they rather made choices.

tönt toll, je nach Charakter vergleichen Kinder sich und fühlen sich dann schlecht oder fühlen sich viel besser und verachten andere etwas. Im Klassenklima durch kreative Tasks muss dies ausgeglichen werden, da jeder nach Respekt strebt.

Súmula 576/STJ - Ausente requerimento administrativo no INSS, o termo inicial para a implantação da aposentadoria por invalidez concedida judicialmente será a data da citação válida.

Como conciliar a Súmula 576 do STJ com a decisão do STF que impõe o prévio requerimento administrativo (RE 631240/MG)?

• Se formos analisar os precedentes que deram origem à Súmula 576-STJ, iremos perceber que eles envolvem processos judiciais iniciados antes da decisão do STF no RE 631240/MG, ou seja, na época em que a jurisprudência majoritária não exigia o prévio requerimento administrativo para que o segurado pudesse ingressar com a ação.
• Portanto, os debates que envolveram a Súmula 576-STJ ocorreram em processos surgidos em dado momento histórico em que o segurado ainda podia escolher se primeiro iria tentar requerer o benefício na via administrativa ou se já queria propor diretamente a ação judicial pleiteando a aposentadoria por invalidez. Assim, a Súmula 576-STJ surgiu principalmente para dirimir estes processos.

(CAVALCANTE, Márcio André Lopes. Súmula 576-STJ. Buscador Dizer o Direito, Manaus. Disponível em: https://buscadordizerodireito.com.br/jurisprudencia/3660/sumula-576-stj.)

Observe que a nulidade relativa à ausência de intimação do MP não é automática. O próprio MP deve se pronunciar a respeito da existência ou não de prejuízo antes da decretação da nulidade.

• O termo inicial do prazo decadencial para o direito de revisar/invalidar a tutela de urgência antecedente se constitui na data da ciência da decisão que extinguiu o processo.

• Ou seja, não se deve contar da data da decisão que extinguiu o processo, nem mesmo da data da sua publicação; mas sim na data em que se tomou ciência inequívoca da referida decisão.

• Informativo nº 731
• 4 de abril de 2022.
• QUARTA TURMA
• Processo: REsp 1.978.138-SP, Rel. Min. Antonio Carlos Ferreira, Quarta Turma, por unanimidade, julgado em 22/03/2022.

Ramo do Direito DIREITO PROCESSUAL CIVIL

TemaPaz, Justiça e Instituições Eficazes <br /> Ação civil pública. Legitimidade ativa ad causam. Administração pública indireta. Pertinência temática. Necessidade.

Destaque - A legitimidade ativa na ação civil pública das pessoas jurídicas da administração pública indireta depende da pertinência temática entre suas finalidades institucionais e o interesse tutelado.

Informações do Inteiro Teor - Inicialmente, a pertinência temática consiste na "harmonização entre as finalidades institucionais das associações civis ou dos órgãos públicos legitimados e o objeto a ser tutelado na ação civil pública. Em outras palavras, mencionadas pessoas somente poderão propor a ação civil pública em defesa de um interesse cuja tutela seja de sua finalidade institucional"

• É fato que o art. 5º da Lei n. 7.347/1985 apenas exige expressamente da associação, pessoa jurídica de direito privado, a comprovação de pertinência temática para propositura de ação civil pública.

• Por conseguinte, em uma interpretação literal, não seria necessária a comprovação da pertinência temática para que as autarquias, empresas públicas, fundações públicas e sociedades de economia mista ajuizassem ações coletivas.

• Nessa perspectiva, os integrantes da administração pública indireta passariam a ter amplos poderes, concorrendo, inclusive, com as finalidades institucionais do Ministério Público e da Defensoria Pública, convertendo-se em verdadeiros "procuradores universais", com legitimidade para ajuizamento das mais variadas demandas coletivas, independentemente de sua área de atuação.

• Tal concepção <u>ignora</u> as competências legais e estatutárias das instituições, as quais delimitam o campo de atuação das pessoas jurídicas integrantes da administração pública indireta. Sob o mesmo raciocínio, a doutrina entende que "*não basta a existência fática de uma pessoa da Administração Pública indireta: necessário se faz o exame de seu regime estatutário* (lei, regulamento, contrato ou ato de constituição etc.). Será o seu estatuto que conferirá legitimidade adequada (ou não) à pessoa jurídica, com densidades diferentes: uma coisa é uma autarquia; outra, uma sociedade de economia mista com capital aberto na bolsa de valores".

• Portanto, não há como considerar titular do interesse, na propositura da ação coletiva, pessoa jurídica da administração pública indireta sem nenhum vínculo com a tese jurídica deduzida, cujo objeto litigioso não se encontra entre aqueles a serem protegidos por sua finalidade institucional.

Indikátory jsou dostatečně popsány v jiné části zprávy, a tady bych to tolik neduplikoval. Nechal bych jen krátký odstavec s tím, jak se to tedy vztahuje k hlavní otázce této části, jaké jsou limity indikátoru v tomto ohledu. A případně je možné dát proklik na jinou část zprávy.

Reviewer #1 (Public review):

Summary:

This preprint from Shaowei Zhao and colleagues presents results that suggest tumorous germline stem cells (GSCs) in the Drosophila ovary mimic the ovarian stem cell niche and inhibit the differentiation of neighboring non-mutant GSC-like cells. The authors use FRT-mediated clonal analysis driven by a germline-specific gene (nos-Gal4, UASp-flp) to induce GSC-like cells mutant for bam or bam's co-factor bgcn. Bam-mutant or bgcn-mutant germ cells produce tumors in the stem cell compartment (the germarium) of the ovary (Figure 1). These tumors contain non-mutant cells - termed SGC for single-germ cells. 75% of SGCs do not exhibit signs of differentiation (as assessed by bamP-GFP) (Figure 2). The authors demonstrate that block in differentiation in SGC is a result of suppression of bam expression (Figure 2). They present data suggesting that in 73% of SGCs, BMP signaling is low (assessed by dad-lacZ) (Figure 3) and proliferation is less in SGCs vs GSCs. They present genetic evidence that mutations in BMP pathway receptors and transcription factors suppress some of the non-autonomous effects exhibited by SGCs within bam-mutant tumors (Figure 4). They show data that bam-mutant cells secrete Dpp, but this data is not compelling (see below) (Figure 5). They provide genetic data that loss of BMP ligands (dpp and gbb) suppresses the appearance of SGCs in bam-mutant tumors (Figure 6). Taken together, their data support a model in which bam-mutant GSC-like cells produce BMPs that act on non-mutant cells (i.e., SGCs) to prevent their differentiation, similar to what is seen in the ovarian stem cell niche. This preprint from Shaowei Zhao and colleagues presents results that suggest tumorous germline stem cells (GSCs) in the Drosophila ovary mimic the ovarian stem cell niche and inhibit the differentiation of neighboring non-mutant GSC-like cells. The authors use FRT-mediated clonal analysis driven by a germline-specific gene (nos-Gal4, UASp-flp) to induce GSC-like cells mutant for bam or bam's co-factor bgcn. Bam-mutant or bgcn-mutant germ cells produce tumors in the stem cell compartment (the germarium) of the ovary (Figure 1). These tumors contain non-mutant cells - termed SGC for single-germ cells. 75% of SGCs do not exhibit signs of differentiation (as assessed by bamP-GFP) (Figure 2). The authors demonstrate that block in differentiation in SGC is a result of suppression of bam expression (Figure 2). They present data suggesting that in 73% of SGCs, BMP signaling is low (assessed by dad-lacZ) (Figure 3) and proliferation is less in SGCs vs GSCs. They present genetic evidence that mutations in BMP pathway receptors and transcription factors suppress some of the non-autonomous effects exhibited by SGCs within bam-mutant tumors (Figure 4). They show data that bam-mutant cells secrete Dpp, but this data is not compelling (see below) (Figure 5). They provide genetic data that loss of BMP ligands (dpp and gbb) suppresses the appearance of SGCs in bam-mutant tumors (Figure 6). Taken together, their data support a model in which bam-mutant GSC-like cells produce BMPs that act on non-mutant cells (i.e., SGCs) to prevent their differentiation, similar to what in seen in the ovarian stem cell niche.

Strengths:

(1) Use of an excellent and established model for tumorous cells in a stem cell microenvironment.

(2) Powerful genetics allow them to test various factors in the tumorous vs non-tumorous cells.

(3) Appropriate use of quantification and statistics.

Weaknesses:

(1) What is the frequency of SGCs in nos>flp; bam-mutant tumors? For example, are they seen in every germarium, or in some germaria, etc, or in a few germaria?

(2) Does the breakdown in clonality vary when they induce hs-flp clones in adults as opposed to in larvae/pupae?

(3) Approximately 20-25% of SGCs are bam+, dad-LacZ+. Firstly, how do the authors explain this? Secondly, of the 70-75% of SGCs that have no/low BMP signaling, the authors should perform additional characterization using markers that are expressed in GSCs (i.e., Sex lethal and nanos).

(4) All experiments except Figure 1I (where a single germarium with no quantification) were performed with nos-Gal4, UASp-flp. Have the authors performed any of the phenotypic characterizations (i.e., figures other than Figure 1) with hs-flp?

(5) Does the number of SGCs change with the age of the female? The experiments were all performed in 14-day-old adult females. What happens when they look at a young female (like 2-day-old). I assume that the nos>flp is working in larval and pupal stages, and so the phenotype should be present in young females. Why did the authors choose this later age? For example, is the phenotype more robust in older females? Or do you see more SGCs at later time points?

(6) Can the authors distinguish one copy of GFP versus 2 copies of GFP in germ cells of the ovary? This is not possible in the Drosophila testis. I ask because this could impact the clonal analyses diagrammed in Figure 4A and 4G and in 6A and B. Additionally, in most of the figures, the GFP is saturated, so it is not possible to discern one vs two copies of GFP.

(7) More evidence is needed to support the claim of elevated Dpp levels in bam or bgcn mutant tumors. The current results with the dpp-lacZ enhancer trap in Figure 5A, B are not convincing. First, why is the dpp-lacZ so much brighter in the mosaic analysis (A) than in the no-clone analysis (B)? It is expected that the level of dpp-lacZ in cap cells should be invariant between ovaries, and yet LacZ is very faint in Figure 5B. I think that if the settings in A matched those in B, the apparent expression of dpp-lacZ in the tumor would be much lower and likely not statistically significant. Second, they should use RNA in situ hybridization with a sensitive technique like hybridization chain reactions (HCR) - an approach that has worked well in numerous Drosophila tissues, including the ovary.

(8) In Figure 6, the authors report results obtained with the bamBG allele. Do they obtain similar data with another bam allele (i.e., bamdelta86)?

Reviewer #2 (Public review):

While the study by Zhang et al. provides valuable insights into how germline tumors can non-autonomously suppress the differentiation of neighboring wild-type germline stem cells (GSCs), several conceptual and technical issues limit the strength of the conclusions.

Major points:

(1) Naming of SGCs is confusing. In line 68, the authors state that "many wild-type germ cells located outside the niche retained a GSC-like single-germ-cell (SGC) morphology." However, bam or bgcn mutant GSCs are also referred to as "SGCs," which creates confusion when reading the text and interpreting the figures. The authors should clarify the terminology used to distinguish between wild-type SGCs and tumor (bam/bgcn mutant) SGCs, and apply consistent naming throughout the manuscript and figure legends.

a) The same confusion appears in Figure 2. It is unclear whether the analyzed SGCs are wild-type or bam mutant cells. If the SGCs analyzed are Bam mutants, then the lack of Bam expression and failure to differentiate would be expected and not informative. However, if the SGCs are wild-type GSCs located outside the niche, then the observation would suggest that Bam expression is silenced in these wild-type cells, which is a significant finding. The authors should clarify the genotype of the SGCs analyzed in Figure 2C, as this information is not currently provided.

b) In Figures 4B and 4E, the analysis of SGC composition is confusing. In the control germaria (bam mutant mosaic), the authors label GFP⁺ SGCs as "wild-type," which makes interpretation unclear. Note, this is completely different from their earlier definition shown in line 68.

c) Additionally, bam⁺/⁻ GSCs (the first bar in Figure 4E) should appear GFP⁺ and Red⁺ (i.e., yellow). It would be helpful if the authors could indicate these bam⁺/⁻ germ cells directly in the image and clarify the corresponding color representation in the main text. In Figure 2A, although a color code is shown, the legend does not explain it clearly, nor does it specify the identity of bam⁺/⁻ cells alone. Figure 4F has the same issue, and in this graph, the color does not match Figure 4A.

(2) The frequencies of bam or bgcn mutant mosaic germaria carrying [wild-type] SGCs or wild-type germ cell cysts with branched fusomes, as well as the average number of wild-type SGCs per germarium and the number of days after heat shock for the representative images, are not provided when Figure 1 is first introduced. Since this is the first time the authors describe these phenotypes, including these details is essential. Without this information, it is difficult for readers to follow and evaluate the presented observations.

(3) Without the information mentioned in point 2, it causes problems when reading through the section regarding [wild-type] SGCs induced by impairment of differentiation or dedifferentiation. In lines 90-97, the authors use the presence of midbodies between cystocytes as a criterion to determine whether the wild-type GSCs surrounded by tumor GSCs arise through dedifferentiation. However, the cited study (Mathieu et al., 2022) reports that midbodies can be detected between two germ cells within a cyst carrying a branched fusome upon USP8 loss.

a) Are wild-type germ cell cysts with branched fusomes present in the bam mutant mosaic germaria? What is the proportion of germaria containing wild-type SGCs versus those containing wild-type germ cell cysts with branched fusomes?

b) If all bam mutant mosaic germaria carry only wild-type GSCs outside the niche and no germaria contain wild-type germ cell cysts with branched fusomes, then examining midbodies as an indicator of dedifferentiation may not be appropriate.

c) If, however, some germaria do contain wild-type germ cell cysts with branched fusomes, the authors should provide representative images and quantify their proportion.

d) In line 95, although the authors state that 50 germ cell cysts were analyzed for the presence of midbodies, it would be more informative to specify how many germaria these cysts were derived from and how many biological replicates were examined.

(4) Note that both bam mutant GSCs and wild-type SGCs can undergo division to generate midbodies (double cells), as shown in Figure 4H. Therefore, the current description of the midbody analysis is confusing. The authors should clarify which cell types were examined and explain how midbodies were interpreted in distinguishing between cell division and differentiation.

(5) The data in Figure 5 showing Dpp expression in bam mutant tumorous GSCs are not convincing. The Dpp-lacZ signal appears broadly distributed throughout the germarium, including in escort cells. To support the claim more clearly, the authors should present corresponding images for Figures 5D and 5E, in which dpp expression was knocked down in the germ cells of bam or bgcn mutant mosaic germaria. Showing these images would help clarify the localization and specificity of Dpp-lacZ expression relative to the tumorous GSCs.

(6) While Figure 6 provides genetic evidence that bam mutant tumorous GSCs produce Dpp to inhibit the differentiation of wild-type SGCs, it should be noted that these analyses were performed in a dpp⁺/⁻ background. To strengthen the conclusion, the authors should include appropriate controls showing [dpp⁺/⁻; bam⁺/⁻] SGCs and [dpp⁺/⁻; bam⁺/⁻] germ cell cysts without heat shock (as referenced in Figures 6F and 6I).

(7) Previous studies have reported that bam mutant germ cells cause blunted escort cell protrusions (e.g., Kirilly et al., Development, 2011), which are known to contribute to germ cell differentiation (e.g., Chen et al., Frontiers in Cell and Developmental Biology, 2022). The authors should include these findings in the Discussion to provide a broader context and to acknowledge how alterations in escort cell morphology may further influence differentiation defects in their model.

(8) Since fusome morphology is an important readout of SGCs vs differentiation. All the clonal analysis should have fusome staining.

(9) Figure arrangement. It is somewhat difficult to identify the figure panels cited in the text due to the current panel arrangement.

(10) The number of biological replicates and germaria analyzed should be clearly stated somewhere in the manuscript-ideally in the Methods section or figure legends. Providing this information is essential for assessing data reliability and reproducibility.

Reviewer #3 (Public review):

Summary:

Zhang et al. investigated how germline tumors influence the development of neighboring wild-type (WT) germline stem cells (GSC) in the Drosophila ovary. They report that germline tumors inhibit the differentiation of neighboring WT GSCs by arresting them in an undifferentiated state, resulting from reduced expression of the differentiation-promoting factor Bam. They find that these tumor cells produce low levels of the niche-associated signaling molecules Dpp and Gbb, which suppress bam expression and consequently inhibit the differentiation of neighboring WT GSCs non-cell-autonomously. Based on these findings, the authors propose that germline tumors mimic the niche to suppress the differentiation of the neighboring stem cells.

Strengths:

This study addresses an important biological question concerning the interaction between germline tumor cells and WT germline stem cells in the Drosophila ovary. If the findings are substantiated, they could provide valuable insights applicable to other stem cell systems.

Weaknesses:

Previous work from Xie's lab demonstrated that bam and bgcn mutant GSCs can outcompete WT GSCs for niche occupancy. Furthermore, a large body of literature has established that the interactions between escort cells (ECs) and GSC daughters are essential for proper and timely germline differentiation (the differentiation niche). Disruption of these interactions leads to arrest of germline cell differentiation in a status with weak BMP signaling activation and low bam expression, a phenotype virtually identical to what is reported here.

Thus, it remains unclear whether the observed phenotype reflects "direct inhibition by tumor cells" or "arrested differentiation due to the loss of the differentiation niche". Because most data were collected at a very late stage (more than 10 days after clonal induction), when tumor cells already dominate the germarium, this question cannot be solved. To distinguish between these two possibilities, the authors could conduct a time-course analysis to examine the onset of the WT GSC-like single-germ-cell (SGC) phenotype and determine whether early-stage tumor clones with a few tumor cells can suppress the differentiation of neighboring WT GSCs with only a few tumor cells present. If tumor cells indeed produce Dpp and Gbb (as proposed here) to inhibit the differentiation of neighboring germline cells, a small cluster or probably even a single tumor cell generated at an early stage might prevent the differentiation of their neighboring germ cells.

The key evidence supporting the claim that tumor cells produce Gpp and Gbb comes from Figures 5 and 6, which suggest that tumor-derived dpp and gbb are required for this inhibition. However, interpretation of these data requires caution.

In Figure 5, the authors use dpp-lacZ to support the claim that dpp is upregulated in tumor cells (Figure 5A and 5B). However, the background expression in somatic cells (ECs and pre-follicular cells) differs noticeably between these panels. In Figure 5A, dpp-lacZ expression in somatic cells in 5A is clearly higher than in 5B, and the expression level in tumor cells appears comparable to that in somatic cells (dpp-lacZ single channel). Similarly, in Figure 5B, dpp-lacZ expression in germline cells is also comparable to that in somatic cells. Providing clear evidence of upregulated dpp and gbb expression in tumor cells (for example, through single-molecular RNA in situ) would be essential.

Most tumor data present in this study were collected from the bam[86] null allele, whereas the data in Figure 6 were derived from a weaker bam[BG] allele. This bam[BG] allele is not molecularly defined and shows some genetic interaction with dpp mutants. As shown in Figure 6E, removal of dpp from homozygous bam[BG] mutant leads to germline differentiation (evidenced by a branched fusome connecting several cystocytes, located at the right side of the white arrowhead). In Figure 6D, fusome is likely present in some GFP-negative bam[BG]/bam[BG] cells. To strengthen their claim that the tumor produces Dpp and Gbb to inhibit WT germline cell differentiation, the authors should repeat these experiments using the bam[86] null allele.

It is well established that the stem niche provides multiple functional supports for maintaining resident stem cells, including physical anchorage and signaling regulation. In Drosophila, several signaling molecules produced by the niche have been identified, each with a distinct function - some promoting stemness, while others regulate differentiation. Expression of Dpp and Gbb alone does not substantiate the claim that these tumor cells have acquired the niche-like property. To support their assertion that these tumors mimic the niche, the authors should provide additional evidence showing that these tumor cells also express other niche-associated markers. Alternatively, they could revise the manuscript title to more accurately reflect their findings.

In the Method section, the authors need to provide details on how dpp-lacZ expression levels were quantified and normalized.

Author response:

Reviewer #1 (Public review):

Summary:

This preprint from Shaowei Zhao and colleagues presents results that suggest tumorous germline stem cells (GSCs) in the Drosophila ovary mimic the ovarian stem cell niche and inhibit the differentiation of neighboring non-mutant GSC-like cells. The authors use FRT-mediated clonal analysis driven by a germline-specific gene (nos-Gal4, UASp-flp) to induce GSC-like cells mutant for bam or bam's cofactor bgcn. Bam-mutant or bgcn-mutant germ cells produce tumors in the stem cell compartment (the germarium) of the ovary (Figure 1). These tumors contain non-mutant cells - termed SGC for single-germ cells. 75% of SGCs do not exhibit signs of differentiation (as assessed by bamP-GFP) (Figure 2). The authors demonstrate that block in differentiation in SGC is a result of suppression of bam expression (Figure 2). They present data suggesting that in 73% of SGCs, BMP signaling is low (assessed by dad-lacZ) (Figure 3) and proliferation is less in SGCs vs GSCs. They present genetic evidence that mutations in BMP pathway receptors and transcription factors suppress some of the non-autonomous effects exhibited by SGCs within bam-mutant tumors (Figure 4). They show data that bam-mutant cells secrete Dpp, but this data is not compelling (see below) (Figure 5). They provide genetic data that loss of BMP ligands (dpp and gbb) suppresses the appearance of SGCs in bam-mutant tumors (Figure 6). Taken together, their data support a model in which bam-mutant GSC-like cells produce BMPs that act on nonmutant cells (i.e., SGCs) to prevent their differentiation, similar to what is seen in the ovarian stem cell niche. 

Strengths:

(1) Use of an excellent and established model for tumorous cells in a stem cell microenvironment.

(2) Powerful genetics allow them to test various factors in the tumorous vs nontumorous cells.

(3) Appropriate use of quantification and statistics.

We greatly appreciate these comments.

Weaknesses:

(1) What is the frequency of SGCs in nos>flp; bam-mutant tumors? For example, are they seen in every germarium, or in some germaria, etc, or in a few germaria?

This is a great question. Because the SGC phenotype depends on the presence of germline tumor clones, our quantification was restricted to germaria that contained them.These quantification data ("SGCs and/or germline cysts per germarium with germline clones") will be presented in the revised Figure 1.

(2) Does the breakdown in clonality vary when they induce hs-flp clones in adults as opposed to in larvae/pupae?

Our initial attempts to induce ovarian hs-flp germline clones by heat-shocking adult flies were unsuccessful, with very few clones being observed. Therefore, we shifted our approach to an earlier developmental stage. Successful induction was achieved by subjecting late-L3/early-pupal animals to a twice-daily heatshock at 37°C for 6 consecutive days (2 hours per session with a 6-hour interval, see Lines 325-329) (Zhao et al., 2018).

(3) Approximately 20-25% of SGCs are bam+, dad-LacZ+. Firstly, how do the authors explain this? Secondly, of the 70-75% of SGCs that have no/low BMP signaling, the authors should perform additional characterization using markers that are expressed in GSCs (i.e., Sex lethal and nanos).

These 20-25% of SGCs are bamP-GFP⁺ dad-lacZ-, not bam⁺ dad-lacZ⁺ (see Figure 2C and 3D). They would be cystoblast-like cells that may have initiated a differentiation program toward forming germline cysts (see Lines 109-117). The 70-75% of SGCs that have low BMP signaling exhibit GSC-like properties, including: 1) dot-like spectrosomes; 2) dad-lacZ positivity; 3) absence of bamP-GFP expression. While additional markers would be beneficial, we think that this combination of properties is sufficient to classify these cells as GSC-like. 

(4) All experiments except Figure 1I (where a single germarium with no quantification) were performed with nos-Gal4, UASp-flp. Have the authors performed any of the phenotypic characterizations (i.e., figures other than Figure 1) with hs-flp?

Yes, we initially identified the SGC phenotype through hs-flp-mediated mosaic analysis of bam or bgcn mutant in ovaries. However, as noted in our response to Weakness (2), this approach was very labor-intensive. Therefore, we switched to using the more convenient nos::flp system for subsequent experiments. To our observation, there was no difference in the SGC phenotype between these two approaches, confirming that the nos::flp system is a valid and more practical alternative for its study. 

(5) Does the number of SGCs change with the age of the female? The experiments were all performed in 14-day-old adult females. What happens when they look at a young female (like 2-day-old). I assume that the nos>flp is working in larval and pupal stages, and so the phenotype should be present in young females. Why did the authors choose this later age? For example, is the phenotype more robust in older females? Or do you see more SGCs at later time points?

These are very good questions. Such time-course analysis data will be provided in revised Figure 1. The SGC phenotype depends on the presence of bam or bgcn mutant germline clones. Germaria from 14-day-old flies contained bigger and more such clones than those from younger flies. This age-dependent increase in clone size and frequency significantly enhanced the efficiency of our quantification (see Lines 129-131). 

(6) Can the authors distinguish one copy of GFP versus 2 copies of GFP in germ cells of the ovary? This is not possible in the Drosophila testis. I ask because this could impact the clonal analyses diagrammed in Figure 4A and 4G and in 6A and B. Additionally, in most of the figures, the GFP is saturated, so it is not possible to discern one vs two copies of GFP.

We greatly appreciate this comment. It was also difficult for us to distinguish 1 and 2 copies of GFP in the Drosophila ovary. In Figure 4A-F, to resolve this problem, we used a triplecolor system, in which red germ cells (RFP^+/+ GFP^-/-) are bam mutant, yellow germ cells (RFP^+/- GFP^+/-) are wild-type, and green germ cells (RFP^-/- GFP^+/+) are punt or med mutant. In Figure 4G-J, we quantified the SGC phenotype only in black germ cells (GFP^-/-), which are wild-type (control) or mad mutant.  In Figure 6, we quantified the SGC phenotype only in green germ cells (both GFP^+/+ and GFP^+/-), all of which are wild-type.

(7) More evidence is needed to support the claim of elevated Dpp levels in bam or bgcn mutant tumors. The current results with the dpp-lacZ enhancer trap in Figure 5A, B are not convincing. First, why is the dpp-lacZ so much brighter in the mosaic analysis (A) than in the no-clone analysis (B)? It is expected that the level of dpplacZ in cap cells should be invariant between ovaries, and yet LacZ is very faint in Figure 5B. I think that if the settings in A matched those in B, the apparent expression of dpp-lacZ in the tumor would be much lower and likely not statistically significant. Second, they should use RNA in situ hybridization with a sensitive technique like hybridization chain reactions (HCR) - an approach that has worked well in numerous Drosophila tissues, including the ovary.

We appreciate this critical comment. The settings of immunofluorescent staining and confocal parameters in Figure 5A were the same as those in 5B. To our observation, the level of dpp-lacZ in cap cells was variable across germaria, even within the same ovary, as quantified in Figure 5C. We will provide RNA in situ hybridization data to further strengthen the conclusion that bam or bgcn mutant germline tumors secret BMP ligands.  

(8) In Figure 6, the authors report results obtained with the bamBG allele. Do they obtain similar data with another bam allele (i.e., bamdelta86)?

No. Given that bam^BG was functionally indistinguishable from bam^Δ86 in inducing the SGC phenotype (compare Figure 6F, I with Figure 6-figure supplement 3C), we believe that repeating these experiments with bam^Δ86 would be redundant and would not alter the key conclusion of our study. Thanks for the understanding!

Reviewer #2 (Public review):

While the study by Zhang et al. provides valuable insights into how germline tumors can non-autonomously suppress the differentiation of neighboring wild-type germline stem cells (GSCs), several conceptual and technical issues limit the strength of the conclusions.

Major points:

(1) Naming of SGCs is confusing. In line 68, the authors state that "many wild-type germ cells located outside the niche retained a GSC-like single-germ-cell (SGC) morphology." However, bam or bgcn mutant GSCs are also referred to as "SGCs," which creates confusion when reading the text and interpreting the figures. The authors should clarify the terminology used to distinguish between wild-type SGCs and tumor (bam/bgcn mutant) SGCs, and apply consistent naming throughout the manuscript and figure legends.

We apologize for any confusion. In our manuscript, the term "SGC" is reserved specifically for wild-type germ cells that maintain a GSC-like morphology outside the niche. bam or bgcn mutant germ cells are referred to as GSC-like tumor cells (Lines 87-88), not SGCs.

(a) The same confusion appears in Figure 2. It is unclear whether the analyzed SGCs are wild-type or bam mutant cells. If the SGCs analyzed are Bam mutants, then the lack of Bam expression and failure to differentiate would be expected and not informative. However, if the SGCs are wild-type GSCs located outside the niche, then the observation would suggest that Bam expression is silenced in these wildtype cells, which is a significant finding. The authors should clarify the genotype of the SGCs analyzed in Figure 2C, as this information is not currently provided.

The SGCs analyzed in Figure 2A-C are wild-type, GSC-like cells located outside the niche. They were generated using the same genetic strategy depicted in Figures 1C and 1E (with the schematic in Figure 1B). The complete genotypes for all experiments are available in Source data 1. 

(b) In Figures 4B and 4E, the analysis of SGC composition is confusing. In the control germaria (bam mutant mosaic), the authors label GFP⁺ SGCs as "wild-type," which makes interpretation unclear. Note, this is completely different from their earlier definition shown in line 68.

The strategy to generate SGCs in Figure 4B-F (with the schematic in Figure 4A) is completely different from that in Figure 1C-F, H, and I (with the schematic in Figure 1B). In Figure 4B-F, we needed to distinguish punt^-/- (or med^-/-) with punt^+/- (or med^+/-) germ cells. As noted in our response to Reviewer #1’s Weakness (6), it was difficult for us to distinguish 1 and 2 copies of GFP in the Drosophila ovary. Therefore, we chose to use the triple-color system to distinguish these germ cells in Figure 4B-F (see genotypes in Source data 1). 

(c) Additionally, bam⁺/⁻ GSCs (the first bar in Figure 4E) should appear GFP⁺ and Red⁺ (i.e., yellow). It would be helpful if the authors could indicate these bam⁺/⁻ germ cells directly in the image and clarify the corresponding color representation in the main text. In Figure 2A, although a color code is shown, the legend does not explain it clearly, nor does it specify the identity of bam⁺/⁻ cells alone. Figure 4F has the same issue, and in this graph, the color does not match Figure 4A.

The color-to-genotype relationships for the schematics in Figures 2A and 4E are provided in Figures 1B and 4A, respectively. Due to the high density of germ cells, it is impractical to label each genotype directly in the images. In contrast to Figure 4E, the colors in Figure 4F do not represent genotypes; instead, blue denotes the percentage of SGCs, and red denotes the percentage of germline cysts, as indicated below the bar chart. 

(2) The frequencies of bam or bgcn mutant mosaic germaria carrying [wild-type] SGCs or wild-type germ cell cysts with branched fusomes, as well as the average number of wild-type SGCs per germarium and the number of days after heat shock for the representative images, are not provided when Figure 1 is first introduced. Since this is the first time the authors describe these phenotypes, including these details is essential. Without this information, it is difficult for readers to follow and evaluate the presented observations.

Thanks for this constructive suggestion. We will include such quantification data in the revised manuscript.

(3) Without the information mentioned in point 2, it causes problems when reading through the section regarding [wild-type] SGCs induced by impairment of differentiation or dedifferentiation. In lines 90-97, the authors use the presence of midbodies between cystocytes as a criterion to determine whether the wild-type GSCs surrounded by tumor GSCs arise through dedifferentiation. However, the cited study (Mathieu et al., 2022) reports that midbodies can be detected between two germ cells within a cyst carrying a branched fusome upon USP8 loss.

Unlike wild-type cystocytes, which undergo incomplete cytokinesis and lack midbodies, those with USP8 loss undergo complete cell division, with the presence of midbodies (white arrow, Figure 1F’ from Mathieu et al., 2022) as a marker of the late cytokinesis stage (Mathieu et al., 2022). 

(a) Are wild-type germ cell cysts with branched fusomes present in the bam mutant mosaic germaria? What is the proportion of germaria containing wild-type SGCs versus those containing wild-type germ cell cysts with branched fusomes?

(b) If all bam mutant mosaic germaria carry only wild-type GSCs outside the niche and no germaria contain wild-type germ cell cysts with branched fusomes, then examining midbodies as an indicator of dedifferentiation may not be appropriate.

We greatly appreciate this critical comment. bam mutant mosaic germaria indeed contained wild-type germline cysts, as evidenced by an SGC frequency of ~70%, rather than 100% (see Figures 2H, 4F, 4J, 6F, 6I, and Figure 6-figure supplement 3C). Since the SGC phenotype depends on the presence of bam or bgcn mutant germline tumors, we quantified it as “the percentage of SGCs relative to the total number of SGCs and germline cysts that are surrounded by germline tumors” (see Lines 124-129). Quantifying the SGC phenotype as "the percentage of germaria with SGCs" would be imprecise. This is because the presence and number of SGCs were highly variable among germaria with bam mutant germline clones, and a small number of germaria entirely lacked these clones. We will provide the data of "SGCs and/or germline cysts per germarium with germline clones" in revised Figure 1.

(c) If, however, some germaria do contain wild-type germ cell cysts with branched fusomes, the authors should provide representative images and quantify their proportion.

Such representative germaria are shown in Figure 2G, 3B, 3C, 6D, 6E, and 6H. The percentage of germline cysts can be calculated by “100% - SGC%”.

(d) In line 95, although the authors state that 50 germ cell cysts were analyzed for the presence of midbodies, it would be more informative to specify how many germaria these cysts were derived from and how many biological replicates were examined.

As noted in our response to points a) and b) above, the germ cells surrounded by germline tumors, rather than germarial numbers, are more precise for analyzing the phenotype. For this experiment, we examined >50 such germline cysts via confocal microscopy. As the analysis was performed on a defined cellular population, this sample size should be sufficient to support our conclusion. 

(4) Note that both bam mutant GSCs and wild-type SGCs can undergo division to generate midbodies (double cells), as shown in Figure 4H. Therefore, the current description of the midbody analysis is confusing. The authors should clarify which cell types were examined and explain how midbodies were interpreted in distinguishing between cell division and differentiation.

We assayed for the presence of midbodies or not specifically within the germline cysts surrounded by bam mutant tumors, not within the tumors themselves (Lines 94-95). As detailed in Lines 88-97, the absence of midbodies was used as a key criterion to exclude the possibility of dedifferentiation.  

(5) The data in Figure 5 showing Dpp expression in bam mutant tumorous GSCs are not convincing. The Dpp-lacZ signal appears broadly distributed throughout the germarium, including in escort cells. To support the claim more clearly, the authors should present corresponding images for Figures 5D and 5E, in which dpp expression was knocked down in the germ cells of bam or bgcn mutant mosaic germaria. Showing these images would help clarify the localization and specificity of Dpp-lacZ expression relative to the tumorous GSCs.

We greatly appreciate this comment. RNA in situ hybridization data will be provided to further strengthen the conclusion that bam or bgcn mutant germline tumors secret BMP ligands.

(6) While Figure 6 provides genetic evidence that bam mutant tumorous GSCs produce Dpp to inhibit the differentiation of wild-type SGCs, it should be noted that these analyses were performed in a dpp⁺/⁻ background. To strengthen the conclusion, the authors should include appropriate controls showing [dpp⁺/⁻; bam⁺/⁻] SGCs and [dpp⁺/⁻; bam⁺/⁻] germ cell cysts without heat shock (as referenced in Figures 6F and 6I).

Schematic cartoons in Figure 6A and 6B demonstrate that these analyses were performed in a dpp^+/- background. Figure 6-figure supplement 1 indicates that dpp^+/- or gbb^+/- does not affect GSC maintenance, germ cell differentiation, and female fly fertility. Figure 6C is the control for 6D and 6E, and 6G is the control for 6H, with quantification in 6F and 6I.  We used nos::flp, not the heat shock method, to induce germline clones in these experiments (see genotypes in Source data 1).

(7) Previous studies have reported that bam mutant germ cells cause blunted escort cell protrusions (e.g., Kirilly et al., Development, 2011), which are known to contribute to germ cell differentiation (e.g., Chen et al., Frontiers in Cell and Developmental Biology, 2022). The authors should include these findings in the Discussion to provide a broader context and to acknowledge how alterations in escort cell morphology may further influence differentiation defects in their model.

Thanks for teaching us! Such discussion will be included in the revised manuscript.

(8) Since fusome morphology is an important readout of SGCs vs differentiation. All the clonal analysis should have fusome staining.

SGC is readily distinguishable from multi-cellular germline cyst based on morphology. In some clonal analysis experiments, fusome staining was not feasible due to technical limitations such as channel saturation or antibody incompatibility. Thanks for the understanding! 

(9) Figure arrangement. It is somewhat difficult to identify the figure panels cited in the text due to the current panel arrangement.

The figure panels were arranged to optimize space while ensuring that related panels are grouped in close proximity for logical comparison. We would be happy to consider any specific suggestions for an alternative layout that could improve clarity. Thanks!

(10) The number of biological replicates and germaria analyzed should be clearly stated somewhere in the manuscript-ideally in the Methods section or figure legends. Providing this information is essential for assessing data reliability and reproducibility.

Thanks for this constructive suggestion. Such information will be included in figure legends in the revised manuscript.

Reviewer #3 (Public review):

Summary:

Zhang et al. investigated how germline tumors influence the development of neighboring wild-type (WT) germline stem cells (GSC) in the Drosophila ovary. They report that germline tumors inhibit the differentiation of neighboring WT GSCs by arresting them in an undifferentiated state, resulting from reduced expression of the differentiation-promoting factor Bam. They find that these tumor cells produce low levels of the niche-associated signaling molecules Dpp and Gbb, which suppress bam expression and consequently inhibit the differentiation of neighboring WT GSCs non-cell-autonomously. Based on these findings, the authors propose that germline tumors mimic the niche to suppress the differentiation of the neighboring stem cells.

Strengths:

This study addresses an important biological question concerning the interaction between germline tumor cells and WT germline stem cells in the Drosophila ovary. If the findings are substantiated, they could provide valuable insights applicable to other stem cell systems.

We greatly appreciate these comments.

Weaknesses:

Previous work from Xie's lab demonstrated that bam and bgcn mutant GSCs can outcompete WT GSCs for niche occupancy. Furthermore, a large body of literature has established that the interactions between escort cells (ECs) and GSC daughters are essential for proper and timely germline differentiation (the differentiation niche). Disruption of these interactions leads to arrest of germline cell differentiation in a status with weak BMP signaling activation and low bam expression, a phenotype virtually identical to what is reported here. Thus, it remains unclear whether the observed phenotype reflects "direct inhibition by tumor cells" or "arrested differentiation due to the loss of the differentiation niche". Because most data were collected at a very late stage (more than 10 days after clonal induction), when tumor cells already dominate the germarium, this question cannot be solved. To distinguish between these two possibilities, the authors could conduct a time-course analysis to examine the onset of the WT GSC-like singlegerm-cell (SGC) phenotype and determine whether early-stage tumor clones with a few tumor cells can suppress the differentiation of neighboring WT GSCs with only a few tumor cells present. If tumor cells indeed produce Dpp and Gbb (as proposed here) to inhibit the differentiation of neighboring germline cells, a small cluster or probably even a single tumor cell generated at an early stage might prevent the differentiation of their neighboring germ cells.

Thanks for this critical comment. Such time-course analysis data will be provided in revised Figure 1.

The key evidence supporting the claim that tumor cells produce Gpp and Gbb comes from Figures 5 and 6, which suggest that tumor-derived dpp and gbb are required for this inhibition. However, interpretation of these data requires caution. In Figure 5, the authors use dpp-lacZ to support the claim that dpp is upregulated in tumor cells (Figure 5A and 5B). However, the background expression in somatic cells (ECs and pre-follicular cells) differs noticeably between these panels. In Figure 5A, dpp-lacZ expression in somatic cells in 5A is clearly higher than in 5B, and the expression level in tumor cells appears comparable to that in somatic cells (dpplacZ single channel). Similarly, in Figure 5B, dpp-lacZ expression in germline cells is also comparable to that in somatic cells. Providing clear evidence of upregulated dpp and gbb expression in tumor cells (for example, through single-molecular RNA in situ) would be essential.

We greatly appreciate this critical comment. In our data, the expression of dpp-lacZ in cap cells was variable across germaria, even within the same ovary, as quantified in Figure 5C. The images in Figures 5A and 5B were selected as representative examples of positive signaling. To directly address the reviewer's point and strengthen our conclusion, we will perform RNA in situ hybridization data in the revised manuscript to visualize the expression of BMP ligands within the bam or bgcn mutant germline tumor cells.

Most tumor data present in this study were collected from the bam[86] null allele, whereas the data in Figure 6 were derived from a weaker bam[BG] allele. This bam[BG] allele is not molecularly defined and shows some genetic interaction with dpp mutants. As shown in Figure 6E, removal of dpp from homozygous bam[BG] mutant leads to germline differentiation (evidenced by a branched fusome connecting several cystocytes, located at the right side of the white arrowhead). In Figure 6D, fusome is likely present in some GFP-negative bam[BG]/bam[BG] cells. To strengthen their claim that the tumor produces Dpp and Gbb to inhibit WT germline cell differentiation, the authors should repeat these experiments using the bam[86] null allele.

Although a structure resembling a "branched fusome" is visible in Figure 6E (right of the white arrowhead), it is an artifact resulting from the cytoplasm of GFP-positive follicle cells, which also stain for α-Spectrin, projecting between germ cells of different clones (see the merged image). In both our previous (Zhang et al., 2023) and current studies, bam^BG was functionally indistinguishable from bam^Δ86 in its ability to block GSC differentiation and induce the SGC phenotype (compare Figure 6F, I with Figure 6-figure supplement 3C). Given this, we believe that repeating the extensive experiments in Figure 6 with the bam^Δ86 allele would be scientifically redundant and would not change the key conclusion of our study. We thank the reviewer for their consideration.

It is well established that the stem niche provides multiple functional supports for maintaining resident stem cells, including physical anchorage and signaling regulation. In Drosophila, several signaling molecules produced by the niche have been identified, each with a distinct function - some promoting stemness, while others regulate differentiation. Expression of Dpp and Gbb alone does not substantiate the claim that these tumor cells have acquired the niche-like property. To support their assertion that these tumors mimic the niche, the authors should provide additional evidence showing that these tumor cells also express other niche-associated markers. Alternatively, they could revise the manuscript title to more accurately reflect their findings.

Dpp and Gbb are the key niche signals from cap cells for maintaining GSC stemness. Our work demonstrates that germline tumors can specifically mimic this signaling function, not the full suite of cap cell properties, to create a non-cell-autonomous differentiation block. The current title “Tumors mimic the niche to inhibit neighboring stem cell differentiation” reflects this precise concept: a partial, functional mimicry of the niche's most relevant activity in this context. We feel it is an appropriate and compelling summary of our main conclusion.

