Con base en el video Object Oriented Programming is not what I thought, la comunicación genera interacción entre los objetos, lo cual le permite al programador contar con los mecanismos necesarios para resolver problemas del mundo real a través de un paradigma que es moldeable, y que de alguna manera, genera relación con otros objetos.

This kind of reminds me of how people try to be "race-blind" when educating students, but in a way, this kind of discounts the experiences that they go through. The cultural aspects and environment that each student goes through gets completely ignored even if it is important to understanding their learning style or behavioral patterns.

Looking at this sentence made me wonder if anything is politically neutral. When beliefs and politics have become so intertwined, there is no separation between politics and every other aspect of society. Being more open-minded and preferring change is linked with being liberal while being more comfortable with the status quo or disliking change is associated with being conservative. Also, the Department of Education is an extension of the government which is by definition, entangled with politics.

Again, Hooks acknowledges the emotional toll of critical pedagogy, where students confront familial or internalized biases. This estrangement is necessary for growth but requires educators to hold space for discomfort (they just got to accept it). It's also ironic to note that transformation often begins with rupture, yet too few institutions prioritize this messy, yet vital work.

O princípio da anterioridade tributária, tanto geral quanto nonagesimal, é aplicável aos casos em que a diminuição ou a extinção de benefícios ou incentivos fiscais acarrete um aumento indireto do valor dos tributos a serem pagos, observadas as determinações e as exceções constitucionais para cada tributo. - O princípio da anterioridade (1) busca assegurar a previsibilidade da relação fiscal, de forma a evitar que os contribuintes sejam surpreendidos por aumentos repentinos de seus encargos tributários e a permitir um planejamento financeiro adequado, em respeito ao princípio da segurança jurídica. - Com base nesse entendimento, o Plenário, por unanimidade, reconheceu a existência de repercussão geral da questão constitucional suscitada (Tema 1.383 da repercussão geral), bem como (i) reafirmou a jurisprudência dominante sobre a matéria (2) para negar provimento ao recurso extraordinário; e (ii) fixou a tese anteriormente citada.

(1) CF/1988: “Art. 150. Sem prejuízo de outras garantias asseguradas ao contribuinte, é vedado à União, aos Estados, ao Distrito Federal e aos Municípios: (...) III - cobrar tributos: (...) b) no mesmo exercício financeiro em que haja sido publicada a lei que os instituiu ou aumentou; (Vide Emenda Constitucional nº 3, de 1993) c ) antes de decorridos noventa dias da data em que haja sido publicada a lei que os instituiu ou aumentou, observado o disposto na alínea b; (Incluído pela Emenda Constitucional nº 42, de 19.12.2003)”

(2) Precedentes citados: RE 564.225 AgR-EDv-AgR-ED, ADI 2.325, ARE 1.339.119 AgR, ARE 1.065.092 AgR, ARE 983.821 AgR, RE 1.081.041 AgR, RE 1.105.918 AgR, RE 1.087.365, RE 1.193.854 AgR-ED, RE 1.214.919 AgR e RE 1.193.858 AgR. RE 1.473.645/PA, relator Ministro Presidente, julgamento finalizado no Plenário Virtual em 21.03.2025 (sexta-feira)

É inconstitucional — pois afronta o dever de proteção ao meio ambiente (CF/1988, art. 225) — dispositivo de lei federal que, ao modificar o processo de compra de ouro, presume a legalidade da aquisição e a boa-fé do adquirente.

• Esta Corte já declarou a inconstitucionalidade de normas que, a pretexto de desburocratizar o licenciamento ambiental, afastam ou enfraquecem o controle prévio de empreendimentos que impactam o meio ambiente (1). Na espécie, as presunções trazidas no diploma legislativo impugnado relativas à legalidade do ouro adquirido e à boa-fé do adquirente sabotam a efetividade do controle de uma atividade inerentemente poluidora.
• Nessa medida, elas deixam de observar o princípio da precaução (2), porque não apenas facilitam, mas servem de incentivo à comercialização de ouro originário de garimpo ilegal. Revelam-se, portanto, opção normativa deficiente quanto à proteção do meio ambiente. A simplificação do processo de compra de ouro da norma impugnada permitiu a expansão do comércio ilegal e fortaleceu as atividades de garimpo ilegal, o que contribuiu com o desmatamento, a contaminação de rios, a violência nas regiões de garimpo e o aumento da criminalidade e da insegurança na região, em detrimento, também, das tribos indígenas.
• Com base nesses e em outros entendimentos, o Plenário, em apreciação conjunta e por unanimidade, conheceu integralmente da ADI 7.273/DF e, em parte, da ADI 7.345/DF e as julgou procedentes, para:

(i) declarar a inconstitucionalidade do § 4º do art. 39 da 10 SUMÁRIO INFORMATIVO STF EDIÇÃO 1170/2025 | Lei nº 12.844/2013 (3);

(ii) determinar ao Poder Executivo federal, em especial à Agência Nacional de Mineração – ANM, ao Banco Central do Brasil – BACEN, ao Instituto Chico Mendes de Conservação da Biodiversidade – ICMBio e à Casa da Moeda do Brasil – CMB, sem prejuízo da atuação de outros órgãos, dentro das respectivas áreas de competência, a adoção de medidas regulatórias e/ou administrativas de forma a inviabilizar a extração e a aquisição de ouro garimpado em áreas de proteção ambiental e terras indígenas, estabelecendo, inclusive, diretrizes normativas para a fiscalização do comércio do ouro, especialmente quanto à verificação da origem legal do ouro adquirido por Distribuidora de Títulos e Valores Mobiliários.

(1 ) Precedente citado: ADI 5.312. (2) Precedente citado: ADI 5.447. (3) Lei 12.844/2013: “Art. 39. A prova da regularidade da primeira aquisição de ouro produzido sob qualquer regime de aproveitamento será feita com base em: (...) § 4º Presumem-se a legalidade do ouro adquirido e a boa-fé da pessoa jurídica adquirente quando as informações mencionadas neste artigo, prestadas pelo vendedor, estiverem devidamente arquivadas na instituição legalmente autorizada a realizar a compra de ouro”. ADI 7.273/DF, relator Ministro Gilmar Mendes, julgamento virtual finalizado em 21.03.2025 (sexta-feira), às 23:59 ADI 7.345/DF, relator Ministro Gilmar Mendes, julgamento virtual finalizado em 21.03.2025 (sexta-feira), às 23:59

• Tema: 1.220
• Processo(s): RE 1.326.559
• Relator: Min. Dias Toffoli
• Título: Declaração de inconstitucionalidade parcial, sem redução de texto, do § 14 do artigo 85 do CPC/2015 para se afastar a possibilidade de ser atribuída preferência de pagamento a honorários advocatícios em relação ao crédito tributário.

O Tribunal fixou a seguinte tese:

• É formalmente constitucional o § 14 do art. 85 do Código de Processo Civil no que diz respeito à preferência dos honorários advocatícios, inclusive contratuais, em relação ao crédito tributário, considerando-se o teor do art. 186 do CTN.

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public review): Summary:

The authors demonstrate that two human preproprotein human mutations in the BMP4 gene cause a defect in proprotein cleavage and BMP4 mature ligand formation, leading to hypomorphic phenotypes in mouse knock-in alleles and in Xenopus embryo assays.

Strengths:

They provide compelling biochemical and in vivo analyses supporting their conclusions, showing the reduced processing of the proprotein and concomitant reduced mature BMP4 ligand protein from impressively mouse embryonic lysates. They perform excellent analysis of the embryo and post-natal phenotypes demonstrating the hypomorphic nature of these alleles. Interesting phenotypic differences between the S91C and E93G mutants are shown with excellent hypotheses for the differences. Their results support that BMP4 heterodimers act predominantly throughout embryogenesis whereas BMP4 homodimers play essential roles at later developmental stages.

Weaknesses:

(1) A control of BMP7 alone in the Xenopus assays seems important to excludeBMP7 homodimer activity in these assays.

We and other have shown that BMP7 homodimers have weak or no activity while BMP4/7 heterodimers single at a much higher level than either BMP4 or BMP7 homodimers in Xenopus ectodermal and mesodermal cells. We have expanded the description of these published findings in the results section (lines 182-187). We have also added representative examples of experiments in which BMP4 and BMP7 alone controls are included (new Fig. S2). Since the level of activity of BMP7 + BMP4 variants is equivalent to that of BMP7 + WT BMP4, this cannot be accounted for by BMP7 homodimers.

(2) The Discussion could be strengthened by more in-depth explanations of how BMP4 homodimer versus heterodimer signaling is supported by the results, so that readers do not have to think it all through themselves. Similarly, a discussion of why the S91C mutant has a stronger phenotype than E93G early in the Discussion would be helpful or least mention that it will be addressed later.

We have revised the discussion as suggested by the reviewer. Please see responses to recommendations 2-4 below.

Reviewer #1 (Recommendations for the authors):

(1) A control of BMP7 injection alone seems missing when comparing the BMP4/7 variants. BMP4 in the embryo assays presented in Fig 1. Is it not possible that the activity observed is BMP7 homodimers, perhaps due to inhibited heterodimer formation by the BMP4 variant?

Multiple published studies have shown that BMP7 homodimers have weak or no activity in Xenopus ectodermal and mesodermal cells, and that ½ dose of RNA encoding BMP4 and BMP7 together signals at a higher level than does a full dose of RNA encoding either BMP4 or BMP7 alone. We have expanded our description of these published findings (lines 182-187), have included additional details about RNA doses that were injected (line 156, 175, 182) and have added representative examples of experiments in which BMP4 and BMP7 controls were included in a new Figure (Fig. S2).

(2) In reading the Discussion, I was continually thinking of the stronger phenotype of the S91C mutant compared to the E93G one, although both are discussed together throughout most of the Discussion. Only at the end of the Discussion is the stronger phenotype of S91C discussed with a compelling explanation for the stronger phenotype, not related to the phosphorylation site function. I wonder if it would be better placed earlier in Discussion or at least mentioned the difference in phenotypes that will be discussed later.

We have moved the possible explanation of differences between Bmp4^S91C and Bmp4^E93G mutants to immediately follow the introductory paragraph of the results section.

(3) Along these same lines, why is it that the E93G exhibits rather normal cleavage at E10.5? Might the mechanisms of cleavage vary in different contexts with phosphorylation-dependent cleavage not functioning at early stages of development? I believe the hypothesis is that it is cleaved due to heterodimerization with BMP7. More discussion of this excellent hypothesis should be provided with clear statements, rather than inferences, if I'm understanding this correctly. For example, I had to read 3 times the first sentence of the last paragraph on p.14 before I understood it. Better to break that sentence down and the one that follows it, so it is easier to understand.

We have rewritten and expanded the paragraphs describing phenotypic and biochemical evidence for defective homodimer but not heterodimer signaling as suggested (lines 343-375). We have also more explicitly stated the possibility that normal cleavage of BMP4^E93G in embryonic lystates may be due to a predominance of BMP4/7 heterodimers in early embryonic stages or spatiotemporal differences in phosphorylation-dependent cleavage of BMP4 homodimers (lines 369-372)

(4) Similarly the last paragraph of the Discussion mentions that the authors provide evidence of BMP4 homodimer signaling. I agree with the authors, but I had to think through the evidence myself. Better if the authors clearly explain the evidence that points to this, as this is a very good point of

See response to point 3, above. Thank you for these useful suggestions.

(5) Last sentence, first paragraph on p.11 should be qualified for the E93G mutant to E13.5, since it was normal at E10.5 regarding Figure 4 results.

Thank you for pointing this out. It has been corrected.

(6) Skip the PC acronym, since it is only repeated once in the text and hard to remember almost 10 pages later when it is used again.

We have corrected this.

(7) In the Discussion, a typo in "a single intramolecular disulfide bond that stabilizes the dimer", should be 'intermolecular'.

Thank you for catching our switch in the use of inter- and intramolecular. We have corrected this (lines 334-335).

(8) At times the E93G mutant is referred to having early lethality, often in conjunction with S91C, while other times it is referred to as late lethality. Considering that the homozygotes die postnatally after weaning, most would consider it late lethality. In contrast S91C is indeed an early lethal.

We have changed the wording in the introduction to state that “mice carrying Bmp4^S91C or Bmp4^E93G knock in mutations show embryonic or enhanced postnatal lethality, respectively,… (lines 141-143)” and have removed the word “early” from the title.

Reviewer #2 (Public review): Summary:

Kim et al. report that two disease mutations in proBMP4, Ser91Cys and Glu93Gly, which disrupt the Ser91 FAM20C phosphorylation site, block the activation of proBMP4 homodimers. Consequently, analysis of DMZ explants from Xenopus embryos expressing the proBMP4 S91C or E93G mutants showed reduced pSmad1 and tbxt1 expression. The block in BMP4 activity caused by the mutations could be overcome by co-expression of BMP7, suggesting that the missense mutations selectively affect the activity of BMP4 homodimers but not BMP4/7 heterodimers. The expert amphibian tissue transplant studies were extended to in vivo studies in Bmp4S91C/+ and Bmp4E93G/+ mice, demonstrating the impact of these mutations on embryonic development, particularly in female mice, in line with patient studies. Finally, studies in MEFs revealed that the mutations did not affect proBMP4 glycosylation or ER-to-Golgi transport but appeared to inhibit the furin-dependent cleavage of proBMP4 to BMP4. Based on these findings and AI (AlphaFold) modeling of proBMP4, the authors speculate that pSer91 influences access of furin to its cleavage site at Arg289AlaLysArg292.

Strengths:

The Xenopus and mouse studies are valuable and elegantly describe the impact of the S91C and E93G disease mutations on BMP signaling and embryonic development.

Weaknesses:

The interpretation of how the mutations may disturb the furin-mediated cleavage of proBMP4 is underdeveloped and does not consider all of their data. Understanding how pS91 influences the furin-dependent cleavage at Arg292 seems to be the crux of this work and thus warrants more consideration. Specifically:

(1) Figure S1 may be significantly more informative than implied. The authors report that BMP4S91D activates pSmad1 only incrementally better than S91C and much less than WT BMP4. However, Fig. S1B does not support the conclusion on page 7 (numbering beginning with title page); "these findings suggest that phosphorylation of S91 is required to generate fully active BMP4 homodimers". The authors rightly note that the S91C change likely has manifold effects beyond inhibiting furin cleavage. The E93G change may also affect proBMP4 beyond disturbing FAM20C phosphorylation. Additional mutation analyses would strengthen the work.

The major goal of generating and comparing the activity of the S91D mutant with S91C was to control for phosphorylation independent defects cause by the deleterious introduction of a cysteine residue, which might cause aberrant disulfide bonding. We opted to introduce S91D since “phosphomimics” can sometimes approximate the phosphorylated state. S91D has significantly higher activity than S91C (p<0.01) and has a less significant loss of activity (p<0.05) than does S91C (<p<0.0001) relative to wild type BMP4 (Fig. S1), consistent with deleterious effects of the cysteine residue and supporting a possible explanation for the more severe phenotype of S91C vs E93G mice. We have rewritten this section to clarify our interpretation (lines 165-174)and have changed our statement that our activity data “suggest the importance of phosphorylation” to a statement that they are consistent with this possibility (lines 179-180). We do not believe that further mutational analysis using activity assays in Xenopus would shed light on how or whether phosphorylation affects proteolytic activation of BMP4.

(2) These findings in Figure S1 are potentially significant because they may inform how proBMP4 is protected from cleavage during transit through the TGN and entry into peripheral cellular compartments. Intriguing modeling studies in Figure 6 suggest that pSer91 is proximal to the furin cleavage site. Based on their presentation, pSer91 may contact Arg289, the critical P4 residue at the furin site. If so, might that suggest how pS91 may prevent furin cleavage, thus explaining why the S91D mutation inhibits processing as presented, and possibly how proBMP4 processing is delayed until transit to distal compartments (perhaps activated by a change in the endosomal microenvironment or a Ser91 phosphatase)? Have the authors considered or ruled out these possibilities? In addition to additional mutation analyses of the FAM20C site, moving the discussion of this model to an "Ideas and Speculation" subsection may be warranted.

The model shown in Fig. 6B proposes the possibility that phosphorylation unmasks (rather than preventing) the furin cleavage motif due to the proximity of Ser91 to the cleavage site (lines 399-402). If S91D truly mimicked phosphorylation, we would predict it would facilitate processing rather than inhibiting it. We do not have data comparing cleavage of S91D relative to wild type BMP4 and have not generated knock in S91D mice to test this idea. While the reviewers questions are intriguing, they cannot be answered by mutational analysis of the FAM20C site and are beyond the scope of the current studies that sought to understand the impact of human pS91C and pE93G mutations and cell biological implications. We have moved the models to an “Ideas and Speculation” subsection as suggested (lines 377-414) since these models are meant to provoke further thought rather than provide definitive answers based on our data.

(3) The lack of an in vitro protease assay to test the effect of the S91 mutations on furin cleavage is problematic.

Although we routinely perform in vitro cleavage assays with recombinant furin, we don’t believe they would be informative on how S91 phosphorylation or mutation of this residue impacts cleavage since in vitro synthesized substrate used in these assays is neither dimerized not post-translationally modified, and cleavage would be tested in isolation from the endogenous trafficking environment that we propose influences cleavage.

Reviewer #2 (Recommendations for the authors):

(1) The impact of BMPS91A should be determined and paired with the S91D phosphomimic data to reveal if it causes proBMP4 to be cleaved prematurely and disturbs pSmad1 expression. Data for S93G should also be included.

Our major goal in comparing the activity of S91D with S91C was to control for phosphorylation independent defects cause by the deleterious introduction of a cysteine residue in S91C, which might cause aberrant disulfide bonding. We opted to introduce S91D since “phosphomimics” can sometimes approximate the phosphorylated state. We note that S91D has significantly higher activity than S91C, consistent with deleterious effects of the cysteine residue and supporting a possible explanation for the more severe phenotype of S91C vs E93G mice. We have revised the wording of this section to clarify this. Our models predict that S91D would be cleaved more efficiently than S91C or S91A, if it really mimics the endogenous phosphorylated state, rather than being cleaved prematurely. Our biochemical analysis compares cleavage of endogenous BMP4 in wild type and mutant MEFs. Generation of S91D, S91A or S93G mutant mice to compare cleavage is beyond the scope of the current work.

(2) Is the distance between pS91 and Arg289 close enough to form a hydrogen bond? If so, might this interaction influence furin access?

AI modeling does not provide high probability prediction of structures surrounding the furin motif (see Fig. S7) and thus we cannot comment on whether or not these residues are close enough to form a hydrogen bond. We have revised the wording of the discussion to state “This simple model building indicates the possibility of direct contact between pSer91 and Arg289, and that phosphorylation is required for furin to access the cleavage site, although we note that predictions surrounding the furin motif represent low probability conformations (Fig. S7) (lines 399-402).”

(3) The genotypes in Figure 2 are labeled awkwardly. Consider labeling the headers for the three subsections of panels (A-F, G-L, and M-O) differently.

We have revised Fig. 2 to clarify that the three subsections of panels are distinct, and to emphasize that the middle subsection represents views of the right and left side of the same embryo.

(4) The tables should be reformatted. As is, the labeling is frequently cut off, and the numbers of expected and observed progeny should both be stated to aid the reader.

We thank the reviewer for noting the formatting errors in the tables, which we have corrected. We have also changed the tables so that normal or abnormal mendelian distributions are reported as numbers of observed/expected progeny rather than numbers/percent observed progeny.

Reviewer #3 (Public review):

Summary:

The authors describe important new biochemical elements in the synthesis of a class of critical developmental signaling molecules, BMP4. They also present a highly detailed description of developmental anomalies in mice bearing known human mutations at these specific elements.

Strengths:

Exceptionally detailed descriptions of pathologies occurring in mutant mice. Novel findings regarding the interaction of propeptide phosphorylation and convertase cleavage, both of which will move the field forward. Provocative hypothesis regarding furin access to cleavage sites, supported by Alphafold predictions.

Weaknesses:

Figure 6A presents two testable models for pre-release access of furin to cleavage sites since physical separation of enzyme from substrate only occurs in one model; could immunocytochemistry resolve?

Available reagents are not sensitive enough to detect endogenous furin and BMP4 with high resolution. Because PC/substrate interactions are transient, whereas the bulk of furin and BMP4 is distributed throughout the secretory pathway, it is not possible to co-immunolocalize furin and BMP4 in vivo at present. Studies using more advanced cell biological techniques such along with tagged proteins may enable us to test these hypotheses in the future.

Reviewer #3 (Recommendations for the authors):

This interesting paper presents new data on an important family of developmental signaling molecules, BMPs. Mutations at FAM20C consensus sites within BMP prodomains are known to cause birth defects. The authors have here explored differential effects of human mutations on hetero- and homodimer activity and maturation, issues that may well arise during human development. In addition to demonstrating the profound effect of these mutations on development in Xenopus and mice, the authors also show differential processing of BMP4 precursors bearing these mutations in MEF cells prepared from mutant embryos. Finally, they show that FAM20C plays a role in BMP4 prodomain processing with quite differing outcomes in homo- vs heterodimers, which they suggest is due to structural differences impacting furin access. While this latter idea remains speculative due to the lack of crystal structures (models are based on Alphafold) it is a highly promising line of work.

The data are beautifully presented and will be of clear interest to all developmental biologists. Certain cell biology results may also extrapolate to other phosphorylated precursor molecules undergoing the interesting (and as yet unexplained) phenomenon of convertase cleavage immediately before secretion, for example, FGF23. I have only a few minor comments regarding the presentation, which is remarkably clear.

(1) The introduction of BMP7 in the Abstract is abrupt. It should be described as a preferred dimerization partner for BMP4.

Thank you for noting this. We have revised the first sentence of the abstract to better introduce BMP7(lines 49-50).

(2) In Figure 1A, what is the small light green box?

This is a small fragment released from the prodomain by the second cleavage. We have clarified this in the introduction (lines 112-114) and in the legend to Figure 1 (lines 758-759).

(3) In the Discussion it might be relevant to mention that FAM20C propeptide is not cleaved by convertases but by S1P (Chen 2021).

We have added this information to clarify (lines 394-396).

(4) Figure 3, define VSD; Figure 5, Endo H removes sugars only from immature (nonsialylated) sugars, not from all chains as implied. More importantly, EndoH and PNGase remove N-linked sugars, yet Results refer only to O-linked glycosylation.

Thank you for noting these oversights. We have defined VSD in Figure 3. We have also revised the headers for Fig. 5 and for the relevant subsection of the results to include N-linked glycosylation and note in the results that EndoH removes only immature N-linked carbohydrates (lines 301-304).

(5) Figure 5- for clarity, I suggest it be broken up into two larger panels labeled "Embryos" and "MEFs"

Thank you for this suggestion, we have subdivided the Figure into two panels.

(6) Figure 6A presents two testable models for pre-release access of furin to cleavage sites since the physical separation of the enzyme from substrate only occurs in one model; could confocal immunocytochemistry resolve?

Available reagents are not sensitive enough to detect endogenous furin and BMP4 with high resolution and PC/substrate interactions are transient whereas the bulk of both furin and BMP4 is in transit through the secretory pathway. For these reasons it is not possible to co-immunolocalize furin and BMP4 in vivo. Future studies using advanced cell biological techniques may enable us to test these hypotheses in the future.

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public Review):

Summary:

In their manuscript, Kong Fang et al describe a robust pipeline for the isolation of small extracellular vesicles through a combination of size exclusion chromatography and miniaturized density gradient separation. Subsequently, they prove that the method is reproducible and suitable for small-volume operations while at the same time not compromising the quality of vesicles.

Strengths:

The paper narrates a robust method for purifying high-quality sEVs from small amounts of blood plasma. They also demonstrate that through this approach, they can derive sEVs without compromising the protein composition, integrity of the vesicles, or contamination with other proteins or lipids.

Weaknesses:

The paper is a nice summary of how to enrich sEVs from blood samples. Although well performed and substantiated with data, the paper primarily deals with method development and optimisation.

We agree with the reviewer's assessment that this paper primarily focuses on the development and optimization of a method. Using this robust technique for isolating small extracellular vesicles (sEVs) from small blood volumes, our future research will investigate sEVs isolated from clinical samples, with a particular focus on their role in various diseases.

Reviewer #2 (Public Review):

Summary:

In this work, the authors manage to optimize a simple and rapid protocol using SEC followed by DGCU to isolate sEVs with adequate purity and yield from small volumes of plasma. Isolated fractions containing sEVs using SEC, DGCU, SEC-DGCU, and DGCU-SEC are compared in terms of their yield, purity surface protein profile, and RNA content. Although the combined use of these methodologies has already been evaluated in previous works, the authors manage to adapt them for the use of small volumes of plasma, which allows working in 1.5 mL tubes and reducing the centrifugation time to 2 hours.

The authors finally find that although both the SEC-DGCU and DGCU-SEC combinations achieve isolates with high purity, the SEC-DGCU combination results in higher yields.

This work provides an interesting tool for the rapid obtention of sEVs with sufficient yield and purity for detailed characterization which could be very useful in research and clinical therapy.

Strengths:

- The work is well-written and organized.

- The authors clearly state the problem they want to address, that is, optimizing a method that allows sEV to be isolated from small volumes of plasma.

- Although these methodologies have been tested in previous works, the authors manage to isolate sEVs of high purity and good performance through a simple and fast methodology.

- The characteristics of all isolated fractions are exhaustively analyzed through various state-of-the-art methodologies.

- They present a good interpretation of the results obtained through the methodologies used.

Weaknesses:

- Lack of references that support some of the results obtained.

- Although this work focuses on comparing different techniques and their combinations to find an optimal option, the authors do not use any statistical method that reliably shows the differences between these techniques, except when repeatability is measured.

We appreciate the reviewer's insightful comments and will incorporate the suggested missing references. We acknowledge that we did not perform statistical analyses when comparing the differences among the three methods. Nevertheless, the superiority of the SEC-DGUC method is evident from observations based on several independent characterization methods, including Cryo-EM, TEM, western blot, and total RNA quantification.

Firstly, repeated Cryo-EM observations consistently confirm that the SEC-alone method shows severe lipoprotein contamination while the SEC-DGUC method drastically reduces such lipoprotein contamination. In comparing the SEC-DGUC and DGUC-SEC methods, multiple independent characterization methods showed that the SEC-DGUC method yields significantly greater quantity of sEVs: 1) The western blot experiment showed much higher signal intensity for all four tested sEV markers (CD9, CD63, CD81, and TSG101), with estimated concentrations approximately 2.1, 2.1, 4.7, and 4.2 times higher than the DGUC-SEC method. 2) The total RNA analysis showed that SEC-DGUC-1 contained more than 4 times the total amount of RNA compared to DGUC-SEC-PF. 3) Establishing the normalization baseline, particle size distributions in SEC-DGUC-1 and DGUC-SEC-PF measured by TEM were found to be similar, suggesting comparable purity and distribution of the captured sEVs. For comparison purposes, within each independent characterization method, the same plasma source and total plasma volume were used, while across different methods, different plasma sources were used. These independent characterization methods have consistently demonstrated the superiority of the SEC-DGUC method over the DGUC-SEC or SEC-alone methods.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

In my opinion, this work is elegantly designed and supported by data, which would motivate more studies related to blood-derived microvesicles in the context of infectious and systemic diseases. Overall, the manuscript is well-written and explained in sufficient detail. I only have minor comments.

(1) Recruitment of volunteers for blood/plasma collection: there is a need for a statement that this was in accordance with ethical and biosafety regulations of the Institute/Clinic.

We added two sentences at the beginning of the Blood Collection section (under Materials and methods): “All procedures involving peripheral blood specimens were approved by the Singapore National Health Group Domain Specific Review Board (the central ethics committee) and were mutually recognized by the Nanyang Technological University Institutional Review Board (IRB#2018/00671). All blood specimens were de-identified prior to their use in the experiments.”

(2) Since this is a method development and validation article, it would be good to include an image of the iodixanol gradient with the high-density sEV zone, after centrifugation.

We have incorporated an image after centrifugation in Supplementary Figure 3.

(3) Although several sEV markers are shown in Figure 7A, flotillin is missing in this figure which was part of Figure 6B. Does flotillin show a different pattern? Flotillin is a DRM-associated marker, and hence may behave differently, would be interesting to add any insights.

We appreciate the reviewer’s careful observation. In Figure 6B, Flotillin was used to confirm the presence of sEVs in different density zones. However, for the purpose of comparing the yield between the SEC-DGUC and DGUC-SEC methods, as shown in Figure 7A, Flotillin was not included in the western blot analysis. No obvious pattern changes were observed in other sEV markers tested in both Figures 6B and 7A.   

(4) Methods section of LC/MS analysis- which protein database was used for protein identification?

We added the following sentence at the end of the LC/MS analysis section: “The protein database used for protein identification was Uniprot Human.”

Reviewer #2 (Recommendations For The Authors):

In line 43 some references are needed.

We added this reference: EL Andaloussi, S., Mäger, I., Breakefield, X. et al. Extracellular vesicles: biology and emerging therapeutic opportunities. Nat Rev Drug Discov 12, 347–357 (2013). https://doi.org/10.1038/nrd3978

In line 107, please avoid using short forms such as "it's".

We have revised that to “it is.”

In line 153: "...separates low-density particles from those of high density, but a considerable amount of..." the word "but" should not be in the sentence.

We have removed “but” in this sentence.

In line 181 the authors establish that "Notably, SEC-PF exhibited a high level of ApoB and low expression of sEV markers." Is there any explanation for this?