In the Method section, the authors need to provide details on how dpp-lacZ expression levels were quantified and normalized.

Thanks for this suggestion. Such information will be included in the revised manuscript.

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

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Reply to the reviewers

We thank all the reviewers for their comments and suggestions, which has helped in revising the manuscript for a broader audience. Some of the experiments that was suggested by the reviewers has been performed and included in the revised manuscript. The response to reviewers is provided below their comments.

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

MprF proteins exist in many bacteria to synthesize aminoacyl phospholipids that have diverse biological functions, e.g. in the defense against small cationic peptides. They integrate two functions, the aminoacylation of lipids, i.e. the transfer of Lys, Arg or Ala from tRNAs to the head group, and the flipping of these modified lipids to the membrane outer leaflet. The authors present structures of MprF from Pseudomonas aeruginosa and describe these structures in great detail. As MprF enzymes confer antibiotic resistance and are therefore highly important, studying them is significant and interesting. Consequently, their structures have been substantially characterized in recent years, including the publication of the dimeric full-length MpfR from Rhizobium (Song et al., 2021).

While the structural work appears to be solid and carried out well on the technical part, one big criticism is how the data are presented in the manuscript, how they are analyzed and how they are put into relation to previous work. As structures of Mpfr from Rhizobium have been published, it is not required and rather distracting to explain the methodological details and the structure of Pseudomonas MprF in such great detail. Instead, the manuscript would benefit very strongly from reaching the interesting and novel parts, the comparison with the previous structures, as early as possible. Overall, the manuscript should be substantially shortened to not divert the reader's attention away from the novel parts by drowning them in miniscule description of the structural features such as secondary structure elements or lipid molecule positions where it remains completely unclear what their relevance is to the story and the message of the paper. Finally, during this revision, care should be taken to improve the language and maybe involve a native speaker in doing so.

It is true that we have described the experimental details of PaMprF in detail including the constructs. We had reconstructed the map of dimeric PaMprF in 2020 but with the publication of the homologues structures (Song et al 2021 and the unpublished Rhizobium etli structure), we had to make sure the PaMprF dimer is not an artefact. Hence, our attempts to rule out this with different constructs and extensive testing with various detergents. Thus, we would like to keep this in the manuscript. We realise the importance of focusing on novel/interesting parts and have reshuffled sections (comparing structures and validating the dimer interface) followed by description of modelling of lipid molecules.

Even more importantly, since the authors observe a dimer interface which strongly deviates from the previously presented arrangement of another species, the most important thing would be to properly characterize this interface and experimentally validate it, both of which has not been done sufficiently. When also taking into account that there were significant differences in the arrangement of the dimer between their structures in GDN and nanodisc, and that in the GDN structure, the cholesterol backbone of GDN appears to be involved in the interface (there should not be any cholesterol in native bacterial membranes!), there is a realistic chance that the observed dimer is an artefact. If the authors cannot convincingly rule out this possibility, all their conclusions are meaningless.

The trials with cholesterol hemisuccinate stems more of out of curiosity (we are aware that no cholesterol is present in bacterial membranes). We had started the initial analysis of PaMprF with DDM and by itself it was largely monomeric (unpublished observation and supported by recent publication of PaMprF in DDM – Hankins et al 2025). When we observed that GDN was essential for the stability of the dimer (and not even LMNG), we asked if a combination of CHS with DDM will keep the dimer intact, which didn’t work and GDN was found to be important. The use of CHS for prokaryotic membrane protein studies has now been reported in few different systems and a recent one includes – Caliseki et al., 2025. We would like to keep the observation with CHS in the manuscript, and we have moved this figure to Appendix Fig. S3C.

In addition, in a recent report on MgtA, a magnesium transporter (Zeinert et al., 2025), it was observed that DDM/LMNG resulted in monomeric enzyme, while GDN resulted in dimeric enzyme albeit, the dimer interface was in the soluble domain. We have added this reference and observation of MgtA in the discussion (page 13, lines 407-411).

We like to think that the milder GDN tends to keep the membrane proteins or oligomers of membrane proteins more stable but further studies on multiple labile membrane protein systems will be required to substantiate this.

Hence, while I think that the data presented here would be worth publishing. However, a major drawback is that the authors do not sufficiently analyse, characterise and validate the dimer interface and fail to show that the dimer is biologically relevant.

Further major points: - The authors always jump between their structures in detergent and nanodisc during all the descriptions, which makes following the story even more difficult. Please first describe one of the structures and then (briefly) discuss relevant similarities and differences afterwards.

The flow and description of the structures is now modified and the figures have now been rearranged to make it easier to follow. The panel in figure 2 describing the overlay of the GDN and nanodisc is now moved to Appendix Fig. S2B. Thus, figure 2 has only description of salient features of the structures (the interacting residues between the membrane and soluble domain) and the terminal helix.

• The difference in dimerization between Pseudomonas and Rhizobium is the most interesting and surprising feature (if true) of the new structures. However, it is not really presented as such. The authors should put more emphasis on making clear that this is a complete rotation of the monomers with respect to each other (by how many degrees?) and they should visualize it even more clearly in Figure 4 (and label the figure so that it is possible to understand it without having to read the text or the legend first).

We thought the colouring of the TM helices should make the difference in interface more obvious (the N and C-terminal TM helices in different colours). Now, we have also labelled the TM helices, so that it is easier to follow (this was also shown in panel E). The rotation is ~180° and this is now mentioned in the figure legend.

• P. 10: The authors insinuate that only one of the dimer interfaces, either Pseudomonas or Rhizobium could be real, but disregard the possibility that both might be the biologically relevant interfaces of the respective species and that there might have been a switch of interfaces during evolution. They should also mention and discuss this possibility.

We didn’t imply that one of the interfaces is real but clearly mentioned that it could also be different conformational state (page 7, lines 226-228). In the revised version, we have included a multiple sequence alignment (we had not included in the initial draft as it had been presented in several previous publications). The MSA (Appendix Fig. S6) reveals that neither of the interfaces are highly conserved.

• Fig. 5G: The authors claim that the higher molecular band that appears in the mutant is a "dimer with aberrant migration" of >250 kDa as opposed to the expected 150 kDa. They should explain how they came to this conclusion and how they can be sure that the band does not correspond to a higher oligomer (trimer or tetramer). They could show, by extraction and purification scheme similar to the wildtype using first LMNG and then GDN, followed by at least a preliminary EM analysis, that the crosslinked mutant MprF is indeed a dimer, or use other biophysical methods to do the same, otherwise this experiment does not show much. Furthermore, they should also include a cysteine mutant in the part of Pseudomonas MprF that would be involved in a Rhizobium-like interface in their crosslinking experiments to check whether they could also stabilize dimers in this case.

The band of the double mutant after crosslinking (or even without crosslinking) migrates at higher molecular weight than that expected for a dimer, and could potentially be a higher molecular band that a dimer. We also note that in the previous publication by Song et al 2021, the crosslinking of RtMprF also resulted in a higher molecular weight band (shown also by Western blot).

We now substantiate the dimer of PaMprF with different approaches. We employed blue-native gel and also SDS-PAGE of the purified protein. This clearly shows that the higher molecular band after crosslinking is a dimer (Figure 4B and Fig. EV4D). In particular, in the BN-PAGE, the treatment of mutants with crosslinkers revealed a dimeric band even in the presence of SDS. Further, we have performed cryoEM analysis of the mutants - H386C/F389C and H566C. The images, classes and reconstruction show that the enzyme forms a dimer similar to the WT. Interestingly, we also observe in H566C mutant in nanodisc, a small population that has similar architecture to the Rhizobium-like interface (classes shown in Fig. EV7 and Appendix Fig. S5). This prompted us to look closely at other datasets and it is clear that during the process of reconstitution in nanodisc, we observe both kinds of dimer interface but the PaMprF dimer is predominant. We also observe higher order oligomers (tetramer) in GDN but as only few views are visible, a reconstruction could not be obtained (Appendix Fig. S5). In addition, we also introduced two cysteines on the Rhizobium-like interface and no crosslinking on the membranes were observed (Figure 4B). But it is possible that these chosen mutants are not accessible to the crosslinker. Thus, we conclude that the oligomers of PaMprF is sensitive to nature of detergents and labile.

• As the question whether the observed interface is real or an artefact is very central to the value of the structural data and the drawn conclusions from it, the authors should make more effort to analyze and try to validate the interface. First, an analysis of interface properties (buried surface area, nature of the interactions, conservation) should be performed for the interface as observed in the Pseudomonas structure but also for a (hypothetical) Rhizobium-like interface of two Pseudomonas monomers (such a model of a dimer should be easily obtainable by AlphaFold using the available Rhizobium structures as models). Then, experimental methods such as FRET or crosslinking-MS would allow to draw more solid conclusions on the distances between potential interface residues. While these experiments are a certain effort, the question whether the dimer interface is real is so central to the paper that it would be worthwhile to make this effort.

We have included the interface area and nature of interactions in the revised manuscript (page 7, lines 221-223).

We attempted AlphaFold for predicting the dimeric structure of PaMprF (and included RtMprF also). Some of the attempts from the predictions is summarised in figure 1.

The prediction of monomer is of high confidence but the oligomer (here dimer) is of low confidence (from ipTM values). Even the prediction for Rhizobium enzyme has low confidence, and gives a complete different architecture (and in some trials with lipids, it gives an inverted or non-physiological dimer). Only when the monomer of PaMprF with lipids and tRNA was given as input (requested by reviewer 2 and described below), it predicts oligomeric structure with some confidence but rest were not informative.

• As it seems that detergents might disrupt or modify the dimer interface, it might be an alternative to solubilize the protein in a more native environment by polymer-stabilized nanodiscs using DIBMA or similar molecules.

We have tried to use SMALPs for extraction of PaMprF. We were able to solubilise but unable to enrich the enzyme sufficient for structural studies currently and will require further optimisation.

• Since parts of the Discussion are mostly repetitions of the Results part and other parts of the Discussion also contain a large extend of structure analysis one would usually rather expect in the Results part instead of the Discussion, the authors should consider condensing both to a combined (and overall much shorter) Results & Discussion section.

We have rewritten much of the discussion section and removed any repetition from the results sections. We would prefer to keep the results and discussion separate.

Minor points: - Explain abbreviations the first time they appear in the text, e.g. TTH

This is now expanded in the first instance

• Figure labels are very minimalistic. This should be improved, e.g. by putting labels to important structural features that appear in the text, otherwise the figures are not an adequate support for the text.

The font size for the labels have been increased.

• Figure 5: Label where the different oligomers run on the gels

Labelled.

Reviewer #1 (Significance (Required)):

While the structural work appears to be solid and carried out well on the technical part, one big criticism is how the data are presented in the manuscript, how they are analyzed and how they are put into relation to previous work. As structures of Mpfr from Rhizobium have been published, it is not required and rather distracting to explain the methodological details and the structure of Pseudomonas MprF in such great detail. Instead, the manuscript would benefit very strongly from reaching the interesting and novel parts, the comparison with the previous structures, as early as possible. Overall, the manuscript should be substantially shortened to not divert the reader's attention away from the novel parts by drowning them in miniscule description of the structural features such as secondary structure elements or lipid molecule positions where it remains completely unclear what their relevance is to the story and the message of the paper. Finally, during this revision, care should be taken to improve the language and maybe involve a native speaker in doing so.

Even more importantly, since the authors observe a dimer interface which strongly deviates from the previously presented arrangement of another species, the most important thing would be to properly characterize this interface and experimentally validate it, both of which has not been done sufficiently. When also taking into account that there were significant differences in the arrangement of the dimer between their structures in GDN and nanodisc, and that in the GDN structure, the cholesterol backbone of GDN appears to be involved in the interface (there should not be any cholesterol in native bacterial membranes!), there is a realistic chance that the observed dimer is an artefact. If the authors cannot convincingly rule out this possibility, all their conclusions are meaningless.

Hence, while I think that the data presented here would be worth publishing. However, a major drawback is that the authors do not sufficiently analyse, characterise and validate the dimer interface and fail to show that the dimer is biologically relevant.

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

Shaileshanand J. et al., reported the structures of Multiple Peptide Resistance Factor, MprF, which is a bi-functional enzyme in bacteria responsible for aminoacylation of lipid head groups. The authors purified MprF from Pseudomonas aeruginosa in GDN micelles and nanodiscs, and by applying cryo-EM single particle method, they successfully reached near-atomic resolution, and built corresponding atomic models. By applying structural analysis as well as biochemistry methods, the authors demonstrated dimeric formation of MprF, exhibited the dynamic nature of the catalytic domain of this enzyme, and proposed a possible model on tRNA binding and aminoacylation.

Major comments 1. In abstract, the authors stated 'Several lipid-like densities are observed in the cryoEM maps, which might indicate the path taken by the lipids and the coupling function of the two functional domains. Thus, the structure of a well characterised PaMprF lays a platform for understanding the mechanism of amino acid transfer to a lipid head group and subsequent flipping across the leaflet that changes the property of the membrane.' Firstly, those lipid-like densities were demonstrated in Fig 3A, since densities of lipids of purified membrane proteins often exist within regions of relatively low local resolution, or low quality, I think more detailed description on how the authors defined which part of the density belongs to lipid and how they acquired the modeling of some of the lipids is required. And the authors modeled phosphatidylglycerol into the GDN MprF, I would require additional experiment, for instance, mass spectrometry over the purified sample, to demonstrate the existence of this specific lipid with the sample. Secondly, regarding the last sentence in the abstract, how these structures lay a platform for further understanding was poorly discussed in both result section and discussion section, since the authors clearly stated 'This cavity perhaps provides a path for holding lipids...', then the statement in the next sentence 'Taken together... the vicinity to the cavities described above indicates the possible path taken by the lipids to enter and exit the enzyme' does not have a reliable evidence to support this conclusion, I would suggest the authors move these statements into discussion section, and elaborate more over this issue since it is an important part in the abstract, or make a more solid proof using other approaches, such as molecular dynamics simulation, to make these statements solid in the result section.

The membranes of E. coli have predominantly phosphatidyl ethanolamine (PE) and phosphatidyl glycerol (PG) as the next abundant lipid with cardiolipin though smaller in number, plays an important role in functioning of many membrane proteins. In our map, the non-protein density are unambiguous and they can be observed as long density reflective of acyl chains (note that GDN used in purification has no acyl chain) and hence attributed these densities to lipids (Fig. EV4E/F and Figure 5A). Only in few of these densities, head group could be modelled and the identity of the lipid as PG at the dimer interface is based on the requirement of negatively charged lipids for oligomerisation of membrane proteins in general (for example – KcsA tetramer formation requires PG, Marius et al., 2005; Valiyaveetil et al., 2002;2004). It is true that the lipid densities are at the peripheral regions of the map but here only acyl chains have been modelled. Within the membrane domain, one reasonably ordered lipid is observed and by analogy with R. tropici structure, it is possible to build a modified-PG (in PaMprF here ala-PG). However, the density of the head group is not unambiguous (unlike lysine in the R. tropici, whose density stands out) and hence we have modelled it as PG alone. In the methods (page 20, lines 649-650), the identification and modelling of lipid densities is described.

We agree that mass spectrometry analysis of purified lipids will be useful but it will not be able to tell the position of the lipid in the map (model) and for this we still require a map at higher resolution with better ordered lipids. We have recently built/developed the workflow for native MS and we plan to initiate analysis of PaMprF in the near future, which will provide details for the lipid purified with the enzyme.

We had initiated molecular dynamics simulation during the review process, and we had included tRNA molecules (shorter version) as we felt the connection between tRNA binding and lipid modification was important. This would have also explained the path taken by lipids (performed by Hankins et al., 2025 in their publication). However, this is likely to require more work (and computing resources) and both mass spectrometry and molecular dynamics will be part of the future work.

We have rewritten the discussion and changed the last line of the abstract to the following

“From the structures, the binding modes of tRNA and lipid transport can be postulated and the mobile secondary structural elements in the synthase domain might play a mechanistic role”.

(in the abstract, lines 24-26).

Fig 2B, it seems the H566 sidechains were overlapping in the zoom-in figure of distance measurement between H566 residues, to clarify this, authors should either present another figure with rotation, to better demonstrate their relative locations, or swap this zoom-in figure with another figure with rotations. Also, could the authors briefly commenting on why they chose H566 for distance measurement specifically?

The side chain of residue H566 in the nanodisc model face towards each other at the interface, hence this residue was chosen to shown the proximity.

Related to previous comment, I see one additional green square in Fig. 2A and an additional green square in Fig. 2B, without any zoom-in images provided on these regions. Besides, they're focusing on two different domains with same color, any particular reason why they're there? If so, please provide the information in figure legends.

The green squares in panels 2A and 2B are the regions that have been zoomed in panels 2D and 2E showing the interactions of the TTH. This is now made clear in the legend as well as in the figure.

Related to previous comment, authors should also provide distance measurement over electrostatic interaction sites in Fig. 2A, since distance plays as an important factor in these forces.

The electrostatic interactions have been included.

For Fig. 2C, since in Fig. 1, the authors have already indicated the differences between reconstruction of the GDN and nanodisc datasets, this information provided here seems to be a bit abundant, I suggest either move this panel to Fig. 1, to make a visualization on both electron densities as well as atomic models, or move this panel to supplementary figures.

We thank the reviewer for the suggestion. The panel, figure 2C is moved to Appendix Fig. S2B.

Fig. 3B, some of the spheres of the lipids were also marked as red, any particular reason why they're red? Do they indicate they're phosphate heads? If so, could the authors provide evidences how they define these orientations of the lipid heads? If not, any particular reason why they're red?

Although, there are non-protein densities (i.e., density beyond noise that remain after modelling of protein residues and found individually) have been modelled as lipids (In Fig. EV4E, these additional densities are shown). Except for few, all these densities have been modelled only as acyl chain. The lipids modelled with head group and phosphate (that have oxygen) and the fit of the density are shown in both figure 3A and EV4F. Hence, the red (oxygen) is seen in the space filling model of lipids (the density for few lipids are shown, also in the response to the comment below).

Fig. 3C, the fitted model of lipid and its corresponding density should be added to Fig. S4, to give more detailed view on the quality of the fitting.

The figure 3 has now been reorganised and the new figure (fig. 5) has only 3 panels. We have provided an enlarged view of the lipids in the membrane domain along with unmodelled densities in 3A. In addition, in fig. EV4F, fit of the lipid to density (select lipids) are shown.

Fig. 4D and 4E, could the authors also indicate the RMSD values when comparing the differences of RtMprF, PaMprF, ReMprF, this information would be helpful to understand how big of a difference within these three models.

The RMSD values of the structural comparison is given in the text.

Fig. 6E, the coloring used for CCA-Ala were similar to the blue part of soluble domain, could the authors change the coloring a bit? Also, for Fig. 6F, I would suggest the authors provide a prediction model, such as using AlphaFold3, of this tRNA interaction site, to further validate this proposed model.

The colour of the CCA part is changed in the revised figure. Following the suggestion of the reviewer, we used AlphaFold3 to predict the complex formation of PaMprF with tRNA (or shorter version) (Figure 2). As mentioned above in response to reviewer 1, the prediction of dimeric enzyme was of low confidence and this is also reflected when a combination of tRNA, lipids and enzyme sequence are given. Instead of full-length tRNA, if only the CCA end is provided, then the prediction program does position this in the postulated cavity. Only with the monomeric enzyme and tRNA does one get a reasonable model. With respect to the proposed model in 6F, currently we don’t have any evidence and this remains a postulate. In the revised manuscript, we have replaced this with conservation figure, which we thought is more relevant.

In Supplementary Figures S1 and S3, the angular distribution of maps exhibited preferred orientation to certain extent, 3D FSC estimation should also be supplied for these maps, as an indication of whether the reconstructed densities were affected or not.

We have included the 3DFSC plots for all the data sets (including the new ones in figures EV1, 2, 5, 6, 7). It is evident that the nanodisc datasets in general are slightly anisotropic.

For Fig S3B, could the authors switch to another image with better contrast?

This is now replaced with an image to show the particles.

Minor comments 1. Fig. 2E and 2F, distance measurement should also be supplied to these two panels.

We have now included the distance measurement in both the panels, which are now Fig. 2D and 2E.

Fig. 5D, since in Fig. 4F and 4G already mentioned the skeleton of GDN, this modeling part should be presented before exhibit it in dimer interface, the authors should rearrange the sequence over these three panels.

The figures in the revised manuscript has been rearranged. Figure 5 (now figure 4) has been modified to include the biochemical analysis (crosslinking studies) and the panel 5D has been removed.

In Supplementary Figure S3, which density was shown for the PaMprF local resolution estimation result? Authors should provide this information as two maps were shown in this figure.

The local resolution is for C2 symmetrised map and this is now mentioned in the panel.

CROSS-REFEREE COMMENTS Both Reviewer #1 and #3 made comments over technical issue, their evaluation over functional aspects of this protein is what I was lacking over my comments, also, their evaluation of the biological narrative, relevance toward previous research is also more insightful. Finally, they offer valuable suggestions on how to adjust the article to make it more readable, and better describing the biological story which I would suggest the authors to pay attention to.

Reviewer #2 (Significance (Required)):

Significance The authors mainly focused on the structure of MprF in Pseudomonas aeruginosa, this protein is essential for the resistance to cationic antimicrobial peptides. A combination of structural and biochemical analysis provided evidences to the dimeric formation to this enzyme, and the analysis over differences of purified proteins using GDN and nanodisc was particular interesting, which provide new insight regarding the flexible nature of this enzyme, and potentially could be beneficial to the membrane protein community, as it demonstrates the differences in detergent/nanodisc of choice could affect the assembly of the protein of interest. Still, some of the statements in the manuscript, for instance, the assignment of lipids was over-claimed and could be benefited from additional approaches to support the issue. I would suggest some refinement in the discussion section as well as some of the figures.

My expertise: cryo-EM single particle analysis; cryo-ET; sub-tomo averaging; cryo-FIB;

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

Jha and Vinothkumar characterize the cryoEM structure of the alanyl-phosphatidylglycerol producing multiple peptide resistance factor (MprF) of Pseudomonas aeruginosa. MprF proteins mediate the transfer of amino acids from aminoacyl-tRNAs to negatively charged phospholipids resulting in reduced membrane interactions with cationic antimicrobial peptides (produced by the host and competing microorganisms). The phospholipid modifications involve in most cases the transfer of lysine or alanine to phosphatidylglycerol. MprF proteins are membrane proteins consisting of a soluble and hydrophobic domain. Multiple functional studies have shown that the soluble domain of MprF mediates the aminoacylation of phosphatidylglycerol, while the hydrophobic domain mediates the "flipping" of aminoacylated phospholipids across the membrane, a process that is crucial to repulse or prevent the interaction of antimicrobial peptides encountered at the outer leaflet of bacterial membranes. Aside from its role in conferring antimicrobial peptide resistance, other roles of MprF have been described including more physiological roles such as improving growth under acidic conditions. Interestingly, MprF proteins are also found in Gram-negative bacteria which are already protected by an additional membrane that includes LPS. However, in Pseudomonas aeruginosa, MprF confers phenotypes that are similar to those observed in Gram-positive bacteria. Importantly, crystal structures of the soluble domain have led to important insights into aminoacyl phospholipid synthesis and recent studies on the cryoEM structure of Rhizobium tropici have confirmed functional and preliminary structural studies with other MprF proteins. The cryoEM structure from R. tropici confirmed the dimeric structure of MprF and supported a role of the hydrophobic domain in flipping lysyl-phosphatidylglycerol across the membrane. A comparison of the structures of lysyl-phosphatidylglycerol with alanyl-phosphatidylglycerol producing MprFs could reveal new insights into the mechanism of transferring aminoacyl-phospholipids from the soluble domain to the hydrophobic domain and translocation of alanyl- vs lysyl-phosphatidylglycerol across the membrane.

Major concerns

1. The study by Jha and Vinothkumar provides the cryoEM structure of an alanyl-phosphatidylglycerol producing MprF protein which is in principle an important milestone in gaining a better understanding of the mechanism of aminoacyl-phospholipid synthesis and flipping, including the potentially different requirements of accommodating different aminoacyl -tRNAs and aminoacyl-phospholipid species. However, this is not addressed. The authors present a "distinct architecture" compared to the structure of R. tropici- MprF, without providing functional insights and the focus of the study shifts to the role of detergents in determining MprF structures via cryoEM. Thus, after fundamental discoveries have been made with crystal structures of the soluble domain and cryoEM structure of R. tropici, this study -while valuable as a resource- seems to offer only an incremental advance in understanding the mode of action of MprF and the potential different requirements for transferring alanyl-phosphatidylglycerol to the hydrophobic domain and flipping across the membrane. The reader is left with the finding of a distinct architecture with no further explanation or hypothesis.

We thank the reviewer for his/her comments. It is true that the crystal structures of soluble domains of MprF (from 3 species) and the cryoEM structures are now available (two Rhizobium species). However, the cryoEM maps that we have obtained has several salient features including the distinct dimeric interface and the position of the C-terminal helix of the soluble domain. This in particular is important. In the previous study, Hebecker et al 2011 had reported that the terminal helix of PaMprF was important for the activity and the construct without the TM domain can also function in modifying the lipids. The full-length cryoEM map of PaMprF in GDN now provides an idea how this occurs, with the terminal helix buried at the interface. Further, the proposed tRNA binding site (from Hebecker et al 2015, lysine amide bound structure) face other in the dimeric architecture of R. tropici and it is not clear how the full-length tRNA will bind without disrupting the dimer. In contrast, the dimer architecture observed for PaMprF has the tRNA binding site facing away and they can bind to the enzyme without any constraints. We think the mobile/dynamic elements (or secondary structure) of the synthase domain play a major role in interaction with substrates and mechanism. The current structures provide some evidence for this and form the basis of future studies. Instead of cartoon description, we have now included a conservation plot of the molecule in explaining the possible mechanism along with the surface representation in figure 6.

Differences to R.tropici MprF and other studies are difficult to follow as only a topological map of the Pseudomonas MprF is provided and conserved amino acids that have been shown to be crucial in mediating synthesis and flipping are not highlighted in the text or in the figures, specifically addressed, or discussed. Conserved amino acids in the presented cryoEM structure could provide important mechanistic insights and could address substrate specificity/requirements for aminoacyl phospholipid synthesis, transfer to the hydrophobic domain and flipping.

The conservation of residues across MprF homologues have been presented in previous published articles and hence, initially we had not included in the manuscript. We have now included multiple sequence alignment of select homologues of MprF highlighting conserved residues (Appendix Fig. S6) as well a figure (Fig. 6F) colouring the molecule with conservation scores with CONSURF. In figure 6F, zoomed in version, we highlight the many of the conserved residues in the synthase domain as they play a role in substrate selectivity.

Authors characterize an alanyl-phosphatidylglycerol producing MprF but do not detect the lipid in the cryoEM structure. Thus, the potential path taken by alanyl-phosphatidylglycerol remains unclear. Authors model the detected lipids as phosphatidylglycerol, which may be an interesting finding as it would indicate that MprF is generally capable of flipping phospholipids (this is however not discussed). While it is plausible that MprF flippases may be able to flip phosphatidyglycerol it could have a different path and structural requirements. It is also difficult to follow what the suggested pathway of flipping is in the Pseudomonas-MprF flippase (compared to R.tropici). Authors could provide a similar overview figure as in Song et al. and indicate what the potential differences are.

We modelled phosphatidylglycerol as the lipid as the current density doesn’t allow to model ala-PG ambiguously though it is found in the same position as the lys-PG in the R. tropici maps. The recent in-vitro assay by Hankins et al 2025 shows that PaMprF is able to flip wide range of lipids and we would also like to point out that PG from outer leaflet can be flipped, whose headgroup can be modified at the inner leaflet and flipped back. As shown by Song et al 2021 and Hebecker et al 2011, the specificity for the substrates is in the synthase domain (by mutagenesis and swapping). We don’t think there will be any difference between the lys-PG and Ala-PG path but in our opinion the positional relation between the soluble and membrane domain is the most important and has remained the focus of the manuscript along with the dimeric architecture. The figure 6 in the manuscript is descriptive of this and provides a summary of the structural observation from the presented structures.

Minor concerns

• Page 13: the following sentence should be rephrased: "Among the missing links in the current cryoEM maps is the lack of well-ordered density for lipid molecules on the inner leaflet closer to the re-entrant helices but it is reasonable to assume from the cluster of positive charge that there will be lipid molecules and are dynamic. "

This is has been rephrased.

• Page 4: Klein et al do not show that the Pseudomonas aeruginosa MprF mediates flipping

Corrected to reflect only the modification of lipid and not flipping.

Reviewer #3 (Significance (Required)):

General assessment: see review

Advance: Minor

Audience: Specialized

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

Learn more at Review Commons

Referee #2

Evidence, reproducibility and clarity

Shaileshanand J. et al., reported the structures of Multiple Peptide Resistance Factor, MprF, which is a bi-functional enzyme in bacteria responsible for aminoacylation of lipid head groups. The authors purified MprF from Pseudomonas aeruginosa in GDN micelles and nanodiscs, and by applying cryo-EM single particle method, they successfully reached near-atomic resolution, and built corresponding atomic models. By applying structural analysis as well as biochemistry methods, the authors demonstrated dimeric formation of MprF, exhibited the dynamic nature of the catalytic domain of this enzyme, and proposed a possible model on tRNA binding and aminoacylation.

Major comments:

1. In abstract, the authors stated 'Several lipid-like densities are observed in the cryoEM maps, which might indicate the path taken by the lipids and the coupling function of the two functional domains. Thus, the structure of a well characterised PaMprF lays a platform for understanding the mechanism of amino acid transfer to a lipid head group and subsequent flipping across the leaflet that changes the property of the membrane.' Firstly, those lipid-like densities were demonstrated in Fig 3A, since densities of lipids of purified membrane proteins often exist within regions of relatively low local resolution, or low quality, I think more detailed description on how the authors defined which part of the density belongs to lipid and how they acquired the modeling of some of the lipids is required. And the authors modeled phosphatidylglycerol into the GDN MprF, I would require additional experiment, for instance, mass spectrometry over the purified sample, to demonstrate the existence of this specific lipid with the sample. Secondly, regarding the last sentence in the abstract, how these structures lay a platform for further understanding was poorly discussed in both result section and discussion section, since the authors clearly stated 'This cavity perhaps provides a path for holding lipids...', then the statement in the next sentence 'Taken together... the vicinity to the cavities described above indicates the possible path taken by the lipids to enter and exit the enzyme' does not have a reliable evidence to support this conclusion, I would suggest the authors move these statements into discussion section, and elaborate more over this issue since it is an important part in the abstract, or make a more solid proof using other approaches, such as molecular dynamics simulation, to make these statements solid in the result section.

2. Fig 2B, it seems the H566 sidechains were overlapping in the zoom-in figure of distance measurement between H566 residues, to clarify this, authors should either present another figure with rotation, to better demonstrate their relative locations, or swap this zoom-in figure with another figure with rotations. Also, could the authors briefly commenting on why they chose H566 for distance measurement specifically?

3. Related to previous comment, I see one additional green square in Fig. 2A and an additional green square in Fig. 2B, without any zoom-in images provided on these regions. Besides, they're focusing on two different domains with same color, any particular reason why they're there? If so, please provide the information in figure legends.

4. Related to previous comment, authors should also provide distance measurement over electrostatic interaction sites in Fig. 2A, since distance plays as an important factor in these forces.

5. For Fig. 2C, since in Fig. 1, the authors have already indicated the differences between reconstruction of the GDN and nanodisc datasets, this information provided here seems to be a bit abundant, I suggest either move this panel to Fig. 1, to make a visualization on both electron densities as well as atomic models, or move this panel to supplementary figures.

6. Fig. 3B, some of the spheres of the lipids were also marked as red, any particular reason why they're red? Do they indicate they're phosphate heads? If so, could the authors provide evidences how they define these orientations of the lipid heads? If not, any particular reason why they're red?

7. Fig. 3C, the fitted model of lipid and its corresponding density should be added to Fig. S4, to give more detailed view on the quality of the fitting.

8. Fig. 4D and 4E, could the authors also indicate the RMSD values when comparing the differences of RtMprF, PaMprF, ReMprF, this information would be helpful to understand how big of a difference within these three models.

9. Fig. 6E, the coloring used for CCA-Ala were similar to the blue part of soluble domain, could the authors change the coloring a bit? Also, for Fig. 6F, I would suggest the authors provide a prediction model, such as using AlphaFold3, of this tRNA interaction site, to further validate this proposed model.

10. In Supplementary Figures S1 and S3, the angular distribution of maps exhibited preferred orientation to certain extent, 3D FSC estimation should also be supplied for these maps, as an indication of whether the reconstructed densities were affected or not.

11. For Fig S3B, could the authors switch to another image with better contrast?

Minor comments:

1. Fig. 2E and 2F, distance measurement should also be supplied to these two panels.

2. Fig. 5D, since in Fig. 4F and 4G already mentioned the skeleton of GDN, this modeling part should be presented before exhibit it in dimer interface, the authors should rearrange the sequence over these three panels.

3. In Supplementary Figure S3, which density was shown for the PaMprF local resolution estimation result? Authors should provide this information as two maps were shown in this figure.

CROSS-REFEREE COMMENTS

Both Reviewer #1 and #3 made comments over technical issue, their evaluation over functional aspects of this protein is what I was lacking over my comments, also, their evaluation of the biological narrative, relevance toward previous research is also more insightful. Finally, they offer valuable suggestions on how to adjust the article to make it more readable, and better describing the biological story which I would suggest the authors to pay attention to.

Significance

Significance

The authors mainly focused on the structure of MprF in Pseudomonas aeruginosa, this protein is essential for the resistance to cationic antimicrobial peptides. A combination of structural and biochemical analysis provided evidences to the dimeric formation to this enzyme, and the analysis over differences of purified proteins using GDN and nanodisc was particular interesting, which provide new insight regarding the flexible nature of this enzyme, and potentially could be beneficial to the membrane protein community, as it demonstrates the differences in detergent/nanodisc of choice could affect the assembly of the protein of interest. Still, some of the statements in the manuscript, for instance, the assignment of lipids was over-claimed and could be benefited from additional approaches to support the issue. I would suggest some refinement in the discussion section as well as some of the figures.

My expertise: cryo-EM single particle analysis; cryo-ET; sub-tomo averaging; cryo-FIB;

For models 3a and 3b I cannot identify how you generate stratum-specific estimates of your main effect as reported in Table 2 of your main submission.

This makes me wonder whether you accurately represent the quantities reported in Table 2...

This code section seems unnecessarily complicated given the tools available from the tableone or gtsummary or the course-specific svyTable1 packages, usage of which would be less prone to user-error.

Thực tế cho thấy, nghiên cứu của Norton cho biết có đến 68% người dùng Wi-Fi công cộng đã trở thành nạn nhân của tội phạm mạng, chủ yếu do mất cắp dữ liệu nhạy cảm . Con số này phản ánh rõ mức độ nguy hiểm thực tế của các cuộc tấn công như Evil Twin Ngoài ra, cảnh sát liên bang Úc cáo buộc người đàn ông này đã tạo ra mạng wifi "song sinh độc ác" - mô phỏng các mạng wifi hợp pháp - để lừa người dùng nhập thông tin cá nhân của họ.

Tấn công MITM sử dụng ARP Poisoning là kỹ thuật được sử dụng phổ biến nhất để thực hiện các cuộc tấn công MITM và điều này là do tính bảo mật kém của giao thức ARP và cũng vì đây là cách đơn giản nhất để thực hiện cuộc tấn công

Từ góc độ người dùng, các kết nối Wi-Fi công cộng miễn phí được khuyến nghị nên tránh xa. Chúng là một cách dễ dàng nhưng hiệu quả để thực hiện các cuộc tấn công MITM và khó phát hiện hơn nhiều, đặc biệt là từ góc độ người dùng và việc thiếu các biện pháp phòng ngừa thích hợp.

No contexto de transmissão de obrigação, no que tange à assunção de dívida, o silêncio protege o credor. Isto é, acaso não manifeste expressa concordância quanto à assunção da dívida, será interpretado como recusa.

Se comprador ordenar o transporte da coisa, será por sua conta e risco. A exceção é quando o vendedor não cumprir com as determinações e causar prejuízo.

Contrato de compra e venda que não estipular o local da tradição, será presumido que esse local é onde a está a coisa.

PROCESSUAL CIVIL. RECURSO ESPECIAL. AÇÃO REVISIONAL DE CONTRATO DE ALUGUEL ENTRE SHOPPING CENTER E LOJISTA. SUPERVENIÊNCIA DA PANDEMIA DECORRENTE DA COVID-19. CONTRATOS PARITÁRIOS. REGRA GERAL. PRINCÍPIO DO PACTA SUNT SERVANDA. POSSIBILIDADE DE REVISÃO. HIPÓTESES EXCEPCIONAIS. PREVISÃO DO ART. 317 DO CÓDIGO CIVIL. TEORIA DA IMPREVISÃO. ART. 478 DO CÓDIGO CIVIL. TEORIA DA ONEROSIDADE EXCESSIVA. RESOLUÇÃO. INTERPRETAÇÃO SISTEMÁTICA E TELEOLÓGICA DO DISPOSITIVO QUE AUTORIZA TAMBÉM A REVISÃO. PANDEMIA DA COVID-19 QUE CONFIGURA, EM TESE, EVENTO IMPREVISÍVEL E EXTRAORDINÁRIO APTO A POSSIBILITAR A REVISÃO DO CONTRATO DE ALUGUEL, DESDE QUE PREENCHIDOS OS DEMAIS REQUISITOS LEGAIS. HIPÓTESE DOS AUTOS. AUSÊNCIA DE COMPROVAÇÃO. MANUTENÇÃO DA DECISÃO RECORRIDA.

• 1. Ação revisional de contrato de aluguel entre shopping center e lojista, ajuizada em 20/4/2020, da qual foi extraído o presente recurso especial, interposto em 30/8/2022 e concluso ao gabinete em 20/10/2022.

• 2. O propósito recursal consiste em decidir se é cabível a revisão de contrato de aluguel firmado entre shopping center e lojista, com fundamento nas teorias da imprevisão (art. 317 do CC) e onerosidade excessiva (art. 478 do CC), em razão da superveniência da pandemia do coronavírus.

• 3. Nos contratos empresariais deve ser conferido especial prestígio aos princípios da liberdade contratual e do pacta sunt servanda, diretrizes positivadas no art. 421, caput, e 421-A do Código Civil, incluídas pela Lei nº 13.874/2019.

• 4. Nada obstante, o próprio diploma legal consolidou hipóteses de revisão e resolução dos contratos (317, 478, 479 e 480 do CC). Com amparo doutrinário, verifica-se que o art. 317 configura cláusula geral de <u>revisão</u> da prestação contratual e que a interpretação sistêmica e teleológica dos arts. 478, 479 e 480 autorizam também a revisão judicial do pactuado.

• 5. A <u>Teoria da Imprevisão</u> (art. 317 do CC), de matriz francesa, exige a comprovação dos seguintes requisitos: (I) obrigação a ser adimplida em momento posterior ao de sua origem; (II) superveniência de evento imprevisível; (III) que acarrete desproporção manifesta entre o valor da prestação devida e o do momento de sua execução. A pedido da parte, o juiz poderá corrigir o valor da prestação, de modo a assegurar, quanto possível, o seu valor real.

• 6. A <u>Teoria da Onerosidade Excessiva</u> (art. 478 do CC), de origem italiana, pressupõe (I) contratos de execução continuada ou diferida; (II) superveniência de acontecimento extraordinário e imprevisível; (III) que acarrete prestação excessivamente onerosa para uma das partes; (IV) extrema vantagem para a outra; e (V) inimputabilidade da excessiva onerosidade da prestação ao lesado.

Possibilidade de flexibilização da "extrema vantagem".

• 7. A pandemia da Covid-19 configura crise sanitária sem precedentes, que não apenas colocou em risco, mas também resultou, lamentavelmente, na perda de incontáveis vidas. Diante do cenário emergencial, garantiu-se às autoridades públicas, no âmbito de suas competências, a adoção de medidas necessárias para tentar preservar, ao máximo, a saúde e a vida das pessoas (Lei nº 13.979/2020). Nesse contexto, entes da Federação decretaram a suspensão de atividades e do funcionamento de estabelecimentos comerciais e industriais (lockdown), entre os quais se destacam, por exemplo, o atendimento ao público em shopping centers - excepcionados, muitas vezes, os supermercados, laboratórios, clínicas de saúde e farmácias neles existentes.

• 8. A situação de pandemia não constitui, por si só, justificativa para o inadimplemento da obrigação, mas é circunstância que, por sua imprevisibilidade, extraordinariedade e por seu grave impacto na situação socioeconômica mundial, não pode ser desprezada pelos contratantes, tampouco pelo Poder Judiciário. Desse modo, a revisão de contratos paritários com fulcro nos eventos decorrentes da pandemia não pode ser concebida de maneira abstrata, mas depende, sempre, da análise da relação contratual estabelecida entre as partes, sendo imprescindível que a pandemia tenha interferido de forma substancial e prejudicial na relação negocial.

• 9. A superveniência de doença disseminada mundialmente, que, na tentativa de sua contenção, ocasionou verdadeiro lockdown econômico e isolamento social, qualifica-se como evento imprevisível, porquanto não foi prevista, conhecida ou examinada pelos contratantes quando da celebração do negócio jurídico, e extraordinário, pois distante da álea e das consequências ínsitas e objetivamente vinculadas ao contrato.

• 10. Conclui-se que a pandemia ocasionada pela Covid-19 pode ser qualificada como evento imprevisível e extraordinário apto a autorizar a revisão dos aluguéis em contratos estabelecidos pelo shopping center e seus lojistas, desde que verificados os demais requisitos legais estabelecidos pelo art. 317 ou 478 do Código Civil.

• 11. Na mesma linha de raciocínio, esta Corte permitiu a revisão proporcional de aluguel em razão das consequências particulares da pandemia da Covid-19 em relação à empresa de coworking, cujo faturamento foi drasticamente reduzido no período pandêmico (REsp 1.984.277/DF, Quarta Turma, DJe 9/9/2022).

• 12. Hipótese em que o contexto fático delineado pelo Tribunal de origem, soberano no exame do acervo fático-probatório, demostra não estar caracterizado o desequilíbrio na relação locatícia no contrato estabelecido entre o shopping center (recorrido) e o lojista (recorrente), pois não verificada a desproporção (art. 317) ou a excessiva onerosidade (art. 478) na prestação in concreto. Ao contrário, o acórdão estadual afirma que o recorrido concedeu desconto substancial no valor do aluguel em razão do cenário pandêmico de suspensão das atividades econômicas. Ausentes os requisitos legais, não há possibilidade de revisão do contrato. Necessidade de manutenção da decisão.