SEC-PF represents the eluate from the SEC step, collected before the DGUC step. This fraction contains a mixture of lipoproteins and sEVs. Due to the overwhelming abundance of lipoproteins compared to sEVs, the western blot predictably shows a high level of ApoB with minimal expression of sEV markers. This highlights that SEC alone effectively reduces plasma protein content but does not efficiently remove lipoproteins. Figure 6C further illustrates this point, as cryo-EM images of SEC-PF reveal the presence of sEVs, which are vastly outnumbered by lipoproteins.

In line 198, the sentence "Theoretically, the DGUC-SEC protocol should also effectively isolate sEVs from plasma" need to be supported by references.

See for instance:

- Holcar M, Ferdin J, Sitar S, Tušek-Žnidarič M, Dolžan V, Plemenitaš A, Žagar E, Lenassi M. 2020. Enrichment of plasma extracellular vesicles for reliable quantification of their size and concentration for biomarker discovery. Sci Rep 10:21346. doi:10.1038/s41598-020-78422-y.

- Jia Y, Yu L, Ma T, Xu W, Qian H, Sun Y, Shi H. 2022. Small extracellular vesicles isolation and separation: Current techniques, pending questions and clinical applications. Theranostics 12:6548-6575. doi:10.7150/thno.74305

- Vergauwen G, Dhondt B, Van Deun J, De Smedt E, Berx G, Timmerman E, Gevaert K, Miinalainen I, Cocquyt V, Braems G, Van den Broecke R, Denys H, De Wever O, Hendrix A. 2017. Confounding factors of ultrafiltration and protein analysis in extracellular vesicle research. Sci Rep 7:2704. doi:10.1038/s41598-017-02599-y

We have added this reference: Holcar M, Ferdin J, Sitar S, Tušek-Žnidarič M, Dolžan V, Plemenitaš A, Žagar E, Lenassi M. 2020. Enrichment of plasma extracellular vesicles for reliable quantification of their size and concentration for biomarker discovery. Sci Rep 10:21346. https://doi.org/10.1038/s41598-020-78422-y.  

In line 309 the authors establish that "NTA measured size distributions displayed well-overlapped histograms of particles". It is possible for the authors to analyze this overlapping using some statistical test as a chi-squared test?

We have conducted a statistical analysis of the histogram similarities using the Jensen-Shannon Divergence (JSD) method. This is reflected in the manuscript under the results section, “Repeatability and reliability of the SEC-DGUC protocol”, where we state: “We then compared size distributions for each plasma fraction using Jensen-Shannon Divergence (JSD). The JSD values, which are well below 0.1 (Figure 10B), indicate a consistent population of isolated particles, as further supported by Supplementary Figure 8.” Additionally, we included JSD values in the legend of Figure 10B: “JSD values for SEC-DGUC-1 to 4 are 0.015, 0.006, 0.001, and 0.002, indicating strong similarities among the histograms.” These additions demonstrate the robustness and repeatability of the SEC-DGUC protocol.

In line 360, "lasts ~ 16 hours or more." This statement needs a reference that supports this time.

We have added this reference: Vergauwen, G. et al. Robust sequential biophysical fractionation of blood plasma to study variations in the biomolecular landscape of systemically circulating extracellular vesicles across clinical conditions. J Extracell Vesicles 10, e12122 (2021).

In line 399, the reference format is different from the previously used format.

This is corrected. We thank the reviewer for this careful examination.

Line 466: This sentence is not quite clear. It can be understood that for every 0.5 mL of plasma, 2 mL of particle fraction are obtained and that for 6 mL of plasma, this method will give a total volume of 24 mL. However, it is not clear what is meant by the fact that it has been concentrated to 6 mL. While one can assume that those final 6 mL concentrates come from the initial 24 mL, perhaps the way this sentence was worded was not appropriate. I would recommend rewriting it for a simpler interpretation of how this method was performed.

We have changed the sentence to: “For the DGUC experiment using the 12 ml tube, 24 ml of PFs were obtained from 6 ml of plasma and subsequently concentrated to 6 ml. The 6 ml of concentrated PFs were then transferred to a Beckman Coulter ultra-clear centrifuge tube (344059, Beckman Coulter, USA) for further processing.”

Line 519: The authors established a second dilution to avoid absorbance values above 1.2. Is there any justification for this value, taking into account that the Lambert-Beer law presents more precision in the absorbance range of 0.2 to 0.8?

We have added this reference: https://diagnostic.serumwerk.com/wp-content/uploads/2021/05/V05-Serumwerk.pdf

Line 519-520: "Also included were water and 0.25 M sucrose as blanks". Perhaps authors could consider rephrasing this sentence.

We have changed the sentence to: “The absorbance measurements were made against water and 0.25 M sucrose blanks.”

In line 520, the sentence must say "each sample was made by triplicate".

We have changed the sentence to: “Each sample was prepared by triplicate to reduce error.” We thank the reviewer for this suggestion.

Line 673: The phrase "0.1% formic acid in 100% ACN" would be better, in my opinion, if it said "0.1% formic acid in ACN".

Yes, these two expressions have the same meaning. However, to ensure clarity, we have updated the description to “0.1% formic acid in ACN.”. We thank reviewer for this suggestion.

Supplementary Figure 1: in the Figure caption there is an error in the numbering: at the end, where it is written (E), it should be (F). Please, correct this.

We have made the necessary correction and sincerely appreciate the reviewer’s attentiveness.

Supplementary Figure 5: Some sEVs are hard to visualize due to poor image resolution. Is there any possibility for the authors to enhance these images?

We thank the reviewer for this valuable comment. To improve the visual clarity of the images, we have opted to display four sub-figures instead of nine.

DOI: 10.1172/jci.insight.183327

Resource: (IMSR Cat# JAX_002287,RRID:IMSR_JAX:002287)

Curator: @scibot

SciCrunch record: RRID:IMSR_JAX:002287

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Reply to the reviewers

Reply to the Reviewers

I would like to thank the reviewers for their comments and interest in the manuscript and the study.

Reviewer #1

1. I would assume that there are RNA-seq and/or ChIP-seq data out there produced after knockdown of one or more of these DBPs that show directional positioning.

The directional positioning of CTCF-binding sites at chromatin interaction sites was analyzed by CRISPR experiment (Guo Y et al. Cell 2015). We found that the machine learning and statistical analysis showed the same directional bias of CTCF-binding motif sequence and RAD21-binding motif sequence at chromatin interaction sites as the experimental analysis of Guo Y et al. (lines 229-253, Figure 3b, c, d and Table 1). Since CTCF is involved in different biological functions (Braccioli L et al. Essays Biochem. 2019 ResearchGate webpage), the directional bias of binding sites may be reduced in all binding sites including those at chromatin interaction sites (lines 68-73). In our study, we investigated the DNA-binding sites of proteins using the ChIP-seq data of DNA-binding proteins and DNase-seq data. We also confirmed that the DNA-binding sites of SMC3 and RAD21, which tend to be found in chromatin loops with CTCF, also showed the same directional bias as CTCF by the computational analysis.

__2. Figure 6 should be expanded to incorporate analysis of DBPs not overlapping CTCF/cohesin in chromatin interaction data that is important and potentially more interesting than the simple DBPs enrichment reported in the present form of the figure. __

Following the reviewer's advice, I performed the same analysis with the DNA-binding sites that do no overlap with the DNA-binding sites of CTCF and cohesin (RAD21 and SMC3) (Fig. 6 and Supplementary Fig. 4). The result showed the same tendency in the distribution of DNA-binding sites. The height of a peak on the graph became lower for some DNA-binding proteins after removing the DNA-binding sites that overlapped with those of CTCF and cohesin. I have added the following sentence on lines 435 and 829: For the insulator-associated DBPs other than CTCF, RAD21, and SMC3, the DNA-binding sites that do not overlap with those of CTCF, RND21, and SMC3 were used to examine their distribution around interaction sites.

3. Critically, I would like to see use of Micro-C/Hi-C data and ChIP-seq from these factors, where insulation scores around their directionally-bound sites show some sort of an effect like that presumed by the authors - and many such datasets are publicly-available and can be put to good use here.

As suggested by the reviewer, I have added the insulator scores and boundary sites from the 4D nucleome data portal as tracks in the UCSC genome browser. The insulator scores seem to correspond to some extent to the H3K27me3 histone marks from ChIP-seq (Fig. 4a and Supplementary Fig. 3). We found that the DNA-binding sites of the insulator-associated DBPs were statistically overrepresented in the 5 kb boundary sites more than other DBPs (Fig. 4d). The direction of DNA-binding sites on the genome can be shown with different colors (e.g. red and green), but the directionality of insulator-associated DNA-binding sites is their overall tendency, and it may be difficult to notice the directionality from each binding site because the directionality may be weaker than that of CTCF, RAD21, and SMC3 as shown in Table 1 and Supplementary Table 2. We also observed the directional biases of CTCF, RAD21, and SMC3 by using Micro-C chromatin interaction data as we estimated, but the directionality was more apparent to distinguish the differences between the four directions of FR, RF, FF, and RR using CTCF-mediated ChIA-pet chromatin interaction data (lines 287 and 288).

 I found that the CTCF binding sites examined by a wet experiment in the previous study may not always overlap with the boundary sites of chromatin interactions from Micro-C assay (Guo Y et al. *Cell* 2015). The chromatin interaction data do not include all interactions due to the high sequencing cost of the assay, and include less long-range interactions due to distance bias. The number of the boundary sites may be smaller than that of CTCF binding sites acting as insulators and/or some of the CTCF binding sites may not be locate in the boundary sites. It may be difficult for the boundary location algorithm to identify a short boundary location. Due to the limitations of the chromatin interaction data, I planned to search for insulator-associated DNA-binding proteins without using chromatin interaction data in this study.

 I discussed other causes in lines 614-622: Another reason for the difference may be that boundary sites are more closely associated with topologically associated domains (TADs) of chromosome than are insulator sites. Boundary sites are regions identified based on the separation of numerous chromatin interactions. On the other hand, we found that the multiple DNA-binding sites of insulator-associated DNA-binding proteins were located close to each other at insulator sites and were associated with distinct nested and focal chromatin interactions, as reported by Micro-C assay. These interactions may be transient and relatively weak, such as tissue/cell type, conditional or lineage-specific interactions.

 Furthermore, I have added the statistical summary of the analysis in lines 372-395 as follows: Overall, among 20,837 DNA-binding sites of the 97 insulator-associated proteins found at insulator sites identified by H3K27me3 histone modification marks (type 1 insulator sites), 1,315 (6%) overlapped with 264 of 17,126 5kb long boundary sites, and 6,137 (29%) overlapped with 784 of 17,126 25kb long boundary sites in HFF cells. Among 5,205 DNA-binding sites of the 97 insulator-associated DNA-binding proteins found at insulator sites identified by H3K27me3 histone modification marks and transcribed regions (type 2 insulator sites), 383 (7%) overlapped with 74 of 17,126 5-kb long boundary sites, 1,901 (37%) overlapped with 306 of 17,126 25-kb long boundary sites. Although CTCF-binding sites separate active and repressive domains, the limited number of DNA-binding sites of insulator-associated proteins found at type 1 and 2 insulator sites overlapped boundary sites identified by chromatin interaction data. Furthermore, by analyzing the regulatory regions of genes, the DNA-binding sites of the 97 insulator-associated DNA-binding proteins were found (1) at the type 1 insulator sites (based on H3K27me3 marks) in the regulatory regions of 3,170 genes, (2) at the type 2 insulator sites (based on H3K27me3 marks and gene expression levels) in the regulatory regions of 1,044 genes, and (3) at insulator sites as boundary sites identified by chromatin interaction data in the regulatory regions of 6,275 genes. The boundary sites showed the highest number of overlaps with the DNA-binding sites. Comparing the insulator sites identified by (1) and (3), 1,212 (38%) genes have both types of insulator sites. Comparing the insulator sites between (2) and (3), 389 (37%) genes have both types of insulator sites. From the comparison of insulator and boundary sites, we found that (1) or (2) types of insulator sites overlapped or were close to boundary sites identified by chromatin interaction data.

4. The suggested alternative transcripts function, also highlighted in the manuscripts abstract, is only supported by visual inspection of a few cases for several putative DBPs. I believe this is insufficient to support what looks like one of the major claims of the paper when reading the abstract, and a more quantitative and genome-wide analysis must be adopted, although the authors mention it as just an 'observation'.

According to the reviewer's comment, I performed the genome-wide analysis of alternative transcripts where the DNA-binding sites of insulator-associated proteins are located near splicing sites. The DNA-binding sites of insulator-associated DNA-binding proteins were found within 200 bp centered on splice sites more significantly than the other DNA-binding proteins (Fig. 4e and Table 2). I have added the following sentences on lines 405 - 412: We performed the statistical test to estimate the enrichment of insulator-associated DNA-binding sites compared to the other DNA-binding proteins, and found that the insulator-associated DNA-binding sites were significantly more abundant at splice sites than the DNA-binding sites of the other proteins (Fig 4e and Table 2; Mann‒Whitney U test, p value 5. Figure 1 serves no purpose in my opinion and can be removed, while figures can generally be improved (e.g., the browser screenshots in Figs 4 and 5) for interpretability from readers outside the immediate research field.

I believe that the Figure 1 would help researchers in other fields who are not familiar with biological phenomena and functions to understand the study. More explanation has been included in the Figures and legends of Figs. 4 and 5 to help readers outside the immediate research field understand the figures.

6. Similarly, the text is rather convoluted at places and should be re-approached with more clarity for less specialized readers in mind.

Reviewer #2's comments would be related to this comment. I have introduced a more detailed explanation of the method in the Results section, as shown in the responses to Reviewer #2's comments.

Reviewer #2

1. Introduction, line 95: CTCF appears two times, it seems redundant.

On lines 91-93, I deleted the latter CTCF from the sentence "We examine the directional bias of DNA-binding sites of CTCF and insulator-associated DBPs, including those of known DBPs such as RAD21 and SMC3".

2. Introduction, lines 99-103: Please stress better the novelty of the work. What is the main focus? The new identified DPBs or their binding sites? What are the "novel structural and functional roles of DBPs" mentioned?

Although CTCF is known to be the main insulator protein in vertebrates, we found that 97 DNA-binding proteins including CTCF and cohesin are associated with insulator sites by modifying and developing a machine learning method to search for insulator-associated DNA-binding proteins. Most of the insulator-associated DNA-binding proteins showed the directional bias of DNA-binding motifs, suggesting that the directional bias is associated with the insulator.

 I have added the sentence in lines 96-99 as follows: Furthermore, statistical testing the contribution scores between the directional and non-directional DNA-binding sites of insulator-associated DBPs revealed that the directional sites contributed more significantly to the prediction of gene expression levels than the non-directional sites. I have revised the statement in lines 101-110 as follows: To validate these findings, we demonstrate that the DNA-binding sites of the identified insulator-associated DBPs are located within potential insulator sites, and some of the DNA-binding sites in the insulator site are found without the nearby DNA-binding sites of CTCF and cohesin. Homologous and heterologous insulator-insulator pairing interactions are orientation-dependent, as suggested by the insulator-pairing model based on experimental analysis in flies. Our method and analyses contribute to the identification of insulator- and chromatin-associated DNA-binding sites that influence EPIs and reveal novel functional roles and molecular mechanisms of DBPs associated with transcriptional condensation, phase separation and transcriptional regulation.

3. Results, line 111: How do the SNPs come into the procedure? From the figures it seems the input is ChIP-seq peaks of DNBPs around the TSS.

On lines 121-124, to explain the procedure for the SNP of an eQTL, I have added the sentence in the Methods: "If a DNA-binding site was located within a 100-bp region around a single-nucleotide polymorphism (SNP) of an eQTL, we assumed that the DNA-binding proteins regulated the expression of the transcript corresponding to the eQTL".

4. Again, are those SNPs coming from the different cell lines? Or are they from individuals w.r.t some reference genome? I suggest a general restructuring of this part to let the reader understand more easily. One option could be simplifying the details here or alternatively including all the necessary details.

On line 119, I have included the explanation of the eQTL dataset of GTEx v8 as follows: " The eQTL data were derived from the GTEx v8 dataset, after quality control, consisting of 838 donors and 17,382 samples from 52 tissues and two cell lines". On lines 681 and 865, I have added the filename of the eQTL data "(GTEx_Analysis_v8_eQTL.tar)".

5. Figure 1: panel a and b are misleading. Is the matrix in panel a equivalent to the matrix in panel b? If not please clarify why. Maybe in b it is included the info about the SNPs? And if yes, again, what is then difference with a.

The reviewer would mention Figure 2, not Figure 1. If so, the matrices in panels a and b in Figure 2 are equivalent. I have shown it in the figure: The same figure in panel a is rotated 90 degrees to the right. The green boxes in the matrix show the regions with the ChIP-seq peak of a DNA-binding protein overlapping with a SNP of an eQTL. I used eQTL data to associate a gene with a ChIP-seq peak that was more than 2 kb upstream and 1 kb downstream of a transcriptional start site of a gene. For each gene, the matrix was produced and the gene expression levels in cells were learned and predicted using the deep learning method. I have added the following sentences to explain the method in lines 133 - 139: Through the training, the tool learned to select the binding sites of DNA-binding proteins from ChIP-seq assays that were suitable for predicting gene expression levels in the cell types. The binding sites of a DNA-binding protein tend to be observed in common across multiple cell and tissue types. Therefore, ChIP-seq data and eQTL data in different cell and tissue types were used as input data for learning, and then the tool selected the data suitable for predicting gene expression levels in the cell types, even if the data were not obtained from the same cell types.

6. Line 386-388: could the author investigate in more detail this observation? Does it mean that loops driven by other DBPs independent of the known CTCF/Cohesin? Could the author provide examples of chromatin structural data e.g. MicroC?

As suggested by the reviewer, to help readers understand the observation, I have added Supplementary Fig. S4c to show the distribution of DNA-binding sites of "CTCF, RAD21, and SMC3" and "BACH2, FOS, ATF3, NFE2, and MAFK" around chromatin interaction sites. I have modified the following sentence to indicate the figure on line 501: Although a DNA-binding-site distribution pattern around chromatin interaction sites similar to those of CTCF, RAD21, and SMC3 was observed for DBPs such as BACH2, FOS, ATF3, NFE2, and MAFK, less than 1% of the DNA-binding sites of the latter set of DBPs colocalized with CTCF, RAD21, or SMC3 in a single bin (Fig. S4c).

 In Aljahani A et al. *Nature Communications* 2022, we find that depletion of cohesin causes a subtle reduction in longer-range enhancer-promoter interactions and that CTCF depletion can cause rewiring of regulatory contacts. Together, our data show that loop extrusion is not essential for enhancer-promoter interactions, but contributes to their robustness and specificity and to precise regulation of gene expression. Goel VY et al. *Nature Genetics* 2023 mentioned in the abstract: Microcompartments frequently connect enhancers and promoters and though loss of loop extrusion and inhibition of transcription disrupts some microcompartments, most are largely unaffected. These results suggested that chromatin loops can be driven by other DBPs independent of the known CTCF/Cohesin.

I added the following sentence on lines 569-577: The depletion of cohesin causes a subtle reduction in longer-range enhancer-promoter interactions and that CTCF depletion can cause rewiring of regulatory contacts. Another group reported that enhancer-promoter interactions and transcription are largely maintained upon depletion of CTCF, cohesin, WAPL or YY1. Instead, cohesin depletion decreased transcription factor binding to chromatin. Thus, cohesin may allow transcription factors to find and bind their targets more efficiently. Furthermore, the loop extrusion is not essential for enhancer-promoter interactions, but contributes to their robustness and specificity and to precise regulation of gene expression.

 FOXA1 pioneer factor functions as an initial chromatin-binding and chromatin-remodeling factor and has been reported to form biomolecular condensates (Ji D et al. *Molecular Cell* 2024). CTCF have also found to form transcriptional condensate and phase separation (Lee R et al. *Nucleic acids research* 2022). FOS was found to be an insulator-associated DNA-binding protein in this study and is potentially involved in chromatin remodeling, transcription condensation, and phase separation with the other factors such as BACH2, ATF3, NFE2 and MAFK. I have added the following sentence on line 556: FOXA1 pioneer factor functions as an initial chromatin-binding and chromatin-remodeling factor and has been reported to form biomolecular condensates.

7. In general, how the presented results are related to some models of chromatin architecture, e.g. loop extrusion, in which it is integrated convergent CTCF binding sites?

Goel VY et al. Nature Genetics 2023 identified highly nested and focal interactions through region capture Micro-C, which resemble fine-scale compartmental interactions and are termed microcompartments. In the section titled "Most microcompartments are robust to loss of loop extrusion," the researchers noted that a small proportion of interactions between CTCF and cohesin-bound sites exhibited significant reductions in strength when cohesin was depleted. In contrast, the majority of microcompartmental interactions remained largely unchanged under cohesin depletion. Our findings indicate that most P-P and E-P interactions, aside from a few CTCF and cohesin-bound enhancers and promoters, are likely facilitated by a compartmentalization mechanism that differs from loop extrusion. We suggest that nested, multiway, and focal microcompartments correspond to small, discrete A-compartments that arise through a compartmentalization process, potentially influenced by factors upstream of RNA Pol II initiation, such as transcription factors, co-factors, or active chromatin states. It follows that if active chromatin regions at microcompartment anchors exhibit selective "stickiness" with one another, they will tend to co-segregate, leading to the development of nested, focal interactions. This microphase separation, driven by preferential interactions among active loci within a block copolymer, may account for the striking interaction patterns we observe.

 The authors of the paper proposed several mechanisms potentially involved in microcompartments. These mechanisms may be involved in looping with insulator function. Another group reported that enhancer-promoter interactions and transcription are largely maintained upon depletion of CTCF, cohesin, WAPL or YY1. Instead, cohesin depletion decreased transcription factor binding to chromatin. Thus, cohesin may allow transcription factors to find and bind their targets more efficiently (Hsieh TS et al. *Nature Genetics* 2022). Among the identified insulator-associated DNA-binding proteins, Maz and MyoD1 form loops without CTCF (Xiao T et al. *Proc Natl Acad Sci USA* 2021 ; Ortabozkoyun H et al. *Nature genetics* 2022 ; Wang R et al. *Nature communications* 2022). I have added the following sentences on lines 571-575: Another group reported that enhancer-promoter interactions and transcription are largely maintained upon depletion of CTCF, cohesin, WAPL or YY1. Instead, cohesin depletion decreased transcription factor binding to chromatin. Thus, cohesin may allow transcription factors to find and bind their targets more efficiently. I have included the following explanation on lines 582-584: Maz and MyoD1 among the identified insulator-associated DNA-binding proteins form loops without CTCF.

 As for the directionality of CTCF, if chromatin loop anchors have some structural conformation, as shown in the paper entitled "The structural basis for cohesin-CTCF-anchored loops" (Li Y et al. *Nature* 2020), directional DNA binding would occur similarly to CTCF binding sites. Moreover, cohesin complexes that interact with convergent CTCF sites, that is, the N-terminus of CTCF, might be protected from WAPL, but those that interact with divergent CTCF sites, that is, the C-terminus of CTCF, might not be protected from WAPL, which could release cohesin from chromatin and thus disrupt cohesin-mediated chromatin loops (Davidson IF et al. *Nature Reviews Molecular Cell Biology* 2021). Regarding loop extrusion, the 'loop extrusion' hypothesis is motivated by in vitro observations. The experiment in yeast, in which cohesin variants that are unable to extrude DNA loops but retain the ability to topologically entrap DNA, suggested that in vivo chromatin loops are formed independently of loop extrusion. Instead, transcription promotes loop formation and acts as an extrinsic motor that extends these loops and defines their final positions (Guerin TM et al. *EMBO Journal* 2024). I have added the following sentences on lines 543-547: Cohesin complexes that interact with convergent CTCF sites, that is, the N-terminus of CTCF, might be protected from WAPL, but those that interact with divergent CTCF sites, that is, the C-terminus of CTCF, might not be protected from WAPL, which could release cohesin from chromatin and thus disrupt cohesin-mediated chromatin loops. I have included the following sentences on lines 577-582: The 'loop extrusion' hypothesis is motivated by in vitro observations. The experiment in yeast, in which cohesin variants that are unable to extrude DNA loops but retain the ability to topologically entrap DNA, suggested that in vivo chromatin loops are formed independently of loop extrusion. Instead, transcription promotes loop formation and acts as an extrinsic motor that extends these loops and defines their final positions.

 Another model for the regulation of gene expression by insulators is the boundary-pairing (insulator-pairing) model (Bing X et al. *Elife* 2024) (Ke W et al. *Elife* 2024) (Fujioka M et al. *PLoS Genetics* 2016). Molecules bound to insulators physically pair with their partners, either head-to-head or head-to-tail, with different degrees of specificity at the termini of TADs in flies. Although the experiments do not reveal how partners find each other, the mechanism unlikely requires loop extrusion. Homologous and heterologous insulator-insulator pairing interactions are central to the architectural functions of insulators. The manner of insulator-insulator interactions is orientation-dependent. I have summarized the model on lines 559-567: Other types of chromatin regulation are also expected to be related to the structural interactions of molecules. As the boundary-pairing (insulator-pairing) model, molecules bound to insulators physically pair with their partners, either head-to-head or head-to-tail, with different degrees of specificity at the termini of TADs in flies (Fig. 7). Although the experiments do not reveal how partners find each other, the mechanism unlikely requires loop extrusion. Homologous and heterologous insulator-insulator pairing interactions are central to the architectural functions of insulators. The manner of insulator-insulator interactions is orientation-dependent.

8. Do the authors think that the identified DBPs could work in that way as well?

The boundary-pairing (insulator-pairing) model would be applied to the insulator-associated DNA-binding proteins other than CTCF and cohesin that are involved in the loop extrusion mechanism (Bing X et al. Elife 2024) (Ke W et al. Elife 2024) (Fujioka M et al. PLoS Genetics 2016).

 Liquid-liquid phase separation was shown to occur through CTCF-mediated chromatin loops and to act as an insulator (Lee, R et al. *Nucleic Acids Research* 2022). Among the identified insulator-associated DNA-binding proteins, CEBPA has been found to form hubs that colocalize with transcriptional co-activators in a native cell context, which is associated with transcriptional condensate and phase separation (Christou-Kent M et al. *Cell Reports* 2023). The proposed microcompartment mechanisms are also associated with phase separation. Thus, the same or similar mechanisms are potentially associated with the insulator function of the identified DNA-binding proteins. I have included the following information on line 554: CEBPA in the identified insulator-associated DNA-binding proteins was also reported to be involved in transcriptional condensates and phase separation.

9. Also, can the authors comment about the mechanisms those newly identified DBPs mediate contacts by active processes or equilibrium processes?

Snead WT et al. Molecular Cell 2019 mentioned that protein post-transcriptional modifications (PTMs) facilitate the control of molecular valency and strength of protein-protein interactions. O-GlcNAcylation as a PTM inhibits CTCF binding to chromatin (Tang X et al. Nature Communications 2024). I found that the identified insulator-associated DNA-binding proteins tend to form a cluster at potential insulator sites (Supplementary Fig. 2d). These proteins may interact and actively regulate chromatin interactions, transcriptional condensation, and phase separation by PTMs. I have added the following explanation on lines 584-590: Furthermore, protein post-transcriptional modifications (PTMs) facilitate control over the molecular valency and strength of protein-protein interactions. O-GlcNAcylation as a PTM inhibits CTCF binding to chromatin. We found that the identified insulator-associated DNA-binding proteins tend to form a cluster at potential insulator sites (Fig. 4f and Supplementary Fig. 3c). These proteins may interact and actively regulate chromatin interactions, transcriptional condensation, and phase separation through PTMs.

10. Can the author provide some real examples along with published structural data (e.g. the mentioned micro-C data) to show the link between protein co-presence, directional bias and contact formation?

Structural molecular model of cohesin-CTCF-anchored loops has been published by Li Y et al. Nature 2020. The structural conformation of CTCF and cohesin in the loops would be the cause of the directional bias of CTCF binding sites, which I mentioned in lines 539 - 543 as follows: These results suggest that the directional bias of DNA-binding sites of insulator-associated DBPs may be involved in insulator function and chromatin regulation through structural interactions among DBPs, other proteins, DNAs, and RNAs. For example, the N-terminal amino acids of CTCF have been shown to interact with RAD21 in chromatin loops.

 To investigate the principles underlying the architectural functions of insulator-insulator pairing interactions, two insulators, Homie and Nhomie, flanking the *Drosophila even skipped *locus were analyzed. Pairing interactions between the transgene Homie and the eve locus are directional. The head-to-head pairing between the transgene and endogenous Homie matches the pattern of activation (Fujioka M et al. *PLoS Genetics* 2016).

Reviewer #3

Major Comments:

1. Some of these TFs do not have specific direct binding to DNA (P300, Cohesin). Since the authors are using binding motifs in their analysis workflow, I would remove those from the analysis.