• 13. Recurso especial conhecido e desprovido.

(REsp n. 2.032.878/GO, relatora Ministra Nancy Andrighi, Terceira Turma, julgado em 18/4/2023, DJe de 20/4/2023.)

CONDIÇÕES IMPOSSÍVEIS

• Se suspensi<u>V</u>as: in<u>V</u>alidam o negócio jurídico;
• Se resolu<u>T</u>ivas: são inexis<u>T</u>en<u>T</u>es no negócio jurídico.

Essa lógica faz todo o sentido, considerando que:

• A cláusula resolutiva não impede o exercício ou a aquisição do direito. Implementada a condição resolutiva, o negócio é extinto. Com isso, se a cláusula resolutiva é impossível, apenas se risca do negócio jurídico, já que não o afetará e jamais ocorrerá de fato.

• Situação é outra quanto à cláusula suspensiva, a qual impede a aquisição do direito até a sua implementação. Logo, se negócio jurídico é subordinado à cláusula suspensiva impossível, ele jamais se concretizará, razão pela qual a lei decreta a invalidade da avença.

• Informativo 1143
• ADPF 1011 / PE
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. GILMAR MENDES
• Julgamento: 28/06/2024 (Virtual)
• Ramo do Direito: Processual Civil, Constitucional
• Matéria: Execução; Multa Simples; Tribunal De Contas; Legitimidade Ad Causam/Fiscalização Contábil, Financeira e Orçamentária; Controle Externo; Tribunal de Contas

Multas aplicadas pelo Tribunal de Contas estadual: legitimidade dos entes públicos para executá-las

Tese fixada - 1. O Município prejudicado é o legitimado para a execução de crédito decorrente de multa aplicada por Tribunal de Contas estadual a agente público municipal, em razão de danos causados ao erário municipal.

• 2. Compete ao Estado-membro a execução de crédito decorrente de multas simples, aplicadas por Tribunais de Contas estaduais a agentes públicos municipais, em razão da inobservância das normas de Direito Financeiro ou, ainda, do descumprimento dos deveres de colaboração impostos, pela legislação, aos agentes públicos fiscalizados.

Resumo - Os estados possuem legitimidade ativa para executar multas meramente sancionatórias aplicadas por seus Tribunais de Contas em face de agentes públicos municipais que, por seus atos, infrinjam as normas de Direito Financeiro ou violem os deveres de colaboração com o órgão de controle, impostos pela legislação.

• A Constituição Federal de 1988 confere aos Tribunais de Contas em todo o País a competência para aplicar as sanções previstas em lei aos responsáveis por ilegalidades de despesas ou irregularidades nas contas (1).

• Consoante o julgamento que originou a fixação da tese do Tema 642 da repercussão geral, o que determina o ente competente para executar a multa aplicada pelas Cortes de Contas estaduais é a natureza jurídica dessa sanção. A multa simples imposta ao agente público municipal — que diz respeito à modalidade sancionatória de responsabilidade financeira — em razão da grave inobservância de normas financeiras, contábeis e orçamentárias, ou como consequência direta da violação de deveres de colaboração que os agentes fiscalizados devem guardar com o órgão de controle (obrigações acessórias), configura ferramenta de desincentivo à prática de futuras transgressões dessas normas e, em certos casos, de reafirmação da autoridade das decisões ou diligências determinadas pelos Tribunais de Contas.

• Por outro lado, as penalidades de imputação de débito e de multa proporcional ao dano abrangem a modalidade reintegratória de responsabilidade financeira, eis que visam recompor o erário em virtude de desvio, pagamento indevido ou falta de cobrança ou liquidação, nos termos da lei.

• Nesse contexto, quando as sanções aplicadas pelo Tribunal de Contas estadual a agente público municipal referirem-se ao ressarcimento ao erário, a legitimidade para executá-las é do município cujo patrimônio público foi atingido (2), ao passo que é o próprio estado o legitimado ativo para executar as multas que decorrem do poder sancionador da Corte de Contas (sanção pecuniária e que não possui qualquer relação com a existência de dano ao erário) (3).

• Com base nesses e em outros entendimentos, o Plenário, por unanimidade, julgou procedente a ação, bem como (i) assentou que a presente decisão não afeta automaticamente a coisa julgada formada em momento anterior à publicação da ata deste julgamento; e (ii) determinou o acréscimo de uma nova proposição (item 2) à tese do Tema 642 da repercussão geral, a fim de abranger o novo entendimento do Tribunal.

• Informativo 1029
• RE 1003433 / RJ - Tema 642
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. MARCO AURÉLIO
• Redator(a) do acórdão: Min. ALEXANDRE DE MORAES
• Julgamento: 14/09/2021 (Virtual)
• Ramo do Direito: Processual Civil, Administrativo
• Matéria: Execução; Controle externo

Legitimidade para executar multa por danos causados a erário municipal

Tese fixada - O Município prejudicado é o legitimado para a execução de crédito decorrente de multa aplicada por Tribunal de Contas estadual a agente público municipal, em razão de danos causados ao erário municipal.

Resumo - Os estados não têm legitimidade ativa para a execução de multas aplicadas, por Tribunais de Contas estaduais, em face de agentes públicos municipais, que, por seus atos, tenham causado prejuízos a municípios.

• Se a multa aplicada pelo Tribunal de Contas decorre da prática de atos que causaram prejuízo ao erário municipal, o legitimado ativo para a execução do crédito fiscal é o município lesado, e não o estado (1). Entendimento diverso caracterizaria hipótese de enriquecimento sem causa.

• Com base nesse entendimento, o Plenário, por maioria, ao julgar o Tema 642 da RG, negou provimento a recurso extraordinário. Vencidos os ministros Marco Aurélio (relator) e Edson Fachin.

Precedentes: RE 525.663 AgR e RE 223.037

• Informativo 1007
• ADI 1668 / DF
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. EDSON FACHIN
• Julgamento: 27/02/2021 (Virtual)
• Ramo do Direito: Administrativo
• Matéria: AGÊNCIAS REGULADORAS

Serviços de telecomunicações: criação da ANATEL e competências do órgão regulador

Resumo - A competência atribuída ao chefe do Poder Executivo para expedir decreto em ordem a instituir ou eliminar a prestação do serviço em regime público, em concomitância ou não com a prestação no regime privado, aprovar o plano geral de outorgas do serviço em regime público e o plano de metas de universalização do serviço prestado em regime público está em perfeita consonância com o poder regulamentar previsto no art. 84, IV, parte final, e VI, da Constituição Federal (CF). O art. 18, I, II e III da Lei 9.472/1997 é compatível com os arts. 21, XI, e 48, XII, da Constituição Federal (CF).

• A competência da Agência Nacional de Telecomunicações (ANATEL) para expedir normas subordina-se aos preceitos legais e regulamentares que regem a outorga, prestação e fruição dos serviços de telecomunicações no regime público e no regime privado. O art. 19, IV e X, da Lei 9.472/1997, desse modo, é constitucional.

• A busca e posterior apreensão efetuada sem ordem judicial, com base apenas no poder de polícia de que é investida a ANATEL, mostra-se <u>inconstitucional</u> diante da violação ao disposto no princípio da inviolabilidade de domicílio, à luz do art. 5º, XI, da Constituição Federal. Logo, o art. 19, XV, da Lei 9.472/1997 é inconstitucional. A competência atribuída ao Conselho Diretor da ANATEL para editar normas próprias de licitação e contratação (Lei 9.472/1997, art. 22, II) deve observar o arcabouço normativo atinente às licitações e aos contratos, em respeito ao princípio da legalidade.

• Diante da especificidade dos serviços de telecomunicações, é válida a criação de novas modalidades licitatórias por <u>lei de mesma hierarquia</u> da Lei Geral de Licitações (Lei 8.666/1993). Portanto, sua disciplina deve ser feita por meio de lei, e não de atos infralegais, em obediência aos artigos 21, XI, e 22, XXVII, do texto constitucional. Em razão disso, é inconstitucional a expressão “serão disciplinados pela Agência” contida no art. 55 da Lei 9.472/1997.

• A contratação, a que se refere o art. 59 da Lei 9.472/1997, de técnicos ou empresas especializadas, inclusive consultores independentes e auditores externos, para executar atividades de competência da ANATEL, deve observar o regular procedimento licitatório previsto pelas leis de regência.

• A possibilidade de concomitância de regimes público e privado de prestação do serviço, assim como a definição das modalidades do serviço são questões estritamente técnicas, da alçada da agência, a quem cabe o estabelecimento das bases normativas de cada matéria relacionada à execução, à definição e ao estabelecimento das regras peculiares a cada serviço.

• A ANATEL não pode disciplinar procedimento licitatório simplificado por meio de norma de hierarquia inferior à Lei Geral de Licitações, sob pena de ofensa ao princípio da reserva legal. Por isso, são inconstitucionais as expressões “simplificado” e “nos termos por ela regulados” do art. 119, da Lei 9.472/1997.

• A competência atribuída ao chefe do Poder Executivo para expedir decreto em ordem a instituir ou eliminar a prestação do serviço em regime público, em concomitância ou não com a prestação no regime privado, aprovar o plano geral de outorgas do serviço em regime público e o plano de metas de universalização do serviço prestado em regime público está em perfeita consonância com o poder regulamentar previsto no art. 84, IV, parte final, e VI, da Constituição Federal (CF). O art. 18, I, II e III da Lei 9.472/1997 (1) é compatível com os arts. 21, XI, e 48, XII, da Constituição Federal (CF) (2).

• De fato, as medidas previstas no art. 18 são atinentes à execução da política de telecomunicações definidas no corpo da Lei 9.472/1997 e estão condicionadas por várias normas desse diploma.

• O caput do art. 18 da Lei 9.472/1997 observa, portanto, esses dispositivos constitucionais, que atribuem ao Presidente da República a competência para expedir decretos e regulamentos destinados à fiel execução de lei, e a ele outorgam o poder de dispor, mediante decreto, sobre a organização e funcionamento da administração federal.

• É ínsito ao poder regulamentar atuar secundum legem e intra legem. Assim, atendidos os limites da legislação que rege a matéria, a Lei 9.472/1997, ao tempo em que confere tal poder ao Presidente da República, também fixa parâmetros para o seu exercício.

• A competência da Agência Nacional de Telecomunicações (ANATEL) para expedir normas subordina-se aos preceitos legais e regulamentares que regem a outorga, prestação e fruição dos serviços de telecomunicações no regime público e no regime privado. O art. 19, IV e X, da Lei 9.472/1997 (3), desse modo, é constitucional.

• Na esteira da jurisprudência do Supremo Tribunal Federal (STF) (4), cabe às agências reguladoras, como a ANATEL, desempenhar a tarefa ordenadora e fiscalizatórias dos setores a elas submetidos. E, para a adequada execução dessa função, exsurge o poder de expedir normas como imanente à atividade regulatória das agências, a quem compete, no âmbito de sua atuação e nos limites do arcabouço normativo sobre o tema, disciplinar a prestação dos serviços.

• Não se trata, portanto, de delegação de poderes legislativos, pois a expedição de normas regulatórias é sempre exercida com fundamento na lei, que também lhe serve de limite, mas que não esgota as possibilidades de mediação dos interesses diversos colocados para composição pelos órgãos reguladores.

• A busca e posterior apreensão efetuada sem ordem judicial, com base apenas no poder de polícia de que é investida a ANATEL, mostra-se inconstitucional diante da violação ao disposto no princípio da inviolabilidade de domicílio, à luz do art. 5º, XI, da Constituição Federal (5). Logo, o art. 19, XV, da Lei 9.472/1997 (6) é inconstitucional.

• A possibilidade de promoção de interdição de estabelecimentos, instalações ou equipamentos, e apreensão de bens ou produtos, nos termos do art. 3º, parágrafo único, da Lei 10.871/2004 (que dispõe sobre a criação de carreiras e organização de cargos efetivos das autarquias especiais, denominadas agências reguladoras), constitui exercício do poder de polícia da Administração Pública, dotado de autoexecutoriedade, inerente ao exercício dessa função (7).

• Ocorre que o art. 19, XV, da Lei 9.472/1997, que estabelece a busca e apreensão de bens, tem uma dimensão distinta. Frise-se que, segundo orientação do STF, o conceito de domicílio não está limitado à residência domiciliar, mas abarca também qualquer compartimento privado onde alguém exerce profissão ou atividade (8).

• A competência atribuída ao Conselho Diretor da ANATEL para editar normas próprias de licitação e contratação (Lei 9.472/1997, art. 22, II) (9) deve observar o arcabouço normativo atinente às licitações e aos contratos, em respeito ao princípio da legalidade.

• Com efeito, as agências reguladoras não possuem a prerrogativa de legislar em matéria de licitação. Primeiro, porque isso viola a competência legislativa privativa da União (CF, art. 22, XXVII). Segundo, porque inovar no ordenamento jurídico não se encontra dentre os atributos que a função regulatória desses órgãos detêm, uma vez que eles colmatam lacunas propositais de natureza técnica na legislação, mas não podem estabelecer, de forma originária e primária, deveres e obrigações aos particulares, menos ainda exercer atividade criativa no que concerne a modalidades licitatórias e contratuais.

• Diante da especificidade dos serviços de telecomunicações, é válida a criação de novas modalidades licitatórias por lei de mesma hierarquia da Lei Geral de Licitações (Lei 8.666/1993). Portanto, sua disciplina deve ser feita por meio de lei, e não de atos infralegais, em obediência aos artigos 21, XI, e 22, XXVII, do texto constitucional. Em razão disso, é inconstitucional a expressão “serão disciplinados pela Agência” contida no art. 55 da Lei 9.472/1997 (10).

• A inserção, no ordenamento jurídico, de novas modalidades licitatórias, por lei que tem o mesmo status que a Lei Geral de Licitações não viola a Carta Magna. Todavia, para que seja respeitado o princípio da reserva legal e, ainda, tendo em vista que a consulta é instituto que não está restrito à ANATEL, mas cuja aplicação foi estendida, por meio do art. 37 da Lei 9.986/2000, a todas as agências reguladoras, a disciplina deve dar-se mediante lei.

• A contratação, a que se refere o art. 59 da Lei 9.472/1997 (11), de técnicos ou empresas especializadas, inclusive consultores independentes e auditores externos, para executar atividades de competência da ANATEL, deve observar o regular procedimento licitatório previsto pelas leis de regência.

• Efetivamente, a contratação sem o procedimento licitatório previsto pelas leis de regência fere o art. 22, XXVII, da CF.

• A possibilidade de concomitância de regimes público e privado de prestação do serviço, assim como a definição das modalidades do serviço são questões estritamente técnicas, da alçada da agência, a quem cabe o estabelecimento das bases normativas de cada matéria relacionada à execução, à definição e ao estabelecimento das regras peculiares a cada serviço.

• Diante da existência de parâmetros definidores na legislação, e da permissão constitucional para a prestação do serviço de telecomunicações pelo regime privado, por meio de autorização, não se vislumbra inconstitucionalidade nos artigos 65, III, §§ 1º e 2º, 66 e 69 da Lei 9.472/1997 (12).

• A atribuição à agência da competência para definir os serviços não desborda dos limites de seu poder regulatório.

• A previsão constitucional do art. 21, XI, permite a exploração “diretamente ou mediante autorização, concessão ou permissão, os serviços de telecomunicações, nos termos da lei ”.

• Portanto, a despeito da previsão mais genérica do art. 175 da CF (13), no caso dos serviços de telecomunicações, é o texto constitucional que permite a exploração por meio de autorização, o que significa conferir à Administração a faculdade de instituir um regime privado, submetido à livre concorrência, ainda que derrogado parcialmente pela regulação estabelecida pela ANATEL (14).

• A ANATEL não pode disciplinar procedimento licitatório simplificado por meio de norma de hierarquia inferior à Lei Geral de Licitações, sob pena de ofensa ao princípio da reserva legal. Por isso, são inconstitucionais as expressões “simplificado” e “nos termos por ela regulados” do art. 119, da Lei 9.472/1997 (15).

• As normas licitatórias são cogentes, não viabilizando atuação livre deste ou daquele administrador, por maior que lhe seja a envergadura.

• Com base nesse entendimento, o Plenário, por maioria, julgou parcialmente procedente pedido formulado em ação direta ajuizada contra dispositivos da Lei 9.472/1997, que dispõe sobre a organização dos serviços de telecomunicações, a criação e o funcionamento de um órgão regulador e outros aspectos institucionais, nos termos da Emenda Constitucional 8/1995. Vencido o ministro Roberto Barroso.

• ADI 429
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. LUIZ FUX
• Julgamento: 20/08/2014
• Publicação: 30/10/2014

AÇÃO DIRETA DE INCONSTITUCIONALIDADE. TRIBUTÁRIO. NORMAS GERAIS DE DIREITO TRIBUTÁRIO. ICMS. CONSTITUIÇÃO DO ESTADO DO CEARÁ. IMPUGNAÇÃO AOS ARTIGOS 192, §§ 1º E 2º; 193 E SEU PARÁGRAFO ÚNICO; 201 E SEU PARÁGRAFO ÚNICO; 273, PARÁGRAFO ÚNICO; E 283, III, DA CONSTITUIÇÃO ESTADUAL. ADEQUADO TRATAMENTO TRIBUTÁRIO AO ATO COOPERATIVO E ISENÇÃO DE TRIBUTOS ESTADUAIS ÀS PEQUENAS E MICROEMPRESAS; PEQUENOS E MICROPRODUTORES RURAIS; BEM COMO PARA AS EMPRESAS QUE ABSORVAM CONTINGENTES DE DEFICIENTES NO SEU QUADRO FUNCIONAL OU CONFECCIONE E COMERCIALIZE APARELHOS DE FABRICAÇÃO ALTERNATIVA PARA PORTADORES DE DEFICIÊNCIA. DISPOSIÇÕES PREVISTAS NA CONSTITUIÇÃO ESTADUAL. VIOLAÇÃO AO DISPOSTO NO ARTIGO 146, INCISO III, ALÍNEA “C”, DA CRFB/88. COMPETÊNCIA CONCORRENTE DA UNIÃO, ESTADOS E DISTRITO FEDERAL PARA LEGISLAR SOBRE DIREITO TRIBUTÁRIO. ARTIGO 24, INCISO I, DA CRFB/88. AUSÊNCIA DE INCONSTITUCIONALIDADE. DEMAIS DISPOSITIVOS OBJURGADOS. CONCESSÃO UNILATERAL DE BENEFÍCIOS E INCENTIVOS FISCAIS. ICMS. AUSÊNCIA DE CONVÊNIO INTERESTADUAL. AFRONTA AO DISPOSTO NO ARTIGO 155, § 2º, INCISO XII, “G”, DA CRFB/88. CAPUT DO ART. 193 DA CONSTITUIÇÃO ESTADUAL. INTERPRETAÇÃO CONFORME À CONSTITUIÇÃO SEM DECLARAÇÃO DE NULIDADE. EXCLUSÃO DO ICMS DO SEU CAMPO DE INCIDÊNCIA. - 1. O Federalismo brasileiro exterioriza-se, dentre outros campos, no segmento tributário pela previsão de competências legislativo-fiscais privativas dos entes políticos, reservada à Lei Complementar estabelecer normas gerais.

• 2. A concessão de benefícios fiscais não é matéria relativa à inciativa legislativa privativa do Chefe do Poder Executivo, nos termos do estabelecido no artigo 61, § 1º, inciso II, alínea b, da CRFB/88.

• 3. O poder de exonerar corresponde a uma derivação do poder de tributar, assim, presente este, não há impedimentos para que as entidades investidas de competência tributária, como o são os Estados-membros, definam hipóteses de isenção ou de não-incidência das espécies tributárias em geral, à luz das regras de competência tributária, o que não interdita a Constituição estadual de dispor sobre o tema.

• 4. O art. 146, III, “c”, da CRFB/88 determina que lei complementar estabeleça normas gerais sobre matéria tributária e, em especial, quanto ao adequado tratamento tributário a ser conferido ao ato cooperativo praticado pelas sociedades cooperativas.

• 5. Não há a alegada inconstitucionalidade da Constituição estadual, porquanto a competência para legislar sobre direito tributário é concorrente, cabendo à União estabelecer normas gerais, aos Estados-membros e o Distrito Federal suplementar as lacunas da lei federal sobre normas gerais, afim de afeiçoá-las às particularidades locais, por isso que inexistindo lei federal de normas gerais, acerca das matérias enunciadas no citado artigo constitucional, os Estados podem exercer a competência <u>legislativa plena</u> (§ 3º, do art. 24 da CRFB/88).

• 6. Consectariamente, o § 1º do artigo 192 da Constituição cearense que estabelece que “o ato cooperativo, praticado entre o associado e sua cooperativa, não implica em operação de mercado”, não é inconstitucional.

• 7. É que a Suprema Corte, ao apreciar situação análoga, assentou que, enquanto não promulgada a lei complementar a que se refere o art. 146, III, “c”, da CRFB/88, não se pode pretender que, com base na legislação local, não possa o Estado-membro, que tem competência concorrente em se tratando de direito tributário (artigo 24, I e § 3º, da Carta Magna), dê às cooperativas o tratamento que julgar adequado, até porque tratamento adequado <u>não significa necessariamente tratamento privilegiado</u>, verbis: “Inexiste, no caso, ofensa ao artigo 146, III, ‘c’, da Constituição, porquanto esse dispositivo constitucional não concedeu às cooperativas imunidade tributária, razão por que, enquanto não for promulgada a lei complementar a que ele alude, não se pode pretender que, com base na legislação local mencionada no aresto recorrido, não possa o Estado-membro, que tem competência concorrente em se tratando de direito tributário (artigo 24, I e § 3º, da Carta Magna), dar às Cooperativas o tratamento que julgar adequado, até porque tratamento adequado não significa necessariamente tratamento privilegiado.”(RE 141.800, Rel. Min. MOREIRA ALVES, DJ de 30.10.97).

• 8. A concessão unilateral de benefícios fiscais relativos ao ICMS, sem a prévia celebração de convênio intergovernamental, nos termos do que dispõe a LC nº 24/75, recepcionada inequivocamente consoante jurisprudência da Corte, afronta ao disposto no artigo 155, § 2º, XII, “g”, da CRFB/88.

• 9. O comando constitucional contido no art. 155, § 2º, inciso “g”, que reserva à lei complementar federal “regular a forma como, mediante deliberação dos Estados e do Distrito Federal, isenções, incentivos e benefícios fiscais serão concedidos e revogados” aplicado, in casu, revela manifesta a inconstitucionalidade material dos dispositivos da Constituição cearense que outorga incentivo fiscal incompatível com a CRFB/88. Precedentes: ADI 84, Rel. Min. ILMAR GALVÃO, Tribunal Pleno, julgado em 15/02/1996, DJ 19-04-1996).

• 10. A outorga de benefícios fiscais relativos ao ICMS, sem a prévia e necessária celebração de convênio entre os Estados e o Distrito Federal é manifestamente inconstitucional. Precedentes: ADI 2906/RJ, rel. Min. Marco Aurélio, 1º.6.2011; ADI 2376/RJ, rel. Min. Marco Aurélio, 1º.6.2011; ADI 3674/RJ, rel. Min. Marco Aurélio, 1º.6.2011; ADI 3413/RJ, rel. Min. Marco Aurélio, 1º.6.2011; ADI 4457/PR, rel. Min. Marco Aurélio, 1º.6.2011; ADI 3794/PR, rel. Min. Joaquim Barbosa, 1º.6.2011; ADI 2688/PR, rel. Min. Joaquim Barbosa, 1º.6.2011; ADI 1247/PA, rel. Min. Dias Toffolli, 1º.6.2011; ADI 3702/ES, rel. Min. Dias Toffoli, 1º.6.2011; ADI 4152/SP, rel. Min. Cezar Peluso, 1º.6.2011; ADI 3664/RJ, rel. Min. Cezar Peluso, 1º.6.2011; ADI 3803/PR, rel. Min. Cezar Peluso, 1º.6.2011; ADI 2549/DF, rel. Min. Ricardo Lewandowski, 1º.6.2011.

• 11. Calcado nessas premissas, forçoso concluir que: a) O § 2º do art. 192 da Constituição cearense concede isenção tributária de ICMS aos implementos e equipamentos destinados aos deficientes físicos auditivos, visuais, mentais e múltiplos, bem como aos veículos automotores de fabricação nacional com até 90 HP de potência adaptados para o uso de pessoas portadoras de deficiência, o que acarreta a declaração de sua inconstitucionalidade, sem a pronúncia de nulidade, por um prazo de doze meses. b) O caput do artigo 193 da Constituição cearense isenta as microempresas de tributos estaduais, ao passo que seu parágrafo único estende a isenção, de forma expressa, ao ICMS, o que acarreta a declaração de inconstitucionalidade do parágrafo único e do caput, este por interpretação conforme para excluir de seu âmbito de incidência o ICMS. c) A Inconstitucionalidade do artigo 201 e seu parágrafo único, da Constituição cearense é manifesta, porquanto pela simples leitura dos dispositivos verifica-se que o imposto estadual com tal campo de incidência é o ICMS, verbis: “Art. 201. Não incidirá imposto, conforme a lei dispuser, sobre todo e qualquer produto agrícola pertencente à cesta básica , produzido por pequenos e microprodutores rurais que utilizam apenas a mão-de-obra familiar, vendido diretamente aos consumidores finais. Parágrafo único. A não-incidência abrange produtos oriundos de associações e cooperativas de produção e de produtores, cujos quadros sociais sejam compostos exclusivamente por pequenos e microprodutores e trabalhadores rurais sem terra. d) O parágrafo único do art. 273 e o inciso III do art. 283, da Constituição cearense incidem na mesma inconstitucionalidade, verbis: “Art. 273. Toda entidade pública ou privada que inclua o atendimento à criança e ao adolescente, inclusive os órgãos de segurança, tem por finalidade prioritária assegurar-lhes os direitos fundamentais. Parágrafo único. As empresas privadas que absorvam contingentes de até cinco por cento de deficientes no seu quadro funcional gozarão de incentivos fiscais de redução de um por cento no ICMS. (…) Art. 283. Para estimular a confecção e comercialização de aparelhos de fabricação alternativa para as pessoas portadoras de deficiência, o Estado concederá: (…) III - isenção de cem por cento do ICMS.

• 12. Pedido de inconstitucionalidade julgado parcialmente procedente para declarar: (i) inconstitucional o parágrafo 2º do art. 192, sem a pronúncia de nulidade, por um prazo de doze meses (ii) parcialmente inconstitucional o caput do art. 193, dando-lhe interpretação conforme para excluir de seu âmbito de incidência o ICMS; (iii) inconstitucional o parágrafo único do artigo 193; (iv) inconstitucional o artigo 201, caput, e seu parágrafo único; (v) inconstitucional o parágrafo único do artigo 273; (vi) inconstitucional o inciso III do artigo 283; julgar improcedente o pedido quanto ao caput e §1º do artigo 192, todos os artigos da Constituição cearense.

Observação - Acórdão(s) citado(s): (COMPETÊNCIA, LEI COMPLEMENTAR FEDERAL, REGULAÇÃO, BENEFÍCIO FISCAL, ICMS) ADI 84 (TP). (INCONSTITUCIONALIDADE, ESTADO-MEMBRO, CONCESSÃO UNILATERAL, BENEFÍCIO FISCAL, ICMS) ADI 1247 (TP), ADI 2376 (TP), ADI 2549 (TP), ADI 2688 (TP), ADI 2906 (TP), ADI 3413 (TP), ADI 3664 (TP), ADI 3674 (TP), ADI 3702 (TP), ADI 3794 (TP), ADI 3803 (TP), ADI 3809 (TP), ADI 4152 (TP), ADI 4457 (TP). (COMPETÊNCIA, ESTADO-MEMBRO, REGULAÇÃO, TRATAMENTO TRIBUTÁRIO, COOPERATIVA) RE 141800 (2ªT). (ISENÇÃO TRIBUTÁRIA, ICMS, TEMPLO RELIGIOSO) ADI 3421 (TP). Número de páginas: 44. Análise: 12/12/2014, RAF.

Doutrina Ferreira. BRANCO, Paulo Gustavo Gonet. Curso de Direito Constitucional. 8. ed. São Paulo: Saraiva, 2013. p. 803/804. PYRRHO, Sérgio. Soberania, ICMS e isenções os convênios e os tratados internacionais. Rio de Janeiro: Lumen Juris, 2008. p. 32. TORRES, Ricardo Lobo. Tratado de direito constitucional financeiro

Obs.: Muito embora a CF preveja lei complementar federal para conferir qual será o tratamento adequado ao ato cooperativo, isso não significa que os Estados-Membros estarão impedidos de legislar plenamente quanto à matéria enquanto não houve a necessária lei complementar federal. Isso é, segue-se a regra geral do art. 24, § 3º, CF que estabelece competência legislativa plena aos Estados acaso União não edite regras gerais.

• Informativo 969
• RE 828040 / DF
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. ALEXANDRE DE MORAES
• Julgamento: 12/03/2020 (Presencial)
• Ramo do Direito: Civil, Constitucional
• Matéria: Responsabilidade Civil - Direitos Sociais

Responsabilidade civil objetiva e acidente de trabalho

• O artigo 927, parágrafo único, do Código Civil é compatível com o artigo 7º, XXVIII, da Constituição Federal, sendo constitucional a responsabilização objetiva do empregador por danos decorrentes de acidentes de trabalho, nos casos especificados em lei, ou quando a atividade normalmente desenvolvida, por sua natureza, apresentar exposição habitual a risco especial, com potencialidade lesiva e implicar ao trabalhador ônus maior do que aos demais membros da coletividade.

Resumo - É admissível — nos casos especificados por lei, ou em razão do risco inerente à própria atividade — a responsabilização objetiva do empregador por danos decorrentes de acidentes de trabalho.

• O art. 927, parágrafo único, do Código Civil (CC) (1) é compatível com o art. 7º, XXVIII, da Constituição Federal (CF) (2), sendo constitucional a responsabilização objetiva do empregador por danos decorrentes de acidentes de trabalho nos casos especificados em lei ou quando a atividade normalmente desenvolvida, por sua natureza, apresentar exposição habitual a risco especial, com potencialidade lesiva, e implicar ao trabalhador ônus maior do que aos demais membros da coletividade.

• Essa é a tese do Tema 932 da repercussão geral, fixada pelo Plenário, por maioria, ao negar provimento a recurso extraordinário (Informativo 950).

Vencido o ministro Marco Aurélio.(1) CC/2002: “Art. 927. Aquele que, por ato ilícito (arts. 186 e 187), causar dano a outrem, fica obrigado a repará-lo. 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.” (2) CF/1988: “Art. 7º São direitos dos trabalhadores urbanos e rurais, além de outros que visem à melhoria de sua condição social: (...) XXVIII – seguro contra acidentes de trabalho, a cargo do empregador, sem excluir a indenização a que este está obrigado, quando incorrer em dolo ou culpa;”

Legislação: CC/2002, art. 927. CF, art. 7º, XXVIII.

Consultar todos os resumos relacionados ao processo (2)

• Informativo 991
• RE 784439 / DF
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. ROSA WEBER
• Julgamento: 26/06/2020 (Virtual)
• Ramo do Direito: Tributário
• Matéria: ISS; Impostos

Natureza taxativa da lista do rol de serviços sujeitos a ISS

Tese fixada

• É taxativa a lista de serviços sujeitos ao ISS a que se refere o art. 156, III, da Constituição Federal, admitindo-se, contudo, a incidência do tributo sobre as atividades inerentes aos serviços elencados em lei em razão da interpretação extensiva.

Resumo - As listas de serviços preveem ser irrelevante a nomenclatura dada ao serviço e trazem expressões para permitir a interpretação extensiva de alguns de seus itens, notadamente se socorrendo da fórmula “e congêneres”. Não existe obstáculo constitucional contra esta sistemática legislativa e excessos interpretativos que venham a ocorrer serão dirimíveis pelo Poder Judiciário.

Legislação: CF/1988, art. 5º, LV, art. 156, III.

Precedentes: RE 592.905/SC, relator Min. Eros Grau, DJe de 5.3.2010 (Tema 125 RG)RE 651.703/PR, relator Min. Luiz Fux, DJe de 26.4.2017 (Tema 581 RG)

Observação: Clipping das sessões virtuais. Acórdão publicado no DJe de 15.9.2020.

• Informativo 1110
• ADI 5674 / DF
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. ANDRÉ MENDONÇA
• Julgamento: 29/09/2023 (Virtual)
• Ramo do Direito: Tributário, Constitucional
• Matéria: Impostos; ISS; Hipóteses de Incidência; Hospedagem / Sistema Tributário Nacional; Impostos dos Municípios; ISS

ISS: incidência sobre atividades relativas à hospedagem

Resumo - É constitucional a incidência do Imposto sobre Serviços de Qualquer Natureza (ISS) sobre as atividades relativas à hospedagem de qualquer natureza, prevista no subitem 9.01 da lista de serviços anexa à Lei Complementar 116/2003.

• Os contratos que veiculam hospedagem de qualquer natureza, nos meios dispostos na referida lista, são preponderantemente de serviços. Ademais, o ISS incide sobre as atividades que representam obrigações de fazer e obrigações mistas, que incluem obrigação de dar (1).

• Não se pode fazer confusão entre a relação negocial de hospedagem e o contrato de locação de bem imóvel, de modo que é indevido excluir da base de cálculo desse tributo municipal a parcela da locação da unidade habitacional, visto que a circulação de serviço prevista contratualmente tem caráter singular e ganha sentido econômico com sua visualização unitária.

• Assim, dada a prevalência da uniformização da legislação federal, reforça-se o entendimento do STJ de que todas as parcelas que integram o preço do serviço de hotelaria compõem a base de cálculo do ISS.

• Com base nesses e em outros entendimentos, o Plenário, por unanimidade, julgou improcedente a ação, para assentar a constitucionalidade do subitem 9.01 da lista de serviços anexa à Lei Complementar 116/2003 (2).(1) Precedentes citados: RE 651.703 (Tema 581 RG); RE 603.136 (Tema 300 RG) e RE 784.439 (Tema 296 RG). (2) Lista de serviços anexa à Lei Complementar 116/2003: “9 – Serviços relativos a hospedagem, turismo, viagens e congêneres. 9.01 – Hospedagem de qualquer natureza em hotéis, apart-service condominiais, flat, apart-hotéis, hotéis residência, residence-service, suite service, hotelaria marítima, motéis, pensões e congêneres; ocupação por temporada com fornecimento de serviço (o valor da alimentação e gorjeta, quando incluído no preço da diária, fica sujeito ao Imposto Sobre Serviços).”

Legislação: Lista de serviços anexa à Lei Complementar 116/2003: subitem 9.01.

Precedentes: RE 651.703 (Tema 581 RG); RE 603.136 (Tema 300 RG) e RE 784.439 (Tema 296 RG).

Com efeito, vide do acórdão da ADI 5938, que o afastamento deve ser automático e incondicionado. Determinar à gestante que apresente laudo médico para afastamento vulnera a proteção à maternidade e à integral proteção à criança, criando hipóteses em que - por desconhecimento, receio de demissão ou algo do gênero - a mulher tenha contato com ambiente potencialmente insalubre em grau máximo, inclusive durante a amamentação.

• Informativo 942
• ADI 5938 / DF
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. ALEXANDRE DE MORAES
• Julgamento: 29/05/2019 (Presencial)
• Ramo do Direito: Trabalho
• Matéria: PROTEÇÃO À MATERNIDADE

CLT, art. 394-A: atividade insalubre e afastamento de gestante e de lactante

Resumo - O Plenário, por maioria, confirmou medida cautelar deferida pelo ministro Alexandre de Moraes (relator) em decisão monocrática e julgou parcialmente procedente pedido formulado em ação direta para declarar a inconstitucionalidade da expressão “quando apresentar atestado de saúde, emitido por médico de confiança da mulher, que recomende o afastamento”, contida nos incisos II e III do art. 394-A da Consolidação das Leis do Trabalho (CLT) (1), inseridos pelo art. 1º da Lei 13.467/2017.

• O colegiado registrou que, na redação anterior, o preceito estabelecia que a empregada gestante ou lactante seria afastada, enquanto durasse a gestação e a lactação, de quaisquer atividades, operações ou locais insalubres e deveria exercer suas atividades em local salubre.

• Com a alteração implementada pela Lei 13.467/2017, que promoveu a “Reforma Trabalhista” de 2017, o art. 394-A passou a permitir que a mulher gestante continuasse a realizar suas atividades mesmo em condições insalubres em grau mínimo ou médio. Ainda mais grave, no caso da lactação, que ela permanecesse a desempenhá-las inclusive em grau máximo de insalubridade. Ademais, criou o ônus à gestante ou à lactante da apresentação de atestado de saúde, emitido por médico de sua confiança, que certificasse a necessidade do afastamento. Essa mudança trouxe a exposição dessas trabalhadoras a atividades insalubres.

• A Corte assinalou que a Constituição Federal (CF) proclama, no caput do art. 6º, a proteção à maternidade como direito social, ligado à dignidade da pessoa humana. Essa proteção é a ratio para inúmeros outros direitos sociais instrumentais, como a licença-gestante, o direito à segurança no emprego, que compreende a tutela da relação de emprego contra dispensa arbitrária sem justa causa da gestante, e, nos termos do art. 7º, a proteção do mercado de trabalho da mulher, mediante incentivos específicos (inciso XX), e a redução dos riscos inerentes ao trabalho, por meio de normas de saúde, higiene e segurança (inciso XXII).

• Sob essa ótica, a proteção da mulher grávida ou lactante contra o trabalho insalubre caracteriza-se como importante direito social instrumental protetivo tanto da mulher quanto da criança. Trata-se de normas de salvaguarda dos direitos sociais da mulher e de efetivação de integral proteção ao recém-nascido, possibilitando sua convivência com a mãe, nos primeiros meses de vida, de maneira harmônica e segura, sem os perigos de um ambiente insalubre. A imprescindibilidade da máxima eficácia desse direito social também decorre da absoluta prioridade que o art. 227 do texto constitucional (2) estabelece à integral proteção à criança, inclusive ao nascituro e ao recém-nascido lactente.

• Há, na hipótese, direito de dupla titularidade. A proteção à maternidade e a integral proteção à criança são direitos irrenunciáveis e não podem ser afastados pelo desconhecimento, pela impossibilidade decorrente da distância de centros médicos ou pela própria negligência da gestante ou lactante em apresentar atestado médico, sob pena de prejudicá-la e de prejudicar o recém-nascido. Outras razões poderiam levar a mulher a não apresentar o documento, como, por exemplo, o medo de vir a ser demitida posteriormente ou a pressão para não entregar o atestado.

• Dessa forma, as expressões impugnadas não estão em consonância com os dispositivos constitucionais. A previsão do <u>afastamento automático</u> da mulher gestante ou lactante do ambiente insalubre está de acordo com a jurisprudência do Supremo Tribunal Federal (STF) em relação à integral proteção à maternidade e à saúde da criança.

• Na espécie, a mudança trazida pela lei pretendeu a inversão do ônus da demonstração probatória e documental da circunstância insalubre, a inversão da proteção à maternidade e ao nascituro ou recém-nascido. Partiu-se erroneamente da lógica de que, em regra, a insalubridade mínima e a média, durante a gestação, e mesmo a máxima, durante a lactação, não causam riscos. Isso desfavorece a plena proteção do interesse constitucionalmente protegido, na medida em que sujeita a empregada a maior embaraço para o exercício de seus direitos. O caso guarda relação com julgado recente em que apreciado o Tema 497 da repercussão geral (RE 629.053) sobre a estabilidade de empregada gestante.

• Naquele julgamento, o STF consignou que o conjunto dos direitos sociais foi consagrado constitucionalmente como uma das espécies de direitos fundamentais, caracterizando-se como verdadeiras liberdades positivas, de observância obrigatória em um Estado Social de Direito, visando à melhoria das condições de vida dos hipossuficientes e à concretização da igualdade social.

• O ministro Edson Fachin frisou que não se trata de reconhecer às mulheres qualquer benesse do ponto de vista constitucional. Por sua vez, o ministro Roberto Barroso acrescentou que a exigência viola o princípio da precaução, que vale também para o ambiente do trabalho, pelo qual, sempre que houver risco ou incerteza, deve ser favorecida a posição mais conservadora e protetiva.

• A ministra Rosa Weber expôs o histórico do direito e os principais instrumentos internacionais a respeito. Aduziu que a alteração implica inegável retrocesso social, uma vez que revoga anterior norma proibitória desse trabalho da gestante e lactante, além do menoscabo ao direito fundamental à saúde da mãe trabalhadora, pois transfere ao próprio sujeito tutelado a responsabilidade pela conveniência de atestado indicando a necessidade de afastamento do trabalho. Por seu turno, o ministro Luiz Fux também apontou a inconstitucionalidade por violação à igualdade de gênero, acompanhando o que destacado pelo ministro Alexandre de Moraes (relator) e pela ministra Rosa Weber.

• Já o ministro Celso de Mello reforçou os fundamentos trazidos e registrou que a cláusula que proíbe o retrocesso em matéria social traduz, no processo de sua concretização, verdadeira dimensão negativa pertinente aos direitos sociais, a impedir que os níveis de concretização dessas prerrogativas, uma vez atingidos, venham a ser reduzidos, degradados ou suprimidos.

• Vencido o ministro Marco Aurélio, que reputou improcedente o pleito formulado na ação. A seu ver, os preceitos encerram tão somente liberdade da mulher prestadora dos serviços, no que prevista a possibilidade de afastamento do ambiente insalubre, e visam atender às exigências do mercado de trabalho para não se criarem óbices à contratação da mão de obra feminina. O ministro afirmou não ser desarrazoada a imposição do atestado médico.

(1) CLT: “Art. 394-A. Sem prejuízo de sua remuneração, nesta incluído o valor do adicional de insalubridade, a empregada deverá ser afastada de: (...) II – atividades consideradas insalubres em grau médio ou mínimo, quando apresentar atestado de saúde, emitido por médico de confiança da mulher, que recomende o afastamento durante a gestação; III – atividades consideradas insalubres em qualquer grau, quando apresentar atestado de saúde, emitido por médico de confiança da mulher, que recomende o afastamento durante a lactação.” (2) CF/1988: “Art. 227. É dever da família, da sociedade e do Estado assegurar à criança, ao adolescente e ao jovem, com absoluta prioridade, o direito à vida, à saúde, à alimentação, à educação, ao lazer, à profissionalização, à cultura, à dignidade, ao respeito, à liberdade e à convivência familiar e comunitária, além de colocá-los a salvo de toda forma de negligência, discriminação, exploração, violência, crueldade e opressão.” (3) CF/1988: “Art. 1º A República Federativa do Brasil, formada pela união indissolúvel dos Estados e Municípios e do Distrito Federal, constitui-se em Estado Democrático de Direito e tem como fundamentos: (...) IV – os valores sociais do trabalho e da livre iniciativa;”

Legislação: CF, arts. 1º, IV e 227. CLT, art. 394-A, II e III.

Precedentes: RE 629.053

AQUI UNA OBSERVACION

El cuestionario de la ENM utiliza en todas sus preguntas el supuesto neutro masculino: “los extranjeros”, por lo que, de nueva cuenta, es imposible analizar la variación de las respuestas en función del género.

El lenguaje hace que pensemos en hombres, no en mujeres migrantes. Podría ser importante ajustar las preguntas de los cuestionarios para un análisis con perspectiva de genero.

Las preguntas omitidas en un cuestiona- rio son tan importantes como las preguntas que sí se hacen (Westmarland, 2001).

Esto es muy cierto lo que no se pregunta también importa, porque deja fuera parte de la realidad y no se logra un análisis realmente crítico.

De los muchos mitos que existen respecto a México y su sociedad, quizá dos de los más perversos son que el racismo no existe y que somos un país de puertas abiertas frente a la inmigración extranjera.

Estoy de acuerdo es un mito que no existe el racismo, aunque muchas personas creen esto o están escépticas a la idea , es importante verlo de manera mas critica ya que en México sí hay racismo y discriminación.

La falta de datos sobre las mujeres migrantes deja fuera sus necesidades e ignora que el género interactúa con variables como la edad, el origen y la condición social. Hay una ausencia de preguntas en las encuestas sobre las diferencias que podrían existir entre los dos géneros. Esto solo refleja la complejidad del tema y que los programas de protección no están especializados para atender adecuadamente a las mujeres y sus distintas situaciones de vulnerabilidad.

La ceguera de género nos habla de toda la falta de consideración e información sobre migración y discriminación dependiendo del sexo, lo que provoca la invisibilización de las experiencias femeninas en estos temas. Se asume al instante al migrante como masculino; esto limita la comprensión total del fenómeno, creando un problema donde no se incluyen las variables de género ni se reconocen como algo relevante.

El texto menciona que se asume que todos los migrantes son únicamente del sexo masculino, por lo que se refuerza la ignorancia hacia las experiencias femeninas. Esta escasa perspectiva impide que se reconozcan en las investigaciones particularidades de la migración femenina como los distintos riesgos y las estrategias de supervivencia específicas de cada mujer. En consecuencia, las mujeres migrantes no reciben la suficiente protección.

Mediante simples aditamento, o Presidente, por despacho, poderá estender os efeitos da suspensão a liminares supervenientes cujo objeto seja idêntico. Ou seja, em virtude da celeridade processual, é prescindível processos autônomos para a suspensão de liminares análogas, desde que haja aditamento da inicial.

Observe que, assim, a suspensão, em regra, não é para liminares futuras, para as quais necessita-se de <u>aditamento</u> à inicial.

La nada no sería nada, no puede ser un estado porque la nada no tiene atributos. Esa parte del argumento no tiene sentido, no merece ser abordada. No hay posibilidad de que la nada exista porque su existencia implicaría que contiene el atributo del ser.

Leibniz se da un tiro en el pie. Si Dios es necesario pero también es perfecto, el mundo como creación suya no pudo ser de otra manera y no pudo no existir porque Dios no pudo no haberlo creado ya que su decisión de crear el mundo es perfecta y no hay otra decisión posible derivada de su perfección, por lo tanto el mundo es necesario, no contingente ya que es una consecuencia necesaria de un ser necesario por lo tanto ambos existen necesariamente. Además, si no pudo haber momento ni instancia en la que el mundo no existiera porque dios no pudo haber permanecido en un estado de imperfección (ya que crear el mundo y coexistir con el es perfecto, entonces su contrario es imperfecto) el mundo tiene que existir desde siempre con Dios mismo por lo que no comenzó a existir.

Heidegger se apropia del concepto de "nada" para dar su explicación de la experiencia mental humana de imaginar la nada y sus consecuencias, derivando en un aprecio profundo por el ser. Parlotea sobre el concepto de nada deformandolo dificultando la comprensión de su idea. El lector de por sí siempre da su toque de deformación de la idea, pero elegir el parloteo por sobre la expresión explícita añade una capa inecesaria mas sobre la interpretación de la idea.

Pas sûr que réexpliquer la mécanique historique de l'expansion des pêcheries du Nord global soit une super intro pour un papier.

O locatário tem direito de preferência na compra do imóvel que locatário pretenda vender. Acaso haja preterição, o locatário ainda possui meios para obter a propriedade do bem.

Para tanto, os requisitos para a constituição de Direito Real a favor do locatário:

• Requerer, em até 6 (seis) meses, o imóvel para si, contado da data do registro em cartório de imóveis;
• Depositar o valor do imóvel e demais despesas de transferências;
• Averbação junto à matrícula do imóvel do contrato de locação, desde que averbado em, no mínimo, 30 (trinta) dias da data da alienação junto à matrícula;

Observe que a lei estabelece que a averbação do contrato de locação na matrícula do imóvel tem 2 importantes efeitos: - Assegurar que eventual novo locatário observe o prazo de locação, proibindo a denúncia do contrato sem antes decorrer o prazo contratual; - Assegurar a aquisição do imóvel acaso haja preterição do locador quanto ao direito de preferência do locatário.

Por fim, cabível destacar que a averbação, enquanto manifestação da publicidade dos atos relativos a direitos reais, é essencial para geração de efeito erga omnes. Com efeito, para garantir o direito real de aquisição, é imprescindível a averbação do contrato de locação.

Lado outro, tratando-se da outra hipótese referente ao prejuízo do direito de preferência do locatário, o pleito de perdas e danos não se submete a registro público como condição.

STF Súmula 442 A inscrição do contrato de locação no Registro de Imóveis, para a validade da cláusula de vigência contra o adquirente do imóvel, ou perante terceiros, dispensa a transcrição no Registro de Títulos e Documentos.

Observe que o direito de vigência da locação, na hipótese de alienação do imóvel a terceiros, tem 3 requisitos: - Existir no contrato de locação a cláusula de vigência; - Haver averbação do contrato de locação na matrícula do imóvel. - Locação por prazo determinado.

Com isso, inexistindo algum dos requisitos acima, não haverá direito à vigência.

Zeker — hier is dezelfde uitleg, maar dan eenvoudiger verwoord:

| Kenmerk | België: Internering | Nederland: TBS | | -------------- | ------------------------------------------------------------------------------------------------------------------- | ---------------------------------------------------------------------------------------------------------- | | Wat het is | Een maatregel, geen straf. | Ook een maatregel, maar onderdeel van het strafrecht. | | Voor wie | Alleen voor mensen met een psychische stoornis of mentale beperking die een strafbaar feit hebben gepleegd. | Voor mensen die een ernstig misdrijf hebben gepleegd en (gedeeltelijk) ontoerekeningsvatbaar zijn. | | Doel | 1. Maatschappij beschermen tegen gevaar.<br>2. Zorg en behandeling bieden aan de persoon. | 1. Maatschappij beschermen.<br>2. Behandeling en terugkeer in de samenleving mogelijk maken. | | Duur | Onbepaalde tijd – kan in theorie levenslang duren. | Bepaalde tijd, maar kan verlengd worden na evaluatie. | | Nadruk | Vooral op zorg en behandeling. | Op veiligheid én resocialisatie (terugkeer in de maatschappij). |

Kort gezegd:

• In België draait internering vooral om zorg voor mensen met een psychische stoornis die iets strafbaars hebben gedaan.
• In Nederland ligt bij TBS meer nadruk op veiligheid en geleidelijke terugkeer in de maatschappij.

Wil je dat ik dit ook netjes opmaak als tabel met randen (zodat je het direct in je samenvatting kunt plakken)?

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from

O jaká kritéria se, například, jedná?

Napadá mě jen "logoterapeut" a "filosofický poradce". Odhadl jsem správně, jaká povolání mělo na mysli autorstvo? Jaká povolání napadla nebo napadají vás?

Autorstvo zde, zdá se, předpokládá, že filosofování vede ke kladnému hodnocení integrity; k určité "křesťanské" (pro momentální neschopnost najít vhodnější označení) etice. Rád bych souhlasil a zároveň doufám, že směr etického snažení navržený tímto manifestem by měl smysl i v případě, že by filosofování mohlo vést i k méně obvyklým etickým závěrům. Jinými slovy, nepovažuji filosofování za samospásné. Podobně riskantní, mimochodem, mi připadá spoléhat se na evoluční výhody "křesťanské" etiky.

"et" à la place de "mais"

Le style du formulaire dans cette version est différente de celui dans P4C4-exercice.

DOI: 10.14814/phy2.70602

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What is this?

DOI: 10.12688/f1000research.169502.1

Resource: (Thermo Fisher Scientific Cat# 65-6120, RRID:AB_2533967)

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What is this?

DOI: 10.1016/j.cell.2025.09.029

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What is this?

C'est dit ! Il n'y a plus que la fédération et les pays n'ont plus rien à dire. Ni l'Italie, ni la France, ni l'Allemagne. L'ex de Goldman Sachs a parlé.

Cette Europe là doit mourir et le plus tôt sera le mieux. Vae victis.

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

Learn more at Review Commons

Reply to the reviewers

Manuscript number: RC-2025-03130

Corresponding author(s): Ellie S. Heckscher

[The "revision plan" should delineate the revisions that authors intend to carry out in response to the points raised by the referees. It also provides the authors with the opportunity to explain their view of the paper and of the referee reports.

• *

The document is important for the editors of affiliate journals when they make a first decision on the transferred manuscript. It will also be useful to readers of the reprint and help them to obtain a balanced view of the paper.

• *

If you wish to submit a full revision, please use our "Full Revision" template. It is important to use the appropriate template to clearly inform the editors of your intentions.]

1. General Statements [optional]

We thank all three reviewers for their feedback on the paper. Reviewers stated that the paper was of broad interest to developmental biologists and neurobiologists. However, we want to ensure that our two key conceptual contributions are clear. We clarify in the following paragraph and include a revised abstract. We will update the introduction and paper to better reflect these advances. We also attach a supplemental table 1, which was inadvertently omitted from the previous submission due to our error.

The first advance is that serially homologous neuroblasts follow a multimodal production model: In principle, stem cells can divide any number of times, from once to throughout the entire lifetime of the animal. And, on each division, a stem cell can generate either a proliferative daughter cell or a post-mitotic neuron. Together, therefore, there is a vast potential number of neurons any given stem cell could produce. From the literature on the vertebrate neocortex, we had the following models: (1) "random production" model, in which any number of neurons could be made by a stem cell; or (2) "unitary production" model, in which the same number of neurons (~eight) is produced by a stem cell regardless of context. Our data revealed an entirely new "multi-modal production" model, which could not have been predicted by prior literature. In the context of serially homologous neuroblasts arrayed along the Drosophila larval body axis, sets of five to seven neurons are produced in increments of one, two, or four. These increments correspond to units called temporal cohorts. Temporal cohorts are lineage fragments, or small set of neurons that share synaptic partners, making them lineage-based units of circuit assembly. Thus, in a multimodal production model, serially homologous stem cells produce different numbers of temporal cohorts depending on location. Our data advance the field by showing that stem cells produce circuit-relevant sets of neurons by adding or omitting temporal cohorts from a region, to meet regional needs.

Key to understanding the second advance is that there are multiple types of temporal cohorts: early-born Notch OFF, early-born Notch ON, late-born Notch OFF, and late-born Notch ON. One temporal cohort type, the early-born Notch OFF, is found in every segment, which we term the "ubiquitous" temporal cohort. The other temporal cohort types can be produced in various combinations depending on the stem cell division pattern and segmental location. In a result that could not have been predicted, we found that the ubiquitous temporal cohorts are refined both in terms of the number of neurons and their connectivity, depending on body region. In contrast, when other temporal cohort types are produced, they are not refined to the same degree.

The impact of this work is to advance how we think about stem cell-based circuit assembly.

2. Description of the planned revisions

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

*Summary: The study by Vasudevan et al intends to address how serially homologous neural progenitors generate different numbers and types of neurons depending on their location along the body axis. *

Investigation of full repertoire of neurogenesis for these progenitors necessitates a precise ability to track the fates of both progenitors and their neuronal progeny making it extremely difficult in vertebrate paradigm. The authors used NB3-3 in the developing fly embryo as a model to investigate the full extent of the flexibility in neurogenesis from a single type of serially homologous stem cell. Previous work showed NB3-3 generates neurons including lateral interneurons that can be positively labeled by Even-skipped, but detailed characterization of the NB3-3 lineage mainly focused on 3 segments during embryogenesis. The authors defined the number of EL neurons in all segments of the central nervous system in early larvae after the completion of circuit formation and carried out clonal analyses to determine the proliferation pattern of NB3-3. They described the failure to express Eve in Notch OFF/B neurons as a new mechanism for controlling the number of EL neurons and PCD limits EL neurons in terminal segments.

• *Thank you! In addition to the contributions highlighted by the reviewer, we also showed that all segments have ELs with early-born molecular identities, but only a subset have ELs with late-born identities (Figure 5). And we showed that early-born temporal cohorts can be mapped into different circuits depending on the axial region (Figure 6).

*Major comments: The authors performed careful analyses of the NB3-3 lineage using EL neurons. My main concerns are limited applicability of their findings and lack of mechanisms as how NB3-3 generate various numbers of EL neurons. Their findings are exclusively relevant to the NB3-3 lineage despite their effort in highlighting that other NB lineages also generate temporal cohorts of EL neurons. *

  Thank you for raising these points. First, to clarify, as Reviewer 4 also mentioned, NB3-3 is the only lineage to produce EL neurons. We will ensure that this is clearly stated in the revised text.

We agree that our findings might not apply beyond the NB3-3 lineage. However, as this is the first study of its kind, it is impossible to know a priori to what extent the concepts surfaced here are generalizable. In our opinion, this speaks to the novelty and impact of the study. A contribution is to motivate a need for future studies. We will make this explicit in our updated manuscript in the Discussion section.

  Our manuscript provides cell biological mechanisms that explain how stem cells give rise to different numbers of EL neurons in different regions, including stem cell division duration and type, neural cell death, identity gene expression, and differentiation state. If the reviewer is interested in genetic or molecular mechanisms, this is an interesting point. Several prior studies using NB3-3 as a model (e.g., Tsuji et al., 2008, Birkholz et al., 2013, Baumgardt et al., 2014) have elucidated the genetic regulation of specific cell biological processes. However, these studies provided fragmentary insight with regard to serially homologous stem cell development along the body axis. A comprehensive understanding of how the NB3-3 lineage, or any other serially homologous lineage, develops was missing. This is what makes our study both novel and needed. Without an analysis that both examines every segment and assays multiple cell biological processes, we would have missed key insights: that there is a ubiquitous type of temporal cohort, and that neurons within the ubiquitous temporal cohort are selectively refined post-mitotically (See General Statements for more details).

*I disagreed with their conclusion that failure to express Eve as a mechanism for controlling EL neuron numbers when Eve serves as the marker for these neurons. Are there any other strategy to assess the fates and functions of these cells beside relying solely on Eve expression? I am not familiar with the significance of Eve expression on the functions of these neurons. Is it possible to perform clonal analyses of NB3-3 mutant for Eve and see if these neurons adopt different functionalities/identities? *

• We agree that if Eve were only a marker, our logic would be circular. The Eve homolog, Evx1/2 is crucial for vertebrate interneuron cell fate (Moran-Rivard et al., 2001). Eve is essential for motor neuron morphology in Drosophila *(Fujioka et al., 2003). Eve is critical in Even-skipped for both the morphology and function of Even-skipped interneurons (Marshall et al., 2022). Hence, ELs cannot fully differentiate or incorporate into circuits without Eve. Thus, we use the failure to express Eve as a mechanism for controlling EL number. Furthermore, our prior study (Wang et al., 2022) showed that NB3-3 Notch OFF neurons in A1 that fail to express Eve have small soma and "stick-like" neurite projections that are typical of undifferentiated neurons. We will be sure to add this context to the revised manuscript.

*If NB3-3 in the SEZ continually generate GMCs based on the interpretation of clonal analyses and depicted in Fig. 2A, why is the percent of clones that are 1:0 virtually at or near 100% from division 6-11 shown in 2G? *

Admittedly, the ts-MARCM heat-shock-based lineage tracing experiments are inherently messy. This is part of the reason why we included the G-TRACE lineage tracing experiments in Figure 3. In Figure 3E, one can see that the number of Notch ON/A neurons in SEZ3 is equal to the number of ELs in that segment (Figure 1E). This is a second independent method that supports the assertion that in SEZ, NB3-3 stem cells continually generate GMCs. Given this independent observation, it leads us to believe that this question is most likely explained by technical issues inherent in ts-MARCM. These issues include but are not limited to: cell-type specific accessibility/success of heat-shock induced recombination; variably effective RNAi; and idiosyncrasies of the EL-GAL4 line used to detect recombination events. If the question is why the data is only reported for division 6-11, the answer is that the ts-MARCM dataset, which included SEZ clones only used later heat-shock time points (line from the paper "for the SEZ-containing dataset, inductions started at NB3-3's 5th division"). Along with this revision plan, we will include Supplemental Table 1, which was inadvertently omitted from the previous submission due to our error. This table shows all of the clonal data. We will include a section in the discussion to describe limitations in ts-MARCM.

The authors also indicate that NB3-3 in the abdomen directly generate Notch OFF/B cells that assume EL neuronal identity. In this scenario, shouldn't the percent of 1:0 clones be 100% in later divisions in Fig. 2G? Based on the number of clones in abdomen shown in Fig. 2E, I cannot seem to understand how the authors come to the percent of 1:0 clones shown in Fig. 2G

  We agree that one might expect the 12th division to be 100% 1:0 clones in the abdomen. Unfortunately, we didn't sample that late in our dataset, and even when we sampled the inferred 11th division, we had a small sample size (Figure 2E). Other studies suggest that NB3-3 in the abdomen directly generates Notch OFF/B neurons (Baumgardt et al., 2014), which served as our starting point. We will revise the text to make this clearer. As you can see from Figure 3E, there is only one NB3-3 Notch ON/ A neuron produced in each abdominal segment in comparison to the number of NB3-3 Notch OFF/B/EL neurons (Figure 1E). According to two independent assessments, Figure 3 and Baumgardt et al., 2014, the data support the conclusion that NB3-3 in the abdomen directly generates Notch OFF/B cells that assume EL identity for all but one of their divisions. Again, we believe technical issues make the ts-MARCM dataset messy. We will include a section in the discussion to describe limitations in ts-MARCM.

*There are many potentially interesting questions related to this study that can significantly broaden the impact of this study. For example, are other NB lineages that also generate distinct temporal cohorts of EL neurons display similar proliferation patterns (type 1 division in SEZ, early termination of cell division in thoracic segments and type 0 division in abdomen)? *

• *NB3-3 is the only lineage that makes ELs; Many lineages switch proliferation fates along the body axis. Previous studies have described how this switch in division patterns produces the wedge-shaped CNS: Cobeta et al., 2017. In the revision, we will be sure to clarify both points.

*Why does NB3-3 in the thoracic segment become quiescence so much sooner than SEZ and abdominal segments? *

• *NB3-3 in the thorax enters quiescence due to Hox genes and temporal transcription factors (Tsuji et al., 2008). In the revision, we will be sure to clarify this point.

The authors' observations suggest that NB3-3 in SEZ and abdomen generate a similar number of EL neurons despite the difference in their division patterns (type 1 vs type 0). Are the mechanisms that promote EL neuron generate in NB3-3 in SEZ and abdomen the same? Anything else is known beside Notch OFF?

• We agree this is an interesting point. Previous work has detailed NB3-3 division patterns, showing Type 1 divisions in the thorax, and Type 1 to Type 0 switch in the abdomen (Baumgardt et al., 2014). However, the proliferation pattern of NB3-3 in the SEZ had not been addressed until our study. Figures 2 and 3 suggest the following (1) SEZ proliferates for the duration of embryonic neurogenesis; (2) It produces a GMC on each division; (3) the GMC divides to produce one EL Notch OFF neuron and one Notch ON neuron. In our revision, we will manipulate the Notch pathway using two mutants, sanpodo, which produces two Notch OFF cells, and numb*, which produces two Notch ON cells (Skeath et al., 1998), to specifically test how ELs in the SEZ are regulated by Notch signaling. The other difference we know of between the SEZ, and abdomen is Hox gene expression. In Figure S2, we show that a subset of ELs in the SEZ express the anterior Hox genes, Sex combs reduced (Scr). The role of Hox genes in this lineage is an interesting question, as addressed in the discussion. This is an important future direction that merits in-depth study and is beyond the scope of what of this study is trying to accomplish.

Minor commentsThe authors' writing style is highly unusual especially in the result section. There is an overwhelming large amount of background information in the result section but very thin description on their observations. The background information portion also includes previously published observations. Since the nature of this study is not hypothesis-driven, it is very confusing to read in many places and difficult to distinguish their original observations from previously published results and making. One easily achievable improvement is to insert relevant figure numbers into the text more often.

Thank you for this comment. It is invaluable. In the revision, we will expand the background into a more comprehensive introduction and present the results more clearly. We will certainly insert relevant figure numbers. In responding to the reviewer's comments above, we can see where our writing lacked clarity and will improve these areas. Thank you again.

Reviewer #1 (Significance (Required)):

The study by Vasudevan et al intends to address how serially homologous neural progenitors generate different numbers and types of neurons depending on their location along the body axis. Investigation of full repertoire of neurogenesis for these progenitors necessitates a precise ability to track the fates of both progenitors and their neuronal progeny making it extremely difficult in vertebrate paradigm. The authors used NB3-3 in the developing fly embryo as a model to investigate the full extent of the flexibility in neurogenesis from a single type of serially homologous stem cell. Previous work showed NB3-3 generates neurons including lateral interneurons that can be positively labeled by Even-skipped, but detailed characterization of the NB3-3 lineage mainly focused on 3 segments during embryogenesis. The authors defined the number of EL neurons in all segments of the central nervous system in early larvae after the completion of circuit formation and carried out clonal analyses to determine the proliferation pattern of NB3-3. They described the failure to express Eve in Notch OFF/B neurons as a new mechanism for controlling the number of EL neurons and PCD limits EL neurons in terminal segments.

Because this text is the same as the summary, please see our response to that section.

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

In this manuscript, Vasudevan et al provide a detailed characterisation of the different numbers and temporal birthdates of Even-skipped Lateral (EL) neurons produced at in different segments from the same neuroblast, NB3-3. The work highlights the differences in EL neuronal generation across segments is achieved through a combination of different division patterns, failure to upregulate EL marker Eve and segment-specific program cell death. For neurons born within the same window and segment, the authors describe additional heterogeneity in their circuit formation. The work underscores the large diversity that the same neuroblast can generate across segments.

Thank you!

Major comments:

- Based on the ts-MARCM 1:0 clones representing 100% of the SEZ clones at any given inferred cell division, the authors conclude "NB3-3 neuroblasts generate proliferative daughter GMCs in the SEZ and thorax on most divisions". Figure 2G does not have any data for SEZ before inferred division 5, whereas there is data in other regions. The authors also state "In the SEZ and abdomen, ELs were labelled regardless of induction time." In reference to Fig 2F, which seems inaccurate given there are no SEZ clones before inferred division 5. There is no comment on this fact, which is surprising give their focus on temporal cohorts. The authors should explain this discrepancy, if known, or modify their statements to reflect the data.

• *Thank you for raising this point. The reason is because we produced two ts-MARCM datasets. One had SEZ clones, the other did not. The dataset with SEZ clones used heat shock protocols only for later time points, because those were most informative. The text from the paper is "We combined a published ts-MARCM (Wang et al., 2022) dataset with a new one (Table S1). The differences between the datasets are (1) CNSs were imaged either at low resolution for all regions (SEZ to terminus) or higher resolution for nerve cords (thorax to terminus); (2) for the SEZ-containing dataset, inductions started at NB3-3's 5th division. The combined data includes ~12 different heat shock protocols, 80 CNS, and 234 clones (Table S2)". In response to this comment, however, we will further clarify this point. In addition, we are submitting Supplemental table 1, which contains all the clonal data, as you can see experiments a-h lack SEZ data and experiments i-k contain SEZ data.

- The temporal cohort (early-born vs late-born) identity is exclusively examined based on markers. Given the absence of SEZ clones from early NB3-3 divisions, a time course showing that the SEZ generate early-born Els or some other complementary method would be desirable.

Thank you for raising this point. We show early-born versus late-born identity using markers in Figure 5. We conducted the time-course experiment as suggested and can confirm that there are early-born ELs in the SEZ at stage 13. We will include a new Supplemental Figure that includes a time course of EL number at stages 11, 13, 15, and 17 for segments SEZ3 to Te2 in the revision. See figure below.

- The authors repeatedly refer to their work as showing how a stem cell type can have "flexibility". Flexibility would imply that NB3-3 from one segment could adopt a different behaviour (different division pattern, or cell death or connectivity) if it were placed in a different segment. This is not what is being shown. In my opinion, "heterogeneity" of the same neuroblast across different segments would be more appropriate.

• *Thank you for this comment. We will change the wording to heterogeneity in the revision.

Minor comments:

- Figure 2A depicts a combination of known data and conclusions from their own (mainly SEZ). The authors might consider editing the figure to highlight what is new. A possibility would be for figure A to be a diagram of the experimental design and their summary division pattern to be shown after the new data instead of being panel A.

Thank you for this suggestion. We will make the suggested change.

- The authors state that they combined published ts-MARCM with their new one, which differed in a number ways that they list, but they don't specify which limitations are associated with the published vs new dataset. Could the authors please clarify?

  We now include Supplemental Table 1, which shows the complete combined datasets. In the first dataset, experiments a-h, the CNS was imaged at high resolution, but in a smaller region. The limitation is that the SEZ is missing. In the second dataset, i-k, inductions started at NB3-3's 5th division. The limitation is that we fail to sample early time points. This was a strategic decision. There were two possible scenarios: (1) in the SEZ, NB3-3 divided early, made GMCs, but both daughters expressed Eve. (2) in the SEZ, NB3-3 divided for the entirety of the embryonic neurogenesis, making GMCs, with only the Notch OFF daughters expressing Eve-our data support (2). Only late heat shocks were needed to distinguish between these possibilities. As these experiments are labor-intensive, we focused our efforts on the later time points. We will make this clearer in our revised text.

- The title refers exclusively to "temporal cohorts", which in the manuscript are defined quite narrowly and do not seem to apply to all segments.

• *Thank you! This, in our opinion, is a central, not a minor point to raise, because the impact of this study involves temporal cohort biology. We outlined the essential concepts in Part 1 "general statements" section of this revision plan. We did not mean to use "temporal cohort" in a limited sense, and we can see how the writing of our results section led to this comment. We will revise to make this clear.

- Several cited references are missing from the Reference list at the end. Could the authors please double check this? (e.g. Matsushita, 1997; Sweeney et al., 2018)

• *Thank you, we will remedy this!

- Legend for figure 2 is a bit confusing, there is a "(A)" within the legend for (D), which indicates that segments A1-A7 are shown (this seems inaccurate, as it only goes to A6).

Thank you, we will remedy this!

Reviewer #3 (Significance (Required)):

This study provides a comprehensive analysis of different cell biological scenarios for a neuroblast to generate distinct progeny across repeating axial units. The strength is the detailed and systematic approach across segments and possible scenarios: different division patterns, cell death, molecular marker expression. While it focuses on one specific neuroblast of the ventral nerve cord of Drosophila, the authors have done extensive work to place their findings and interpretation in the context of other cell types and across model organisms both in the introduction and discussion. This makes the work of interest for developmental biologists in general, neurodevelopment research in particular and those interested in circuit assembly, beyond their specialised community. This point of view comes from someone working in vertebrate CNS development.

Thank you!

Reviewer #4 (Evidence, reproducibility and clarity (Required)):

Summary

This manuscript addresses the question of how the number of neurons produced by each progenitor in the nervous system is determined. To address this question the authors use the Drosophila embryo model. They focus on a single type of neural stem cell (neuroblast), with homologues in each hemisegment along the anterior-posterior axis.

Using a combination of clonal labelling, antibody stainings, and blockade of programmed cell death, they provide a detailed description of segment-specific differences in the proliferation patterns of these neuroblasts, as well as in the fate and survival of their neuronal progeny.

Furthermore, by employing trans-synaptic labelling, they demonstrate that neurons derived from the same progenitor type receive distinct patterns of synaptic input depending on their segmental origin, in part due to their temporal window origin.

Overall this work shows that different mechanisms contribute to the final number and identity of the neuronal progeny arising from a single progenitor, even within homologous progenitors along the anterior posterior body axis.

Thank you!

Major Comments

I would suggest adding line numbers to the text for future submissions, this massively helps providing comments.

  Thank you for this comment. We will definitely add line numbers to the revised manuscript. We also thank you for providing comments despite this oversight on our part. We appreciate your time, and did not mean to make extra work.

*The authors propose that all neuroblasts produce the same type of temporal cohort (early born) and that, by changing the pattern of cell division, different temporal cohorts can be added. The way this this presented in the abstract sounds like an obvious thing, what would be the alternative scenario/s? *

  Thank you for raising the point that the abstract should be updated. We have included a revised abstract. The things that are obvious are: (1) changing a neuroblast's division pattern will change the number of neurons produced, and (2) if you have late-born neurons, the stem cell must at some point, have made early-born neurons. However, within those bounds is an extremely large parameter space. Each stem cell can choose to divide or not, and it can also choose to produce a proliferative daughter or not. The stem cell must navigate these choices at every division. The field had two models for what a stem cell might do - a "random production" model and a "unitary production" model. Our data support a third "multimodal production" model, which could not have been predicted based on prior literature or data.

We had raised these points in the discussion as follows-

"Under a null model, the durations and types of proliferation would vary stochastically across segments, resulting in a continuous and unstructured distribution of neuron numbers (Llorca et al., 2019). In a unitary production model, based on the vertebrate neocortex, there is a fixed neurogenic output of ~8-9 neurons per progenitor (Gao et al., 2014). However, our data support a third model, a multimodal production model. In a multimodal model, serially homologous neuroblasts generate different numbers of neurons depending on the segment."

We will now update the text to address this concern.

Here it's the late born neurons that lack in thoracic segments because of early NB quiescence, but it cannot be excluded that different neuroblast types adopt a different strategy.

• *True. Neural development is complex. Other lineages could easily employ alternative strategies. Our study presents a new conceptual framework that should inspire future research.

I found the ts-MARCM results confusing for 2 reasons:

1- It's not clear to me why there are so many single cell clones in div 3 and 4 in abdominal segments. This is not compatible with the division model depicted for abdominal segments, unless GMCs are produced in those division window and the MARCM hits the GMC, as also mentioned in the legend for G. This aspect is important because, either the previous model by Baumgardt et al. - please correct cit. currently Gunnar et al. 2026 - is wrong, or something strange happens in this experiment, or the relative temporal order is incorrect.

Thank you for raising this point. Having multiple single-cell (i.e., 1:0) clones in divisions 3 and 4 is not precisely what would be predicted by the model in Figure 2C. In part because heat-shock-based recombination methods in fly are stochastic and inherently "messy", we also conducted a second set of lineage tracing experiments, as shown in Figure 3, using G-TRACE. Figure 3E shows one Notch ON/A neuron in each abdominal segment, suggesting there is only one GMC present during lineage progression. But Figure 3E's result does not localize the GMC to any particular division. One possibility is that the GMC is generated once, but randomly throughout lineage progression. This possibility is consistent with the idea that the relative temporal order is incorrect and suggests that Baumgardt is erroneous. However, the Baumgardt data are strong, so we do not favor this idea. A second possibility, which we favor, is that something strange happened in this experiment. Here is how we envision the strange occurrence: heterogeneity in the EL driver. Ts-MARCM's recombination timing dictates the upper limit for the number of cells within a clone. However, recombination is detected by GAL4. So, if the GAL4 driver for some reason detects fewer cells than one expects, then one would see unusually small clones as is the case in question. To detect Ts-MARCM recombination in Figure 2, we used the EL-GAL4 driver. The EL-GAL4 driver is an enhancer fragment, ~400KB, meaning that it does not capture the full regulatory context of the eve locus. In our experience (e.g., Manning et al., 2012), drivers using small enhancers tend to give highly-specific, but somewhat variable expression, and this is the case for EL-GAL4 in our experience. We will update the discussion to discuss the ts-MARCM dataset and its limitations. And, we will correct the citation to Baumgardt et al., 2014, not Gunnar. Thank you!

2- In segments other than abdomen, it is quite rare to hit proper clones, it appears that only GMCs are hit by recombination, with very few exceptions. Could the author please provide an explanation for this or at least mention this aspect?

• *This is true. We cannot explain it. It could have something to do with the RNAi cassettes that are used in ts-MARCM, because in the original paper they mention that RNAi can be differently regulated in GMCs versus neuroblasts (Yu et al., 2009). We will mention it in the revised discussion about ts-MARCM limitations.

It is also unclear whether in F the graph includes all types of clones (including 1:0 clones). This is important, because the timing of division for NBs and GMCs is different, and inclusion of 1:0 might lead to a wrong estimate of the NB proliferation window (longer than it actually is because GMCs divide for longer). This is particularly important for the SEZ, where most clones in normalised division 10 and 11 are with ratio 1:0, thus compatible with both terminal division as well as GMC division.

• *The graph in F does include all types of clones. We provide Supplemental Table 1, which shows the full dataset. Unfortunately, we do not have enough data to analyze only NB clones. We agree that the estimate of the NB proliferation window is coarse using this analysis method and could overrepresent the division time by one cell division. We will mention this in the discussion and make sure that our results text is free from any overreaching claims about the precision of these measurements.

To obtain an estimate of the timing of division, the authors normalise clone size to the size of the bigger clone in the abdomen. What happened to those samples where no abdominal clones were hit? Were they simply excluded from the analysis?

  From the analysis in Figure 2, we excluded the clones that were SEZ, thorax, or terminus only. They were rare. They are shown in Supplemental Table 1, which will now be added in our revision plan.

It is proposed that in the thorax late temporal cohort neurons are not produced, yet the ts-MARCM experiment detects some 1:0 clones. What is the fate of these cells? Are they all derived from GMC division and therefore decoupled from the temporal identity window? Or is this a re-activation of division?

Figure 2F shows at the inferred 11th NB3-3 division, 100% of thoracic clones are of the 1:0 type. This is an n=1 observation (Supplemental Table 1, row f-Jan20-2). When we look at the morphology of this thoracic EL, we can see that it is a fully differentiated neuron that crosses the midline and ascends to the CNS, which is similar to EL morphologies in A1, so we don't think it's a whole new cell type. We have no way of determining whether this neuron was derived from a GMC division. It is also possible that this is an infrequent event or a technical anomaly. To address the question of reactivation of the thoracic NB3-3 division, we plan to include a Supplemental Figure of EL number over developmental time (stages 11, 13, 15, 17) for segments SEZ3 to Te2. This is the same data that we mentioned to Reviewer 3. This will reveal the extent to which the thorax produces late-born ELs.

*"in A1, a majority of segments had one Notch OFF/B neuron that failed to label with Eve" does "the majority" in this sentence mean that there were cases where all B neurons were labelled with Eve? If yes, where would this stochasticity come from? *

• • Yes, "the majority" in this sentence means that there were cases where all B neurons were labeled by Eve. In Figure 3F, for segment A1, that number is four. In contrast, there are 6 cases where B neurons failed to label with Eve. We can only speculate about the origin of the stochasticity. It could be biological (e.g., low level of Eve expression) or technical (e.g., poor antibody penetration). We plan to mention this in the discussion.

Additionally, there is no evidence that it's the first born NotchOFF neuron in A1 that does not express Eve. The authors should clarify where this speculation comes from.

• *The evidence that the first-born Notch OFF neuron in A1 does not express Eve comes from our ts-MARCM data: "So far, our ts-MARCM analyses grouped segments into regions (Figure 2A-C), however, EL number varies on a segment-by-segment basis (Figure 1). Therefore, we looked for segment-by-segment differences in ts-MARCM data (Table S1). The only detectable difference was between A1 and the other abdominal segments: When both A1 and another abdominal segment were labeled in a single CNS, a majority had smaller A1 clones. These data suggest that the production of ELs by NB3-3 neuroblasts lags in A1 compared to A2-A7." We will add a representation of these data to the ts-MARCM figure. As we stated above, we will add a Supplemental Figure of EL number over developmental time (stages 11, 13, 15, 17) for segments SEZ3 to Te2, which could strengthen this point.

When discussing trends shared with other phyla:

A- "In the mammalian spinal cord, more neurons are present in regions that control limbs (Francius et al., 2013). Analogously, EL numbers do not smoothly taper from anterior to posterior; instead, the largest number of ELs is found in two non-adjacent regions, SEZ and the abdomen." It's unclear what is the link between the figure in the mammalian spinal cord and the Drosophila embryo. The embryo doesn't even have limbs and the number of neurons measured here refer only to a single lineage, while there could be (and in fact there are) lineage-to-lineage differences that could depict a different scenario.

Thank you for this comment. We will rewrite this sentence, "in the mammalian spinal cord, more neurons are present in regions that control limbs (Francius et al., 2013)" to more accurately reflect the data in the Francius paper, and make the parallel more explicit. We will say "the size of columns of V3, V1, V2a, V2b, and V0v neurons differ at brachial compared to lumbar levels in the developing spinal cord." This removes the confusion about limbs and somewhat mitigates the concern about lineage-to-lineage differences, at least from the perspective of the spinal cord.

B- The parallelism between V1 mouse neurons and EL Drosophila neurons is also unclear to me. The similarity in fold change across segments could be a pure coincidence and, from what I understand, the two cell types are not functionally linked.

  Thank you for this comment. We believe this is the sentence in question (sorry about no line numbers). "(3) In the mouse spinal cord, ~10-fold differences in molecular subtypes for V1 neurons (Sweeney et al., 2018). In *Drosophila*, NB3-3 neuroblasts show differences in EL number, depending on region, with similar fold changes, suggesting this trait is shared across phyla."  The emphasis was intended to be on the fold-changes, not cell types. Coincidence or not, it is parallel. We will update the sentence to say "(3) In the mouse spinal cord, ~10-fold differences in molecular subtypes for V1 neurons (Sweeney et al., 2018). Although V1 neurons are not direct homologs of EL neurons, the number also varies ~10-fold depending on the region. One possibility is that this trait is shared across phyla." And, we will remove the final part of the paragraph, which distracts from the point "Thus, for this study and future research, NB3-3 development now offers a uniquely tractable, detailed, and comprehensive model for studying how stem cells flexibly produce neurons."

Minor comments:

I found the manuscript somewhat difficult to follow, even though I am familiar with both the model and the topic. For non-specialist readers, I expect it will be even more challenging. The presentation of the results often feels fragmented, at times resembling a sequence of brief statements rather than a continuous narrative. I would encourage the authors to provide more synthesis and interpretation, for example by summarising key findings, rather than listing in detail the number of neurons labelled in each segment for every experiment. This would make the results more accessible and easier to digest.

• *Thank you for this comment. We will provide more synthesis and interpretation in results by summarizing key findings.

From the way the MS is written it's not clear from the beginning that the work focuses exclusively on embryonic-born neurons. Since in Drosophila neuronal stem cells undergo two rounds of neurogenesis, one in the embryo and one in the larva, this omission could lead to confusion.

  Thank you for this comment. We will mention this in the abstract, introduction and discussion.

In the abstract, what would be the other temporal cohorts generated in specific regions? (ref to: "In specific regions, NB3-3 neuroblasts produce additional types of temporal cohorts, including but not limited to the late-born EL temporal cohort.")

  In this manuscript, we use lineage tracing to identify four types of temporal cohorts- early-born Notch ON, early-born Notch OFF, late-born Notch ON, and late-born Notch OFF. This is now reflected in the revised abstract. ELs are early-born Notch OFF and/or late-born Notch OFF.

This sentence in the introduction is inaccurate: "The Drosophila CNS is

organized into an anterior hindbrain-like subesophageal zone (SEZ) and a posterior spinal cord-like nerve cord". The anterior hindbrain-like portion of the CNS is in fact the supraesophageal ganglion (or cerebrum), while the SEZ is a posterior-like region.

  Thank you. We will change this sentence to: "The *Drosophila* CNS is

organized into a hindbrain-like subesophageal zone (SEZ) and a spinal cord-like nerve cord".

Fig 1E: the encoding of the significance is not immediately clear. In the legend the 4 stars could also be arranged in the same way for clarity.

• *Thank you. We will change it for clarity.

Fig 2E legend: it is mentioned that B corresponds to a 1:4 clone, however the MARCM example is shown for C and it's a 1:5.

Thank you. We will fix this.

The occurrence of "undifferentiated" neurons in Th segments is in less than 10% of the clones, I wonder if this a stochastic or deterministic event and to what extent small cell bodies could just be the consequence of local differences in tissue architecture.

• Because we are using a stochastic technique, it is difficult for us to determine whether the occurrence of neurons with small somas is a stochastic or deterministic event. Several papers suggest neurons with small axons are found across insect species (Pearson and Fourtner, 1975; Burrows, 1996). Neurons with a small soma and short axons/ axonless are found in the Drosophila embryonic abdominal nerve cord (Lacin et al., 2009). In our unpublished work from the Drosophila* nerve cord at a first instar larval stage, we found small somas with short axons in segment A1 (see Figure 4.6 below). This leads us to believe it is not a consequence of local tissue architecture.

Fig 2I: it's unclear what the purple means (I suppose it might be Eve expression) and why in J there should be one purple cell not labelled by the ts-MARCM when this is not present in H and I.

Purple is Eve. We will add labels for stains used in H and I, and remove the extra purple cell from the illustration in J.

"When synapses do occur, they are numerically similar from segment to segment". It's unclear where the evidence for this statement comes from, please clarify or remove the sentence.

We calibrated our trans-Tango data against available connectomic data using segment A1 as a reference. We learned that the trans-tango method only identifies strongly (>15 synapses) connected neurons.

"First, we calibrated trans-Tango for use in larval Drosophila, focusing on segment A1, where connectome data are available (Wang et al., 2022). In the connectome, of the five early-born ELs in A1, three are strongly connected to CHOs (>15 synapses), two are weakly connected (15 synapses) connected to somatosensory neurons."

  We will modify this sentence to say "when synapses do occur they are of similar strengths from segment to segment"

"In SEZ2, NB3-3 divides 10 times (Figure 2F)". Figure 2F does not support this statement and Figure 7 shows 12 divisions. Possibly SEZ2 and 3 have been inverted in this statement, please clarify.

Thank you for pointing this out. We will correct it!

**Referees cross-commenting**

I agree with most of the comments/suggestions provided by the other two reviewers.

In particular:

I agree with reviewer #1's comment about failure to express Eve being a mechanism for controlling neurons number, as this is a circular argument.