When a protein complex binds to DNA, one protein of the complex binds to the DNA directory, and the other proteins may not bind to DNA. However, the DNA motif sequence bound by the protein may be registered as the DNA-binding motif of all the proteins in the complex. The molecular structure of the complex of CTCF and Cohesin showed that both CTCF and Cohesin bind to DNA (Li Y et al. Nature 2020). I think there is a possibility that if the molecular structure of a protein complex becomes available, the previous recognition of the DNA-binding ability of a protein may be changed. Therefore, I searched the Pfam database for 99 insulator-associated DNA-binding proteins identified in this study. I found that 97 are registered as DNA-binding proteins and/or have a known DNA-binding domain, and EP300 and SIN3A do not directory bind to DNA, which was also checked by Google search. I have added the following explanation in line 257 to indicate direct and indirect DNA-binding proteins: Among 99 insulator-associated DBPs, EP300 and SIN3A do not directory interact with DNA, and thus 97 insulator-associated DBPs directory bind to DNA. I have updated the sentence in line 20 of the Abstract as follows: We discovered 97 directional and minor nondirectional motifs in human fibroblast cells that corresponded to 23 DBPs related to insulator function, CTCF, and/or other types of chromosomal transcriptional regulation reported in previous studies.

2. I am not sure if I understood correctly, by why do the authors consider enhancers spanning 2Mb (200 bins of 10Kb around eSNPs)? This seems wrong. Enhancers are relatively small regions (100bp to 1Kb) and only a very small subset form super enhancers.

As the reviewer mentioned, I recognize enhancers are relatively small regions. In the paper, I intended to examine further upstream and downstream of promoter regions where enhancers are found. Therefore, I have modified the sentence in lines 929 - 931 of the Fig. 2 legend as follows: Enhancer-gene regulatory interaction regions consist of 200 bins of 10 kbp between -1 Mbp and 1 Mbp region from TSS, not including promoter.

3. I think the H3K27me3 analysis was very good, but I would have liked to see also constitutive heterochromatin as well, so maybe repeat the analysis for H3K9me3.

Following the reviewer's advice, I have added the ChIP-seq data of H3K9me3 as a truck of the UCSC Genome Browser. The distribution of H3K9me3 signal was different from that of H3K27me3 in some regions. I also found the insulator-associated DNA-binding sites close to the edges of H3K9me3 regions and took some screenshots of the UCSC Genome Browser of the regions around the sites in Supplementary Fig. 3b. I have modified the following sentence on lines 974 - 976 in the legend of Fig. 4: a Distribution of histone modification marks H3K27me3 (green color) and H3K9me3 (turquoise color) and transcript levels (pink color) in upstream and downstream regions of a potential insulator site (light orange color). I have also added the following result on lines 356 - 360: The same analysis was performed using H3K9me3 marks, instead of H3K27me3 (Fig. S3b). We found that the distribution of H3K9me3 signal was different from that of H3K27me3 in some regions, and discovered the insulator-associated DNA-binding sites close to the edges of H3K9me3 regions (Fig. S3b).

4. I was not sure I understood the analysis in Figure 6. The binding site is with 500bp of the interaction site, but micro-C interactions are at best at 1Kb resolution. They say they chose the centre of the interaction site, but we don't know exactly where there is the actual interaction. Also, it is not clear what they measure. Is it the number of binding sites of a specific or multiple DBP insulator proteins at a specific distance from this midpoint that they recover in all chromatin loops? Maybe I am missing something. This analysis was not very clear.

The resolution of the Micro-C assay is considered to be 100 bp and above, as the human nucleome core particle contains 145 bp (and 193 bp with linker) of DNA. However, internucleosomal DNA is cleaved by endonuclease into fragments of multiples of 10 nucleotides (Pospelov VA et al. Nucleic Acids Research 1979). Highly nested focal interactions were observed (Goel VY et al. Nature Genetics 2023). Base pair resolution was reported using Micro Capture-C (Hua P et al. Nature 2021). Sub-kilobase (20 bp resolution) chromatin topology was reported using an MNase-based chromosome conformation capture (3C) approach (Aljahani A et al. Nature Communications 2022). On the other hand, Hi-C data was analyzed at 1 kb resolution. (Gu H et al. bioRxiv 2021). If the resolution of Micro-C interactions is at best at 1 kb, the binding sites of a DNA-binding protein will not show a peak around the center of the genomic locations of interaction edges. Each panel shows the number of binding sites of a specific DNA-binding protein at a specific distance from the midpoint of all chromatin interaction edges. I have modified and added the following sentences in lines 593-597: High-resolution chromatin interaction data from a Micro-C assay indicated that most of the predicted insulator-associated DBPs showed DNA-binding-site distribution peaks around chromatin interaction sites, suggesting that these DBPs are involved in chromatin interactions and that the chromatin interaction data has a high degree of resolution. Base pair resolution was reported using Micro Capture-C.

Minor Comments:

1. PIQ does not consider TF concentration. Other methods do that and show that TF concentration improves predictions (e.g., ____https://www.biorxiv.org/content/10.1101/2023.07.15.549134v2____or ____https://pubmed.ncbi.nlm.nih.gov/37486787____/). The authors should discuss how that would impact their results.

The directional bias of CTCF binding sites was identified by ChIA-pet interactions of CTCF binding sites. The analysis of the contribution scores of DNA-binding sites of proteins considering the binding sites of CTCF as an insulator showed the same tendency of directional bias of CTCF binding sites. In the analysis, to remove the false-positive prediction of DNA-binding sites, I used the binding sites that overlapped with a ChIP-seq peak of the DNA-binding protein. This result suggests that the DNA-binding sites of CTCF obtained by the current analysis have sufficient quality. Therefore, if the accuracy of prediction of DNA-binding sites is improved, although the number of DNA-binding sites may be different, the overall tendency of the directionality of DNA-binding sites will not change and the results of this study will not change significantly.

 As for the first reference in the reviewer's comment, chromatin interaction data from Micro-C assay does not include all chromatin interactions in a cell or tissue, because it is expensive to cover all interactions. Therefore, it would be difficult to predict all chromatin interactions based on machine learning. As for the second reference in the reviewer's comment, pioneer factors such as FOXA are known to bind to closed chromatin regions, but transcription factors and DNA-binding proteins involved in chromatin interactions and insulators generally bind to open chromatin regions. The search for the DNA-binding motifs is not required in closed chromatin regions.

2. DeepLIFT is a good approach to interpret complex structures of CNN, but is not truly explainable AI. I think the authors should acknowledge this.

In the DeepLIFT paper, the authors explain that DeepLIFT is a method for decomposing the output prediction of a neural network on a specific input by backpropagating the contributions of all neurons in the network to every feature of the input (Shrikumar A et al. ICML 2017). DeepLIFT compares the activation of each neuron to its 'reference activation' and assigns contribution scores according to the difference. DeepLIFT calculates a metric to measure the difference between an input and the reference of the input.

 Truly explainable AI would be able to find cause and reason, and to make choices and decisions like humans. DeepLIFT does not perform causal inferences. I did not use the term "Explainable AI" in our manuscript, but I briefly explained it in Discussion. I have added the following explanation in lines 623-628: AI (Artificial Intelligence) is considered as a black box, since the reason and cause of prediction are difficult to know. To solve this issue, tools and methods have been developed to know the reason and cause. These technologies are called Explainable AI. DeepLIFT is considered to be a tool for Explainable AI. However, DeepLIFT does not answer the reason and cause for a prediction. It calculates scores representing the contribution of the input data to the prediction.

 Furthermore, to improve the readability of the manuscript, I have included the following explanation in lines 159-165: we computed DeepLIFT scores of the input data (i.e., each binding site of the ChIP-seq data of DNA-binding proteins) in the deep leaning analysis on gene expression levels. DeepLIFT compares the importance of each input for predicting gene expression levels to its 'reference or background level' and assigns contribution scores according to the difference. DeepLIFT calculates a metric to measure the difference between an input and the reference of the input.

• Informativo 1150
• RE 1366243 / SC
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. GILMAR MENDES
• Julgamento: 13/09/2024 (Virtual)
• Ramo do Direito: Administrativo, Processual Civil
• Matéria: Serviços Públicos; Saúde; Fornecimento de Medicamentos; Registro na Anvisa; Incorporação no SUS/Solução de Conflitos; Autocomposição; Diálogo Interfederativo; Comissão Especial

Critérios para o fornecimento de medicamentos não incorporados na política pública do SUS: homologação de acordo firmado entre os entes federativos

Tese fixada

I – Competência.

1) Para fins de fixação de competência, as demandas relativas a medicamentos não incorporados na política pública do SUS, mas com registro na ANVISA, tramitarão perante a Justiça Federal, nos termos do art. 109, I, da Constituição Federal, quando o valor do tratamento anual específico do fármaco ou do princípio ativo, com base no Preço Máximo de Venda do Governo (PMVG – situado na alíquota zero), divulgado pela Câmara de Regulação do Mercado de Medicamentos (CMED - Lei 10.742/2003), for igual ou superior ao valor de 210 salários mínimos, na forma do art. 292 do CPC. - 1.1) Existindo mais de um medicamento do mesmo princípio ativo e não sendo solicitado um fármaco específico, considera-se, para efeito de competência, aquele listado no menor valor na lista CMED (PMVG, situado na alíquota zero). - 1.2) No caso de inexistir valor fixado na lista CMED, considera-se o valor do tratamento anual do medicamento solicitado na demanda, podendo o magistrado, em caso de impugnação pela parte requerida, solicitar auxílio à CMED, na forma do art. 7º da Lei 10.742/2003. - 1.3) Caso inexista resposta em tempo hábil da CMED, o juiz analisará de acordo com o orçamento trazido pela parte autora. - 1.4) No caso de cumulação de pedidos, para fins de competência, será considerado apenas o valor do(s) medicamento(s) não incorporado(s) que deverá(ão) ser somado(s), independentemente da existência de cumulação alternativa de outros pedidos envolvendo obrigação de fazer, pagar ou de entregar coisa certa.

II – Definição de Medicamentos Não Incorporados.

2.1) Consideram-se medicamentos não incorporados aqueles que não constam na política pública do SUS; medicamentos previstos nos PCDTs para outras finalidades; medicamentos sem registro na ANVISA; e medicamentos off label sem PCDT ou que não integrem listas do componente básico. - 2.1.1) Conforme decidido pelo Supremo Tribunal Federal na tese fixada no tema 500 da sistemática da repercussão geral, é mantida a competência da Justiça Federal em relação às ações que demandem fornecimento de medicamentos sem registro na Anvisa, as quais deverão necessariamente ser propostas em face da União, observadas as especificidades já definidas no aludido tema.

III – Custeio.

3) As ações de fornecimento de medicamentos incorporados ou não incorporados, que se inserirem na competência da Justiça Federal, serão custeadas integralmente pela União, cabendo, em caso de haver condenação supletiva dos Estados e do Distrito Federal, o ressarcimento integral pela União, via repasses Fundo a Fundo (FNS ao FES), na situação de ocorrer redirecionamento pela impossibilidade de cumprimento por aquela, a ser implementado mediante ato do Ministério da Saúde, previamente pactuado em instância tripartite, no prazo de até 90 dias. - 3.1) Figurando somente a União no polo passivo, cabe ao magistrado, se necessário, promover a inclusão do Estado ou Município para possibilitar o cumprimento efetivo da decisão, o que não importará em responsabilidade financeira nem em ônus de sucumbência, devendo ser realizado o ressarcimento pela via acima indicada em caso de eventual custo financeiro ser arcado pelos referidos entes. - 3.2) Na determinação judicial de fornecimento do medicamento, o magistrado deverá estabelecer que o valor de venda do medicamento seja limitado ao preço com desconto, proposto no processo de incorporação na Conitec (se for o caso, considerando o venire contra factum proprium/tu quoque e observado o índice de reajuste anual de preço de medicamentos definido pela CMED), ou valor já praticado pelo ente em compra pública, aquele que seja identificado como menor valor, tal como previsto na parte final do art. 9º na Recomendação 146, de 28.11.2023, do CNJ. Sob nenhuma hipótese, poderá haver pagamento judicial às pessoas físicas/jurídicas acima descritas em valor superior ao teto do PMVG, devendo ser operacionalizado pela serventia judicial junto ao fabricante ou distribuidor. - 3.3) As ações que permanecerem na Justiça Estadual e cuidarem de medicamentos não incorporados, as quais impuserem condenações aos Estados e Municípios, serão ressarcidas pela União, via repasses Fundo a Fundo (FNS ao FES ou ao FMS). Figurando somente um dos entes no polo passivo, cabe ao magistrado, se necessário, promover a inclusão do outro para possibilitar o cumprimento efetivo da decisão.

3.3.1) O ressarcimento descrito no item 3.3 ocorrerá no percentual de 65% (sessenta e cinco por cento) dos desembolsos decorrentes de condenações oriundas de ações cujo valor da causa seja superior a 7 (sete) e inferior a 210 (duzentos e dez) salários mínimos, a ser implementado mediante ato do Ministério da Saúde, previamente pactuado em instância tripartite, no prazo de até 90 dias. - 3.4) Para fins de ressarcimento interfederativo, quanto aos medicamentos para tratamento oncológico, as ações ajuizadas previamente a 10 de junho de 2024 serão ressarcidas pela União na proporção de 80% (oitenta por cento) do valor total pago por Estados e por Municípios, independentemente do trânsito em julgado da decisão, a ser implementado mediante ato do Ministério da Saúde, previamente pactuado em instância tripartite, no prazo de até 90 dias. O ressarcimento para os casos posteriores a 10 de junho de 2024 deverá ser pactuado na CIT, no mesmo prazo.

IV – Análise judicial do ato administrativo de indeferimento de medicamento pelo SUS.

4) Sob pena de nulidade do ato jurisdicional (art. 489, § 1º, V e VI, c/c art. 927, III, § 1º, ambos do CPC), o Poder Judiciário, ao apreciar pedido de concessão de medicamentos não incorporados, deverá obrigatoriamente analisar o ato administrativo comissivo ou omissivo da não incorporação pela Conitec e da negativa de fornecimento na via administrativa, tal como acordado entre os Entes Federativos em autocomposição no Supremo Tribunal Federal. - 4.1) No exercício do controle de legalidade, o Poder Judiciário não pode substituir a vontade do administrador, mas tão somente verificar se o ato administrativo específico daquele caso concreto está em conformidade com as balizas presentes na Constituição Federal, na legislação de regência e na política pública no SUS. - 4.2) A análise jurisdicional do ato administrativo que indefere o fornecimento de medicamento não incorporado restringe-se ao exame da regularidade do procedimento e da legalidade do ato de não incorporação e do ato administrativo questionado, à luz do controle de legalidade e da teoria dos motivos determinantes, não sendo possível incursão no mérito administrativo, ressalvada a cognição do ato administrativo discricionário, o qual se vincula à existência, à veracidade e à legitimidade dos motivos apontados como fundamentos para a sua adoção, a sujeitar o ente público aos seus termos. - 4.3) Tratando-se de medicamento não incorporado, é do autor da ação o ônus de demonstrar, com fundamento na Medicina Baseada em Evidências, a segurança e a eficácia do fármaco, bem como a inexistência de substituto terapêutico incorporado pelo SUS. - 4.4) Conforme decisão da STA 175-AgR, não basta a simples alegação de necessidade do medicamento, mesmo que acompanhada de relatório médico, sendo necessária a demonstração de que a opinião do profissional encontra respaldo em evidências científicas de alto nível, ou seja, unicamente ensaios clínicos randomizados, revisão sistemática ou meta-análise.