• *We address this earlier and direct you to that text. Briefly, Eve is not just a marker, but a key differentiation gene for ELs.

I agree with reviewer #2's concern about the use of the word "flexibility"; "heterogeneity" would be a more appropriate term, as I would associate the word "flexibility" to the ability of a single neuroblast in a single segment to produce neurons with different fates under, for example, unusual growth conditions. Here no genetic/epigenetic manipulations were performed to address flexibility and the observed (stereotypical) differences result from axial patterning.

• *We will change this, thank you.

*As a note, Reviewer #1 asks about other temporal cohorts of EL neurons produced by other lineages, but these neurons are specifically generated from NB3-3. *

• *Thank you for adding this clarification.

To generalise the observations reported in this study, the authors would need to focus on other molecularly defined temporal cohorts or, more generally, on other lineages, which, however, are likely to adopt different combinations of mecahnisms to tune progeny number across segments.

• *We agree that further studies are needed to assess the generalizability of our findings.

Reviewer #4 (Significance (Required)):

In Drosophila melanogaster, the relationship between neural progenitors and their neuronal progeny has been studied in great detail. This work has provided a comprehensive description of the number of progenitors present in each embryonic segment, their molecular identities, the number of neurons they produce, and the temporal transcriptional cascades that couple progenitor temporal identity to neuronal fate.

This work adds to the existing knowledge a detailed characterisation of intersegmental differences in the pattern of proliferation of a single type of neuronal progenitor as well as in post-divisional fate depending on anterior-posterior position in the body axis (i.e. programmed cell death and Notch signalling activation). This is a first step towards understanding the cellular and molecular mechanisms underlying such differences, but it's not disclosing them.

We have disclosed the cellular mechanisms- stem cell division duration and type, neural cell death, identity gene expression, and differentiation state -unless something else is envisaged by this comment. The molecular mechanisms are beyond the scope of this paper.

That homologous neuroblasts can generate variable numbers of progeny neurons depending on their segmental position has been established previously. What this manuscript adds is the demonstration that these differences arise through a combination of altered division patterns and differential programmed cell death, thereby revealing a more complex and less predictable scenario than could have been anticipated from existing knowledge in other contexts. The advance provided by this study is therefore incremental, refining rather than overturning our understanding of how segmental diversity in neuroblast lineages is achieved.

The key conceptual advances provided by this study are described in the General Statements section above. We don't overturn, but we advance the field.

By touching on the general question of how progenitors generate diversity, this work could be of broad interest to developmental neuroscientists beyond the fly field. However, the way it is currently written does not make it very accessible to non-specialists.

Thank you for this comment. We will endeavor to make it more accessible in the revised manuscript. Reviewer 3, an expert in vertebrate neurobiology, agreed that our work was of broad interest.

My expertise: Drosophila neurodevelopment, nerve cord, cell types specification

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

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

With this Revision Plan, we submit a revised abstract, and a supplemental table 1. We plan to address every point raised by the reviewers.

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

Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. This can be due to time or resource limitations or in case of disagreement about the necessity of such additional data given the scope of the study. Please leave empty if not applicable.

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Referee #2

Evidence, reproducibility and clarity

In this manuscript, Vasudevan et al provide a detailed characterisation of the different numbers and temporal birthdates of Even-skipped Lateral (EL) neurons produced at in different segments from the same neuroblast, NB3-3. The work highlights the differences in EL neuronal generation across segments is achieved through a combination of different division patterns, failure to upregulate EL marker Eve and segment-specific program cell death. For neurons born within the same window and segment, the authors describe additional heterogeneity in their circuit formation. The work underscores the large diversity that the same neuroblast can generate across segments.

Major comments:

• Based on the ts-MARCM 1:0 clones representing 100% of the SEZ clones at any given inferred cell division, the authors conclude "NB3-3 neuroblasts generate proliferative daughter GMCs in the SEZ and thorax on most divisions". Figure 2G does not have any data for SEZ before inferred division 5, whereas there is data in other regions. The authors also state "In the SEZ and abdomen, ELs were labelled regardless of induction time." In reference to Fig 2F, which seems inaccurate given there are no SEZ clones before inferred division 5. There is no comment on this fact, which is surprising give their focus on temporal cohorts. The authors should explain this discrepancy, if known, or modify their statements to reflect the data.
• The temporal cohort (early-born vs late-born) identity is exclusively examined based on markers. Given the absence of SEZ clones from early NB3-3 divisions, a time course showing that the SEZ generate early-born Els or some other complementary method would be desirable.
• The authors repeatedly refer to their work as showing how a stem cell type can have "flexibility". Flexibility would imply that NB3-3 from one segment could adopt a different behaviour (different division pattern, or cell death or connectivity) if it were placed in a different segment. This is not what is being shown. In my opinion, "heterogeneity" of the same neuroblast across different segments would be more appropriate.

Minor comments:

• Figure 2A depicts a combination of known data and conclusions from their own (mainly SEZ). The authors might consider editing the figure to highlight what is new. A possibility would be for figure A to be a diagram of the experimental design and their summary division pattern to be shown after the new data instead of being panel A.
• The authors state that they combined published ts-MARCM with their new one, which differed in a number ways that they list, but they don't specify which limitations are associated with the published vs new dataset. Could the authors please clarify?
• The title refers exclusively to "temporal cohorts", which in the manuscript are defined quite narrowly and do not seem to apply to all segments.
• Several cited references are missing from the Reference list at the end. Could the authors please double check this? (e.g. Matsushita, 1997; Sweeney et al., 2018)
• Legend for figure 2 is a bit confusing, there is a "(A)" within the legend for (D), which indicates that segments A1-A7 are shown (this seems inaccurate, as it only goes to A6).

Significance

This study provides a comprehensive analysis of different cell biological scenarios for a neuroblast to generate distinct progeny across repeating axial units. The strength is the detailed and systematic approach across segments and possible scenarios: different division patterns, cell death, molecular marker expression. While it focuses on one specific neuroblast of the ventral nerve cord of Drosophila, the authors have done extensive work to place their findings and interpretation in the context of other cell types and across model organisms both in the introduction and discussion. This makes the work of interest for developmental biologists in general, neurodevelopment research in particular and those interested in circuit assembly, beyond their specialised community. This point of view comes from someone working in vertebrate CNS development.

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Referee #1

Evidence, reproducibility and clarity

Summary: The study by Vasudevan et al intends to address how serially homologous neural progenitors generate different numbers and types of neurons depending on their location along the body axis. Investigation of full repertoire of neurogenesis for these progenitors necessitates a precise ability to track the fates of both progenitors and their neuronal progeny making it extremely difficult in vertebrate paradigm. The authors used NB3-3 in the developing fly embryo as a model to investigate the full extent of the flexibility in neurogenesis from a single type of serially homologous stem cell. Previous work showed NB3-3 generates neurons including lateral interneurons that can be positively labeled by Even-skipped, but detailed characterization of the NB3-3 lineage mainly focused on 3 segments during embryogenesis. The authors defined the number of EL neurons in all segments of the central nervous system in early larvae after the completion of circuit formation and carried out clonal analyses to determine the proliferation pattern of NB3-3. They described the failure to express Eve in Notch OFF/B neurons as a new mechanism for controlling the number of EL neurons and PCD limits EL neurons in terminal segments.

Major comments: The authors performed careful analyses of the NB3-3 lineage using EL neurons. My main concerns are limited applicability of their findings and lack of mechanisms as how NB3-3 generate various numbers of EL neurons. Their findings are exclusively relevant to the NB3-3 lineage despite their effort in highlighting that other NB lineages also generate temporal cohorts of EL neurons. I disagreed with their conclusion that failure to express Eve as a mechanism for controlling EL neuron numbers when Eve serves as the marker for these neurons. Are there any other strategy to assess the fates and functions of these cells beside relying solely on Eve expression? I am not familiar with the significance of Eve expression on the functions of these neurons. Is it possible to perform clonal analyses of NB3-3 mutant for Eve and see if these neurons adopt different functionalities/identities? If NB3-3 in the SEZ continually generate GMCs based on the interpretation of clonal analyses and depicted in Fig. 2A, why is the percent of clones that are 1:0 virtually at or near 100% from division 6-11 shown in 2G? The authors also indicate that NB3-3 in the abdomen directly generate Notch OFF/B cells that assume EL neuronal identity. In this scenario, shouldn't the percent of 1:0 clones be 100% in later divisions in Fig. 2G? Based on the number of clones in abdomen shown in Fig. 2E, I cannot seem to understand how the authors come to the percent of 1:0 clones shown in Fig. 2G

There are many potentially interesting questions related to this study that can significantly broaden the impact of this study. For example, are other NB lineages that also generate distinct temporal cohorts of EL neurons display similar proliferation patterns (type 1 division in SEZ, early termination of cell division in thoracic segments and type 0 division in abdomen)? Why does NB3-3 in the thoracic segment become quiescence so much sooner than SEZ and abdominal segments? The authors' observations suggest that NB3-3 in SEZ and abdomen generate a similar number of EL neurons despite the difference in their division patterns (type 1 vs type 0). Are the mechanisms that promote EL neuron generate in NB3-3 in SEZ and abdomen the same? Anything else is known beside Notch OFF?

Minor comments:

The authors' writing style is highly unusual especially in the result section. There is an overwhelming large amount of background information in the result section but very thin description on their observations. The background information portion also includes previously published observations. Since the nature of this study is not hypothesis-driven, it is very confusing to read in many places and difficult to distinguish their original observations from previously published results and making. One easily achievable improvement is to insert relevant figure numbers into the text more often.

Significance

The study by Vasudevan et al intends to address how serially homologous neural progenitors generate different numbers and types of neurons depending on their location along the body axis. Investigation of full repertoire of neurogenesis for these progenitors necessitates a precise ability to track the fates of both progenitors and their neuronal progeny making it extremely difficult in vertebrate paradigm. The authors used NB3-3 in the developing fly embryo as a model to investigate the full extent of the flexibility in neurogenesis from a single type of serially homologous stem cell. Previous work showed NB3-3 generates neurons including lateral interneurons that can be positively labeled by Even-skipped, but detailed characterization of the NB3-3 lineage mainly focused on 3 segments during embryogenesis. The authors defined the number of EL neurons in all segments of the central nervous system in early larvae after the completion of circuit formation and carried out clonal analyses to determine the proliferation pattern of NB3-3. They described the failure to express Eve in Notch OFF/B neurons as a new mechanism for controlling the number of EL neurons and PCD limits EL neurons in terminal segments.

Author response:

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

Reviewer #1 (Public review): 

Summary: 

The authors report the structure of the human CTF18-RFC complex bound to PCNA. Similar structures (and more) have been reported by the O'Donnell and Li labs. This study should add to our understanding of CTF18-RFC in DNA replication and clamp loaders in general. However, there are numerous major issues that I recommend the authors fix. 

Strengths: 

The structures reported are strong and useful for comparison with other clamp loader structures that have been reported lately. 

Weaknesses: 

The structures don't show how CTF18-RFC opens or loads PCNA. There are recent structures from other groups that do examine these steps in more detail, although this does not really dampen this reviewer's enthusiasm. It does mean that the authors should spend their time investigating aspects of CTF18-RFC function that were overlooked or not explored in detail in the competing papers. The paper poorly describes the interactions of CTF18-RFC with PCNA and the ATPase active sites, which are the main interest points. The nomenclature choices made by the authors make the manuscript very difficult to read. 

Reviewer #2 (Public review): 

Summary 

Briola and co-authors have performed a structural analysis of the human CTF18 clamp loader bound to PCNA. The authors purified the complexes and formed a complex in solution. They used cryo-EM to determine the structure to high resolution. The complex assumed an auto-inhibited conformation, where DNA binding is blocked, which is of regulatory importance and suggests that additional factors could be required to support PCNA loading on DNA. The authors carefully analysed the structure and compared it to RFC and related structures. 

Strength & Weakness 

Their overall analysis is of high quality, and they identified, among other things, a human-specific beta-hairpin in Ctf18 that flexibly tethers Ctf18 to Rfc2-5. Indeed, deletion of the beta-hairpin resulted in reduced complex stability and a reduction in a primer extension assay with Pol ε. This is potentially very interesting, although some more work is needed on the quantification. Moreover, the authors argue that the Ctf18 ATP-binding domain assumes a more flexible organisation, but their visual representation could be improved. 

The data are discussed accurately and relevantly, which provides an important framework for rationalising the results. 

All in all, this is a high-quality manuscript that identifies a key intermediate in CTF18dependent clamp loading. 

Reviewer #3 (Public review): 

Summary: 

CTF18-RFC is an alternative eukaryotic PCNA sliding clamp loader that is thought to specialize in loading PCNA on the leading strand. Eukaryotic clamp loaders (RFC complexes) have an interchangeable large subunit that is responsible for their specialized functions. The authors show that the CTF18 large subunit has several features responsible for its weaker PCNA loading activity and that the resulting weakened stability of the complex is compensated by a novel beta hairpin backside hook. The authors show this hook is required for the optimal stability and activity of the complex. 

Relevance: 

The structural findings are important for understanding RFC enzymology and novel ways that the widespread class of AAA ATPases can be adapted to specialized functions. A better understanding of CTF18-RFC function will also provide clarity into aspects of DNA replication, cohesion establishment, and the DNA damage response. 

Strengths: 

The cryo-EM structures are of high quality enabling accurate modelling of the complex and providing a strong basis for analyzing differences and similarities with other RFC complexes. 

Weaknesses: 

The manuscript would have benefitted from more detailed biochemical analysis to tease apart the differences with the canonical RFC complex. 

I'm not aware of using Mg depletion to trap active states of AAA ATPases. Perhaps the authors could provide a reference to successful examples of this and explain why they chose not to use the more standard practice in the field of using ATP analogues to increase the lifespan of reaction intermediates. 

Overall appraisal: 

Overall the work presented here is solid and important. The data is sufficient to support the stated conclusions and so I do not suggest any additional experiments. 

Reviewer #1 (Recommendations for the authors): 

We thank the reviewer for their positive comments and for their thorough review. All raised points have been addressed below.

Major points 

(1) The nomenclature used in the paper is very confusing and sometimes incorrect. The authors refer to CTF18 protein as "Ctf18", and the entire CTF18-RFC complex as "CTF18". This results in massive confusion because it is hard to ascertain whether the authors are discussing the individual subunits or the entire complex. Because these are human proteins, each protein name should be fully capitalized (i.e. CTF18, RFC4 etc). The full complex should be referred to more clearly with the designation CTF18-RFC or CTF18-RLC (RFC-like complex). Also, because the yeast and human clamp loader complexes use the same nomenclature for different subunits, it would be best for the authors to use the "A, B, C, D, E subunit" nomenclature that has been standard in the field for the past 20 years. Finally, the authors try to distinguish PCNA subunits by labeling them "PCNA2" or "PCNA1" (see Page 8 lines 180,181 for an example). This is confusing because the names of the RFC subunits have similar formats (RFC2, RFC3, RFC4, etc). In the case of RFC this denotes unique genes, whereas PCNA is a homotrimer. Could the authors think of another way to denote the different subunits, such as super/subscript? PCNA-I, PCNA-II, PCNA-III? 

We thank the reviewer for pointing out the confusing nomenclature. Following the referee suggestion, we now refer to the CTF18 full complex as “CTF18-RFC”. We prefer keeping the nomenclature used for CTFC18 subunits as RFC2, RFC3 etc., as recently used in Yuan et al, Science, 2024. However, we followed the referee’s suggestion for PCNA subunits, now referred to as PCNA-I, PCNA-II and PCNA-III.

(2) I believe that the authors are over-interpreting their data in Figure 1. The claim that "less sharp definition" of the map corresponding to the AAA+ domain of Ctf18 supports a relatively high mobility of this subunit is largely unsubstantiated. There are several reasons why one could get varying resolution in a cryo-EM reconstruction, such as compositional heterogeneity, preferred orientation artifacts, or how the complex interacts with the air-water interface. If other data were presented that showed this subunit is flexible, this evidence would support that data but cannot alone as justification for subunit mobility. Along these lines, how was the buried surface area (2300 vs 1400 A2) calculated? Is this the total surface area or only the buried surface area involving the AAA+ domains? It is surprising that these numbers are so different considering that the subunits and complexes look so similar (Figures 1c and 2b). 

We respectfully disagree with the suggestion that our interpretation of local flexibility in the AAA+ domain of Ctf18 is overreaching. Several lines of evidence support this interpretation. First, compositional heterogeneity is unlikely, as the A′ domain of Ctf18 is well-resolved and forms stable interactions with RFC3, indicating that Ctf18 is consistently incorporated into the complex. Second, preferred orientation artifacts are excluded, as the particle distribution shows excellent angular coverage (Fig. S9a). Third, we now include a 3D variability analysis (3DVA; Supplementary Video 1), which reveals local conformational heterogeneity centered around the AAA+ domain of Ctf18, consistent with intrinsic flexibility.

Regarding the buried surface area values, the reported numbers refer specifically to the interfaces between the AAA+ domain of Ctf18 and RFC2, and are derived from buried surface area calculations performed with PISA. The smaller interface (~1400 Ų) compared to RFC1–RFC2 (~2300 Ų) reflects low sequence identity (~26%) and divergent structural features, including the absence of conserved elements such as the canonical PIP-box in Ctf18. We have clarified and expanded this explanation in the revised manuscript (Page 7).

(3) The authors very briefly discuss interactions with PCNA and how the CTF18-RFC complex differs from the RFC complex. This is amongst the most interesting results from their work, but also not well-developed. Moreover, Figure 3D describing these interactions is extremely unclear. I feel like this observation had potential to be interesting, but is largely ignored by the authors. 

We thank the referee for pointing this out. We have expanded the section describing the interactions of CTF18-RFC and PCNA (Page 9 in the new manuscript), and made a new panel figure with further details (Fig. 3D).  

(4) The authors make the observation that key ATP-binding residues in RFC4 are displaced and incompatible with nucleotide binding in their CTF18-RFC structure compared to the hRFC structure. This should be a main-text figure showing these displacements and how it is incompatible with ATP binding. Again, this is likely an interesting finding that is largely glossed over by the authors. 

We now discuss this feature in detail (Pag 11 in the new manuscript), and added two figure insets (Fig. 4c) describing the incompatibility of RFC4 with nucleotide binding.

(5) The authors claim that the work of another group (citation 50) "validate(s) our predictions regarding the significant similarities between CTF18-RFC and canonical RFC in loading PCNA onto a ss/dsDNA junction." However, as far as this reviewer can tell the work in citation 50 was posted online before the first draft of this manuscript appeared on biorxiv, so it is dubious to claim that these were "predictions." 

We agree with the referee about this claim. We have now revised the text as follows:

“While our work was being finalized, several cryo-EM structures of human CTF18-RFC bound to PCNA and primer/template DNA were reported by another group (He et al, PNAS, 2024). These findings are consistent with the distinct features of CTF18-RFC observed in our structures and independently support the notion of significant mechanistic similarity between CTF18-RFC and canonical RFC in loading PCNA onto a ss/dsDNA junction”.

(6) The authors use a primer extension assay to test the effects of truncating the Nterminal beta hairpin of CTF18. However, this assay is only a proxy for loading efficiency and the observed effects of the mutation are rather subtle. The authors could test their hypothesis more clearly if they performed an ATPase assay or even better a clamp loading assay. 

We thank the referee for this valuable suggestion. In response, we have performed clamp loading assays comparing the activities of human RFC, wild-type CTF18-RFC, and the β-hairpin–truncated CTF18-RFC mutant. The results, now presented in Fig. 6 and Table 1 of the revised manuscript, clearly show that truncation of the N-terminal βhairpin results in a slower rate of PCNA loading. We propose that this reduced loading rate likely contributes to the diminished Pol ε–mediated DNA synthesis observed in the primer extension assays.

Minor points 

(1) Page 3 line 53 the introduction suggests that ATP hydrolysis prompts clamp closure. While this may be the case, to my knowledge all recent structural work shows that closure can occur without ATP hydrolysis. It may be better to rephrase it to highlight that under normal loading conditions, ATP hydrolysis occurs before clamp closure. 

The text now reads (Page 3): 

“DNA binding prompts the closure of the clamp and hydrolysis of ATP induces the concurrent disassembly of the closed clamp loader from the sliding clamp-DNA complex, completing the cycle necessary for the engagement of the replicative polymerases to start DNA synthesis.”

(2) Page 3 line 60, I do not see how the employment of alternative loaders highlights the specificity of the loading mechanism - would it not be possible for multiple loaders to have promiscuous clamp loading? 

We thank the referee for this comment. The text now reads (Page 3):

“However, eukaryotes also employ alternative loaders (20), including CTF18-RFC (6, 21-24), which likely use a conserved loading mechanism but are functionally specialized through specific protein interactions and context-dependent roles in DNA replication.”

(3) Page 4 line 75 could you please cite a study that shows Ctf8 and Dcc1 bind to the Ctf18 C-terminus and that a long linker is predicted to be flexible? 

Two references have been added (Stokes et al, NAR, 2020 and Grabarczyk et al, Structure, 2018)

(4) Figure 2A has the N-terminal region of Ctf18 as bound to RFC3 but should likely be labeled as bound to RFC5. This caused significant confusion while trying to parse this figure. Further, the inclusion of "X" as a sequence - does this refer to a sequence that was not buildable in the cryo-EM map? I would be surprised that density immediately after the conserved DEXX box motif is unbuildable. If this is the case, it should be clearly stated in the figure legend that "X" denotes an unbuildable sequence. For the conserved beta-hairpin in the sequence, could the authors superimpose the AlphaFold prediction onto their structure? It would be more informative than just looking at the sequence. 

We apologize for this confusion. The error in Figure 2A has been corrected. The figure caption now explicitely says that “X” refers to amino acid residues in the sequence which were not modelled. A superposition of the cryo-EM model of the N-terminal Beta hairpin in human Ctf18 and AlphaFold predictions for this feature in drosophila and yeast Ctf18 is now presented in Figure 2A.

(5) Page 8 line 168, the use of the term "RFC5" here feels improper, since the "C" subunit is not RFC5 in all lower eukaryotes (see comment above about nomenclature). For instance, in S cerevisiae, the C subunit is RFC3. I would expect this interaction to be maintained in all C subunits, not all RFC5 subunits. 

The text now reads (Page 8):

“Therefore, lower eukaryotes may use a similar b-hairpin motif to bind the corresponding subunit of the RFC-module complex (RFC5 in human, Rfc3 in S. cerevisiae), emphasizing its importance.”  

(6) Page 10 line 228, the authors claim that hydrolysis is dispensable at the Ctf18/RFC2 interface based on evidence from RFC1/RFC2 interface, by analogy that this is the "A/B" interface in both loaders. However, the wording makes it sound as if the cited data were collected while studying Ctf18 loaders. The authors should clarify this point. 

The text has been modified as follows (Pag 11): 

“Prior research has indicated that hydrolysis at the large subunit/RFC2 interface is not essential for clamp loading by various loaders (48-51), while the others are critical for the clamp-loading activity of eukaryotic RFCs. “

(7) Page 11 line 243/244 the authors introduce the separation pin. Could they clarify whether Ctf18 contains any aromatic residues in this structural motif that would suggest it serves the same functional purpose? Also, the authors highlight this is similar to yeast RFC, which makes it sound like this is not conserved in human RFC, but the structural motif is also conserved in human RFC. 

We thank the reviewer for this helpful comment. We have clarified in the revised text (Page 12) that the separation pin is conserved not only in yeast RFC but also in human RFC, and now note that human Ctf18 also harbors aromatic residues at the corresponding positions. This observation is supported by the new panel in Figure 4e.

Minutia 

(1) Page 2 line 37 please remove the word "and" before PCNA. 

This has been corrected.

(2) Please define AAA+ and update the language to clarify that not all pentameric AAA+ ATPases are clamp loaders. 

AAA+ has been now defined (Page 3).

(3) Page 4 line 86 Given the relatively weak interaction of Pol ε. 

This has been corrected.

(4) Page 8 line 204 the authors likely mean "leucine" and not "lysine". 

We thank the reviewer for catching this. The error has been corrected.

(5) Page 14 line 300, the authors claim that CTF18 utilizes three subunits but then list four. 

We have corrected this.

Reviewer #2 (Recommendations for the authors): 

We thank the reviewer for their positive comments and valuable suggestions. The points raised by the referee have been addressed below.

Major point: 

(1) Please quantify Figure 6 and S9 from 3 independent repeats and determine the standard deviation to show the variability of the Ctf18 beta hairpin deletion.  The authors suggest that a suboptimal Ctf18 complex interaction with PCNA impacts the stability of the complex, but do not test this hypothesis. Could the suboptimal PIP motif in Ctf18 be changed to an improved motif and the impact tested in the primer extension assay? Although not essential, it would be a nice way to explore the mechanism. 

We thank the reviewer for the suggestion. However, we note that Figure 6b (now 7b) already presents the quantification of the primer extension assay from three independent replicates, with error bars showing standard deviations, and includes the calculated rate of product accumulation. These data clearly indicate a 42% reduction in primer synthesis rate upon deletion of the Ctf18 β-hairpin.

We agree that we do not provide direct evidence of impaired complex stability upon deletion of the Ctf18 β-hairpin. However, the 2D classification of the cryo-EM dataset (Figure S9) shows a marked reduction in the number of particles corresponding to intact CTF18-RFC–PCNA complexes in the β-hairpin deletion sample, with the majority of particles corresponding to free PCNA. This contrasts with the wild-type dataset, where complex particles are predominant. These findings indirectly suggest that deletion of the β-hairpin compromises the stability or assembly of the clamp-loader–clamp complex.

We thank the reviewer for the valuable suggestion to mutate the weak PIP-box of Ctf18. While an interesting direction, we instead sought to directly test the mechanism by performing quantitative clamp loading assays. These assays revealed a significant reduction in the rate of PCNA loading by the CTF18^Δ165–194-RFCmutant (Figure 6), supporting the conclusion that the β-hairpin contributes to productive PCNA loading. This loading delay likely underlies the reduced rate of primer extension observed in the Pol ε assay (Figure 7), consistent with impaired formation of processive polymerase– clamp complexes.

(2) I did not see the method describing how the 2D classes were quantified to evaluate the impact of the Ctf18 beta hairpin deletion on complex formation. Please add the relevant information. 

The relevant information has been added to the Method section:

“For quantification of complex stability, the number of particles contributing to each 2D class was extracted from the classification metadata (Datasets 1 and 3). All classes showing isolated PCNA rings were summed and compared to the total number of particles in classes representing intact CTF18-RFC–PCNA complexes. This analysis was performed for both wild-type and β-hairpin deletion mutant datasets. Notably, no 2D classes corresponding to free PCNA were observed in the wild-type dataset, whereas in the mutant dataset, a substantial fraction of particles corresponded to isolated PCNA, suggesting reduced stability of the mutant complex.”

Minor point: 

(1) Page 2, line 25. Detail what type of mobility is referred to. Do you mean flexibility in the EM-map? 

We have clarified this. The text now reads:

“The unique RFC1 (Ctf18) large subunit of CTF18-RFC, which based on the cryo-EM map shows high relative flexibility, is anchored to PCNA through an atypical low-affinity PIP box”

(2) Page 4, line 82. Please introduce CMGE, or at least state what the abbreviation stands for. 

This has been addressed.

(3) Page 4, line 89. Specify that the architecture of the HUMAN CTF18-RFC module is not known, as the yeast one has been published. 

At the time our study was initiated, the architecture of the human CTF18-RFC module was unknown. A structure of the human complex was published by another group during the final stages of our work and is now properly acknowledged in the Discussion.

(4) Page 6. Is it possible to illustrate why the autoinhibited state cannot bind to DNA? A visual representation would be nice. 

We thank the reviewer for this suggestion. Figure 4b in the original manuscript already illustrates why the autoinhibited, overtwisted conformation of the CTF18-RFC pentamer cannot accommodate DNA. In this state, the inner chamber of the loader is sterically occluded, precluding the binding of duplex DNA.

Reviewer #3 (Recommendations for the authors): 

We thank Reviewer #3 for their constructive feedback and positive overall assessment of our work.

We also thank the reviewer for their remarks on the use of Mg depletion to halt hydrolysis. Magnesium is an essential cofactor for ATP hydrolysis, and its depletion is expected to effectively prevent catalysis by destabilizing the transition state, possibly more completely than the use of slowly hydrolysable analogues such as ATPγS. We have recently employed Mg^²+ depletion to successfully trap a pre-hydrolytic intermediate in a replicative AAA+ helicase engaged in DNA unwinding (Shahid et al., Nature, 2025). This precedent supports the rationale for our choice, and the reference has now been included in the revised manuscript.

I think the authors deposited the FSC curve for the +Mg structure in the -Mg structure PDB/EMDB entry according to the validation report. 

We thank the reviewer for their careful inspection of the deposition materials. The discrepancy in the deposited FSC curve has now been corrected, and the appropriate FSC curves have been assigned to the correct PDB/EMDB entries.

Beaucoup de texte

Beaucoup de texte

Tady bych doplnila i skutečnost, že pro mladé lidi představuje problém zejména skutečnost, že jako noví účastníci systému nemají přístup k historicky uzavíraným, výhodnějším nájemním smlouvám, včetně těch obecních.

Navrhuju včlenit tuto větu:

K významnému vývoji dochází v posledních dvou dekádách také v oblasti nájemního bydlení, kdy rozšiřující se sektor soukromého nájemního bydlení, financializace a spekulativní formy investování stěžují přístup mladým lidem i do nájemního bydlení, zejména z pohledu jeho finanční dostupnosti. Pro mladé lidi představuje problém zejména skutečnost, že jako noví účastníci systému nemají přístup k historicky uzavíraným, výhodnějším nájemním smlouvám, včetně těch obecních, a jsou tak ve větší míře odkázaní na nabídku soukromého nájemního bydlení. Soukromé nájemní bydlení s sebou navíc nese další omezení, jako je rostoucí nejistota a prekarita bydlení, diskriminace v přístupu a stále se prohlubující nerovnosti mezi mladými, které mají významné dopady na jejich schopnost usadit se.

doplnila bych "došlo"

na straně druhé došlo ke...

Může působit jako vlastnické bydlení.

Dá se nahradit:

Samostatné bydlení je součástí procesu dospívání. Osamostatnění a vytvoření nové domácnosti je tedy významným milníkem v životě každého jedince.

Případně "Založení vlastní domácnosti..." To by bylo sjednocené s dalšími částmi textu, kde píšeš "...žilo xy osob ve vlastní domácnosti"

navrhuju upravit na:

Finanční a sociální podpora rodiny je tak v tomto období značná a často je také jedním z faktorů, který ovlivňuje budoucí bytové možnosti mladé generace.

technickém? ve špatném fyzickém stavu jsem já :-D

Z porovnání mladých domácností v nájemním bydlení s domácnostmi v produktivním věku v nájemním bydlení je zřejmé, že zejména v krajských městech se odlišný vývojový trend těchto dvou skupin neustále prohlubuje; zatímco podíl domácností v produktivním věku, které jsou v nájemním bydlení, se mezi lety 2008 a 2024 snížil z 24 na 17 % (snížení o 7 p.b.), u mladých domácností se ve stejném období zvýšil z 43 na 59 % (zvýšení o 16 p.b.).

Tady bych to formulačně sjednotila:

Při pohledu na strukturu bydlení v krajských městech ve srovnání s ostatními obcemi Česka je patrné, že právě v krajských městech je výrazně vyšší podíl mladých domácností v nájemním bydlení než ve vlastnickém a tento rozdíl se od roku 2008 nadále zvyšuje; v roce 2024 žilo v krajských městech 30 % mladých domácností ve vlastnickém a 60 % v nájemním bydlení. Téměř opačná situace je v ostatních obcích, kde naopak dlouhodobě převládá vlastnické bydlení nad nájemním. Od roku 2018 se tento rozdíl ještě zvyšuje, a v roce 2024 tak v obcích mimo krajská města žilo 60 % mladých domácností ve vlastnickém oproti 24 % v nájemním bydlení.

Tady bych zvážila, jestli graf nezjednodušit a nedat pouze záložku na domácnosti. Viz předchozí komentář. Tiskla jsem a vytisklo se mi automaticky pro osoby a právě mě to mátlo s tím předchozím grafem, než jsem to pochopila.

Zároveň by mi tady přišlo jednodušší mluvit o domácnostech a ne o těch osobách, protože

a) to je formulačně trochu krkolomné

b) o osobách mluvíš v tom předchozím grafu/textu a čtenář musí být superpozorný, aby odlišil mladé osoby a osoby žijící v domácnostech mladých ;)

formulaci v předchozím komentu už jsem upravila na domácnosti - zvaž podle sebe

V odstavci výše to popisuješ hlavně v procentech - nebylo by přehlednější tady dát škálu v % a ne v abs? Naopak bych dala hned první graf kapitoly mladých, kde je struktura podle věku, v abs číslech - tam by se to podle mě skvěle hodilo, vč. toho, že uvidíme, že v abs číslech se ten počet mladých dramaticky snižuje.

Tady v té části už bychom věděli, že se počet ladých snižuje (viz ten první graf) a podívali bychom se na tu strukturu podle právního důvodu.

Jenom návrh :-)

Tato část tvrzení mi už přijde trochu moc. Buď bych to zúžila na ty, kterých se to týká, nebo to vypustila.

En este extracto busca un sinónimo de ¨soportable¨.

Encuentra en este extracto un sinónimo de amenazar, adversario, misión, enganchar.

Preguntas de Comprensión

¿Qué experiencia tiene Alex Rawlings con el aprendizaje de idiomas?

Según el texto, ¿por qué algunas personas se desaniman al aprender un nuevo idioma?

¿Cuál es uno de los principales consejos de Rawlings para aprender un nuevo idioma?

¿Qué método recomienda Rawlings para comenzar a aprender un nuevo idioma?

¿Qué beneficios adicionales menciona el texto sobre hablar más de un idioma?

Preguntas de Reflexión ¿Por qué crees que es importante no desanimarse al empezar a aprender un nuevo idioma, sin importar la edad? ¿Qué métodos de aprendizaje de idiomas te parecen más efectivos y por qué?

¿Qué quiere decir esta frase? ¿Te parece importante?

¿Cómo explicas esta cita de Rawlings en tus propias palabras?

Responde a las siguientes preguntas de vocabulario y comprensión y después contrasta tus respuestas con las de la clave que encontrarás más abajo.

1. Preguntas de vocabulario: Define las siguientes palabras según el contexto del texto: Gastronomía Platillo Cocción Mortero Marinada

2. Encuentra sinónimos en el texto para las siguientes palabras: Únicos Fama Tradicional

3. Preguntas de comprensión:

3.1 ¿Cuántos platillos de Latinoamérica están entre los más populares del mundo según TasteAtlas?

3.2 ¿Qué técnica de cocción se asocia con la barbacoa mexicana?

3.3 ¿Cuál es la base del mole mexicano?

3.4 Describe el método de preparación del churrasco brasileño.

3.5 ¿Qué ingredientes básicos se utilizan en el ceviche peruano?

3.6 Explica el origen del guacamole.

3.7 ¿Qué diferencia a las quesadillas de Ciudad de México respecto al resto del país?

3.8 ¿De qué están hechos los tamales y cómo se cocinan?

3.9 ¿En qué consisten los burritos y dónde surgieron?

3.10 ¿Qué ingredientes llevan los nachos tradicionales?

3.11 ¿Qué importancia tiene la tortilla en la gastronomía mexicana?

3.12 ¿Cómo se definen los tacos y cuál es su origen histórico según el texto?

Relaciona estas frases con los siguientes platos: quesadilla, tamal, burrito, tacos:

1. Este plato tiene su origen en las minas de plata del siglo dieciocho en México.
2. Este plato consiste en tortillas que se pueden doblar y rellenar de frijoles, guacamole y queso.
3. Este plato tradicionalmente se envuelve en hojas de maíz y se basa en una masa de maíz rellena.
4. Este plato consiste en tortillas de harina dobladas por la mutad con papas, frijoles o carne y queso fundido por dentro.

Encuentra en este extracto: un verbo de cambio y un verbo que significa ¨que tiene raices en¨

Encuentra un sinónimo de: trozos, destemplado, aderezar, adobar

Intenta responder a las siguientes preguntas de comprensión. Después de intentarlo, comprueba tus respuestas con las claves.

Encuentra sinónimos en el texto para las siguientes palabras: Impresionante Declarada Ruinas

Preguntas de comprensión:

¿Cuántos saltos tienen las Cataratas del Iguazú y cuál es su altura aproximada?

Describe la región de la Amazonia y menciona cuántos países abarca.

¿Cuál era la importancia de Chichén Itzá en la península de Yucatán?

¿Qué representan los moáis en la Isla de Pascua según la tradición?

¿Qué tipo de figuras componen las líneas de Nazca?

¿Cuántas islas forman el conjunto de las Islas Galápagos y qué tipo de especies albergan?

¿Dónde se encuentra el Salto Ángel y qué lo hace especial?

¿Qué simboliza el Cristo Redentor en Río de Janeiro y cuándo fue inaugurado?

Explica el significado de Machu Picchu y por qué es famoso.

Describe las ruinas de Teotihuacan y menciona algunas de sus estructuras más destacadas.

**Preguntas: **

1. Menciona alguna ventaja del teletrabajo para el trabajador y para la empresa explicándola con tus propias palabras.

2. Menciona alguna desventaja del teletrabajo para el trabajador y para la empresa con tus propias palabras.

3. ¿Cómo se pueden atenuar/suavizar muchas de las desventajas del teletrabajo? Menciona algunos ejemplos explicándolos en la medida de lo posible con tus propias palabras.

4. Según el texto, ¿cómo está impactando el teletrabajo en la economía de América Latina y el Caribe?. Describe algún ejemplo.

¿Cómo se pueden atenuar/suavizar muchas de las desventajas del teletrabajo? Menciona algunos ejemplos explicándolos en la medida de lo posible con tus propias palabras.

Según el texto, ¿cómo está impactando el teletrabajo en la economía de América Latina y el Caribe?. Describe algún ejemplo.

Menciona alguna desventaja del teletrabajo para el trabajador y para la empresa mencionada en el texto con tus propias palabras.

Menciona alguna ventaja del teletrabajo para el trabajador y para la empresa mencionada en el texto con tus propias palabras.

Preguntas de comprensión:

1. "Cambiar el rumbo, transformar la educación". ¿Cómo explicarías este lema en tus propias palabras?

2. ¿Verdadero o falso? Canadá es líder entre las potencias mundiales en destinar más dinero para la educación.

3. ¿Verdadero o falso? En Finlandia los padres intervienen con frecuencia en la toma de decisiones escolares.

4. ¿Verdadero o falso? En Hong Kong se pone énfasis en la memorización y en la creatividad.

¿Verdadero o falso? En Finlandia los padres intervienen con frecuencia en la toma de decisiones escolares.

¿Verdadero o falso? En Hong Kong se pone énfasis en la memorización y en la creatividad.

¿Cómo explicarías este lema en tus propias palabras?

¿Verdadero o falso? Canadá es líder entre las potencias mundiales en destinar más dinero para la educación.

Preguntas de comprensión:

1. ¿En qué situación la autora cogió accidentalmente la mano de una viejecilla en Salzburgo?

2. ¿Por qué el grupo de seis personas tuvo problemas al pagar la cuenta en el Hard Rock Pekín?

3. ¿Qué decisión tomó la autora durante la excursión en bicicleta por las Salinas de Maras y cómo afectó esto al resto de la expedición?

4. ¿Qué botón apretó la autora por error en el baño de la estación de tren de Osaka y cuál fue la consecuencia?

Busca en el texto una expresión idiomática que significa lo siguiente: Acabar con una situación de indiferencia, desconfianza o tensión con otra persona, iniciando la conversación con ella y procurando crear un ambiente agradable.

Presta atención a los conectores que dan coherencia a este texto argumentativo (si bien, además, en este caso). ¿Cuál es tu opinión sobre el tema? Escribe un párrafo sobre el tema usando adverbios y conectores para estructurarlo.

Presta atención a los conectores que dan coherencia a este texto argumentativo (si bien, sin embargo, en primer lugar, en segundo lugar, en cambio, en resumen). ¿Cuál es tu opinión sobre el tema? Escribe un párrafo sobre el tema usando adverbios y conectores para estructurarlo.

Presta atención a los conectores que dan coherencia a este texto argumentativo (aunque, ya que, si bien, por otro lado, además, dado que). ¿Cuál es tu opinión sobre el tema? Escribe un párrafo sobre el tema usando adverbios y conectores para estructurarlo.

Presta atención a los conectores que dan coherencia a este texto argumentativo (debido a, por otro lado, ya que). ¿Cuál es tu opinión sobre el tema? Escribe un párrafo sobre el tema usando adverbios y conectores para estructurarlo.

Aquí tienes diez ejemplos de breves textos argumentativos para practicar la expresión de la opinión crítica sobre cualquier tema haciendo uso de los marcadores discursivos que estamos aprendiendo. Fíjate en los ejemplos y, siguiendo los modelos, reacciona con tu opinión sobre los temas que más te interesen.

Presta atención a los conectores y adverbios que dan coherencia a este texto argumentativo (pero, de manera similar, además de, por lo general, por otro lado, en su mayoría, lamentablemente). ¿Cuál es tu opinión sobre el tema? Escribe un párrafo sobre el tema usando adverbios y conectores para estructurarlo.

Ještě bych možná data odkaz přímo na tu část Demografie - bydlení mladých nebo na daný graf

problém je, že jednočlenná domácnost se nerovná jeden byt, v jednom bytě může být více jednočlenných domácností.

Author response:

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

Reviewer 1:

The authors frequently refer to their predictions and theory as being causal, both in the manuscript and in their response to reviewers. However, causal inference requires careful experimental design, not just statistical prediction. For example, the claim that "algorithmic differences between those with BPD and matched healthy controls" are "causal" in my opinion is not warranted by the data, as the study does not employ experimental manipulations or interventions which might predictably affect parameter values. Even if model parameters can be seen as valid proxies to latent mechanisms, this does not automatically mean that such mechanisms cause the clinical distinction between BPD and CON, they could plausibly also refer to the effects of therapy or medication. I recommend that such causal language, also implicit to expressions like "parameter influences on explicit intentional attributions", is toned down throughout the manuscript.

Thankyou for this chance to be clearer in the language. Our models and paradigm introduce a from of temporal causality, given that latent parameter distributions are directly influenced by latent parameter estimates at a previous point in time (self-uncertainty and other uncertainty directly governs social contagion). Nevertheless, we appreciate the reviewers perspective and have now toned down the language to reflect this.

Abstract:

‘Our model makes clear predictions about the mechanisms of social information generalisation concerning both joint and individual reward.’

Discussion:

‘We can simulate this by modelling a framework that incorporates priors based on both self and a strong memory impression of a notional other (Figure S3).’

‘We note a strength of this work is the use of model comparison to understand algorithmic differences between those with BPD and matched healthy controls.’

Although the authors have now much clearer outlined the stuy's aims, there still is a lack of clarity with respect to the authors' specific hypotheses. I understand that their primary predictions about disruptions to self-other generalisation processes underlying BPD are embedded in the four main models that are tested, but it is still unclear what specific hypotheses the authors had about group differences with respect to the tested models. I recommend the authors specify this in the introduction rather than refering to prior work where the same hypotheses may have been mentioned.

Thankyou for this further critique which has enabled us to more cleary refine our introduction. We have now edited our introduction to be more direct about our hypotheses, that these hypotheses are instantiated into formal models, and what our predictions were. We have also included a small section on how previous predictions from other computational assessments of BPD link to our exploratory work, and highlighted this throughout the manuscript.

‘This paper seeks to address this gap by testing explicitly how disruptions in self-other generalization processes may underpin interpersonal disruptions observed in BPD. Specifically, our hypotheses were: (i) healthy controls will demonstrate evidence for both self-insertion and social contagion, integrating self and other information during interpersonal learning; and (ii) individuals with BPD will exhibit diminished self-other integration, reflected in stronger evidence for observations that assume distinct self-other representations.

We tested these hypotheses by designing a dynamic, sequential, three-phase Social Value Orientation (Murphy & Ackerman, 2014) paradigm—the Intentions Game—that would provide behavioural signatures assessing whether BPD differed from healthy controls in these generalization processes (Figure 1A). We coupled this paradigm with a lattice of models (M1-M4) that distinguish between self-insertion and social contagion (Figure 1B), and performed model comparison:

M1. Both self-to-other (self-insertion) and other-to-self (social contagion) occur before and after learning M2. Self-to-other transfer only occurs M3. Other-to-self transfer only occurs M4. Neither transfer process, suggesting distinct self-other representations

We additionally ran exploratory analysis of parameter differences and model predictions between groups following from prior work demonstrating changes in prosociality (Hula et al., 2018), social concern (Henco et al., 2020), belief stability (Story et al., 2024a), and belief updating (Story, 2024b) in BPD to understand whether discrepancies in self-other generalisation influences observational learning. By clearly articulating our hypotheses, we aim to clarify the theoretical contribution of our findings to existing literature on social learning, BPD, and computational psychiatry.’

Caveats should also be added about the exploratory nature of the many parameter group comparisons. If there are any predictions about group differences that can be made based on prior literature, the authors should make such links clear.

Thank you for this. We have now included caveats in the text to highlight the exploratory nature of these group comparisons, and added direct links to relevant literature where able:

Introduction

‘We additionally ran exploratory analysis of parameter differences and model predictions between groups following from prior work demonstrating changes in prosociality (Hula et al., 2018), social concern (Henco et al., 2020), belief stability (Story et al., 2024a), and belief updating (Story, 2024b) in BPD to understand whether discrepancies in self-other generalisation influences observational learning. By clearly articulating our hypotheses, we aim to clarify the theoretical contribution of our findings to existing literature on social learning, BPD, and computational psychiatry.’

Model Comparison

‘We found that CON participants were best fit at the group level by M1 (Frequency = 0.59, Exceedance Probability = 0.98), whereas BPD participants were best fit by M4 (Frequency = 0.54, Exceedance Probability = 0.86; Figure 2A). This suggests CON participants are best fit by a model that fully integrates self and other when learning, whereas those with BPD are best explained as holding disintegrated and separate representations of self and other that do not transfer information back and forth.

We first explore parameters between separate fits (see Methods). Later, in order to assuage concerns about drawing inferences from different models, we examined the relationships between the relevant parameters when we forced all participants to be fit to each of the models (in a hierarchical manner, separated by group). In sum, our model comparison is supported by convergence in parameter values when comparisons are meaningful (see Supplementary Materials). We refer to both types of analysis below.’

Phase 2 analysis

‘Prior work predicts those with BPD should focus more intently on public social information, rather than private information that only concerns one party (Henco et al., 2020). In BPD participants, only new beliefs about the relative reward preferences – mutual outcomes for both player - of partners differed (see Fig 2E): new median priors were larger than median preferences in phase 1 (mean = -0.47; = -6.10, 95%HDI: -7.60, -4.60).’

‘Models of moral preference learning (Story et al., 2024) predicts that BPD vs non-BPD participants have more rigid beliefs about their partners. We found that BPD participants were equally flexible around their prior beliefs about a partner’s relative reward preferences (= -1.60, 95%HDI: -3.42, 0.23), and were less flexible around their beliefs about a partner’s absolute reward preferences (=-4.09, 95%HDI: -5.37, -2.80), versus CON (Figure 2B).’

Phase 3 analysis

‘Prior work predicts that human economic preferences are shaped by observation (Panizza, et al., 2021; Suzuki et al. 2016; Yu et al, 2021), although little-to-no work has examined whether contagion differs for relative vs. absolute preferences. Associative models predict that social contagion may be exaggerated in BPD (Ereira et al., 2018).… As a whole, humans are more susceptible to changing relative preferences more than selfish, absolute reward preferences, and this is disrupted in BPD.’

Psychometric and Intentional Attribution analysis

‘Childhood trauma, persecution, and poor mentalising in BPD are all predicted to disrupt one’s ability to change (Fonagy & Luyten, 2009).’

‘Prior work has also predicted that partner-participant preference disparity influences mental state attributions (Barnby et al., 2022; Panizza et al., 2021).’

I'm not sure I understand why the authors, after adding multiple comparison correction, now list two kinds of p-values. To me, this is misleading and precludes the point of multiple comparison corrections, I therefore recommend they report the FDR-adjusted p-values only. Likewise, if a corrected p-value is greater than 0.05 this should not be interpreted as a result.

We have now adjusted the exploratory results to include only the FDR corrected values in the text.

‘We assessed conditional psychometric associations with social contagion under the assumption of M3 for all participants. We conducted partial correlation analyses to estimate relationships conditional on all other associations and retained all that survived bootstrapping (5000 reps), permutation testing (5000 reps), and subsequent FDR correction. When not controlled for group status, RGPTSB and CTQ scores were both moderately associated with MZQ scores (RGPTSB r = 0.41, 95%CI: 0.23, 0.60, p[fdr]=0.043; CTQ r = 0.354 95%CI: 0.13, 0.56, p[fdr]=0.02). This was not affected by group correction. CTQ scores were moderately and negatively associated with shifts in individualistic reward preferences (; r = -0.25, 95%CI: -0.46, -0.04, p[fdr]=0.03). This was not affected by group correction. MZQ scores were in turn moderately and negatively associated with shifts in prosocial-competitive preferences () between phase 1 and 3 (r = -0.26, 95%CI: -0.46, -0.06, p[fdr]=0.03). This was diminished when controlled for group status (r = 0.13, 95%CI: -0.34, 0.08, p[fdr]=0.20). Together this provides some evidence that self-reported trauma and self-reported mentalising influence social contagion (Fig S11). Social contagion under M3 was highly correlated with contagion under M1 demonstrating parsimony of outcomes across models (Fig S12).

Prior work has predicted that partner-participant preference disparity influences mental state attributions (Barnby et al., 2022; Panizza et al., 2021). We tested parameter influences on explicit intentional attributions in Phase 2 while controlling for group status. Attributions included the degree to which they believed their partner was motived by harmful intent (HI) and self-interest (SI). According with prior work (Barnby et al., 2022), greater disparity of absolute preferences before learning was associated on a trend level with reduced attributions of SI (<= -0.23, p[fdr]=0.08), and greater disparity of relative preferences before learning exaggerated attributions of HI = 0.21, p[fdr]=0.08), but did not survive correction (Figure S4B). This is likely due to partners being significantly less individualistic and prosocial on average compared to participants (= -5.50, 95%HDI: -7.60, -3.60; = 12, 95%HDI: 9.70, 14.00); partners are recognised as less selfish and more competitive.’

Can the authors please elaborate why the algorithm proposed to be employed by BPD is more 'entropic', especially given both their self-priors and posteriors about partners' preferences tended to be more precise than the ones used by CON? As far as I understand, there's nothing in the data to suggest BPD predictions should be more uncertain. In fact, this leads me to wonder, similarly to what another reviewer has already suggested, whether BPD participants generate self-referential priors over others in the same way CON participants do, they are just less favourable (i.e., in relation to oneself, but always less prosocial) - I think there is currently no model that would incorporate this possibility? It should at least be possible to explore this by checking if there is any statistical relationship between the estimated θ_ppt^m and 〖p(θ〗_par |D^0).

Thank you for this opportunity to be clearer in our wording. We belief the reviewer is referring to this line in the discussion: ‘In either case, the algorithm underlying the computational goal for BPD participants is far higher in entropy and emphasises a less stable or reliable process of inference.’

We note in the revised Figure 2 panel E and in the results that those with BPD under M4 show insertion along absolute reward (they still expect diminished selfishness in others), but neutral priors over relative reward (around 0, suggesting expectations of neither prosocial or competitive tendencies of others). Thus, θ_ppt^m (self preference) and θ_par^m (other preference) are tightly associated for absolute, but not relative reward.

In our wording, we meant that whether under model M4 or M1, those with BPD either show a neutral prior over relative reward (M4) or a prior with large variance over relative reward (M1), showing expectations of difference between themselves and their partner. In both cases, expectation about a partner’s absolute reward preferences is diminished vs. CON participants. We have strengthened our language in the discussion to clarify this:

‘In either case, the algorithm underlying the computational goal for BPD participants is far higher in uncertainty, whether through a neutral central tendency (M4) or large variance (M1) prior over relative reward in phase 2, and emphasises a less certain and reliable expectation about others.’

To note, social contagion under M3 was highly correlated with contagion under M1 (see Fig S11). This provides some preliminary evidence that trauma impacts beliefs about individualism directly, whereas trauma and persecutory beliefs impact beliefs about prosociality through impaired trait mentalising" - I don't understand what the authors mean by this, can they please elaborate and add some explanation to the main text?

We have now clarified this in the text:

‘Together this provides some evidence that self-reported trauma and self-reported mentalising influence social contagion (Fig S11). Social contagion under M3 was highly correlated with contagion under M1 demonstrating parsimony of outcomes across models (Fig S12).’

I noted that at least some of the newly added references have not been added to the bibliography (e.g., Hitchcock et al. 2022).

Thankyou for noticing this omission. We have now ensured all cited works are in the reference list.

Reviewer 2:

The paper is not based on specific empirical hypotheses formulated at the outset, but, rather, it uses an exploratory approach. Indeed, the task is not chosen in order to tackle specific empirical hypotheses. This, in my view, is a limitation since the introduction reads a bit vague and it is not always clear which gaps in the literature the paper aims to fill. As a further consequence, it is not always clear how the findings speak to previous theories on the topic.’

As I wrote in the public review, however, I believe that an important limitation of this work is that it was not based on testing specific empirical hypotheses formulated at the outset, and on selecting the experimental paradigm accordingly. This is a limitation because it is not always clear which gaps in the literature the paper aims to fill. As a consequence, although it has improved substantially compared to the previous version, the introduction remains a bit vague. As a further consequence, it is not always clear how the findings speak to previous theories on the topic. Still, despite this limitation, the paper has many strengths, and I believe it is now ready for publication

Thank you for this further critique. We appreciate your appraisal that the work has improved substantially and is ready for publication. We nevertheless have opted to clarify our introduction and aprior predictions throughout the manuscript (please see response to Reviewer 1).

Reviewer 3:

Although the authors note that their approach makes "clear and transparent a priori predictions," the paper could be improved by providing a clear and consolidated statement of these predictions so that the results could be interpreted vis-a-vis any a priori hypotheses.

In line with comments from both Reviewer 1 and 2, we have clarified our introduction to make it clear what our aprior predictions and hypotheses are about our core aims and exploratory analyses (see response to Reviewer 1).

The approach of using a partial correlation network with bootstrapping (and permutation) was interesting, but the logic of the analysis was not clearly stated. In particular, there are large group (Table 1: CON vs. BPD) differences in the measures introduced into this network. As a result, it is hard to understand whether any partial correlations are driven primarily by mean differences in severity (correlations tend to be inflated in extreme groups designs due to the absence of observation in middle of scales forming each bivariate distribution). I would have found these exploratory analyses more revealing if group membership was controlled for.

Thank you for this chance to be clearer in our methods. We have now written a more direct exposition of this exploratory method:

‘Exploratory Network Analysis

To understand the individual differences of trait attributes (MZQ, RGPTSB, CTQ) with other-to-self information transfer () across the entire sample we performed a network analysis (Borsboom, 2021). Network analysis allows for conditional associations between variables to be estimated; each association is controlled for by all other associations in the network. It also allows for visual inspection of the conditional relationships to get an intuition for how variables are interrelated as a whole (see Fig S11). We implemented network analysis with the bootNet package in r using the ‘estimateNetwork’ function with partial correlations (Epskamp, Borsboom & Fried, 2018). To assess the stability of the partial correlations we further implemented bootstrap resampling with 5000 repetitions using the ‘bootnet’ function. We then additionally shuffled the data and refitted the network 5000 times to determine a p_permuted value; this indicates the probability that a conditional relationship in the original network was within the null distribution of each conditional relationship. We then performed False Discovery Rate correction on the resulting p-values. We additionally controlled for group status for all variables in a supplementary analysis (Table S4).’

We have also further corrected for group status and reported these results as a supplementary table, and also within the main text alongside the main results. We have opted to relegate Figure 4 into a supplementary figure to make the text clearer.

‘We explored conditional psychometric associations with social contagion under the assumption of M3 for all participants (where everyone is able to be influenced by their partner). We conducted partial correlation analyses to estimate relationships conditional on all other associations and retained all that survived bootstrapping (5000 reps), permutation testing (5000 reps), and subsequent FDR correction. When not controlled for group status, RGPTSB and CTQ scores were both moderately associated with MZQ scores (RGPTSB r = 0.41, 95%CI: 0.23, 0.60, p[fdr]=0.043; CTQ r = 0.354 95%CI: 0.13, 0.56, p[fdr]=0.02). This was not affected by group correction. CTQ scores were moderately and negatively associated with shifts in individualistic reward preferences (; r = -0.25, 95%CI: -0.46, -0.04, p[fdr]=0.03). This was not affected by group correction. MZQ scores were in turn moderately and negatively associated with shifts in prosocial-competitive preferences () between phase 1 and 3 (r = -0.26, 95%CI: -0.46, -0.06, p[fdr]=0.03). This was diminished when controlled for group status (r = 0.13, 95%CI: -0.34, 0.08, p[fdr]=0.20). Together this provides some evidence that self-reported trauma and self-reported mentalising influence social contagion (Fig S11). Social contagion under M3 was highly correlated with contagion under M1 demonstrating parsimony of outcomes across models (Fig S12).’

Discussion first para: "effected -> affected"

Thanks for spotting this. We have now changed it.

Add "s" to "participant: "Notably, despite differing strategies, those with BPD achieved similar accuracy to CON participant."

We have now changed this.

Author response:

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

Reviewer #1 (Public review):

Summary:

Measurement of BOLD MR imaging has regularly found regions of the brain that show reliable suppression of BOLD responses during specific experimental testing conditions. These observations are to some degree unexplained, in comparison with more usual association between activation of the BOLD response and excitatory activation of the neurons (most tightly linked to synaptic activity) in the same brain location. This paper finds two patients whose brains were tested with both non-invasive functional MRI and with invasive insertion of electrodes, which allowed the direct recording of neuronal activity. The electrode insertions were made within the fusiform gyrus, which is known to process information about faces, in a clinical search for the sites of intractable epilepsy in each patient. The simple observation is that the electrode location in one patient showed activation of the BOLD response and activation of neuronal firing in response to face stimuli. This is the classical association. The other patient showed an informative and different pattern of responses. In this person, the electrode location showed a suppression of the BOLD response to face stimuli and, most interestingly, an associated suppression of neuronal activity at the electrode site.

Strengths:

Whilst these results are not by themselves definitive, they add an important piece of evidence to a long-standing discussion about the origins of the BOLD response. The observation of decreased neuronal activation associated with negative BOLD is interesting because, at various times, exactly the opposite association has been predicted. It has been previously argued that if synaptic mechanisms of neuronal inhibition are responsible for the suppression of neuronal firing, then it would be reasonable

Weaknesses:

The chief weakness of the paper is that the results may be unique in a slightly awkward way. The observation of positive BOLD and neuronal activation is made at one brain site in one patient, while the complementary observation of negative BOLD and neuronal suppression actually derives from the other patient. Showing both effects in both patients would make a much stronger paper.

We thank reviewer #1 for their positive evaluation of our paper. Obviously, we agree with the reviewer that the paper would be much stronger if BOTH effects – spike increase and decrease – would be found in BOTH patients in their corresponding fMRI regions (lateral and medial fusiform gyrus) (also in the same hemisphere). Nevertheless, we clearly acknowledge this limitation in the (revised) version of the manuscript (p.8: Material and Methods section).

Note that with respect to the fMRI data, our results are not surprising, as we indicate in the manuscript: BOLD increases to faces (relative to nonface objects) are typically found in the LatFG and BOLD decreases in the medialFG (in the revised version, we have added the reference to an early neuroimaging paper that describes this dissociation clearly:

Pelphrey, K. A., Mack, P. B., Song, A., Güzeldere, G., & McCarthy, G. Faces evoke spatially differentiated patterns of BOLD activation and deactivation. Neuroreport 14, 955–959 (2003).

This pattern of increase/decrease in fMRI can be appreciated in both patients on Figure 2, although one has to consider both the transverse and coronal slices to appreciate it.

Regarding electrophysiological data, in the current paper, one could think that P1 shows only increases to faces, and P2 would show only decreases (irrespective of the region). However, that is not the case since 11% of P1’s face-selective units are decreases (89% are increases) and 4% of P2’s face-selective units are increases. This has now been made clearer in the revised manuscript (p.5).

As the reviewer is certainly aware, the number and positions of the electrodes are based on strict clinical criteria, and we will probably never encounter a situation with two neighboring (macro-micro hybrid electrodes), one with microelectrodes ending up in the lateral MidFG, the other in the medial MidFG, in the same patient. If there is no clinical value for the patient, this cannot be done.

The only thing we can do is to strengthen these results in the future by collecting data on additional patients with an electrode either in the lateral or the medial FG, together with fMRI. But these are the only two patients we have been able to record so far with electrodes falling unambiguously in such contrasted regions and with large (and comparable) measures.

While we acknowledge that the results may be unique because of the use of 2 contrasted patients only (and this is why the paper is a short report), the data is compelling in these 2 cases, and we are confident that it will be replicated in larger cohorts in the future.

Finally, information regarding ethics approval has been provided in the paper.

Reviewer #2 (Public review):

Summary:

This is a short and straightforward paper describing BOLD fMRI and depth electrode measurements from two regions of the fusiform gyrus that show either higher or lower BOLD responses to faces vs. objects (which I will call face-positive and facenegative regions). In these regions, which were studied separately in two patients undergoing epilepsy surgery, spiking activity increased for faces relative to objects in the face-positive region and decreased for faces relative to objects in the face-negative region. Interestingly, about 30% of neurons in the face-negative region did not respond to objects and decreased their responses below baseline in response to faces (absolute suppression).

Strengths:

These patient data are valuable, with many recording sessions and neurons from human face-selective regions, and the methods used for comparing face and object responses in both fMRI and electrode recordings were robust and well-established. The finding of absolute suppression could clarify the nature of face selectivity in human fusiform gyrus since previous fMRI studies of the face-negative region could not distinguish whether face < object responses came from absolute suppression, or just relatively lower but still positive responses to faces vs. objects.

Weaknesses:

The authors claim that the results tell us about both 1) face-selectivity in the fusiform gyrus, and 2) the physiological basis of the BOLD signal. However, I would like to see more of the data that supports the first claim, and I am not sure the second claim is supported.

(1) The authors report that ~30% of neurons showed absolute suppression, but those data are not shown separately from the neurons that only show relative reductions. It is difficult to evaluate the absolute suppression claim from the short assertion in the text alone (lines 105-106), although this is a critical claim in the paper.

We thank reviewer #2 for their positive evaluation of our paper. We understand the reviewer’s point, and we partly agree. Where we respectfully disagree is that the finding of absolute suppression is critical for the claim of the paper: finding an identical contrast between the two regions in terms of RELATIVE increase/decrease of face-selective activity in fMRI and spiking activity is already novel and informative. Where we agree with the reviewer is that the absolute suppression could be more documented: it wasn’t, due to space constraints (brief report). We provide below an example of a neuron showing absolute suppression to faces (P2), as also requested in the recommendations to authors. In the frequency domain, there is only a face-selective response (1.2 Hz and harmonics) but no significant response at 6 Hz (common general visual response). In the time-domain, relative to face onset, the response drops below baseline level. It means that this neuron has baseline (non-periodic) spontaneous spiking activity that is actively suppressed when a face appears.

Author response image 1.

(2) I am not sure how much light the results shed on the physiological basis of the BOLD signal. The authors write that the results reveal "that BOLD decreases can be due to relative, but also absolute, spike suppression in the human brain" (line 120). But I think to make this claim, you would need a region that exclusively had neurons showing absolute suppression, not a region with a mix of neurons, some showing absolute suppression and some showing relative suppression, as here. The responses of both groups of neurons contribute to the measured BOLD signal, so it seems impossible to tell from these data how absolute suppression per se drives the BOLD response.

It is a fact that we find both kinds of responses in the same region. We cannot tell with this technique if neurons showing relative vs. absolute suppression of responses are spatially segregated for instance (e.g., forming two separate sub-regions) or are intermingled. And we cannot tell from our data how absolute suppression per se drives the BOLD response. In our view, this does not diminish the interest and originality of the study, but the statement "that BOLD decreases can be due to relative, but also absolute, spike suppression in the human brain” has been rephrased in the revised manuscript: "that BOLD decreases can be due to relative, or absolute (or a combination of both), spike suppression in the human brain”.

Reviewer #3 (Public review):

In this paper the authors conduct two experiments an fMRI experiment and intracranial recordings of neurons in two patients P1 and P2. In both experiments, they employ a SSVEP paradigm in which they show images at a fast rate (e.g. 6Hz) and then they show face images at a slower rate (e.g. 1.2Hz), where the rest of the images are a variety of object images. In the first patient, they record from neurons over a region in the mid fusiform gyrus that is face-selective and in the second patient, they record neurons from a region more medially that is not face selective (it responds more strongly to objects than faces). Results find similar selectivity between the electrophysiology data and the fMRI data in that the location which shows higher fMRI to faces also finds face-selective neurons and the location which finds preference to non faces also shows non face preferring neurons.

Strengths:

The data is important in that it shows that there is a relationship between category selectivity measured from electrophysiology data and category-selective from fMRI. The data is unique as it contains a lot of single and multiunit recordings (245 units) from the human fusiform gyrus - which the authors point out - is a humanoid specific gyrus.

Weaknesses:

My major concerns are two-fold:

(i) There is a paucity of data; Thus, more information (results and methods) is warranted; and in particular there is no comparison between the fMRI data and the SEEG data.

We thank reviewer #3 for their positive evaluation of our paper. If the reviewer means paucity of data presentation, we agree and we provide more presentation below, although the methods and results information appear as complete to us. The comparison between fMRI and SEEG is there, but can only be indirect (i.e., collected at different times and not related on a trial-by-trial basis for instance). In addition, our manuscript aims at providing a short empirical contribution to further our understanding of the relationship between neural responses and BOLD signal, not to provide a model of neurovascular coupling.

(ii) One main claim of the paper is that there is evidence for suppressed responses to faces in the non-face selective region. That is, the reduction in activation to faces in the non-face selective region is interpreted as a suppression in the neural response and consequently the reduction in fMRI signal is interpreted as suppression. However, the SSVEP paradigm has no baseline (it alternates between faces and objects) and therefore it cannot distinguish between lower firing rate to faces vs suppression of response to faces.

We understand the concern of the reviewer, but we respectfully disagree that our paradigm cannot distinguish between lower firing rate to faces vs. suppression of response to faces. Indeed, since the stimuli are presented periodically (6 Hz), we can objectively distinguish stimulus-related activity from spontaneous neuronal firing. The baseline corresponds to spikes that are non-periodic, i.e., unrelated to the (common face and object) stimulation. For a subset of neurons, even this non-periodic baseline activity is suppressed, above and beyond the suppression of the 6 Hz response illustrated on Figure 2. We mention it in the manuscript, but we agree that we do not present illustrations of such decrease in the time-domain for SU, which we did not consider as being necessary initially (please see below for such presentation).

(1) Additional data: the paper has 2 figures: figure 1 which shows the experimental design and figure 2 which presents data, the latter shows one example neuron raster plot from each patient and group average neural data from each patient. In this reader's opinion this is insufficient data to support the conclusions of the paper. The paper will be more impactful if the researchers would report the data more comprehensively.

We answer to more specific requests for additional evidence below, but the reviewer should be aware that this is a short report, which reaches the word limit. In our view, the group average neural data should be sufficient to support the conclusions, and the example neurons are there for illustration. And while we cannot provide the raster plots for a large number of neurons, the anonymized data is made available at:

(a) There is no direct comparison between the fMRI data and the SEEG data, except for a comparison of the location of the electrodes relative to the statistical parametric map generated from a contrast (Fig 2a,d). It will be helpful to build a model linking between the neural responses to the voxel response in the same location - i.e., estimate from the electrophysiology data the fMRI data (e.g., Logothetis & Wandell, 2004).

As mentioned above the comparison between fMRI and SEEG is indirect (i.e., collected at different times and not related on a trial-by-trial basis for instance) and would not allow to make such a model.

(b) More comprehensive analyses of the SSVEP neural data: It will be helpful to show the results of the frequency analyses of the SSVEP data for all neurons to show that there are significant visual responses and significant face responses. It will be also useful to compare and quantify the magnitude of the face responses compared to the visual responses.

The data has been analyzed comprehensively, but we would not be able to show all neurons with such significant visual responses and face-selective responses.

(c) The neuron shown in E shows cyclical responses tied to the onset of the stimuli, is this the visual response?

Correct, it’s the visual response at 6 Hz.

If so, why is there an increase in the firing rate of the neuron before the face stimulus is shown in time 0?

Because the stimulation is continuous. What is displayed at 0 is the onset of the face stimulus, with each face stimulus being preceded by 4 images of nonface objects.

The neuron's data seems different than the average response across neurons; This raises a concern about interpreting the average response across neurons in panel F which seems different than the single neuron responses

The reviewer is correct, and we apologize for the confusion. This is because the average data on panel F has been notch-filtered for the 6 Hz (and harmonic responses), as indicated in the methods (p.11): ‘a FFT notch filter (filter width = 0.05 Hz) was then applied on the 70 s single or multi-units time-series to remove the general visual response at 6 Hz and two additional harmonics (i.e., 12 and 18 Hz)’.

Here is the same data without the notch-filter (the 6Hz periodic response is clearly visible):

Author response image 2.

For sake of clarity, we prefer presenting the notch-filtered data in the paper, but the revised version makes it clear in the figure caption that the average data has been notch-filtered.

(d) Related to (c) it would be useful to show raster plots of all neurons and quantify if the neural responses within a region are homogeneous or heterogeneous. This would add data relating the single neuron response to the population responses measured from fMRI. See also Nir 2009.

We agree with the reviewer that this is interesting, but again we do not think that it is necessary for the point made in the present paper. Responses in these regions appear rather heterogenous, and we are currently working on a longer paper with additional SEEG data (other patients tested for shorter sessions) to define and quantify the face-selective neurons in the MidFusiform gyrus with this approach (without relating it to the fMRI contrast as reported here).

(e) When reporting group average data (e.g., Fig 2C,F) it is necessary to show standard deviation of the response across neurons.

We agree with the reviewer and have modified Figure 2 accordingly in the revised manuscript.

(f) Is it possible to estimate the latency of the neural responses to face and object images from the phase data? If so, this will add important information on the timing of neural responses in the human fusiform gyrus to face and object images.

The fast periodic paradigm to measure neural face-selectivity has been used in tens of studies since its original reports:

• in EEG: Rossion et al., 2015: https://doi.org/10.1167/15.1.18

• in SEEG: Jonas et al., 2016: https://doi.org/10.1073/pnas.1522033113

In this paradigm, the face-selective response spreads to several harmonics (1.2 Hz, 2.4 Hz, 3.6 Hz, etc.) (which are summed for quantifying the total face-selective amplitude). This is illustrated below by the averaged single units’ SNR spectra across all recording sessions for both participants.

Author response image 3.

There is no unique phase-value, each harmonic being associated with a phase-value, so that the timing cannot be unambiguously extracted from phase values. Instead, the onset latency is computed directly from the time-domain responses, which is more straightforward and reliable than using the phase. Note that the present paper is not about the specific time-courses of the different types of neurons, which would require a more comprehensive report, but which is not necessary to support the point made in the present paper about the SEEG-fMRI sign relationship.

(g) Related to (e) In total the authors recorded data from 245 units (some single units and some multiunits) and they found that both in the face and nonface selective most of the recoded neurons exhibited face -selectivity, which this reader found confusing: They write “ Among all visually responsive neurons, we found a very high proportion of face-selective neurons (p < 0.05) in both activated and deactivated MidFG regions (P1: 98.1%; N = 51/52; P2: 86.6%; N = 110/127)’. Is the face selectivity in P1 an increase in response to faces and P2 a reduction in response to faces or in both it’s an increase in response to faces

Face-selectivity is defined as a DIFFERENTIAL response to faces compared to objects, not necessarily a larger response to faces. So yes, face-selectivity in P1 is an increase in response to faces and P2 a reduction in response to faces.

Additional methods

(a) it is unclear if the SSVEP analyses of neural responses were done on the spikes or the raw electrical signal. If the former, how is the SSVEP frequency analysis done on discrete data like action potentials?

The FFT is applied directly on spike trains using Matlab’s discrete Fourier Transform function. This function is suitable to be applied to spike trains in the same way as to any sampled digital signal (here, the microwires signal was sampled at 30 kHz, see Methods).

In complementary analyses, we also attempted to apply the FFT on spike trains that had been temporally smoothed by convolving them with a 20ms square window (Le Cam et al., 2023, cited in the paper ). This did not change the outcome of the frequency analyses in the frequency range we are interested in. We have also added one sentence with information in the methods section about spike detection (p.10).

(b) it is unclear why the onset time was shifted by 33ms; one can measure the phase of the response relative to the cycle onset and use that to estimate the delay between the onset of a stimulus and the onset of the response. Adding phase information will be useful.

The onset time was shifted by 33ms because the stimuli are presented with a sinewave contrast modulation (i.e., at 0ms, the stimulus has 0% contrast). 100% contrast is reached at half a stimulation cycle, which is 83.33ms here, but a response is likely triggered before reaching 100% contrast. To estimate the delay between the start of the sinewave (0% contrast) and the triggering of a neural response, we tested 7 SEEG participants with the same images presented in FPVS sequences either as a sinewave contrast (black line) modulation or as a squarewave (i.e. abrupt) contrast modulation (red line). The 33ms value is based on these LFP data obtained in response to such sinewave stimulation and squarewave stimulation of the same paradigm. This delay corresponds to 4 screen refresh frames (120 Hz refresh rate = 8.33ms by frame) and 35% of the full contrast, as illustrated below (please see also Retter, T. L., & Rossion, B. (2016). Uncovering the neural magnitude and spatio-temporal dynamics of natural image categorization in a fast visual stream. Neuropsychologia, 91, 9–28).

Author response image 4.

(2) Interpretation of suppression:

The SSVEP paradigm alternates between 2 conditions: faces and objects and has no baseline; In other words, responses to faces are measured relative to the baseline response to objects so that any region that contains neurons that have a lower firing rate to faces than objects is bound to show a lower response in the SSVEP signal. Therefore, because the experiment does not have a true baseline (e.g. blank screen, with no visual stimulation) this experimental design cannot distinguish between lower firing rate to faces vs suppression of response to faces.

The strongest evidence put forward for suppression is the response of non-visual neurons that was also reduced when patients looked at faces, but since these are non-visual neurons, it is unclear how to interpret the responses to faces.

We understand this point, but how does the reviewer know that these are non-visual neurons? Because these neurons are located in the visual cortex, they are likely to be visual neurons that are not responsive to non-face objects. In any case, as the reviewer writes, we think it’s strong evidence for suppression.

We thank all three reviewers for their positive evaluation of our paper and their constructive comments.

Author response:

The following is the authors’ response to the original reviews

Reviewer #1 (Public review):

Summary:

Zhang et al. addressed the question of whether advantageous and disadvantageous inequality aversion can be vicariously learned and generalized. Using an adapted version of the ultimatum game (UG), in three phases, participants first gave their own preference (baseline phase), then interacted with a "teacher" to learn their preference (learning phase), and finally were tested again on their own (transfer phase). The key measure is whether participants exhibited similar choice preferences (i.e., rejection rate and fairness rating) influenced by the learning phase, by contrasting their transfer phase and baseline phase. Through a series of statistical modeling and computational modeling, the authors reported that both advantageous and disadvantageous inequality aversion can indeed be learned (Study 1), and even be generalised (Study 2).

Strengths:

This study is very interesting, it directly adapted the lab's previous work on the observational learning effect on disadvantageous inequality aversion, to test both advantageous and disadvantageous inequality aversion in the current study. Social transmission of action, emotion, and attitude have started to be looked at recently, hence this research is timely. The use of computational modeling is mostly appropriate and motivated. Study 2, which examined the vicarious inequality aversion in conditions where feedback was never provided, is interesting and important to strengthen the reported effects. Both studies have proper justifications to determine the sample size.

Weaknesses:

Despite the strengths, a few conceptual aspects and analytical decisions have to be explained, justified, or clarified.

INTRODUCTION/CONCEPTUALIZATION

(1) Two terms seem to be interchangeable, which should not, in this work: vicarious/observational learning vs preference learning. For vicarious learning, individuals observe others' actions (and optionally also the corresponding consequence resulting directly from their own actions), whereas, for preference learning, individuals predict, or act on behalf of, the others' actions, and then receive feedback if that prediction is correct or not. For the current work, it seems that the experiment is more about preference learning and prediction, and less so about vicarious learning. The intro and set are heavily around vicarious learning, and later the use of vicarious learning and preference learning is rather mixed in the text. I think either tone down the focus on vicarious learning, or discuss how they are different. Some of the references here may be helpful: (Charpentier et al., Neuron, 2020; Olsson et al., Nature Reviews Neuroscience, 2020; Zhang & Glascher, Science Advances, 2020)

We are appreciative of the Reviewer for raising this question and providing the reference. In response to this comment we have elected to avoid, in most cases, use of the term ‘vicarious’ and instead focus the paper on learning of others’ preferences (without specific commitment to various/observational learning per se). These changes are reflected throughout all sections of the revised manuscript, and in the revised title. We believe this simplified terminology has improved the clarity of our contribution.

EXPERIMENTAL DESIGN

(2) For each offer type, the experiment "added a uniformly distributed noise in the range of (-10 ,10)". I wonder what this looks like? With only integers such as 25:75, or even with decimal points? More importantly, is it possible to have either 70:30 or 90:10 option, after adding the noise, to have generated an 80:20 split shown to the participants? If so, for the analyses later, when participants saw the 80:20 split, which condition did this trial belong to? 70:30 or 90:10? And is such noise added only to the learning phase, or also to the baseline/transfer phases? This requires some clarification.

We thank the Reviewer for pointing this out. The uniformly distributed noise was added to all three phases to make the proposers’ behavior more realistic. This added noise was rounded to integer numbers, constrained from -9 to 9, which means in both 70:30 and 90:10 offer types, an 80:20 split could not occur. We have made this feature of our design clear in the Method section Line 524 ~ 528:

“In all task phases, we added uniformly distributed noise to each trial’s offer (ranging from -9 to 9, inclusive, rounding to the nearest integer) such that the random amount added (or subtracted) from the Proposer’s share was subtracted (or added) to the Receiver’s share. We adopted this manipulation to make the proposers’ behavior appear more realistic. The orders of offers participants experienced were fully randomized within each experiment phase. ”

(3) For the offer conditions (90:10, 70:30, 50:50, 30:70, 10:90) - are they randomized? If so, how is it done? Is it randomized within each participant, and/or also across participants (such that each participant experienced different trial sequences)? This is important, as the order especially for the learning phase can largely impact the preference learning of the participants.

We agree with the Reviewer the order in which offers are experienced could be very important. The order of the conditions was randomized independently for each participant (i.e. each participant experienced different trial sequences). We made this point clear in the Methods part. Line 527 ~ 528:

“The orders of offers participants experienced were fully randomized within each experiment phase.”

STATISTICAL ANALYSIS & COMPUTATIONAL MODELING

(4) In Study 1 DI offer types (90:10, 70:30), the rejection rate for DI-AI averse looks consistently higher than that for DI averse (ie, the blue line is above the yellow line). Is this significant? If so, how come? Since this is a between-subject design, I would not anticipate such a result (especially for the baseline). Also, for the LME results (eg, Table S3), only interactions were reported but not the main results.

We thank the Reviewer for pointing out this feature of the results. Prompted by this comment, we compared the baseline rejection rates between two conditions for these two offer types, finding in Experiment 1 that rejection rates in the DI-AI-averse condition were significantly higher than in the DI-averse condition (DI-AI-averse vs. DI-averse; Offer 90:10, β = 0.13, p < 0.001, Offer 70:30, β = 0.09, p < 0.034). We agree with the Reviewer that there should, in principle, be no difference between the experiences of participants in these two conditions is identical in the Baseline phase. However, we did not observe these difference in baseline preferences in Experiment 2 (DI-AI-averse vs. DI-averse; Offer 90:10, β = 0.07, p < 0.100, Offer 70:30, β = 0.05, p < 0.193). On the basis of the inconsistency of this effect across studies we believe this is a spurious difference in preferences stemming from chance.

Regarding the LME results, the reason why only interaction terms are reported is due to the specification of the model and the rationale for testing.

Taking the model reported in Table S3 as an example—a logistic model which examines Baseline phase rejection rates as a function of offer level and condition—the between-subject conditions (DI-averse and DI-AI-averse) are represented by dummy-coded variables. Similarly, offer types were also dummy-coded, such that each of the five columns (90:10, 70:30, 50:50, 30:70, and 10:90) correspond corresponded to a particular offer type. This model specification yields ten interaction terms (i.e., fixed effects) of interest—for example, the “DI-averse × Offer 90:10” indicates baseline rejection rates for 90:10 offers in DI-averse condition. Thus, to compare rejection rates across specific offer types, we estimate and report linear contrasts between these resultant terms. We have clarified the nature of these reported tests in our revised Results—for example, line189-190: “linear contrasts; e.g. 90:10 vs 10:90, all Ps<0.001, see Table S3 for logistic regression coefficients for rejection rates).

Also in response to this comment that and a recommendation from Reviewer 2 (see below), we have revised our supplementary materials to make each model specification clearer as SI line 25:

“RejectionRate ~ 0 + (Disl + Advl):(Offer10 + Offer30 + Offer50 + Offer70 + Offer90) + (1|Subject)”

(5) I do not particularly find this analysis appealing: "we examined whether participants' changes in rejection rates between Transfer and Baseline, could be explained by the degree to which they vicariously learned, defined as the change in punishment rates between the first and last 5 trials of the Learning phase." Naturally, the participants' behavior in the first 5 trials in the learning phase will be similar to those in the baseline; and their behavior in the last 5 trials in the learning phase would echo those at the transfer phase. I think it would be stronger to link the preference learning results to the change between the baseline and transfer phase, eg, by looking at the difference between alpha (beta) at the end of the learning phase and the initial alpha (beta).

Thanks for pointing this out. Also, considering the comments from Reviewer 2 concerning the interpretation of this analysis, we have elected to remove this result from our revision.

(6) I wonder if data from the baseline and transfer phases can also be modeled, using a simple Fehr-Schimdt model. This way, the change in alpha/beta can also be examined between the baseline and transfer phase.

We agree with the Reviewer that a simplified F-S model could be used, in principle, to characterize Baseline and Transfer phase behavior, but it is our view that the rejection rates provide readers with the clearest (and simplest) picture of how participants are responding to inequity. Put another way, we believe that the added complexity of using (and explaining) a new model to characterize simple, steady-state choice behavior (within these phases) would not be justified or add appreciable insights about participants’ behavior.

(7) I quite liked Study 2 which tests the generalization effect, and I expected to see an adapted computational modeling to directly reflect this idea. Indeed, the authors wrote, "[...] given that this model [...] assumes the sort of generalization of preferences between offer types [...]". But where exactly did the preference learning model assume the generalization? In the methods, the modeling seems to be only about Study 1; did the authors advise their model to accommodate Study 2? The authors also ran simulation for the learning phase in Study 2 (Figure 6), and how did the preference update (if at all) for offers (90:10 and 10:90) where feedback was not given? Extending/Unpacking the computational modeling results for Study 2 will be very helpful for the paper.

We are appreciative of the Reviewer’s positive impression of Experiment 2. Upon reflection, we realize that our original submission was not clear about the modeling done in Experiment 2, and we should clarify here that we did also fit the Preference Inference model to this dataset. As in Experiment 1, this model assumes that the participants have a representation of the teacher’s preference as a Fehr-Schmidt form utility function and infer the Teacher’s Envy and Guilt parameters through learning. The model indicates that, on the basis of experience with the Teacher’s preferences on moderately unfair offers (i.e., offer 70:30 and offer 30:70), participants can successfully infer these guess of these two parameters, and in turn, compute Fehr-Schmidt utility to guide their decisions in the extreme unfair offers (i.e., offer 90:10 and offer 10:90).

In response to this comment, we have made this clearer in our Results (Line 377-382):

“Finally, following Experiment 1, we fit a series of computational models of Learning phase choice behavior, comparing the goodness-of-fit of the four best-fitting models from Experiment 1 (see Methods). As before, we found that the Preference Inference model provided the best fit of participants’ Learning Phase behavior (Figure S1a, Table S12). Given that this model is able to infer the Teacher’s underlying inequity-averse preferences (rather than learns offer-specific rejection preferences), it is unsurprising that this model best describes the generalization behavior observed in Experiment 2.”

and in our revised Methods (Line 551-553)

“We considered 6 computational models of Learning Phase choice behavior, which we fit to individual participants’ observed sequences of choices, in both Experiments 1 and 2, via Maximum Likelihood Estimation”

Reviewer #2 (Public review):

Summary:

This study investigates whether individuals can learn to adopt egalitarian norms that incur a personal monetary cost, such as rejecting offers that benefit them more than the giver (advantageous inequitable offers). While these behaviors are uncommon, two experiments demonstrate that individuals can learn to reject such offers through vicarious learning - by observing and acting in line with a "teacher" who follows these norms. The authors use computational modelling to argue that learners adopt these norms through a sophisticated process, inferring the latent structure of the teacher's preferences, akin to theory of mind.