V – Plataforma Nacional

5) Os Entes Federativos, em governança colaborativa com o Poder Judiciário, implementarão uma plataforma nacional que centralize todas as informações relativas às demandas administrativas e judiciais de acesso a fármaco, de fácil consulta e informação ao cidadão, na qual constarão dados básicos para possibilitar a análise e eventual resolução administrativa, além de posterior controle judicial.

• 5.1) A porta de ingresso à plataforma será via prescrições eletrônicas, devidamente certificadas, possibilitando o controle ético da prescrição, a posteriori, mediante ofício do Ente Federativo ao respectivo conselho profissional.
• 5.2) A plataforma nacional visa a orientar todos os atores ligados ao sistema público de saúde, possibilitando a eficiência da análise pelo Poder Público e compartilhamento de informações com o Poder Judiciário, mediante a criação de fluxos de atendimento diferenciado, a depender de a solicitação estar ou não incluída na política pública de assistência farmacêutica do SUS e de acordo com os fluxos administrativos aprovados pelos próprios Entes Federativos em autocomposição.
• 5.3) A plataforma, entre outras medidas, deverá identificar quem é o responsável pelo custeio e fornecimento administrativo entre os Entes Federativos, com base nas responsabilidades e fluxos definidos em autocomposição entre todos os Entes Federativos, além de possibilitar o monitoramento dos pacientes beneficiários de decisões judiciais, com permissão de consulta virtual dos dados centralizados nacionalmente, pela simples consulta pelo CPF, nome de medicamento, CID, entre outros, com a observância da Lei Geral de Proteção da Dados e demais legislações quanto ao tratamento de dados pessoais sensíveis.
• 5.4) O serviço de saúde cujo profissional prescrever medicamento não incorporado ao SUS deverá assumir a responsabilidade contínua pelo acompanhamento clínico do paciente, apresentando, periodicamente, relatório atualizado do estado clínico do paciente, com informações detalhadas sobre o progresso do tratamento, incluindo melhorias, estabilizações ou deteriorações no estado de saúde do paciente, assim como qualquer mudança relevante no plano terapêutico.

VI – Medicamentos incorporados.

6) Em relação aos medicamentos incorporados, conforme conceituação estabelecida no âmbito da Comissão Especial e constante do Anexo I, os Entes concordam em seguir o fluxo administrativo e judicial detalhado no Anexo I, inclusive em relação à competência judicial para apreciação das demandas e forma de ressarcimento entre os Entes, quando devido. - 6.1) A(o) magistrada(o) deverá determinar o fornecimento em face de qual ente público deve prestá-lo (União, estado, Distrito Federal ou Município), nas hipóteses previstas no próprio fluxo acordado pelos Entes Federativos, anexados ao presente acórdão.”

Resumo - Em autocomposição no STF, os entes federativos acordaram sobre as diretrizes a serem observadas nas ações judiciais de fornecimento de medicamentos pelo Sistema Único de Saúde (SUS), em especial sobre a uniformização da nomenclatura dos medicamentos incorporados ou não incorporados na política pública do SUS, a competência jurisdicional, a responsabilidade pelo custeio dos medicamentos e a implementação de uma plataforma nacional com informações a respeito das demandas de medicamentos.

• Definiu-se, para fins de análise administrativa e judicial, que medicamentos não incorporados são (i) os que não constam na política pública do SUS; (ii) os previstos no Protocolo Clínico e Diretrizes Terapêuticas (PCDT) para outras finalidades; (iii) aqueles sem registro na Agência Nacional de Vigilância Sanitária (Anvisa); e (iv) os denominados “off label” sem PCDT ou que não integrem listas do componente básico.
• Nesse contexto, compete à Justiça Federal o julgamento das demandas relacionadas a medicamentos não incorporados na política pública do SUS, mas com registro na ANVISA, quando o valor do tratamento anual específico do fármaco ou do princípio ativo for igual ou superior ao valor de 210 salários mínimos. Ademais, conforme jurisprudência desta Corte, mantém-se a competência da Justiça Federal em relação às ações que demandem fornecimento de medicamentos sem registro na Anvisa, as quais deverão ser propostas, necessariamente, em face da União, observadas as especificidades definidas na tese fixada para o Tema 500 da repercussão geral (1).
• As ações de fornecimento de medicamentos incorporados ou não, que se inserirem na competência da Justiça Federal, devem ser custeadas integralmente pela União. Desse modo, nos casos de condenação supletiva dos estados e do Distrito Federal, é cabível o ressarcimento integral pela União, via repasses do Fundo Nacional de Saúde (FNS) ao Fundo Estadual de Saúde (FES), quando ocorrer redirecionamento pela sua impossibilidade de cumprimento.
• Ademais, nos pedidos de concessão de medicamentos não incorporados, o Poder Judiciário deverá analisar, obrigatoriamente, o ato administrativo comissivo ou omissivo da não incorporação pela Comissão Nacional de Incorporação de Tecnologias no SUS (Conitec) e da negativa de fornecimento na via administrativa, nos moldes do acordo interfederativo.
• Por fim, em governança colaborativa com o Poder Judiciário, os entes federativos implementarão uma plataforma nacional que centralize todas as informações relativas às demandas administrativas e judiciais de acesso a fármaco. Essa plataforma deverá ser de fácil consulta e informação ao cidadão, e conterá dados básicos que possibilitem a análise e eventual resolução administrativa, sem prejuízo de posterior controle judicial.
• Com base nesses e em outros entendimentos, o Plenário, por unanimidade, ao apreciar o Tema 1.234 da repercussão geral, negou provimento ao recurso extraordinário e homologou, em parte, os termos dos 3 (três) acordos, com as condicionantes e adaptações sintetizadas nas teses anteriormente citadas. Os efeitos da presente decisão foram modulados tão somente quanto ao deslocamento de competência (item 1 do acordo), aplicando-se apenas aos feitos ajuizados após a publicação do julgamento de mérito no DJe, de modo a afastar sua incidência aos processos em tramitação até o referido marco, sem possibilidade de suscitação de conflito negativo de competência a respeito dos processos anteriores.
• Além disso, o Tribunal (i) determinou a transformação das teses em enunciado sintetizado de súmula vinculante com a seguinte redação: “O pedido e a análise administrativos de fármacos na rede pública de saúde, a judicialização do caso, bem ainda seus desdobramentos (administrativos e jurisdicionais), devem observar os termos dos 3 (três) acordos interfederativos (e seus fluxos) homologados pelo Supremo Tribunal Federal, em governança judicial colaborativa, no tema 1.234 da sistemática da repercussão geral (RE 1.366.243)”; bem como (ii) fixou, nos termos da respectiva ata de julgamento, diversas determinações e esclarecimentos.

(1) Precedente citado: RE 657.718 (Tema 500 RG).

Legislação: CPC/2015: art. 292, art. 489, § 1º, V e VI e art. 927, III, § 1º. Lei 10.742/2003.

Precedentes: RE 657.718 (Tema 500 RG).

DOI: 10.1016/j.celrep.2025.115425

Resource: (Thermo Fisher Scientific Cat# 31412, RRID:AB_228265)

Curator: @scibot

SciCrunch record: RRID:AB_228265

What is this?

This provides a complex understanding giving t-1 Co2 from the sentence. That is a another example of how this is meant for a more advanced audience rather than a general audience.

I just thought that this was a super powerful way to summarize and conclude this piece. It makes me wonder how much our obsession with culture with social media especially in western markets of individual brilliance and doing it hustling by yourself. But the truth is in the chaos and the brilliance of "dozens of people". Conan O' Brian used to write for the Simpsons before he was randomly chosen to host the late night show but I think did a great job in his career to highlight that it was more than a one-man show effort.

Se hace enfasis, junto con el parrafo anterior, que en general, las muertes fetales, primero se van a a dar mayormente en embarazos a termino y que la presencia de factores de riesgos, no aumentan mucho el riesgo, el unico que si, es una muerte fetal previa

Se hace una anotación sobre si el SARVS CoV 2 puede ser factor de riesgo o no sobre la muerte fetal, teniendo 2 casos, donde en uno si aumenta y en otro no

Segundo o entendimento do STF, trata-se de hipótese de imunidade tributária, e não de isenção. A banca, no entanto, cobrou a literalidade do texto legal.

TOMBAMENTO QUILOMBOS - Os documentos e sítios que guardem reminiscências dos antigos quilombos foram objeto de tombamento legal-compulsório pela Constituição da República de 1988, no seu art. 216, §5º: Ficam tombados todos os documentos e os sítios detentores de reminiscências históricas dos antigos quilombos. Nessa hipótese, portanto, é desnecessário o processo administrativo ou judicial para constituir o tombamento – que já existe por determinação da CF/88

Art. 40. O regime próprio de previdência social dos servidores titulares de cargos efetivos terá caráter contributivo e solidário, mediante contribuição do respectivo ente federativo, de servidores ativos, de aposentados e de pensionistas, observados critérios que preservem o equilíbrio financeiro e atuarial. (Redação dada pela Emenda Constitucional nº 103, de 2019)

§ 1º O servidor abrangido por regime próprio de previdência social será aposentado: (Redação dada pela Emenda Constitucional nº 103, de 2019)

II - compulsoriamente, com proventos proporcionais ao tempo de contribuição, aos 70 (setenta) anos de idade, ou aos 75 (setenta e cinco) anos de idade, na forma de lei complementar; (Redação dada pela Emenda Constitucional nº 88, de 2015) (Vide Lei Complementar nº 152, de 2015)

O não encaminhamento de projeto de lei de revisão anual dos vencimentos dos servidores públicos, previsto no inciso X do art. 37 da CF/88, não gera direito subjetivo a indenização. Deve o Poder Executivo, no entanto, se pronunciar, de forma fundamentada, acerca das razões pelas quais não propôs a revisão. STF. Plenário. RE 565089 /SP, rel. orig. Min. Marco Aurélio, red. p/ o ac. Min. Roberto Barroso, julgado em 25/9/2019 (repercussão geral – Tema 19) (Info 953)

TETO NACIONAL: Subsídio dos Ministros do STF

Subteto União: Subsídio dos Ministros do STF.

Subteto municipal: Subsídio do Prefeito, excetuados os procuradores municipais

Subteto nos Estados/DF: Existem duas opções:

Opção 1 - subteto único para todos os Poderes: o valor máximo seria o subsídio dos Desembargadores do TJ, limitado a 90,25% do subsídio mensal, em espécie, dos Ministros do STF. O subsídio dos Deputados Estaduais/Distritais seguirá regras próprias (§ 2º do art. 27), não estando sujeito ao subsídio dos Desembargadores mesmo que se adote esta opção. Vale ressaltar que quem define se o Estado-membro adotará subtetos diferentes ou único é a Constituição estadual.

Opção 2 - subtetos diferentes para cada um dos Poderes:

Executivo: subsídio do Governador.

Legislativo: subsídio dos Deputados Estaduais.

Judiciário (inclui MP, Defensoria e Procuradoria): subsídio dos Desembargadores do TJ, limitado a 90,25% do subsídio mensal, em espécie, dos Ministros do STF.*

↪ A inexigibilidade reflexa só atinge o Poder Executivo.↩

Súmula vinculante 18-STF: A dissolução da sociedade ou do vínculo conjugal, no curso do mandato, não afasta a inelegibilidade prevista no § 7º do artigo 14 da Constituição Federal.

Observação: A inelegibilidade do art. 14, § 7º, da Constituição NÃO ALCANÇA o cônjuge supérstite (sobrevivente, viúvo) quando o falecimento tiver ocorrido no primeiro mandato, com regular sucessão do vice-prefeito, e tendo em conta a construção de novo núcleo familiar. A Súmula Vinculante 18 do STF não se aplica aos casos de extinção do vínculo conjugal pela morte de um dos cônjuges. (STF, RE 758.461, Tese RG 678, 2014).

Lei n. 9.394/96 - Diretrizes e bases da educação.

Art. 5º O acesso à educação básica obrigatória é direito público subjetivo, podendo qualquer cidadão, grupo de cidadãos, associação comunitária, organização sindical, entidade de classe ou outra legalmente constituída e, ainda, o Ministério Público, acionar o poder público para exigi-lo.

O Estado de Defesa durará até 30 dias, podendo ser prorrogado somente por uma vez por igual período.

Preceitua a súmula 691:

NÃO COMPETE AO SUPREMO TRIBUNAL FEDERAL CONHECER DE "HABEAS CORPUS" IMPETRADO CONTRA DECISÃO DO RELATOR QUE, EM "HABEAS CORPUS" REQUERIDO A TRIBUNAL SUPERIOR, INDEFERE A LIMINAR.

De acordo com os eminentes ministros do Supremo Tribunal Federal:

“Não obstante esse óbice processual, a jurisprudência desta Suprema Corte, mitigando o rigor da apontada súmula, tem admitido a impetração de habeas corpus nas hipóteses de flagrante ilegalidade ou teratologia”.

• Informativo 1168
• HC 232627 / DF
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. GILMAR MENDES
• Julgamento: 11/03/2025 (Virtual)
• Ramo do Direito: Processual Penal, Constitucional
• Matéria: Jurisdição e Competência; Foro Especial por Prerrogativa de Função; Cessação do Exercício da Função/Direitos e Garantias Fundamentais; Poder Judiciário; Competência do Supremo Tribunal Federal

Foro por prerrogativa de função: competência para julgamento de crimes funcionais após a cessação do cargo

Tese fixada - A prerrogativa de foro para julgamento de crimes praticados no cargo e em razão das funções subsiste mesmo após o afastamento do cargo, ainda que o inquérito ou a ação penal sejam iniciados depois de cessado seu exercício.

Resumo - O STF fixou posição mais abrangente sobre a competência dos tribunais para julgar os crimes funcionais praticados por autoridades com prerrogativa de foro (“foro privilegiado”), no sentido de mantê-la mesmo após o término do exercício das respectivas funções. Aprimorou-se a orientação vigente com o intuito de assegurar a imparcialidade, a independência do julgamento e inibir os deslocamentos que resultam em lentidão, ineficiência e até mesmo prescrição das ações penais.

• O ordenamento jurídico prevê o foro especial por prerrogativa de função (CF/1988, art. 102, I, “b”) para proteger o exercício de cargos ou funções estatais de alta relevância constitucional contra ameaças do próprio acusado, manter a estabilidade das instituições democráticas, preservar o funcionamento do Estado e assegurar um julgamento menos suscetível a influências externas (1).
• Essa prerrogativa assegura que determinadas autoridades sejam julgadas por órgãos colegiados de maior hierarquia do Poder Judiciário. Portanto, o foro especial não constitui um privilégio pessoal, mas uma garantia para o adequado exercício das funções públicas.

• No que concerne à problemática do momento de encerramento do direito ao foro privilegiado, a jurisprudência desta Corte oscilou ao definir a sua extensão, ora pela natureza do delito (regra da contemporaneidade e da pertinência temática), ora pelo exercício atual de funções públicas (regra da atualidade), o que gerou uma indefinição quanto à abrangência do instituto.

• Com o cancelamento da Súmula 394/STF (2) — no julgamento da Questão de Ordem no Inquérito nº 687/SP (3) —, esta Corte realizou uma redução teleológica do foro privilegiado ao limitar sua aplicabilidade, de modo que o foro especial não se manteria após a perda do mandato, mesmo na hipótese de crimes cometidos durante o exercício das funções.

• Posteriormente, na Questão de Ordem na Ação Penal nº 937/RJ (4), o Tribunal entendeu que o referido foro se aplicaria apenas aos crimes cometidos durante o exercício do cargo e relacionados às funções desempenhadas. Assim, com exceção das ações cuja fase da instrução processual esteja concluída — hipótese de manutenção da competência, inclusive nos casos de infrações penais não relacionadas ao cargo ou à função exercida — a cessação do exercício das funções ensejaria o declínio da competência para o Juízo de primeiro grau.
• Nesse contexto, nas hipóteses de crimes funcionais, a imposição da remessa dos autos para a primeira instância com o término do exercício funcional subverte a finalidade do foro por prerrogativa de função. Isso ocorre porque, além de ser contraproducente ao causar flutuações de competência (“sobe e desce”) no decorrer das causas criminais e trazer instabilidade ao sistema de Justiça, permite a alteração da competência absoluta ratione personae ou ratione funcionae por ato voluntário do agente público acusado, ao renunciar ao mandato ou à função antes do final da instrução processual.
• Na espécie, esta Corte firmou a perpetuação da competência para o julgamento de crimes funcionais com base em uma interpretação mais ampla do foro especial, centrada na natureza do crime praticado pelo agente, em vez de critérios temporais relacionados à permanência no cargo ou ao exercício atual do mandato, que podem ser manipulados pelo acusado. Ademais, a saída do cargo somente afasta o foro privativo na hipótese de crimes perpetrados antes da investidura no cargo ou que não possuam relação com o seu exercício.
• Com base nesses e em outros entendimentos, o Plenário, por maioria, concedeu a ordem de habeas corpus para (i) assentar a competência do Supremo Tribunal Federal para processar e julgar a ação penal nº 1033998-13.2020.4.01.3900; e (ii) fixar a tese anteriormente mencionada, com o entendimento de que essa nova linha interpretativa deve aplicar-se imediatamente aos processos em curso, ressalvados todos os atos praticados e decisões proferidas pelo STF e pelos demais Juízos com base na jurisprudência anterior, conforme precedentes firmados no QO no INQ 687 e na QO na AP 937.

(1) CF/1988: “Art. 102. Compete ao Supremo Tribunal Federal, precipuamente, a guarda da Constituição, cabendo-lhe: I - processar e julgar, originariamente: (...) b) nas infrações penais comuns, o Presidente da República, o Vice-Presidente, os membros do Congresso Nacional, seus próprios Ministros e o Procurador-Geral da República”. (2) Súmula 394/STF: “Cometido o crime durante o exercício funcional, prevalece a competência especial por prerrogativa de função, ainda que o inquérito ou a ação penal sejam iniciados após a cessação daquele exercício. (Cancelada)”. (3) Precedente citado: QO no INQ 687. (4) Precedente citado: QO na AP 937.

Legislação: CF/1988: art. 102, I, b. Súmula 394/STF.

Precedentes: QO no INQ 687 e QO na AP 937.

• Processo(s): RE 882.461
• Tema: 816
• Relator: Min. Dias Toffoli

Título:

a) Incidência do ISSQN em operação de industrialização por encomenda, realizada em materiais fornecidos pelo contratante, quando referida operação configura etapa intermediária do ciclo produtivo de mercadoria.

b) Limites para a fixação da multa fiscal moratória, tendo em vista a vedação constitucional ao efeito confiscatório.

O Tribunal fixou a seguinte tese:

1. É inconstitucional a incidência do ISS a que se refere o subitem 14.05 da Lista anexa à LC nº 116/03 se o objeto é destinado à industrialização ou à comercialização;

2. As multas moratórias instituídas pela União, Estados, Distrito Federal e municípios devem observar o teto de 20% do débito tributário", e, no que diz respeito apenas à primeira tese fixada, atribuiu eficácia ex nunc, a contar da data de publicação da ata de julgamento do mérito, para:

a) impossibilitar a repetição de indébito do ISS em favor de quem recolheu esse imposto até a véspera da referida data, vedando, nesse caso, a cobrança do IPI e do ICMS em relação aos mesmos fatos geradores;

b) impedir que os municípios cobrem o ISS em relação aos fatos geradores ocorridos até a véspera daquela data.

1. Ficam ressalvadas (i) as ações judiciais ajuizadas até a véspera da mesma data, inclusive as de repetição de indébito e as execuções fiscais em que se discuta a incidência do ISS, e (ii) as hipóteses de comprovada bitributação relativas a fatos geradores ocorridos até a véspera da mencionada data, casos em que o contribuinte terá direito à repetição do indébito do ISS e não do IPI/ICMS, respeitado o prazo prescricional, independentemente da propositura de ação judicial até esse marco.

2. No caso de não recolhimento nem do ISS nem do IPI/ICMS, o Tribunal entendeu pela incidência do IPI/ICMS em relação aos fatos geradores ocorridos até a véspera da publicação da ata de julgamento do mérito.”

• RE 846854 - Repercussão Geral – Mérito (Tema 544)
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. LUIZ FUX
• Redator(a) do acórdão: Min. ALEXANDRE DE MORAES
• Julgamento: 01/08/2017

• Publicação: 07/02/2018

• As Guardas Municipais executam atividade de segurança pública (art. 144, § 8º, da CF), essencial ao atendimento de necessidades inadiáveis da comunidade (art. 9º, § 1º, CF), pelo que se submetem às restrições firmadas pelo Supremo Tribunal Federal no julgamento do ARE 654.432 (Rel. Min. EDSON FACHIN, redator para acórdão Min. ALEXANDRE DE MORAES, Tribunal Pleno, julgado em 5/4/2017)

• ADPF 995
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. ALEXANDRE DE MORAES
• Julgamento: 28/08/2023
• Publicação: 09/10/2023 ODS 16 - Paz, Justiça e Instituições Eficazes

DIREITO CONSTITUCIONAL E SEGURANÇA PÚBLICA. ART. 144, §8º, DA CONSTITUIÇÃO. RECONHECIMENTO DAS GUARDAS MUNICIPAIS COMO ÓRGÃO DE SEGURANÇA PÚBLICA. LEGÍTIMA OPÇÃO DO CONGRESSO NACIONAL AO INSTITUIR O SISTEMA ÚNICO DE SEGURANÇA PÚBLICA (LEI N° 13.675/18). PRECEDENTES. PROCEDÊNCIA DO PEDIDO. 1. É evidente a necessidade de união de esforços para o combate à criminalidade organizada e violenta, não se justificando, nos dias atuais da realidade brasileira, a atuação separada e estanque de cada uma das Polícias Federal, Civis e Militares e das Guardas Municipais; pois todas fazem parte do Sistema Único de Segurança Pública.

1. Essa nova perspectiva de atuação na área de segurança pública, fez com que o Plenário desta Suprema Corte, no julgamento do RE 846.854/SP, reconhecesse que as Guardas Municipais executam atividade de segurança pública (art. 144, § 8º, da CF), essencial ao atendimento de necessidades inadiáveis da comunidade (art. 9º, § 1º, da CF).
2. O reconhecimento dessa posição institucional das Guardas Municipais possibilitou ao , com CONGRESO NACIONAL, em legítima opção legislativa, no § 7º do artigo 144 da Constituição Federal, editar a Lei nº 13.675, de 11/6/2018, na qual as Guardas Municipais são colocadas como integrantes operacionais do Sistema Único de Segurança Pública (art. 9º, § 1º, inciso VII).
3. O quadro normativo constitucional e jurisprudencial dessa SUPREMA CORTE em relação às Guardas Municipais permite concluir que se trata de órgão de segurança pública, integrante do Sistema Único de Segurança Pública (SUSP).
4. Arguição de Descumprimento de Preceito Fundamental conhecida e julgada procedente para, nos termos do artigo 144, §8º da CF, CONCEDER INTERPRETAÇÃO CONFORME À CONSTITUIÇÃO aos artigo 4º da Lei 13.022/14 e artigo 9º da 13.675/18 DECLARANDO INCONSTITUCIONAL todas as interpretações judiciais que excluam as Guardas Municipais, devidamente criadas e instituídas, como integrantes do Sistema de Segurança Pública.

• Informativo nº 791
• 18 de outubro de 2023.
• TERCEIRA SEÇÃO
• Processo: HC 830.530-SP, Rel. Ministro Rogerio Schietti Cruz, Terceira Seção, por unanimidade, julgado em 27/9/2023, DJe 4/10/2023.

Ramo do Direito DIREITO CONSTITUCIONAL, DIREITO PROCESSUAL PENAL

Paz, Justiça e Instituições EficazesTema <br /> Guardas municipais. Exercício de atividade de segurança pública que não se equipara por completo às polícias. Art. 301 do CPP. Flagrante delito. Tráfico de drogas. Não ocorrência. Art. 244 do CPP. Busca pessoal. Ausência de relação com as finalidades da guarda municipal. Impossibilidade. Prova ilícita.

DESTAQUE - O fato de as guardas municipais não haverem sido incluídas nos incisos do art. 144, caput, da CF não afasta a constatação de que elas exercem atividade de segurança pública e integram o Sistema Único de Segurança Pública. Isso, todavia, não significa que possam ter a mesma amplitude de atuação das polícias.

INFORMAÇÕES DO INTEIRO TEOR - O fato de as guardas municipais não haverem sido incluídas nos incisos do art. 144, caput, da Constituição Federal não afasta a constatação de que elas exercem atividade de segurança pública e integram o Sistema Único de Segurança Pública. Isso, todavia, não significa que possam ter a mesma amplitude de atuação das polícias.

• Bombeiros militares, por exemplo, integram o rol de órgãos de segurança pública previsto nos incisos do art. 144, caput, da Constituição, mas nem por isso se cogita que possam realizar atividades alheias às suas atribuições, como fazer patrulhamento ostensivo e revistar pessoas em via pública à procura de drogas.

• O Supremo Tribunal Federal, apesar de reconhecer em diversos julgados que as guardas municipais integram o Sistema Único de Segurança Pública e exercem atividade dessa natureza, nunca as equiparou por completo aos órgãos policiais para todos os fins.

• Não se pode confundir "poder de polícia" com "poder das polícias" ou "poder policial". "Poder de polícia" é conceito de direito administrativo previsto no art. 78 do Código Tributário Nacional e explicado pela doutrina como "atividade do Estado consistente em limitar o exercício dos direitos individuais em benefício do interesse público". Já o "poder das polícias" ou "poder policial", típico dos órgãos policiais, é marcado pela possibilidade de uso direto da força física para fazer valer a autoridade estatal, o que não se verifica nas demais formas de manifestação do poder de polícia, que somente são legitimadas a se valer de mecanismos indiretos de coerção, tais como multas e restrições administrativas de direitos. Um agente de vigilância sanitária, por exemplo, quando aplica multa e autua um restaurante por descumprimento a normas de higiene, o faz em exercício de seu poder de polícia, mas nem de longe se pode compará-lo com um agente policial que usa a força física para submeter alguém a uma revista pessoal.

• Dessa forma, o "poder das polícias" ou "poder policial" diz respeito a um específico aspecto do poder de polícia relacionado à repressão de crimes em geral pelos entes policiais, de modo que todo órgão policial exerce poder de polícia, mas nem todo poder de polícia é necessariamente exercido por um órgão policial.

• Conquanto não sejam órgãos policiais propriamente ditos, as guardas municipais exercem poder de polícia e também algum poder policial residual e excepcional dentro dos limites de suas atribuições. A busca pessoal - medida coercitiva invasiva e direta - é exemplo desse poder, razão pela qual só pode ser realizada dentro do escopo de atuação da guarda municipal.

• Ao dispor, no art. 301 do CPP, que "qualquer do povo poderá [...] prender quem quer que seja encontrado em flagrante delito", o legislador, tendo em conta o princípio da autodefesa da sociedade e a impossibilidade de que o Estado seja onipresente, contemplou apenas os flagrantes visíveis de plano, como, por exemplo, a situação de alguém que, no transporte público, flagra um indivíduo subtraindo sorrateiramente a carteira do bolso da calça de outrem e o detém. Distinta, no entanto, é a hipótese em que a situação de flagrante só é evidenciada depois de realizar atividades invasivas de polícia ostensiva ou investigativa, como a busca pessoal ou domiciliar, uma vez que não é qualquer do povo que pode investigar, interrogar, abordar ou revistar seus semelhantes.

• A adequada interpretação do art. 244 do Código de Processo Penal é a de que a fundada suspeita de posse de corpo de delito é um requisito necessário, mas não suficiente, por si só, para autorizar a realização de busca pessoal, porque não é a qualquer cidadão que é dada a possibilidade de avaliar a presença dele. Em outras palavras, mesmo se houver elementos concretos indicativos de fundada suspeita da posse de corpo de delito, a busca pessoal só será válida se realizada pelos agentes públicos com atribuição para tanto, a quem compete avaliar a presença de tais indícios e proceder à abordagem e à revista do suspeito.

• Da mesma forma que os guardas municipais não são equiparáveis a policiais, também não são cidadãos comuns, de modo que, se, por um lado, não podem realizar tudo o que é autorizado às polícias, por outro, também não estão plenamente reduzidos à mera condição de "qualquer do povo". Trata-se de agentes públicos que desempenham atividade de segurança pública e são dotados do importante poder-dever de proteger os bens, serviços e instalações municipais, assim como os seus respectivos usuários.

• Dessa forma, é possível e recomendável que exerçam a vigilância, por exemplo, de creches, escolas e postos de saúde municipais, para garantir que não tenham sua estrutura danificada por vândalos, ou que seus frequentadores não sejam vítimas de furto, roubo ou algum tipo de violência, a fim de permitir a continuidade da prestação do serviço público municipal correlato a tais instalações. Nessa linha, guardas municipais podem realizar patrulhamento preventivo na cidade, mas sempre vinculados à finalidade da corporação, sem que lhes seja autorizado atuar como verdadeira polícia para reprimir e investigar a criminalidade urbana ordinária.

• Não é das guardas municipais, mas sim das polícias, como regra, a competência para investigar, abordar e revistar indivíduos suspeitos da prática de tráfico de drogas ou de outros delitos cuja prática não atente de maneira clara, direta e imediata contra os bens, serviços e instalações municipais ou as pessoas que os estejam usando naquele momento.