Strengths:

This paper is well-written and tackles a critical topic relevant to social norms, morality, and justice. The findings, which show that individuals can adopt just and fair norms even at a personal cost, are promising. The study is well-situated in the literature, with clever experimental design and a computational approach that may offer insights into latent cognitive processes. Findings have potential implications for policymakers.

Weaknesses:

Note: in the text below, the "teacher" will refer to the agent from which a participant presumably receives feedback during the learning phase.

(1) Focus on Disadvantageous Inequity (DI): A significant portion of the paper focuses on responses to Disadvantageous Inequitable (DI) offers, which is confusing given the study's primary aim is to examine learning in response to Advantageous Inequitable (AI) offers. The inclusion of DI offers is not well-justified and distracts from the main focus. Furthermore, the experimental design seems, in principle, inadequate to test for the learning effects of DI offers. Because both teaching regimes considered were identical for DI offers the paradigm lacks a control condition to test for learning effects related to these offers. I can't see how an increase in rejection of DI offers (e.g., between baseline and generalization) can be interpreted as speaking to learning. There are various other potential reasons for an increase in rejection of DI offers even if individuals learn nothing from learning (e.g. if envy builds up during the experiment as one encounters more instances of disadvantageous fairness).

We are appreciative of the Reviewer’s insight here and for the opportunity to clarify our experimental logic. We included DI offers in order to 1) expose participants to the full spectrum of offer types, and avoid focusing participants exclusively upon AI offers, which might result in a demand characteristic and 2) to afford exploration of how learning dynamics might differ in DI context s—which was, to some extent, examined in our previous study (FeldmanHall, Otto, & Phelps, 2018)—versus AI contexts. Furthermore, as this work builds critically on our previous study, we reasoned that replicating these original findings (in the DI context) would be important for demonstrating the generality of the learning effects in the DI context across experimental settings. We now remark on this point in our revised Introduction Line 129 ~132:

“In addition, to mechanistically probe how punitive preferences are acquired in Adv-I and Dis-I contexts—in turn, assessing the replicability of our earlier study investigating punitive preference acquisition in the Dis context—we also characterize trial-by-trial acquisition of punitive behavior with computational models of choice.”

(2) Statistical Analysis: The analysis of the learning effects of AI offers is not fully convincing. The authors analyse changes in rejection rates within each learning condition rather than directly comparing the two. Finding a significant effect in one condition but not the other does not demonstrate that the learning regime is driving the effect. A direct comparison between conditions is necessary for establishing that there is a causal role for the learning regime.

We agree with the Reviewer and upon reflection, believe that direct comparisons between conditions would be helpful to support the claim that the different learning conditions are responsible for the observed learning effects. In brief, these specific tests buttress the idea that exposure to AI-averse preferences result in increases in AI punishment rates in the Transfer phase (over and above the rates observed for participants who were only exposed to DI-averse preferences).

Accordingly, our revision now reports statistics concerning the differences between conditions for AI offers in Experiment 1 (Line 198~ 207):

“Importantly, when comparing these changes between the two learning conditions, we observed significant differences in rejection rates for Adv-I offers: compared to exposure to a Teacher who rejected only Dis-I offers, participants exposed to a Teacher who rejected both Dis-I and Adv-I offers were more likely to reject Adv-I offers and rated these offers more unfair. This difference between conditions was evident in both 30:70 offers (Rejection rates: β(SE) = 0.10(0.04), p = 0.013; Fairness ratings: β(SE) = -0.86(0.17), p < 0.001) and 10:90 offers (Rejection rates: β(SE) = 0.15(0.04), p < 0.001, Fairness ratings: β(SE) = -1.04(0.17), p < 0.001). As a control, we also compared rejection rates and fairness rating changes between conditions in Dis-I offers (90:10 and 30:70) and Fair offers (i.e., 50:50) but observed no significant difference (all ps > 0.217), suggesting that observing an Adv-I-averse Teacher’s preferences did not influence participants’ behavior in response to Dis-I offers.”

Line 222 ~ 230:

“A mixed-effects logistic regression revealed a significant larger (positive) effect of trial number on rejection rates of Adv-I offers for the Adv-Dis-I-Averse condition compared to the Dis-I-Averse condition. This relative rejection rate increase was evident both in 30:70 offers (Table S7; β(SE) = -0.77(0.24), p < 0.001) and in 10:90 offers (β(SE) = -1.10(0.33), p < 0.001). In contrast, comparing Dis-I and Fairness offers when the Teacher showed the same tendency to reject, we found no significant difference between the two conditions (90:10 splits: β(SE)=-0.48(0.21),p=0.593;70:30 splits: β(SE)=-0.01(0.14),p=0.150; 50:50 splits: β(SE)=-0.00(0.21),p=0.086). In other words, participants by and large appeared to adjust their rejection choices in accordance with the Teacher’s feedback in an incremental fashion.”

And in Experiment 2 Line 333 ~ 345:

“Similar to what we observed in Experiment 1 (Figure 4a), Compared to the participants in the Dis-I-Averse Condition, participants in the Adv-I-Averse Condition increased their rates of rejection of extreme Adv-I offerers (i.e., 10:90) in the Transfer Phase, relative to the Baseline phase (β(SE) = -0.12(0.04), p < 0.004; Table S9), suggesting that participants’ learned (and adopted) Adv-I-averse preferences, generalized from one specific offer type (30:70) to an offer types for which they received no Teacher feedback (10:90). Examining extreme Dis-I offers where the Teacher exhibited identical preferences across the two learning conditions, we found no difference in the Changes of Rejection Rates from Baseline to Transfer phase between conditions (β(SE) = -0.05(0.04), p < 0.259). Mirroring the observed rejection rates (Figure 4b), relative to the Dis-I-Averse Condition, participants’ fairness ratings for extreme Adv-I offers increased more from the Baseline to Transfer phase in the Adv-Dis-I-Averse Condition than in the Dis-I-Averse condition (β(SE) = -0.97(0.18), p < 0.001), but, importantly, changes in fairness ratings for extreme Dis-I offers did not differ significantly between learning conditions (β(SE) = -0.06(0.18), p < 0.723)”

Line 361 ~ 368:

“Examining the time course of rejection rates in Adv-I-contexts during the Learning phase (Figure 5) revealed that participants learned over time to punish mildly unfair 30:70 offers, and these punishment preferences generalized to more extreme offers (10:90). Specifically, compared to the Dis-I-Averse Condition, in the Adv-Dis-I-Averse condition we observed a significant larger trend of increase in rejections rates for 10:90 (Adv-I) offers (Figure 5, β(SE) = -0.81(0.26), p < 0.002 mixed-effects logistic regression, see Table S10). Again, when comparing the rejection rate increase in the extremely Dis-I offers (90:10), we didn’t find significant difference between conditions (β(SE) = -0.25(0.19), p < 0.707).”

(3) Correlation Between Learning and Contagion Effects:

The authors argue that correlations between learning effects (changes in rejection rates during the learning phase) and contagion effects (changes between the generalization and baseline phases) support the idea that individuals who are better aligning their preferences with the teacher also give more consideration to the teacher's preferences later during generalization phase. This interpretation is not convincing. Such correlations could emerge even in the absence of learning, driven by temporal trends like increasing guilt or envy (or even by slow temporal fluctuations in these processes) on behalf of self or others. The reason is that the baseline phase is temporally closer to the beginning of the learning phase whereas the generalization phase is temporally closer to the end of the learning phase. Additionally, the interpretation of these effects seems flawed, as changes in rejection rates do not necessarily indicate closer alignment with the teacher's preferences. For example, if the teacher rejects an offer 75% of the time then a positive 5% learning effect may imply better matching the teacher if it reflects an increase in rejection rate from 65% to 70%, but it implies divergence from the teacher if it reflects an increase from 85% to 90%. For similar reasons, it is not clear that the contagion effects reflect how much a teacher's preferences are taken into account during generalization.

This comment is very similar to a previous comment made by Reviewer 1, who also called into question the interpretability of these correlations. In response to both of these comments we have elected to remove these analyses from our revision.

(4) Modeling Efforts: The modelling approach is underdeveloped. The identification of the "best model" lacks transparency, as no model-recovery results are provided, and fits for the losing models are not shown, leaving readers in the dark about where these models fail. Moreover, the reinforcement learning (RL) models used are overly simplistic, treating actions as independent when they are likely inversely related (for example, the feedback that the teacher would have rejected an offer provides feedback that rejection is "correct" but also that acceptance is "an error", and the later is not incorporated into the modelling). It is unclear if and to what extent this limits current RL formulations. There are also potentially important missing details about the models. Can the authors justify/explain the reasoning behind including these variants they consider? What are the initial Q-values? If these are not free parameters what are their values?

We are appreciative of the Reviewer for identifying these potentially unaddressed questions.

The RL models we consider in the present study are naïve models which, in our previous study (FeldmanHall, Otto, & Phelps, 2018), we found to capture important aspects of learning. While simplistic, we believed these models serve as a reasonable baseline for evaluating more complex models, such as the Preference Inference model. We have made this point more explicit in our revised Introduction, Line 129 ~ 132:

“In addition, to mechanistically probe how punitive preferences may be acquired in Adv-I and Dis-I contexts—in turn, assessing the replicability of our earlier study investigating punitive preference acquisition in the Dis-I context—we also characterize trial-by-trial acquisition of punitive behavior with computational models of choice.”

Again, following from our previous modeling of observational learning (FeldmanHall et al., 2018), we believe that the feedback the Teacher provides here is ideally suited to the RL formalism. In particular, when the teacher indicates that the participant’s choice is what they would have preferred, the model receives a reward of ‘1’ (e.g., the participant rejects and the Teacher indicates they would preferred rejection, resulting in a positive prediction error) otherwise, the model receives a reward of ‘0’ (e.g., the participant accepts and the Teacher indicates they would preferred rejection, resulting in a negative prediction error), indicating that the participant did not choose in accordance with the Teacher’s preferences. Through an error driven learning process, these models provide a naïve way of learning to act in accordance with the Teacher’s preferences.

Regarding the requested model details: When treating the initial values as free parameters (model 5), we set Q(reject, offertype) as free values in [0,1] and Q(accept,offertype) as 0.5. This setting can capture participants' initial tendency to reject or accept offers from this offer type. When the initial values are fixed, for all offer types we set Q(reject, offertype) = Q(accept,offertype) = 0.5. In practice, when the initial values are fixed, setting them to 0.5 or 0 doesn’t make much difference. We have clarified these points in our revised Methods, Line 275 ~ 576:

“We kept the initial values fixed in this model, that is Q₀(reject,offertype) =0.5, (offertype ∈ 90:10, 70:30, 50:50, 30:70, 10:90)”

And Line 582 ~ 584:

“Formally, this model treats Q₀(reject,offertype) =0.5, (offertype ∈ 90:10, 70:30, 50:50, 30:70, 10:90) as free parameters with values between 0 and 1.”

(5) Conceptual Leap in Modeling Interpretation: The distinction between simple RL models and preference-inference models seems to hinge on the ability to generalize learning from one offer to another. Whereas in the RL models learning occurs independently for each offer (hence to cross-offer generalization), preference inference allows for generalization between different offers. However, the paper does not explore RL models that allow generalization based on the similarity of features of the offers (e.g., payment for the receiver, payment for the offer-giver, who benefits more). Such models are more parsimonious and could explain the results without invoking a theory of mind or any modelling of the teacher. In such model versions, a learner learns a functional form that allows to predict the teacher's feedback based on said offer features (e.g., linear or quadratic form). Because feedback for an offer modulates the parameters of this function (feature weights) generalization occurs without necessarily evoking any sophisticated model of the other person. This leaves open the possibility that RL models could perform just as well or even show superiority over the preference learning model, casting doubt on the authors' conclusions. Of note: even the behaviourists knew that as Little Albert was taught to fear rats, this fear generalized to rabbits. This could occur simply because rabbits are somewhat similar to rats. But this doesn't mean little Alfred had a sophisticated model of animals he used to infer how they behave.

We are appreciative of the Reviewer for their suggestion of an alternative explanation for the observed generalization effects. Our understanding of the suggestion, put simply, put simply, is that an RL model could capture the observed generalization effects if the model were to learn and update a functional form of the Teacher’s rejection preferences using an RL-like algorithm. This idea is similar, conceptually to our account of preference learning whereby the learner has a representation of the teacher’s preferences. In our experiment the offer is in the range of [0-100], the crux of this idea is why the participants should take the functional form (either v-shaped or quadratic) with the minimum at 50. This is important because, at the beginning of the learning phase, the rejection rates are already v-shaped with 50 as its minimum. The participants do not need to adjust the minimum of this functional form. Thus, if we assume that the participants represent the teacher’s rejection rate as a v-shape function with a minimum at [50,50], then this very likely implies that the participants have a representation that the teacher has a preference for fairness. Above all, we agree that with suitable setup of the functional form, one could implement an RL model to capture the generalization effects, without presupposing an internal “model” of the teacher’s preferences.

However, there is another way of modeling the generalization effect by truly “model-free” similarity-based Reinforcement learning. In this approach, we do not assume any particular functional form of the teacher’s preferences, but rather, assumes that experience acquired in one offer type can be generalized to offers that are close (i.e., similar) to the original offer. Accordingly, we implement this idea using a simple RL model in which the action values for each offer type is updated by a learning rate that is scaled by the distance between that offer and the experienced offer (i.e., the offer that generated the prediction error). This learning rate is governed by a Gaussian distribution, similar to the case in the Gaussian process regression (cf. Chulz, Speekenbrink, & Krause, 2018). The initial value of the ‘Reject’ action, for each offer , is set to a free parameter between 0 and 1, and the initial value for the 'Accept’ action was set to 0.5. The results show that even though this model exhibits the trend of increasing rejection rates observed in the AI-DI punish condition, the initial preferences (i.e., starting point of learning) diverges markedly from the Learning phase behavior we observed in Experiment 1:

Author response image 1.

This demonstrated that the participant at least maintains a representation of the teacher’s preference at the beginning. That is, they have prior knowledge about the shape of this preference. We incorporated this property into the model, that is, we considered a new model that assumes v-shaped starting values for rejection with two parameters, alpha and beta, governing the slope of this v-shaped function (this starting value actually mimics the shape of the preference functions of the Fehr-Schmidt model). We found that this new model (which we term the “Model RL Sim Vstart”) provided a satisfactory qualitative fit of the Transfer phase learning curves in Experiment 1 (see below).

Author response image 2.

However, we didn’t adopt this model as the best model for the following reasons. First, this model yielded a larger AIC value (indicating worse quantitative fit) compared to our preference Inference model in both Experiments 1 and 2, likely owing to its increased complexity (5 free parameters versus 4 in the Preference Inference model). Accordingly, we believe that inclusion of this model in our revised submission would be more distracting than helpful on account of the added complexity of explaining and justifying these assumptions, and of course its comparatively poor goodness of fit (relative to the preference inference model).

(6) Limitations of the Preference-Inference Model: The preference-inference model struggles to capture key aspects of the data, such as the increase in rejection rates for 70:30 DI offers during the learning phase (e.g. Figure 3A, AI+DI blue group). This is puzzling.

Thinking about this I realized the model makes quite strong unintuitive predictions that are not examined. For example, if a subject begins the learning phase rejecting the 70:30 offer more than 50% of the time (meaning the starting guilt parameter is higher than 1.5), then overleaning the tendency to reject will decrease to below 50% (the guilt parameter will be pulled down below 1.5). This is despite the fact the teacher rejects 75% of the offers. In other words, as learning continues learners will diverge from the teacher. On the other hand, if a participant begins learning to tend to accept this offer (guilt < 1.5) then during learning they can increase their rejection rate but never above 50%. Thus one can never fully converge on the teacher. I think this relates to the model's failure in accounting for the pattern mentioned above. I wonder if individuals actually abide by these strict predictions. In any case, these issues raise questions about the validity of the model as a representation of how individuals learn to align with a teacher's preferences (given that the model doesn't really allow for such an alignment).

In response to this comment we explain our efforts to build a new model that might be able conceptually resolves the issue identified by the Reviewer.

The key intuition guiding the Preference inference model is a Bayesian account of learning which we aimed to further simplify. In this setting, a Bayesian learner maintains a representation of the teacher’s inequity aversion parameters and updates it according to the teacher’s (observed) behavior. Intuitively, the posterior distribution shifts to the likelihood of the teacher’s action. On this view, when the teacher rejects, for instance, an AI offer, the learner should assign a higher probability to larger values of the Guilt parameter, and in turn the learner should change their posterior estimate to better capture the teacher’s preferences.

In the current study, we simplified this idea, implementing this sort of learning using incremental “delta rule” updating (e.g. Equation 8 of the main text). Then the key question is to define the “teaching signal”. Assuming that the teacher rejects an offer 70:30, based on Bayesian reasoning, the teacher’s envy parameter (α) is more likely to exceed 1.5 (computed as 30/(50-30), per equation 7) than to be smaller than 1.5. Thus, 1.5, which is then used in equation 8 to update α, can be thought of as a teaching signal. We simply assumed that if the initial estimate is already greater than 1.5, which means the prior is consistent with the likelihood, no updating would occur. This assumption raises the question of how to set the learning rate range. In principle, an envy parameter that is larger than 1.5 should be the target of learning (i.e., the teaching signal), and thus our model definition allows the learning rate to be greater than 1, incorporating this possibility.

Our simplified preference inference model has already successfully captured some key aspects of the participants’ learning behavior. However, it may fail in the following case: assume that the participant has an initial estimate of 1.51 for the envy parameter (β). Let’s say this corresponds to a rejection rate of 60%. Thus, no matter how many times the teacher rejects the offer 70:30, the participant’s estimate of the envy parameter remains the same, but observing only one offer acceptance would decrease this estimate, and in turn, would decrease the model’s predicted rejection rate. We believe this is the anomalous behavior—in 70:30 offers—identified by the Reviewer which the model does not appear able to recreate participants’ in these offers.

This issue actually touches the core of our model specification, that is, the choosing of the teaching signal. As we chose 1.5 as the teaching signal—i.e. lower bound on whenever the teacher rejects or accepts an offer of 70:30, a very small deviation of 1.5 would fail one part of updating. One way to mitigate this problem would be to choose a lower bound for α greater than 1.5, such that when the Teacher rejects a 70:30 offer, we assign a number greater than 1.5 (by ‘hard-coding’ this into the model via modification of equation 7). One sensible candidate value could be the middle point between 1.5 and 10 (the maximum value of α per our model definition). Intuitively, the model of this setting could still pull up the value of α to 1.51 when the teacher rejects 70:30, thus alleviating (but not completely eliminating) the anomaly.

We fitted this modified Preference Inference model to the data from Experiment 1 (see Author response image 3 below) and found that even though this model has a smaller AIC (and thus better quantitative fit than the original Preference Inference model), it still doesn’t fully capture the participants’ behavior for 70:30 offers.

Author response image 3.

Accordingly, rather than revising our model to include an unprincipled ‘kludge’ to account for this minor anomaly in the model behavior, we have opted to report our original model in our revision as we still believe it parsimoniously captures our intuitions about preference learning and provides a better fit to the observed behavior than the other RL models considered in the present study.

Reviewer #1 (Recommendations for the authors):

(1) I do not particularly prefer the acronyms AI and DI for disadvantageous inequity and advantageous inequity. Although they have been used in the literature, not every single paper uses them. More importantly, AI these days has such a strong meaning of artificial intelligence, so when I was reading this, I'd need to very actively inhibit this interpretation. I believe for the readability for a wider readership of eLife, I would advise not to use AI/DI here, but rather use the full terms.

We thank the Reviewer for this suggestion. As the full spelling of the two terms are somewhat lengthy, and appear frequently in the figures, we have elected to change the abbreviations for disadvantageous inequity and advantageous inequity to Dis-I and Adv-I, respectively in the main text and the supplementary information. We still use AI/DI in the response letter to make the terminology consistent.

(2) Do "punishment rate" and "rejection rate" mean the same? If so, it would be helpful to stick with one single term, eg, rejection rate.

We thank the Reviewer for this suggestion. As these terms have the same meaning, we have opted to use the term “rejection rate” throughout the main text.

(3) For the linear mixed effect models, were other random effect structures also considered (eg, random slops of experimental conditions)? It might be worth considering a few model specifications and selecting the best one to explain the data.

Thanks for this comment. Following established best practices (Barr, Levy, Scheepers, & Tily, 2013) we have elected to use a maximal random effects structure, whereby all possible predictor variables in the fixed effects structure also appear in the random effects structure.

(4) For equation (4), the softmax temperature is denoted as tau, but later in the text, it is called gamma. Please make it consistent.

We are appreciative of the Reviewer’s attention to detail. We have corrected this error.

Reviewer #2 (Recommendations for the authors):

(1) Several Tables in SI are unclear. I wasn't clear if these report raw probabilities of coefficients of mixed models. For any mixed models, it would help to give the model specification (e.g., Walkins form) and explain how variables were coded.

We are appreciative of the Reviewer’s attention to detail. We have clarified, in the captions accompanying our supplemental regression tables, that these coefficients represent log-odds. Regretfully we are unaware of the “Walkins form” the Reviewer references (even after extensive searching of the scientific literature). However, in our new revision we do include lme4 model syntax in our supplemental information which we believe will be helpful for readers seeking replicate our model specification.

(2) In one of the models it was said that the guilt and envy parameters were bounded between 0-1 but this doesn't make sense and I think values outside this range were later reported.

We are again appreciative of the Reviewer’s attention to detail. This was an error we have corrected— the actual range is [0,10].

(3) It is unclear if the model parameters are recoverable.

In response to this comment our revision now reports a basic parameter recovery analysis for the winning Preference Inference model. This is reported in our revised Methods:

“Finally, to verify if the free parameters of the winning model (Preference Inference) are recoverable, we simulated 200 artificial subjects, based on the Learning Phase of Experiment 1, with free parameters randomly chosen (uniformly) from their defined ranges. We then employed the same model-fitting procedure as described above to estimate these parameter value, observing that parameters. We found that all parameters of the model can be recovered (see Figure S2).”

And scatter plots depicting these simulated (versus recovered) parameters are given in Figure S2 of our revised Supplementary Information:

(4) I was confused about what Figure S2 shows. The text says this is about correlating contagious effects for different offers but the captions speak about learning effects. This is an important aspect which is unclear.

We have removed this figure in response to both Reviewers’ comments about the limited insights that can be drawn on the basis of these correlations.

Synthèse sur les Événements Traumatiques et le Secourisme en Santé Mentale

Résumé

Ce document de synthèse analyse en profondeur la nature des événements traumatiques, le trouble de stress post-traumatique (TSPT) et le rôle crucial des secouristes en santé mentale.

S'appuyant sur des expertises psychiatriques et des témoignages de terrain, il ressort que la compréhension du traumatisme repose sur la distinction fondamentale entre le stress aigu, une réaction adaptative normale, et le TSPT, un trouble chronique pouvant se manifester des mois après l'événement.

L'intervention d'un secouriste PSSM (Premiers Secours en Santé Mentale) est essentielle pour créer un climat de sécurité, d'écoute non-jugeante et de réassurance.

Les études de cas de Fabienne, assistante sociale, et de Laurine, une jeune femme aidant une amie, démontrent l'application pratique des compétences PSSM :

• la patience,
• la capacité à laisser du temps à la personne pour s'exprimer et
• l'importance de la mise en sécurité

sont des piliers de l'intervention.

Le secouriste agit comme un maillon essentiel, guidant la personne en souffrance vers des ressources professionnelles adaptées (thérapies comme l'EMDR, centres médico-psychologiques), tout en apprenant à gérer son propre stress et à reconnaître les limites de son rôle.

La formation PSSM est présentée comme une démarche citoyenne indispensable pour briser les tabous et outiller chacun à apporter un premier soutien efficace.

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I. Définition et Compréhension des Traumatismes Psychiques

A. L'Événement à Potentiel Traumatique

Un événement est considéré comme potentiellement traumatique lorsqu'il confronte une personne, directement ou indirectement, à la mort, à une menace de mort, à la peur de mourir, à de graves blessures ou à une menace pour son intégrité physique ou celle d'un tiers.

• Caractéristiques : Il provoque généralement une détresse intense, un sentiment d'impuissance et d'horreur.

• Exemples : Actes de violence interpersonnelle (viols, agressions), accidents, catastrophes naturelles, guerres, attentats, actes de torture.

• Statistiques clés (selon l'OMS) :

◦ 70 % des personnes dans le monde vivent un événement potentiellement traumatisant au cours de leur vie.    ◦ Environ 4 % de ces personnes sont susceptibles de développer un TSPT.

B. Le Trouble de Stress Post-Traumatique (TSPT)

Le TSPT est un trouble qui peut survenir après un événement traumatique. Il se caractérise par des réactions intenses, désagréables et dysfonctionnelles qui persistent dans le temps.

• Symptômes principaux :

◦ Reviviscence : Flashbacks, cauchemars, intrusions sensorielles.  

◦ Évitement : Efforts pour éviter les lieux, personnes ou pensées liés au trauma.  

◦ Hypervigilance : État d'alerte constant, irritabilité, sursauts.   

◦ Altérations cognitives et de l'humeur : Ruminations, changements d'humeur rapides, sentiment de culpabilité ou de honte.

• Troubles associés : Le TSPT s'accompagne fréquemment de dérégulation émotionnelle, de troubles anxiodépressifs ou de troubles liés à l'usage de substances. Un rétablissement est souvent possible avec une prise en charge adaptée.

C. Distinction entre Stress Aigu et TSPT

Selon le Dr Jean-Michel de Lille, psychiatre, il est crucial de différencier ces deux états :

Caractéristique

Stress Aigu

Trouble de Stress Post-Traumatique (TSPT)

Nature

Réaction adaptative et normale face à un danger imminent.

Trouble chronique et pathologique.

Fonction

Mécanisme de défense (le corps se prépare à fuir ou combattre : cœur qui s'accélère, muscles irrigués).

Le système de stress reste activé longtemps après la disparition du danger.

Durée

Temporaire. La pression redescend en quelques jours ou semaines une fois le danger écarté.

Dure plusieurs mois, voire s'installe durablement. Peut survenir à distance de l'événement (parfois un an après).

Facteurs de risque pour le TSPT

Vulnérabilités génétiques, antécédents de traumatismes infantiles (négligence, abus) qui réactivent un stress plus ancien.

D. Les Portes d'Entrée du TSPT

Le Dr de Lille identifie trois principales manières de développer un TSPT, initialement décrites en psychiatrie militaire :

1. Être victime : Avoir subi directement la violence ou l'événement traumatique.

2. Être témoin : Le fait d'être un "témoin impuissant" est une porte d'entrée majeure.

Les taux de TSPT chez les survivants du Bataclan sont plus élevés chez les témoins que chez les victimes directement blessées.

Ce phénomène est aussi crucial chez les enfants témoins de violences conjugales, dont l'impact neuropsychique est décisif.

3. Être auteur : Plus rare, le fait d'avoir commis des actes de violence peut également générer des retours traumatiques.

II. Manifestations et Symptômes du TSPT

A. La Reviviscence Traumatique (Flashbacks et Cauchemars)

La reviviscence, ou "intrusion", est l'un des symptômes les plus douloureux du TSPT. La personne revit la scène traumatique de manière involontaire et intense.

• Flashbacks : La scène est revécue y compris physiquement (cœur qui s'accélère, panique) en l'absence de danger objectif.

Ils peuvent être déclenchés par des éléments sensoriels anodins (ex: croiser un homme avec des lunettes si l'agresseur portait des lunettes).

• Cauchemars : Le sommeil est un moment où l'évitement est impossible, les cauchemars ramènent la scène traumatique.

L'exemple est donné d'un jeune militaire hanté des années plus tard par les odeurs d'un charnier qu'il avait dû déterrer, le menant à abuser de morphine et d'alcool pour obtenir un "sommeil anesthétique".

B. Les Stratégies d'Évitement et l'Hypervigilance

En réaction à la douleur des intrusions, la personne développe des stratégies d'évitement.

• Comportements : Ne plus prendre les transports en commun, éviter le quartier de l'agression, éviter certaines dates (anniversaires).

• Conséquences : Dans les cas sévères, cela peut conduire à un isolement social complet, où toute interaction est perçue comme une menace potentielle.

• Hypervigilance anxieuse : La personne est constamment sur ses gardes, méfiante, a l'impression que le danger va resurgir, ce qui rend la vie "absolument invivable".

C. L'Impact Émotionnel et Comportemental

Le TSPT affecte profondément la sphère émotionnelle et les relations sociales.

• Culpabilité et Honte : Des sentiments de culpabilité ("pourquoi ai-je survécu et pas d'autres ?") ou de honte (particulièrement dans des métiers où l'expression des émotions est taboue) sont fréquents.

• Irritabilité et Isolement : Comme observé dans le cas de l'agent de la route, la personne peut devenir irritable, s'isoler de ses collègues et de sa famille.

III. Le Rôle du Secouriste en Santé Mentale (PSSM)

A. Principes Fondamentaux de l'Intervention

L'intervention d'un secouriste PSSM repose sur des principes clés pour aider une personne suite à un événement traumatique.

1. Créer la Sécurité : L'élément "absolument décisif" est de recréer un climat de sécurité et d'apaisement, de garantir que l'échange se déroule dans un espace sûr.

2. Adopter une Posture Non-Jugeante : Se positionner sur un terrain de parité, d'empathie et de soin, sans se présenter comme un expert.

3. Accueillir et Laisser le Temps : Laisser le temps à la personne d'exprimer ou de ne pas exprimer ses émotions, sans la presser.

La formation PSSM insiste sur cette "notion de temps" et de pauses.

B. Actions Clés face à une Personne en Crise

Face à une personne qui revit un événement, le secouriste peut :

• Rassurer sur la culpabilité : Aider la personne à comprendre qu'elle n'est pas coupable de ce qui s'est passé et qu'elle est une personne respectable.

• Écouter activement : Permettre à la personne de verbaliser ce qu'elle a vu, ressenti et comment elle se sent aujourd'hui.

• Réorienter en douceur vers le présent : Lors d'un flashback, introduire gentiment le doute pour aider la personne à faire la part des choses entre la réalité passée (l'agression) et la réalité présente (la sécurité actuelle).

Exemple : "Le monsieur avec des lunettes dans le tram n'est pas celui que vous avez vu il y a des années."

• Informer sur les ressources : Même si la personne n'est pas prête à parler, lui fournir des informations sur les professionnels et les lieux où elle peut trouver de l'aide (plateformes téléphoniques, médecin, etc.).

C. Connaître ses Limites et Orienter vers les Professionnels

Le secouriste PSSM n'est pas un professionnel de santé. Il est crucial de reconnaître les limites de son intervention.

Le rôle est d'être un pont, d'accompagner la personne vers une prise en charge adaptée et personnalisée par des professionnels qualifiés.

IV. Études de Cas : Applications Pratiques du Secourisme

A. Cas de Fabienne : L'Agent de la Route Traumatisé

Fabienne, assistante sociale et secouriste PSSM, est intervenue auprès d'un agent de la route mutique après avoir été confronté à une victime décédée.

Aspect

Description

Contexte

Un agent de la route est en retrait et isolé après avoir été témoin d'un accident mortel.

Le public est majoritairement masculin, peu habitué à parler des émotions, avec un sentiment de "honte" à exprimer sa fragilité.

Défi

L'agent reste silencieux lors des deux premières tentatives d'entretien. Il refuse de se livrer.

Approche PSSM

Fabienne fait preuve d'une grande patience. Elle laisse passer près d'un mois entre la première et la troisième rencontre.

Elle lui fournit des informations sur les ressources dès le deuxième entretien, anticipant un besoin urgent. Lors du troisième entretien (initié par l'agent), elle applique les principes PSSM :

elle prend le temps, accueille les émotions avec une "qualité d'écoute différente", et laisse des temps de pause importants.

Résultat

L'agent parvient enfin à s'exprimer, livrant son mal-être (cauchemars, irritabilité).

Fabienne l'oriente précisément vers un centre médico-psychologique spécialisé. Plus tard, l'agent la remercie pour son aide et sa confiance.

B. Cas de Laurine : L'Amie en Pleine Reviviscence

Laurine, secouriste PSSM, a aidé une amie proche, diagnostiquée TSPT suite à une enfance violente, lors d'un flashback.

Aspect

Description

Contexte

L'amie a une enfance marquée par une mère alcoolique et un père violent.

Elle se sent coupable de la mort de ses animaux de compagnie, survenue dans des circonstances troubles.

L'alcool est souvent un "facilitateur d'anxiété" pour elle.

Déclencheur

En sortie de boîte de nuit, la vue d'un chien déclenche une reviviscence intense.

Elle s'effondre en appelant le nom de son chien décédé, persuadée de l'avoir retrouvé.

Intervention

1. Mise en sécurité : Laurine l'isole d'un groupe d'hommes alcoolisés.

1. Écoute et validation : Elle ne la contredit pas frontalement ("je veux bien te croire qu'il ressemble beaucoup") mais tente de la ramener à la réalité.

2. Fermeté bienveillante : Face à l'insistance de son amie, elle reste ferme pour la ramener en lieu sûr ("moi en tant qu'amie je peux pas te laisser y retourner").

Réflexion et Résultat

Avec le recul, Laurine pense qu'elle aurait dû être "plus dans l'écoute" et moins dans la volonté de "raisonner".

Le lendemain, elle encourage son amie à en parler à sa psychologue.

L'amie aborde ce trauma en thérapie EMDR et comprend qu'elle a fait un transfert : son désir de sauver le chien était le reflet de son propre désir d'avoir été sauvée enfant.

V. Prise en Charge Professionnelle et Ressources

A. Thérapies Spécifiques pour le TSPT

Le Dr de Lille souligne des avancées majeures dans les thérapies du TSPT, notamment les thérapies d'exposition prolongée.

L'objectif est de permettre à la personne d'évoquer le souvenir traumatique sans subir les symptômes de panique, afin de "désamorcer le couplage" entre la mémoire et la souffrance physique.

• Méthodes psychothérapeutiques :

◦ EMDR (MDR en français) : Thérapie brève utilisant des mouvements oculaires répétitifs pour "digérer" le souvenir traumatique.    ◦ ICV (Intégration du Cycle de la Vie)

• Approches pharmacologiques :

◦ Méthode Brunet : Utilisation de bêta-bloquants (ex: Vlocardie) pour diminuer la réaction physique lors de l'évocation.   

◦ Expérimentations : Des recherches sont en cours avec de nouvelles molécules comme la MDMA.

B. Ressources et Soutien Disponibles

Le podcast met en avant plusieurs ressources pour les personnes concernées et les aidants :

• Numéros d'urgence : 112, 15, 18 et le 3114 (numéro national de prévention du suicide).

• PSSM France :

◦ Le site internet pour se former aux premiers secours en santé mentale.   

◦ Le "Carnet du secouriste en santé mentale : mieux aider un adulte suite à un événement traumatique" (téléchargement gratuit).

• Centre National de Ressources et de résilience (CN2R) : Chaîne YouTube et témoignages pour améliorer la prise en charge du psychotraumatisme.

• Podcast "Émotion" par Louis Média : L'épisode "Stress post-traumatique : Comment s'inscrit-il dans notre corps ?".

VI. Apports et Valeur de la Formation PSSM France

A. Un Cadre Sécurisant pour l'Aidant

La formation PSSM est décrite comme un outil essentiel qui fournit un "cadre sécurisant" à l'aidant.

Elle permet de savoir quoi faire, mais surtout "ce qu'il ne faut pas faire" et "ne pas dire".

• Elle incite à prendre de la hauteur et à requestionner ses propres pratiques et réflexes.

• Elle aide à gérer son propre stress et à se sentir moins affecté personnellement, ce qui est crucial pour pouvoir aider efficacement sans s'épuiser.

B. Témoignages sur l'Impact de la Formation

• Pour Fabienne : La formation a enrichi son approche, notamment sur "cette notion du temps" et l'importance de "laisser à l'autre la possibilité ou pas de dire".

• Pour Laurine : La formation lui a permis de trouver un moyen d'avoir du recul, de moins culpabiliser et de prendre conscience du réseau de ressources existant (elle ne connaissait pas PSSM avant).

C. Un Enjeu Citoyen pour Briser les Tabous

Le podcast conclut en soulignant que, tout comme les premiers secours physiques, les premiers secours en santé mentale sont une démarche citoyenne.

La formation permet d'acquérir des "clés toutes simples" qui peuvent aider de nombreuses personnes.

Elle encourage chacun à jouer un rôle pour briser les tabous autour des troubles psychiques et à "apprendre à aider".

Ve el vídeo y lee el texto sobre el papel de Samuel Melinao como coordinador de un centro de salud mapuche. ¿Puedes resumir los puntos principales que se mencionan en torno a la medicina mapuche y los problemas a los que se ha enfrentado?

Ve el vídeo y lee el texto sobre el papel de Samuel Melinao como coordinador de un centro de salud mapuche. ¿Puedes resumir los puntos principales que se mencionan en torno a la medicina mapuche y los problemas a los que se ha enfrentado?

Preguntas de Comprensión

¿Qué etiquetas asigna el artista Martin Vargic a algunos países de América Latina en su ilustración?

¿Cómo se perciben los argentinos según los estereotipos mencionados en el estudio de la Agencia Española de Cooperación Internacional?

¿Qué características positivas se mencionan sobre los brasileños en el estudio de 2006?

¿Cómo se describen los estereotipos sobre los bolivianos en el estudio mencionado?

¿Qué problemas menciona Elier Chara García sobre la falta de información en la propia región?

Preguntas de Reflexión ¿Qué impacto crees que tienen los estereotipos en la percepción de las personas de una región específica? ¿Cómo podríamos combatir la formación y perpetuación de estereotipos negativos entre países?

Explica el significado de la frase "no todos pueden ser puestos en el mismo saco" dentro del contexto y contrasta el ejemplo de Ismael con otro ejemplo propio.

¿Puedes explicar esta frase con tus propias palabras y añadir tu opinión al respecto?

¿Puedes explicar la ironía contenida en esta frase?

Author Response

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

Reviewer #1:

Concerns Public Review:

1)The framing of 'infinite possible types of conflict' feels like a strawman. While they might be true across stimuli (which may motivate a feature-based account of control), the authors explore the interpolation between two stimuli. Instead, this work provides confirmatory evidence that task difficulty is represented parametrically (e.g., consistent with literatures like n-back, multiple object tracking, and random dot motion). This parametric encoding is standard in feature-based attention, and it's not clear what the cognitive map framing is contributing.

Suggestion:

1) 'infinite combinations'. I'm frankly confused by the authors response. I don't feel like the framing has changed very much, besides a few minor replacements. Previous work in MSIT (e.g., by the author Zhongzheng Fu) has looked at whether conflict levels are represented similarly across conflict types using multivariate analyses. In the paper mentioned by Ritz & Shenhav (2023), the authors looked at whether conflict levels are represented similarly across conflict types using multivariate analyses. It's not clear what this paper contributes theoretically beyond the connections to cognitive maps, which feel like an interpretative framework rather than a testable hypothesis (i.e., these previous paper could have framed their work as cognitive maps).

Response: We acknowledge the limitations inherent in our experimental design, which prevents us from conducting a strict test of the cognitive space view. In our previous revision, we took steps to soften our conclusions and emphasize these limitations. However, we still believe that our study offers valuable and novel insights into the cognitive space, and the tests we conducted are not merely strawman arguments.

Specifically, our study aimed to investigate the fundamental principles of the cognitive space view, as we stated in our manuscript that “the representations of different abstract information are organized continuously and the representational geometry in the cognitive space is determined by the similarity among the represented information (Bellmund et al., 2018)”. While previous research has applied multivariate analyses to understand cognitive control representation, no prior studies had directedly tested the two key hypotheses associated with cognitive space: (1) that cognitive control representation across conflict types is continuous, and (2) that the similarity among representations of different conflict types is determined by their external similarity.

Our study makes a unique contribute by directly testing these properties through a parametric manipulation of different conflict types. This approach differs significantly from previous studies in two ways. First, our parametric manipulation involves more than two levels of conflict similarity, enabling us to directly test the two critical hypotheses mentioned above. Unlike studies such as Fu et al. (2022) and other that have treated different conflict types categorically, we introduced a gradient change in conflict similarity. This differentiation allowed us to employ representational similarity analysis (RSA) over the conflict similarity, which goes beyond mere decoding as utilized in prior work (see more explanation below for the difference between Fu et al., 2022 and our study [1]).

Second, our parametric manipulation of conflict types differs from previous studies that have manipulated task difficulty, and the modulation of multivariate pattern similarity observed in our study could not be attributed by task difficulty. Previous research, including the Ritz & Shenhav (2023) (see below explanation[2]), has primarily shown that task difficulty modulates univoxel brain activation. A recent work by Wen & Egner (2023) reported a gradual change in the multivariate pattern of brain activations across a wide range of frontoparietal areas, supporting the reviewer’s idea that “task difficulty is represented parametrically”. However, we do not believe that our results reflect the task difficulty representation. For instance, in our study, the spatial Stroop-only and Simon-only conditions exhibited similar levels of difficulty, as indicated by their relatively comparable congruency effects (Fig. S1). Despite this similarity in difficulty, we found that the representational similarity between these two conditions was the lowest (see revised Fig. S4, the most off-diagonal value). This observation aligns more closely with our hypothesis that these two conditions are most dissimilar in terms of their conflict types.

[1] Fu et al. (2022) offers important insights into the geometry of cognitive space for conflict processing. They demonstrated that Simon and flanker conflicts could be distinguished by a decoder that leverages the representational geometry within a multidimensional space. However, their model of cognitive space primarily relies on categorical definitions of conflict types (i.e., Simon versus flanker), rather than exploring a parametric manipulation of these conflict types. The categorical manipulations make it difficult to quantify conceptual similarity between conflict types and hence limit the ability to test whether neural representations of conflict capture conceptual similarity. To the best of our knowledge, no previous studies have manipulated the conflict types parametrically. This gap highlights a broader challenge within cognitive science: effectively manipulating and measuring similarity levels for conflicts, as well as other high-level cognitive processes, which are inherently abstract. We therefore believe our parametric manipulation of conflict types, despite its inevitable limitations, is an important contribution to the literature.

We have incorporated the above statements into our revised manuscript: Methodological implications. Previous studies with mixed conflicts have applied mainly categorical manipulations of conflict types, such as the multi-source interference task (Fu et al., 2022) and color Stroop-Simon task (Liu et al., 2010). The categorical manipulations make it difficult to quantify conceptual similarity between conflict types and hence limit the ability to test whether neural representations of conflict capture conceptual similarity. To the best of our knowledge, no previous studies have manipulated the conflict types parametrically. This gap highlights a broader challenge within cognitive science: effectively manipulating and measuring similarity levels for conflicts, as well as other high-level cognitive processes, which are inherently abstract. The use of an experimental paradigm that permits parametric manipulation of conflict similarity provides a way to systematically investigate the organization of cognitive control, as well as its influence on adaptive behaviors.

[2] The work by Ritz & Shenhav (2023) indeed applied multivariate analyses, but they did not test the representational similarity across different levels of task difficulty in a similar way as our investigation into different levels of conflict types, neither did they manipulated conflict types as our study. They first estimated univariate brain activations that were parametrically scaled by task difficulty (e.g., target coherence), yielding one map of parameter estimates (i.e., encoding subspace) for each of the target coherence and distractor congruence. The multivoxel patterns from the above maps were correlated to test whether the target coherence and distractor congruence share the similar neural encoding. It is noteworthy that the encoding of task difficulty in their study is estimated at the univariate level, like the univariate parametric modulation analysis in our study. The representational similarity across target coherence and distractor congruence was the second-order test and did not reflect the similarity across different difficulty levels. Though, we have found another study (Wen & Egner, 2023) that has directly tested the representational similarity across different levels of task difficulty, and they observed a higher representational similarity between conditions with similar difficulty levels within a wide range of brain regions.

Reference:

Wen, T., & Egner, T. (2023). Context-independent scaling of neural responses to task difficulty in the multiple-demand network. Cerebral Cortex, 33(10), 6013-6027. https://doi.org/10.1093/cercor/bhac479

Fu, Z., Beam, D., Chung, J. M., Reed, C. M., Mamelak, A. N., Adolphs, R., & Rutishauser, U. (2022). The geometry of domain-general performance monitoring in the human medial frontal cortex. Science (New York, N.Y.), 376(6593), eabm9922. https://doi.org/10.1126/science.abm9922

Ritz, H., & Shenhav, A. (2023). Orthogonal neural encoding of targets and distractors supports multivariate cognitive control. https://doi.org/10.1101/2022.12.01.518771 Another issue is suggesting mixtures between two types of conflict may be many independent sources of conflict. Again, this feels like the strawman. There's a difference between infinite combinations of stimuli on the one hand, and levels of feature on the other hand. The issue of infinite stimuli is why people have proposed feature-based accounts, which are often parametric, eg color, size, orientation, spatial frequency. Mixing two forms of conflict is interesting, but the task limitations (i.e., highly correlated features) prevent an analysis of whether these are truly mixed (or eg reflect variations on just one of the conflict types). Without being able to compare a mixture between types vs levels of only one type, it's not clear what you can draw from these results re: how these are combined (and not clear how it reconciles the debate between general and specific).

Response: As the reviewer pointed out, a feature (or a parameterization) is an efficient way to encode potentially infinite stimuli. This is the same idea as our hypothesis: different conflict types are represented in a cognitive space akin to concrete features such as a color spectrum. This concept can be illustrated in the figure below.

Author response image 1.

We would like to clarify that in our study we have manipulated five levels of conflict types, but they all originated from two fundamental sources: vertically spatial Stroop and horizontally Simon conflicts. We agree that the mixture of these two sources does not inherently generate additional conflict sources. However, this mixture does influence the similarity among different conflict conditions, which provides essential variability that is crucial for testing the core hypotheses (i.e., continuity and similarity modulation, see the response above) of the cognitive space view. This clarification is crucial as the reviewer’s impression might have been influenced by our introduction, where we repeatedly emphasized multiple sources of conflicts. Our aim in the introduction was to outline a broader conceptual framework, which might not directly reflect the specific design of our current study. Recognizing the possibility of misinterpretation, we have adjusted our introduction and discussion to place less emphasis on the variety of possible conflict sources. For example, we have removed the expression “The large variety of conflict sources implies that there may be innumerable number of conflict conditions” from the introduction. As we have addressed in the previous response, the observed conflict similarity effect could not be attributed to merely task difficulty. Similarly, the mixture of spatial Stroop and Simon conflicts should not be attributed to one conflict source only; doing so would oversimplify it to an issue of task difficulty, as it would imply that our manipulation of conflict types merely represented varying levels of a single conflict, akin to manipulating task difficulty when everything else being equal. Importantly, the mixed conditions differ from variations along a single conflict source in that they also incorporate components of the other conflict source, thereby introducing difference beyond that would be found within variances of a single conflict source. There are a few additional evidence challenging the single dimension assumption. In our previous revisions, we compared model fittings between the Cognitive-Space model and the Stroop-/Simon-only models, and results showed that the CognitiveSpace model (BIC = 5377093) outperformed the Stroop-Only (BIC = 5377122) and Simon-Only (BIC = 5377096) models. This suggests that mixed conflicts might not be solely reflective of either Stroop or Simon sources, although we did not include these results due to concerns raised by reviewers about the validity of such comparisons, given the high anticorrelation between the two dimensions. Furthermore, Fu et al. (2022) demonstrated that the mixture of Simon and Flanker conflicts (the sf condition) is represented as the vector sum of the Flanker and Simon dimensions within their space model, indicating a compositional nature. Similarly, our mixed conditions are combinations of Stroop and Simon conflicts, and it is plausible that these mixtures represent a fusion of both Stroop and Simon components, rather than just one. Thus, we disagree that the mixture of conflicts is a strawman. In response to this concern, we have included a statement in our limitation section: “Another limitation is that in our design, the spatial Stroop and Simon effects are highly anticorrelated. This constraint may make the five conflict types represented in a unidimensional space (e.g., a circle) embedded in a 2D space. This limitation also means we cannot conclusively rule out the possibility of a real unidimensional space driven solely by spatial Stroop or Simon conflicts. However, this appears unlikely, as it would imply that our manipulation of conflict types merely represented varying levels of a single conflict, akin to manipulating task difficulty when everything else being equal. If task difficulty were the primary variable, we would expect to see greater representational similarity between task conditions of similar difficulty, such as the Stroop and Simon conditions, which demonstrates comparable congruency effects (see Fig. S1). Contrary to this, our findings reveal that the Stroop-only and Simon-only conditions exhibit the lowest representational similarity (Fig. S4). Furthermore, Fu et al. (2022) has shown that the representation of mixtures of Simon and Flanker conflicts was compositional, rather than reflecting single dimension, which also applies to our cases.”

My recommendation would be to dramatically rewrite to reduce the framing of this providing critical evidence in favor of cognitive maps, and being more overt about the limitations of this task. However, the authors are not required to make further revisions in eLife's new model, and it's not clear how my scores would change if they made those revisions (ie the conceptual limitations would remain, the claims would just now match the more limited scope).

Response: With the above rationales and the adjustments we have made in the manuscripts, we believe that we have thoroughly acknowledged and articulated the limitations of our study. Therefore, we have decided against a complete rewrite of the manuscript.

Public Review:

2) The representations within DLPFC appear to treat 100% Stoop and (to a lesser extent) 100% Simon differently than mixed trials. Within mixed trials, the RDM within this region don't strongly match the predictions of the conflict similarity model. It appears that there may be a more complex relationship encoded in this region.

Suggestion:

2) RSMs in the key region of interest. I don't really understand the authors response here either. e.g,. 'It is essential to clarify that our conclusions were based on the significant similarity modulation effect identified in our statistical analysis using the cosine similarity model, where we did not distinguish between the within-Stroop condition and the other four within-conflict conditions (Fig. 7A, now Fig. 8A). This means that the representation of conflict type was not biased by the seemingly disparities in the values shown here'. In Figure 1C, it does look like they are testing this model.

It seems like a stronger validation would test just the mixture trials (i.e., ignoring Simon-only and stroop-only). However, simon/stroop-only conditions being qualitatively different does beg the question of whether these are being represented parametrically vs categorically.

Response: We apologize for the confusion caused by our previous response. To clarify, our conclusions have been drawn based on the robust conflict similarity effect.

The conflict similarity regressor is defined by higher values in the diagonal cells (representing within-conflict similarity) than the off-diagonal cells (indicating between-conflict similarity), as illustrated in Fig. 1C and Fig. 8A (now Fig. 4B). It is important to note that this regressor may not be particularly sensitive to the variations within the diagonal cells. Our previous response aimed to emphasize that the inconsistencies observed along the diagonal do not contradict our core hypothesis regarding the conflict similarity effect.

We recognized that since the visualization in Fig. S4, based on the raw RSM (i.e., Pearson correlation), may have been influenced by other regressors in our model than the conflict similarity effect. To reflect pattern similarity with confounding factors controlled for, we have visualized the RSM by including only the fixed effect of the conflict similarity and the residual while excluding all other factors. As shown in the revised Figure S4, the difference between the within-Stroop and other diagonal cells was greatly reduced. Instead, it revealed a clear pattern where that the diagonal values were higher than the off-diagonal values in the incongruent condition, aligning with our hypothesis regarding the conflict similarity modulator. Although some visual distinctions persist within the five diagonal cells (e.g., in the incongruent condition, the Stroop, Simon, and StMSmM conditions appear slightly lower than StHSmL and StLSmM conditions), follow-up one-way ANOVAs among these five diagonal conditions showed no significant differences. This held true for both incongruent and congruent conditions, with Fs < 1. Thus, we conclude that there is no strong evidence supporting the notion that Simon- and spatial Stroop-only conditions are systematically different from other conflict types. As a result, we decided not to exclude these two conflict types from analysis.

Author response image 2.

The stronger conflict type similarity effect in incongruent versus congruent conditions. Shown are the summary representational similarity matrices for the right 8C region in incongruent (left) and congruent (right) conditions, respectively. Each cell represents the averaged Pearson correlation (after regressing out all factors except the conflict similarity) of cells with the same conflict type and congruency in the 1400×1400 matrix. Note that the seemingly disparities in the values of withinconflict cells (i.e., the diagonal) did not reach significance for either incongruent or congruent trials, Fs < 1.

Public Review:

3) To orthogonalized their variables, the authors need to employ a complex linear mixed effects analysis, with a potential influence of implementation details (e.g., high-level interactions and inflated degrees of freedom).

Suggestion:

3) The DF for a mixed model should not be the number of observations minus the number of fixed effects. The gold standard is to use satterthwaite correction (e.g. in Matlab, fixedEffects(lme,'DFMethod','satterthwaite')), or number of subjects - number of fixed effects (i.e. you want to generalize to new subjects, not just new samples from the same subjects). Honestly, running a 4-way interaction probably is probably using more degrees of freedom than are appropriate given the number of subjects.

Response: We concur with the reviewer’s comment that our previous estimation of degrees of freedom (DFs) was inaccurate. Following your suggestion, we have now applied the “Satterthwaite” approach to approximate the DFs for all our linear mixed effect model analyses. This adjustment has led to the correction of both DFs and p values. In the Methods section, we have mentioned this revision.

“We adjusted the t and p values with the degrees of freedom calculated through the Satterthwaite approximation method (Satterthwaite, 1946). Of note, this approach was applied to all the mixed-effect model analyses in this study.”

The application of this method has indeed resulted in a reduction of our statistical significance. However, our overall conclusions remained robust. Instead of the highly stringent threshold used in our previous version (Bonferonni corrected p < .0001), we have now adopted a relatively more lenient threshold of Bonferonni correction at p < 0.05, which is commonly employed in the literature. Furthermore, it is worth noting that the follow-up criteria 2 and 3 are inherently second-order analyses. Criterion 2 involves examining the interaction effect (conflict similarity effect difference between incongruent and congruent conditions), and criterion 3 involves individual correlation analyses. Due to their second-order nature, these criteria inherently have lower statistical power compared to criterion 1 (Blake & Gangestad, 2020). We thus have applied a more lenient but still typically acceptable false discovery rate (FDR) correction to criteria 2 and 3. This adjustment helps maintain the rigor of our analysis while considering the inherent differences in statistical power across the various criteria. We have mentioned this revision in our manuscript:

“We next tested whether these regions were related to cognitive control by comparing the strength of conflict similarity effect between incongruent and congruent conditions (criterion 2) and correlating the strength to behavioral similarity modulation effect (criterion 3). Given these two criteria pertain to second-order analyses (interaction or individual analyses) and thus might have lower statistical power (Blake & Gangestad, 2020), we applied a more lenient threshold using false discovery rate (FDR) correction (Benjamini & Hochberg, 1995) on the above-mentioned regions.”

With these adjustments, we consistently identified similar brain regions as observed in our previous version. Specifically, we found that only the right 8C region met the three criteria in the conflict similarity analysis. In addition, the regions meeting the criteria for the orientation effect included the FEF and IP2 in left hemisphere, and V1, V2, POS1, and PF in the right hemisphere. We have thoroughly revised the description of our results, updated the figures and tables in both the revised manuscript and supplementary material to accurately reflect these outcomes.

Reference:

Blake, K. R., & Gangestad, S. (2020). On Attenuated Interactions, Measurement Error, and Statistical Power: Guidelines for Social and Personality Psychologists. Pers Soc Psychol Bull, 46(12), 1702-1711. https://doi.org/10.1177/0146167220913363

Minor:

1. Figure 8 should come much earlier (e.g, incorporated into Figure 1), and there should be consistent terms for 'cognitive map' and 'conflict similarity'.

Response: We appreciate this suggestion. Considering that Figure 7 (“The crosssubject RSA model and the rationale”) also describes the models, we have merged Figure 7 and 8 and moved the new figure ahead, before we report the RSA results. Now you could find it in the new Figure 4, see below. We did not incorporate them into Figure 1 since Figure 1 is already too crowded.

Author response image 3.

Fig. 4. Rationale of the cross-subject RSA model and the schematic of key RSMs. A) The RSM is calculated as the Pearson’s correlation between each pair of conditions across the 35 subjects. For 17 subjects, the stimuli were displayed on the top-left and bottom-right quadrants, and they were asked to respond with left hand to the upward arrow and right hand to the downward arrow. For the other 18 subjects, the stimuli were displayed on the top-right and bottom-left quadrants, and they were asked to respond with left hand to the downward arrow and right hand to the upward arrow. Within each subject, the conflict type and orientation regressors were perfectly covaried. For instance, the same conflict type will always be on the same orientation. To de-correlate conflict type and orientation effects, we conducted the RSA across subjects from different groups. For example, the bottom-right panel highlights the example conditions that are orthogonal to each other on the orientation, response, and Simon distractor, whereas their conflict type, target and spatial Stroop distractor are the same. The dashed boxes show the possible target locations for different conditions. (B) and (C) show the orthogonality between conflict similarity and orientation RSMs. The within-subject RSMs (e.g., Group1-Group1) for conflict similarity and orientation are all the same, but the cross-group correlations (e.g., Group2-Group1) are different. Therefore, we can separate the contribution of these two effects when including them as different regressors in the same linear regression model. (D) and (E) show the two alternative models. Like the cosine model (B), within-group trial pairs resemble betweengroup trial pairs in these two models. The domain-specific model is an identity matrix. The domaingeneral model is estimated from the absolute difference of behavioral congruency effect, but scaled to 0 (lowest similarity) – 1 (highest similarity) to aid comparison. The plotted matrices in B-E include only one subject each from Group 1 and Group 2. Numbers 1-5 indicate the conflict type conditions, for spatial Stroop, StHSmL, StMSmM, StLSmH, and Simon, respectively. The thin lines separate four different sub-conditions, i.e., target arrow (up, down) × congruency (incongruent, congruent), within each conflict type.

In our manuscript, the term “cognitive map/space” was used when explaining the results in a theoretical perspective, whereas the “conflict similarity” was used to describe the regressor within the RSA. These terms serve distinct purposes in our study and cannot be interchangeably substituted. Therefore, we have retained them in their current format. However, we recognize that the initial introduction of the “Cognitive-Space model” may have appeared somewhat abrupt. To address this, we have included a brief explanatory note: “The model described above employs the cosine similarity measure to define conflict similarity and will be referred to as the Cognitive-Space model.”

Author Response

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

Thank you and the reviewers for further providing constructive comments and suggestions on our manuscript. On behalf of all the co-authors, I have enclosed a revised version of the above referenced paper. Below, I have merged similar public reviews and recommendations (if applicable) from each reviewer and provided point-by-point responses.

Reviewer #1:

People can perform a wide variety of different tasks, and a long-standing question in cognitive neuroscience is how the properties of different tasks are represented in the brain. The authors develop an interesting task that mixes two different sources of difficulty, and find that the brain appears to represent this mixture on a continuum, in the prefrontal areas involved in resolving task difficulty. While these results are interesting and in several ways compelling, they overlap with previous findings and rely on novel statistical analyses that may require further validation.

Strengths

1. The authors present an interesting and novel task for combining the contributions of stimulus-stimulus and stimulus-response conflict. While this mixture has been measured in the multi-source interference task (MSIT), this task provides a more graded mixture between these two sources of difficulty.

2. The authors do a good job triangulating regions that encoding conflict similarity, looking for the conjunction across several different measures of conflict encoding. These conflict measures use several best-practice approaches towards estimating representational similarity.

3. The authors quantify several salient alternative hypothesis and systematically distinguish their core results from these alternatives.

4. The question that the authors tackle is important to cognitive control, and they make a solid contribution.

The authors have addressed several of my concerns. I appreciate the authors implementing best practices in their neuroimaging stats.

I think that the concerns that remain in my public review reflect the inherent limitations of the current work. The authors have done a good job working with the dataset they've collected.

Response: We would like to thank the reviewer for the positive evaluation of our manuscript and the constructive comments and suggestions. In response to your suggestions and concerns, we have removed the Stroop/Simon-only and the Stroop+Simon models, revised our conclusion and modified the misleading phrases.

We have provided detailed responses to your comments below.

1. The evidence from this previous work for mixtures between different conflict sources makes the framing of 'infinite possible types of conflict' feel like a strawman. The authors cite classic work (e.g., Kornblum et al., 1990) that develops a typology for conflict which is far from infinite. I think few people would argue that every possible source and level of difficulty will have to be learned separately. This work provides confirmatory evidence that task difficulty is represented parametrically (e.g., consistent with the n-back, MOT, and random dot motion literature).

notes for my public concerns.

In their response, the authors say:

'If each combination of the Stroop-Simon combination is regarded as a conflict condition, there would be infinite combinations, and it is our major goal to investigate how these infinite conflict conditions are represented effectively in a space with finite dimensions.'

I do think that this is a strawman. The paper doesn't make a strong case that this position ('infinite combinations') is widely held in the field. There is previous work (e.g., n-back, multiple object tracking, MSIT, dot motion) that has already shown parametric encoding of task difficulty. This paper provides confirmatory evidence, using an interesting new task, that demand are parametric, but does not provide a major theoretical advance.

Response: We agree that the previous expression may have seemed somewhat exaggerative. While it is not “infinite”, recent research indeed suggests that the cognitive control shows domain-specificity across various “domains”, including conflict types (Egner, 2008), sensory modalities (Yang et al., 2017), task-irrelevant stimuli (Spape et al., 2008), and task sets (Hazeltine et al., 2011), to name a few.

These findings collectively support the notion that cognitive control is contextspecific (Bream et al., 2014). That is, cognitive control can be tuned and associated with different (and potentially large numbers of) contexts. Recently, Kikumoto and Mayr (2020) demonstrated that combinations of stimulus, rule and response in the same task formed separatable, conjunctive representations. They further showed that these conjunctive representations facilitate performance. This is in line with the idea that each stimulus-location combination in the present task may be represented separately in a domain-specific manner. Moreover, domain-general task representation can also become domain-specific with learning, which further increases the number of domain-specific conjunctive representations (Mill et al., 2023). In line with the domain-specific account of cognitive control, we referred to the “infinite combinations” in our previous response to emphasize the extreme case of domainspecificity. However, recognizing that the term “infinite” may lead to ambiguity, we have replaced it with phrases such as “a large number of”, “hugely varied”, in our revised manuscript.

We appreciate the reviewer for highlighting the potential connection of our work to existing literature that showed the parametric encoding of task difficulty (e.g., Dagher et al., 1999; Ritz & Shenhav, 2023). For instance, in Ritz et al.’s (2023) study, they parametrically manipulated target difficulty based on consistent ratios of dot color, and found that the difficulty was encoded in the caudal part of dorsal anterior cingulate cortex. Analogically, in our study, the “difficulty” pertains to the behavioral congruency effect that we modulated within the spatial Stroop and Simon dimensions. Notably, we did identify univariate effects in the right dmPFC and IPS associated with the difficulty in the Simon dimension. This parametric effect may lend support to our cognitive space hypothesis, although we exercised caution in interpreting their significance due to the absence of a clear brain-behavioral relevance in these regions. We have added the connection of our work to prior literature in the discussion. The parametric encoding of conflict also mirrors prior research showing the parametric encoding of task demands (Dagher et al., 1999; Ritz & Shenhav, 2023).

However, our analyses extend beyond solely testing the parametric encoding of difficulty. Instead, we focused on the multivariate representation of different conflict types, which we believe is independent from the univariate parametric encoding. Unlike the univariate encoding that relies on the strength within one dimension, the multivariate representation of conflict types incorporates both the spatial Stroop and Simon dimensions. Furthermore, we found that similar difficulty levels did not yield similar conflict representation, as indicated by the low similarity between the spatial Stroop and Simon conditions, despite both showing a similar level of congruency effect (Fig. S1). Additionally, we also observed an interaction between conflict similarity and difficulty (i.e., congruency, Fig. 4B/D), such that the conflict similarity effect was more pronounced when conflict was present. Therefore, we believe that our findings make contribution to the literature beyond the difficulty effect.

Reference:

Egner, T. (2008). Multiple conflict-driven control mechanisms in the human brain. Trends in Cognitive Sciences, 12(10), 374-380. https://doi.org/10.1016/j.tics.2008.07.001

Yang, G., Nan, W., Zheng, Y., Wu, H., Li, Q., & Liu, X. (2017). Distinct cognitive control mechanisms as revealed by modality-specific conflict adaptation effects. Journal of Experimental Psychology: Human Perception and Performance, 43(4), 807-818. https://doi.org/10.1037/xhp0000351

Spapé MM, Hommel B (2008). He said, she said: episodic retrieval induces conflict adaptation in an auditory Stroop task. Psychonomic Bulletin Review,15(6):1117-21. https://doi.org/10.3758/PBR.15.6.1117

Hazeltine E, Lightman E, Schwarb H, Schumacher EH (2011). The boundaries of sequential modulations: evidence for set-level control. Journal of Experimental Psychology: Human Perception & Performance. 2011 Dec;37(6):1898-914. https://doi.org/10.1037/a0024662

Braem, S., Abrahamse, E. L., Duthoo, W., & Notebaert, W. (2014). What determines the specificity of conflict adaptation? A review, critical analysis, and proposed synthesis. Frontiers in Psychology, 5, 1134. https://doi.org/10.3389/fpsyg.2014.01134

Kikumoto A, Mayr U. (2020). Conjunctive representations that integrate stimuli, responses, and rules are critical for action selection. Proceedings of the National Academy of Sciences, 117(19):10603-10608. https://doi.org/10.1073/pnas.1922166117.

Mill, R. D., & Cole, M. W. (2023). Neural representation dynamics reveal computational principles of cognitive task learning. bioRxiv. https://doi.org/10.1101/2023.06.27.546751

Dagher, A., Owen, A. M., Boecker, H., & Brooks, D. J. (1999). Mapping the network for planning: a correlational PET activation study with the Tower of London task. Brain, 122 ( Pt 10), 1973-1987. https://doi.org/10.1093/brain/122.10.1973

Ritz, H., & Shenhav, A. (2023). Orthogonal neural encoding of targets and distractors supports multivariate cognitive control. https://doi.org/10.1101/2022.12.01.518771

1. (Public Reviews) The degree of Stroop vs Simon conflict is perfectly negatively correlated across conditions. This limits their interpretation of an integrated cognitive space, as they cannot separately measure Stroop and Simon effects. The author's control analyses have limited ability to overcome this task limitation. While these results are consistent with parametric encoding, they cannot adjudicate between combined vs separated representations.

(Recommendations) I think that it is still an issue that the task's two features (stroop and simon conflict) are perfectly correlated. This fundamentally limits their ability to measure the similarity in these features. The authors provide several control analyses, but I think these are limited.

Response: We need to acknowledge that the spatial Stroop and Simon components in the five conflict conditions were not “perfectly” correlated, with r = –0.89. This leaves some room for the preliminary model comparison to adjudicate between these models. However, it’s essential to note that conclusions based on these results must be tempered. In line with the reviewer’s observation, we agree that the high correlation between the two conflict sources posed a potential limitation on our ability to independently investigate the contribution of spatial Stroop and Simon conflicts. Therefore, in addition to the limitation we have previously acknowledged, we have now further revised our conclusion and adjusted our expressions accordingly.

Specifically, we now regard the parametric encoding of cognitive control not as direct evidence of the cognitive space view but as preliminary evidence that led us to propose this hypothesis, which requires further testing. Notably, we have also modified the title from “Conflicts are represented in a cognitive space to reconcile domain-general and domain-specific cognitive control” to “Conflicts are parametrically encoded: initial evidence for a cognitive space view to reconcile the debate of domain-general and domain-specific cognitive control”. Also, we revised the conclusion as: In sum, we showed that the cognitive control can be parametrically encoded in the right dlPFC and guides cognitive control to adjust goal-directed behavior. This finding suggests that different cognitive control states may be encoded in an abstract cognitive space, which reconciles the long-standing debate between the domain-general and domain-specific views of cognitive control and provides a parsimonious and more broadly applicable framework for understanding how our brains efficiently and flexibly represents multiple task settings.

From Recommendations The authors perform control analyses that test stroop-only and simon-only models. However, these analyses use a totally different similarity metric, that's based on set intersection rather than geometry. This metric had limited justification or explanation, and it's not clear whether these models fit worse because of the similarity metric. Even here, Simon-only model fit better than Stroop+Simon model. The dimensionality analyses may reflect the 1d manipulation by the authors (i.e. perfectly corrected stroop and simon effects).

Response: The Jaccard measure is the most suitable method we can conceive of for assessing the similarity between two conflicts when establishing the Stroop-only and Simon-only models, achieved by projecting them onto the vertical or horizontal axes, respectively (Author response image 1A). This approach offers two advantages. First, the Jaccard similarity combines both similarity (as reflected by the numerator) and distance (reflected by the difference between denominator and numerator) without bias towards either. Second, the Jaccard similarity in our design is equivalent to the cosine similarity because the denominator in the cosine similarity is identical to the denominator in the Jaccard similarity (both are the radius of the circle, Author response image 1B).

Author response image 1.

Definition of Jaccard similarity. A) Two conflicts (1 and 2) are projected onto the spatial Stroop/Simon axis in the Stroop/Simon-only model, respectively. The Jaccard similarity for Stroop-only and Simon-only model are and respectively. Letters a-d are the projected vectors from the two conflicts to the two axes. Blue and red colors indicate the conflict conditions. Shorter vectors are the intersection and longer vectors are the union. B) According to the cosine similarity model, the similarity is defined as , where e is the projected vector from conflict 1 to conflict 2, and g is the vector of conflict 1. The Jaccard similarity for this case is defined by , where f is the projector vector from conflict 2 to itself. Because f = g in our design, the Jaccard similarity is equivalent to the cosine similarity.

Therefore, we believe that the model comparisons between cosine similarity model and the Stroop/Simon-Only models were equitable. However, we acknowledge the reviewer’s and other reviewers’ concerns about the correlation between spatial Stroop and Simon conflicts, which reduces the space to one dimension (1d) and limits our ability to distinguish between the Stroop-only and Simon-only models, as well as between Stroop+Simon and cosine similarity models. While these distinctions are undoubtedly important for understanding the geometry of the cognitive space, we recognize that they go beyond the major objective of this study, that is, to differentiate the cosine similarity model from domain-general/specific models. Therefore, we have chosen to exclude the Stroop-only, Simon-only and Stroop+Simon models in our revised manuscript.

Something that raised additional concerns are the RSMs in the key region of interest (Fig S5). The pure stroop task appears to be represented very differently from all of the conditions that include simon conflict.

Together, I think these limitations reflect the structure of the task and research goals, not the statistical approach (which has been meaningfully improved).

Response: We appreciate the reviewer for pointing this out. It is essential to clarify that our conclusions were based on the significant similarity modulation effect identified in our statistical analysis using the cosine similarity model, where we did not distinguish between the within-Stroop condition and the other four within-conflict conditions (Fig. 7A, now Fig. 8A). This means that the representation of conflict type was not biased by the seemingly disparities in the values shown here. Moreover, to specifically test the differences between the within-Stroop condition and the other within-conflict conditions, we conducted a mixed-effect model analysis only including trial pairs from the same conflict type. In this analysis, the primary predictor was the cross-condition difference (0 for within-Stroop condition and 1 for other within-conflict conditions). The results showed no significant cross-condition difference in either the incongruent (t = 1.22, p = .23) or the congruent (t = 1.06, p = .29) trials. Thus, we believe the evidence for different similarities is inconclusive in our data and decided not to interpret this numerical difference. We have added this note in the revised figure caption for Figure S5.

Author response image 2.

Fig. S5. The stronger conflict type similarity effect in incongruent versus congruent conditions. (A) Summary representational similarity matrices for the right 8C region in incongruent (left) and congruent (right) conditions, respectively. Each cell represents the averaged Pearson correlation of cells with the same conflict type and congruency in the 1400×1400 matrix. Note that the seemingly disparities in the values of Stroop and other within-conflict cells (i.e., the diagonal) did not reach significance for either incongruent (t = 1.22, p = .23) or congruent (t = 1.06, p = .29) trials. (2) Scatter plot showing the averaged neural similarity (Pearson correlation) as a function of conflict type similarity in both conditions. The values in both A and B are calculated from raw Pearson correlation values, in contrast to the z-scored values in Fig. 4D.

Minor:

• In the analysis of similarity_orientation, the df is very large (~14000). Here, and throughout, the df should be reflective of the population of subjects (ie be less than the sample size).

Response: The large degrees of freedom (df) in our analysis stem from the fact that we utilized a mixed-effect linear model, incorporating all data points (a total of 400×35=14000). In mixed-effect models, the df is determined by subtracting the number of fixed effects (in our case, 7) from the total number of observations. Notably, we are in line with the literature that have reported the df in this manner (e.g., Iravani et al., 2021; Schmidt & Weissman, 2015; Natraj et al., 2022).

Reference:

Iravani B, Schaefer M, Wilson DA, Arshamian A, Lundström JN. The human olfactory bulb processes odor valence representation and cues motor avoidance behavior. Proc Natl Acad Sci U S A. 2021 Oct 19;118(42):e2101209118. https://doi.org/10.1073/pnas.2101209118.

Schmidt, J.R., Weissman, D.H. Congruency sequence effects and previous response times: conflict adaptation or temporal learning?. Psychological Research 80, 590–607 (2016). https://doi.org/10.1007/s00426-015-0681-x.

Natraj, N., Silversmith, D. B., Chang, E. F., & Ganguly, K. (2022). Compartmentalized dynamics within a common multi-area mesoscale manifold represent a repertoire of human hand movements. Neuron, 110(1), 154-174. https://doi.org/10.1016/j.neuron.2021.10.002.

• it would improve the readability if there was more didactic justification for why analyses are done a certain way (eg justifying the jaccard metric). This will help less technically-savvy readers.

Response: We appreciate the reviewer’s suggestion. However, considering the Stroop/Simon-only models in our design may not be a valid approach for distinguishing the contributions of the Stroop/Simon components, we have decided not to include the Jaccard metrics in our revised manuscript.

Besides, to improve the readability, we have moved Figure S4 to the main text (labeled as Figure 7), and added the domain-general/domain-specific schematics in Figure 8.

Author response image 3.

Figure 8. Schematic of key RSMs. (A) and (B) show the orthogonality between conflict similarity and orientation RSMs. The within-subject RSMs (e.g., Group1-Group1) for conflict similarity and orientation are all the same, but the cross-group correlations (e.g., Group2-Group1) are different. Therefore, we can separate the contribution of these two effects when including them as different regressors in the same linear regression model. (C) and (D) show the two alternative models. Like the cosine model (A), within-group trial pairs resemble between-group trial pairs in these two models. The domain-specific model is an identity matrix. The domain-general model is estimated from the absolute difference of behavioral congruency effect, but scaled to 0(lowest similarity)-1(highest similarity) to aid comparison. The plotted matrices here include only one subject each from Group 1 and Group 2. Numbers 1-5 indicate the conflict type conditions, for spatial Stroop, StHSmL, StMSmM, StLSmH, and Simon, respectively. The thin lines separate four different sub-conditions, i.e., target arrow (up, down) × congruency (incongruent, congruent), within each conflict type.

Reviewer #2:

This study examines the construct of "cognitive spaces" as they relate to neural coding schemes present in response conflict tasks. The authors use a novel experimental design in which different types of response conflict (spatial Stroop, Simon) are parametrically manipulated. These conflict types are hypothesized to be encoded jointly, within an abstract "cognitive space", in which distances between task conditions depend only on the similarity of conflict types (i.e., where conditions with similar relative proportions of spatial-Stroop versus Simon conflicts are represented with similar activity patterns). Authors contrast such a representational scheme for conflict with several other conceptually distinct schemes, including a domain-general, domain-specific, and two task-specific schemes. The authors conduct a behavioral and fMRI study to test which of these coding schemes is used by prefrontal cortex. Replicating the authors' prior work, this study demonstrates that sequential behavioral adjustments (the congruency sequence effect) are modulated as a function of the similarity between conflict types. In fMRI data, univariate analyses identified activation in left prefrontal and dorsomedial frontal cortex that was modulated by the amount of Stroop or Simon conflict present, and representational similarity analyses (RSA) that identified coding of conflict similarity, as predicted under the cognitive space model, in right lateral prefrontal cortex.

This study tackles an important question regarding how distinct types of conflict might be encoded in the brain within a computationally efficient representational format. The ideas postulated by the authors are interesting ones and the statistical methods are generally rigorous.

Response: We would like to express our sincere appreciation for the reviewer’s positive evaluation of our manuscript and the constructive comments and suggestions. In response to your suggestions and concerns, we excluded the StroopOnly, SimonOnly and Stroop+Simon models, and added the schematic of domain-general/specific model RSMs. We have provided detailed responses to your comments below.

The evidence supporting the authors claims, however, is limited by confounds in the experimental design and by lack of clarity in reporting the testing of alternative hypotheses within the method and results.

1. Model comparison

The authors commendably performed a model comparison within their study, in which they formalized alternative hypotheses to their cognitive space hypothesis. We greatly appreciate the motivation for this idea and think that it strengthened the manuscript. Nevertheless, some details of this model comparison were difficult for us to understand, which in turn has limited our understanding of the strength of the findings.

The text indicates the domain-general model was computed by taking the difference in congruency effects per conflict condition. Does this refer to the "absolute difference" between congruency effects? In the rest of this review, we assume that the absolute difference was indeed used, as using a signed difference would not make sense in this setting. Nevertheless, it may help readers to add this information to the text.

Response: We apologize for any confusion. The “difference” here indeed refers to the “absolute difference” between congruency effects. We have now clarified this by adding the word “absolute” accordingly.

"Therefore, we defined the domain-general matrix as the absolute difference in their congruency effects indexed by the group-averaged RT in Experiment 2."

Regarding the Stroop-Only and Simon-Only models, the motivation for using the Jaccard metric was unclear. From our reading, it seems that all of the other models --- the cognitive space model, the domain-general model, and the domain-specific model --- effectively use a Euclidean distance metric. (Although the cognitive space model is parameterized with cosine similarities, these similarity values are proportional to Euclidean distances because the points all lie on a circle. And, although the domain-general model is parameterized with absolute differences, the absolute difference is equivalent to Euclidean distance in 1D.) Given these considerations, the use of Jaccard seems to differ from the other models, in terms of parameterization, and thus potentially also in terms of underlying assumptions. Could authors help us understand why this distance metric was used instead of Euclidean distance? Additionally, if Jaccard must be used because this metric seems to be non-standard in the use of RSA, it would likely be helpful for many readers to give a little more explanation about how it was calculated.

Response: We believe that the Jaccard similarity measure is consistent with the Cosine similarity measure. The Jaccard similarity is calculated as the intersection divided by the union. To define the similarity of two conflicts in the Stroop-only and Simon-only models, we first project them onto the vertical or horizontal axes, respectively (as shown in Author response image 1A). The Jaccard similarity in our design is equivalent to the cosine similarity because the denominator in the Jaccard similarity is identical to the denominator in the cosine similarity (both are the radius of the circle, Author response image 1B).

However, it is important to note that a cosine similarity cannot be defined when conflicts are projected onto spatial Stroop or Simon axis simultaneously. Therefore, we used the Jaccard similarity in the previous version of our manuscript.

Author response image 4.

Definition of Jaccard similarity. A) Two conflicts (1 and 2) are projected onto the spatial Stroop/Simon axis in the Stroop/Simon-only model, respectively. The Jaccard similarity for Stroop-only and Simon-only model are and respectively. Letters a-d are the projected vectors from the two conflicts to the two axes. Blue and red colors indicate the conflict conditions. Shorter vectors are the intersection and longer vectors are the union. B) According to the cosine similarity model, the similarity is defined as , where e is the projected vector from conflict 1 to conflict 2, and g is the vector of conflict 1. The Jaccard similarity for this case is defined by , where f is the projector vector from conflict 2 to itself. Because f = g in our design, the Jaccard similarity is equivalent to the cosine similarity.

However, we agree with the reviewer’s and other reviewers’ concern that the correlation between spatial Stroop and Simon conflicts makes it less likely to distinguish the Stroop+Simon from cosine similarity models. While distinguishing them is essential to understand the detailed geometry of the cognitive space, it is beyond our major purpose, that is, to distinguish the cosine similarity model with the domain-general/specific models. Therefore, we have chosen to exclude the Stroop-only, Simon-only and Stroop+Simon models from our revised manuscript.

When considering parameterizing the Stroop-Only and Simon-Only models with Euclidean distances, one concern we had is that the joint inclusion of these models might render the cognitive space model unidentifiable due to collinearity (i.e., the sum of the Stroop-Only and Simon-Only models could be collinear with the cognitive space model). Could the authors determine whether this is the case? This issue seems to be important, as the presence of such collinearity would suggest to us that the design is incapable of discriminating those hypotheses as parameterized.

Response: We acknowledge that our design does not allow for a complete differentiation between the parallel encoding (StroopOnly+SimonOnly) model and the cognitive space model, given their high correlation (r = 0.85). However, it is important to note that the StroopOnly+SimonOnly model introduces more free parameters, making the model fitting poorer than the cognitive space model.

Additionally, the cognitive space model also shows high correlations with the StroopOnly and SimonOnly models (both rs = 0.66). It is crucial to emphasize that our study’s primary goal does not involve testing the parallel encoding hypothesis (through the StroopOnly+SimonOnly model). As a result, we have chosen to remove the model comparison results with the StroopOnly, SimonOnly and StroopOnly+SimonOnly models. Instead, the cognitive space model shows lower correlation with the purely domain-general (r = −0.16) and domain-specific (r = 0.46) models.

1. Issue of uniquely identifying conflict coding

We certainly appreciate the efforts that authors have taken to address potential confounders for encoding of conflict in their original submission. We broach this question not because we wish authors to conduct additional control analyses, but because this issue seems to be central to the thesis of the manuscript and we would value reading the authors' thoughts on this issue in the discussion.

To summarize our concerns, conflict seems to be a difficult variable to isolate within aggregate neural activity, at least relative to other variables typically studied in cognitive control, such as task-set or rule coding. This is because it seems reasonable to expect that many more nuisance factors covary with conflict -- such as univariate activation, level of cortical recruitment, performance measures, arousal --- than in comparison with, for example, a well-designed rule manipulation. Controlling for some of these factors post-hoc through regression is commendable (as authors have done here), but such a method will likely be incomplete and can provide no guarantees on the false positive rate.

Relatedly, the neural correlates of conflict coding in fMRI and other aggregate measures of neural activity are likely of heterogeneous provenance, potentially including rate coding (Fu et al., 2022), temporal coding (Smith et al., 2019), modulation of coding of other more concrete variables (Ebitz et al., 2020, 10.1101/2020.03.14.991745; see also discussion and reviews of Tang et al., 2016, 10.7554/eLife.12352), or neuromodulatory effects (e.g., Aston-Jones & Cohen, 2005). Some of these origins would seem to be consistent with "explicit" coding of conflict (conflict as a representation), but others would seem to be more consistent with epiphenomenal coding of conflict (i.e., conflict as an emergent process). Again, these concerns could apply to many variables as measured via fMRI, but at the same time, they seem to be more pernicious in the case of conflict. So, if authors consider these issues to be germane, perhaps they could explicitly state in the discussion whether adopting their cognitive space perspective implies a particular stance on these issues, how they interpret their results with respect to these issues, and if relevant, qualify their conclusions with uncertainty on these issues.

Response: We appreciate the reviewer’s insightful comments regarding the representation and process of conflict.

First, we agree that the conflict is not simply a pure feature like a stimulus but often arises from the interaction (e.g., dimension overlap) between two or more aspects. For example, in the manual Stroop, conflict emerges from the inconsistent semantic information between color naming and word reading. Similarly, other higher-order cognitive processes such as task-set also underlie the relationship between concrete aspects. For instance, in a face/house categorization task, the taskset is the association between face/house and the responses. When studying these higher-order processes, it is often impossible to completely isolate them from bottomup features. Therefore, methods like the representational similarity analysis and regression models are among the limited tools available to attempt to dissociate these concrete factors from conflict representation. While not perfect, this approach has been suggested and utilized in practice (Freund et al., 2021).

Second, we agree that conflict can be both a representation and an emerging process. These two perspectives are not necessarily contradictory. According to David Marr’s influential three-level theory (Marr, 1982), representation is the algorithm of the process to achieve a goal based on the input. Therefore, a representation can refer to not only a static stimulus (e.g., the visual representation of an image), but also a dynamic process. Building on this perspective, we posit that the representation of cognitive control consists of an array of dynamic representations embedded within the overall process. A similar idea has been proposed that the abstract task profiles can be progressively constructed as a representation in our brain (Kikumoto & Mayr, 2020).

We have incorporated this discussion into the manuscript:

"Recently an interesting debate has arisen concerning whether cognitive control should be considered as a process or a representation (Freund, Etzel, et al., 2021). Traditionally, cognitive control has been predominantly viewed as a process. However, the study of its representation has gained more and more attention. While it may not be as straightforward as the visual representation (e.g., creating a mental image from a real image in the visual area), cognitive control can have its own form of representation. An influential theory, Marr’s (1982) three-level model proposed that representation serves as the algorithm of the process to achieve a goal based on the input. In other words, representation can encompass a dynamic process rather than being limited to static stimuli. Building on this perspective, we posit that the representation of cognitive control consists of an array of dynamic representations embedded within the overall process. A similar idea has been proposed that the representation of task profiles can be progressively constructed with time in the brain (Kikumoto & Mayr, 2020)."

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