• Poderão, todavia, realizar busca pessoal em situações excepcionais - e por isso interpretadas restritivamente - nas quais se demonstre concretamente haver clara, direta e imediata relação com a finalidade da corporação, como instrumento imprescindível para a realização de suas atribuições. Vale dizer, salvo na hipótese de flagrante delito, só é possível que as guardas municipais realizem excepcionalmente busca pessoal se, além de justa causa para a medida (fundada suspeita), houver pertinência com a necessidade de tutelar a integridade de bens e instalações ou assegurar a adequada execução dos serviços municipais, assim como proteger os seus respectivos usuários, o que não se confunde com permissão para desempenharem atividades ostensivas ou investigativas típicas das polícias militar e civil para combate da criminalidade urbana ordinária em qualquer contexto.

• No caso, guardas municipais estavam em patrulhamento quando depararam com o acusado em "atitude suspeita". Por isso, decidiram abordá-lo e, depois de revista pessoal, encontraram certa quantidade de drogas no bolso traseiro e nas vestes íntimas dele, o que ensejou a sua prisão em flagrante delito.

• Ainda que, eventualmente, se considerasse provável que o réu ocultasse objetos ilícitos, isto é, que havia fundada suspeita de que ele escondia drogas, não existia certeza sobre tal situação a ponto de autorizar a imediata prisão em flagrante por parte de qualquer do povo, com amparo no art. 301 do CPP. Tanto que só depois de constatado que havia drogas dentro do bolso e das vestes íntimas do abordado é que se deu voz de prisão em flagrante para ele, e não antes.

• Portanto, por não haver sido demonstrada concretamente a existência de relação clara, direta e imediata com a proteção dos bens, serviços ou instalações municipais, ou de algum cidadão que os estivesse usando, não estavam os guardas municipais autorizados, naquela situação, a avaliar a presença da fundada suspeita e efetuar a busca pessoal no acusado.

Guardas municipais: competência legislativa e exercício de policiamento ostensivo e comunitário - RE 608.588/SP (Tema 656 RG)

TESE FIXADA: - É constitucional, no âmbito dos municípios, o exercício de ações de segurança urbana pelas Guardas Municipais, inclusive policiamento ostensivo e comunitário, respeitadas as atribuições dos demais órgãos de segurança pública previstos no art. 144 da Constituição Federal e excluída qualquer atividade de polícia judiciária, sendo submetidas ao controle externo da atividade policial pelo Ministério Público, nos termos do artigo 129, inciso VII, da CF. Conforme o art. 144, § 8º, da Constituição Federal, as leis municipais devem observar as normas gerais fixadas pelo Congresso Nacional.

• A atuação legislativa local para disciplinar as atribuições das guardas municipais destinadas à proteção de bens, serviços e instalações do município deve estar adequada às especificidades locais e à finalidade constitucional de promoção da segurança pública no âmbito da respectiva competência e em cooperação com os demais órgãos de segurança.
• O poder normativo conferido ao legislador municipal tem de se compatibilizar com a repartição constitucional de competências. Para tanto, as leis municipais que instituem suas respectivas guardas devem se adequar às especificidades locais, que restringem o poder legiferante, e à finalidade constitucional de promoção da segurança pública, além de observar as normas gerais fixadas pelo Congresso Nacional (CF/1988, art. 144, § 8º) (1).
• O texto constitucional não realizou uma escolha categórica sobre a forma de atuação das guardas municipais, apenas estabeleceu as balizas norteadoras e atribuiu sua concretização ao legislador local.
• Nesse contexto, o Estatuto Geral das Guardas Municipais (Lei nº 13.022/2014) foi julgado constitucional por esta Corte (2). Ele contribui para delimitar o espaço normativo dado pela Constituição em respeito ao pacto federativo e evidencia o caráter colaborativo entre os entes que atuam na segurança pública e devem atuar de forma conjunta e harmônica.

É constitucional — e não afronta o pacto federativo — o exercício do policiamento ostensivo e comunitário pela guarda municipal no âmbito local correspondente, desde que respeitadas as atribuições dos outros entes federativos.

• As guardas municipais podem exercer ações de segurança urbana e a atribuição do policiamento ostensivo e comunitário se insere no desenho normativo do federalismo de cooperação em prol da segurança pública, que é um dever do Estado e direito e responsabilidade de todos. Ademais, o policiamento ostensivo não é exclusivo da polícia militar.
• As guardas municipais integram o Sistema Único de Segurança Pública – Susp (Lei nº 13.675/2018) e, por força do art. 144 da CF/1988, atuam diretamente na área de segurança pública, naquilo que tem pertinência com a esfera da municipalidade.
• Por fim, além de a atividade policial exercida pelas guardas municipais se submeter ao controle externo do Ministério Público, cuja fiscalização objetiva evitar eventuais abusos (CF/1988, art. 129, VII), elas não podem realizar atividade de polícia judiciária, pois exclusiva da polícia civil e da Polícia Federal, responsáveis por investigar e apurar infrações penais.
• Na espécie, trata-se de recurso extraordinário interposto contra acórdão do Tribunal de Justiça do Estado de São Paulo que, em ação direta, declarou a inconstitucionalidade do dispositivo de lei municipal em que atribuído à guarda local o exercício de ações de segurança urbana, inclusive policiamento preventivo e comunitário, promovendo a mediação de conflitos.
• A decisão recorrida, em suma, considerou que o mencionado preceito invadiu competência da polícia militar para a realização do policiamento ostensivo, em afronta à norma da Constituição estadual que reproduz o disposto no art. 144, § 8º, da CF/1988.
• Com base nesses e em outros entendimentos, o Plenário, por maioria, ao apreciar o Tema 656 da repercussão geral, (i) deu provimento ao recurso extraordinário para declarar a constitucionalidade do art. 1º, I, da Lei nº 13.866/2004 do Município de São Paulo/SP, em sua redação original (3) e naquela dada pela Lei paulista nº 14.879/2009 (4); e (ii) fixou a tese anteriormente citada.

Author response:

The following is the authors’ response to the original reviews

Reviewer #1 (Public Review):

Summary.

In this meticulously conducted study, the authors show that Drosophila epidermal cells can modulate escape responses to noxious mechanical stimuli. First, they show that activation of epidermal cells evokes many types of behaviors including escape responses. Subsequently, they demonstrate that most somatosensory neurons are activated by activation of epidermal cells, and that this activation has a prolonged effect on escape behavior. In vivo analyses indicate that epidermal cells are mechanosensitive and require stored-operated calcium channel Orai. Altogether, the authors conclude that epidermal cells are essential for nociceptive sensitivity and sensitization, serving as primary sensory noxious stimuli.

Strengths.

The manuscript is clearly written. The experiments are logical and complementary. They support the authors' main claim that epidermal cells are mechanosensitive and that epidermal mechanically evoked calcium responses require the stored-operated calcium channel Orai. Epidermal cells activate nociceptive sensory neurons as well as other somatosensory neurons in Drosophila larvae, and thereby prolong escape rolling evoked by mechanical noxious stimulation.

Weaknesses.

Core details are missing in the protocols, including the level of LED intensity used, which are necessary for other researchers to reproduce the experiments. For most experiments, the epidermal cells are activated for 60 s, which is long when considering that nocifensive rolling occurs on a timescale of milliseconds. It would be informative to know the shortest duration of epidermal cell activation that is sufficient for observing the behavioral phenotype (prolongation of escape behavior) and activation of sensory neurons.

(1) We agree with the reviewer that the LED intensity is an important detail of the experimental paradigm. We updated the methods to include intensity measurements for the stimuli used throughout the manuscript.

(2) The Reviewer asks about the shortest duration of epidermal cell activation sufficient for observing the behavior phenotype. We note in the manuscript that behavioral responses to optogenetic epidermal stimulation are apparent within 2 seconds of stimulus (see Figure 2F); this is consistent with our calcium imaging data in which C4da response reaches its maximum within 2-3 sec of stimulation.

Reviewer #1 (Recommendations):

(1) The epidermal cells in this study are activated for 60 s. In the real world, the nociceptive stimulation (a poke, such as penetration by the ovipositor of a parasitic wasp) that evokes escape rolling is short. Does optogenetic activation of 1 s or less still evoke rolling? For example, it is unclear in Figure 4K how long the epidermal cells need to be activated before the poke stimulus prolongs rolling. Is it possible to test behavior and GCaMP activity in sensory neurons when epidermal cells are briefly (1 second) activated?

As described above, behavioral responses to optogenetic epidermal stimulation are apparent within 2 seconds of stimulus (see Figure 2F); this is consistent with our calcium imaging data in which C4da response reaches its maximum within 2-3 sec of stimulation. The kinetics are consistent with a role for epidermal cells in modulating neuronal responses to nocifensive stimuli, and similar to the response kinetics observed in mammalian epidermal cells that modulate neuronal touch and pain responses  (Maksimovic et al., 2014; Woo et al., 2014; Mikesell et al., 2022).

(2) The protocol for optogenetic screening states that the authors used a 488-nm LED. Why was a 488-nm LED used instead of the 610-nm LED for Chrimson activation? No information (except figure 4K) about the light intensity is provided in the figure legend or the protocol section. Please state the LED intensity used for all optogenetic experiments (GCaMP imaging, behavioral experiments, etc.).

We used 488 nm light for the initial screen for technical reasons. The screen was conducted by students at the MBL Neurobiology course (hence the affiliation; student authors are included in the manuscript), and the only LED available to us at that time delivered insufficient illumination at longer wavelenths to be useful. We chose to include the student’s data because (1) we found that the 488 nm light alone did not induce rolling in our setup, (2) we repeated and extended the studies with the epidermal drivers using a higher resolution imaging platform and longer wavelength stimulation (all studies other than Fig. 1), and (3) we observed qualitatively similar results when we repeated stimulation with all drivers using 561 nm light.

We agree that the LED intensity is an important detail of the experimental paradigm. We updated the methods to include intensity measurements for the stimuli used throughout the manuscript. We also include the intensities here:

- 30 μW/mm^2 for calcium imaging experiments Fig 3B-E, Fig 4A, Fig 3S1A-D, Fig 4S1A

- 300 μW/mm^2 for behavior studies in Fig 2B-E, Fig 1S6, Fig 2S1, Fig 3E-F, Fig 3S2A-C

- 25 μW/mm^2 for behavior studies in Fig 4E-J

- 1.16 μW/mm^2 for behavior studies in Fig 4K

(3) Lines 150 - 152: Although the authors refer to "a stereotyped behavior sequence" in Fig 2D, there are no data supporting this claim in Fig 2. Rather, the data appear to represent proportions of different types of behavior at each time point, rather than behavior sequences. If the authors wish to claim that the data show stereotyped behavior sequences, they should analyze the data using a different method (e.g., Markov models).

We agree that in the absence of additional analysis we should avoid commenting on stereotypy of behavior sequences; we therefore adjusted the text to reflect the tendency of nociceptive behaviors to precede non-nociceptive behaviors. The raster plots shown in Supplemental Fig. 2A illustrate this point: in larvae exhibiting nociceptive behaviors, these behaviors appear first, followed by backing and frequently freezing. As one quantitative readout of this sequence we show that the latency of rolling (nociceptive) is shorter compared with backing or freezing (non-nociceptive) (Fig. 2F, Fig. S2G).

(4) Figure 3A-E: a cursory glance at the data suggests that the most responsive sensory neurons are C1da, with all sensory neurons activated. However, at the behavioral level, only some sensory neurons are activated. If all sensory were activated by Chrimson, what behavioral phenotypes would the authors expect to see? Would it be the same as epidermal activation?

The Reviewer raises an interesting question, but we intentionally avoid comparing the response properties among sensory neurons because of differences in driver strength. Likewise, extrapolating “activation” at the behavioral level is exceedingly difficult if/when multiple sensory neurons are simultaneously activated. In response to the Reviewer’s specific question, when all da neurons are activated simultaneously, larvae largely exhibited hunching rather than rolling (Hwang et al., 2007). We find that epidermal stimulation rarely elicits hunching; instead, epidermal stimulation generally triggers nocifensive behaviors followed by non-nocifensive behaviors such as backing and freezing, suggesting an order or priority in neurons activated by epidermal cells (or different response times). Defining the mechanisms by which epidermal cells communicate with different types of sensory neurons is therefore a top priority for future studies.

(5) Figure 3S2; The behavior phenotypes between Fig. 3E, F and Fig 3S2 seems a slightly different. I suggest adding some comments in different behavior phenotype depending on the different GAL4. Specifically, is there increased freezing in some genotypes (e.g., ppk-LexA or NompC-lexA)? Can you show this without TNT data? Is this a background effect or specific GAL4 phenotype?

We currently do not have the driver-only control for this experiment, but our effector-only control experiment (see Fig. 3S2A) suggests that larvae carrying the AOP-TNT insertion exhibit enhanced nociceptive behavioral responses. This point is addressed in our manuscript by the following (copied from the figure legend):

“We note that although baseline rolling probability is elevated in all genetic backgrounds containing the AOP-LexA-TnT insertion, silencing C4da and C3da neurons significantly attenuates responses to epidermal stimulation.”

(6) Calcium-free solution is used in Figure 3. Why do the authors still observe calcium influx? Does this mean that internal calcium stores are released? If so, does the calcium influx represent an action potential? How do the authors focus their LED stimulation to activate epidermal cells and avoid activation of the imaging laser?

The specimens were imaged in calcium-free solution to minimize movement artifacts. However, the CNS is wrapped by glial cells and over short timescales such as those used for the imaging we speculate that extracellular calcium persists in the CNS.

(7) It is unclear when animals begin to crawl after the epidermal cells are mechanically stimulated. How do the authors distinguish between peristaltic crawling and a poke by Orai receptors? Although the in vitro experiments beautifully show radial tensions, it is unclear to what extent A-P axis tension (peristaltic crawling) and radial tension (poke) differ. It might be helpful to explain in the discussion section how epidermal cells are selectively activated.

The Reviewer raises an interesting question about the types and thresholds of forces required to elicit epidermal responses. We cannot eliminate the possibility that peristaltic crawling (or crawling through a 3D substrate) stimulates epidermal cells to a certain degree. Indeed, our results demonstrate a dose-dependent response of Drosophila epidermal cells and human keratinocytes to radial stretch. However, we do not have any information about selectivity in response to different stimuli, though we agree that this is an intriguing avenue for future studies. For example, we don't know whether stretch-responsive cells are more or less responsive to poke. But, a salient feature of our studies is the recruitment of greater numbers of responders with increasing stimulus intensity, therefore we added the following statement to the discussion to clarify our model:

“Finally, we find that epidermal cells exhibit a dose-dependent response to radial stretch; we therefore anticipate that the output of epidermal cells is likewise dependent on the stimulus intensity.  Hence, rather than a fixed threshold beyond which epidermal cells are selectively activated, we hypothesize that increasing stimulus intensities drive increasing signal outputs to neurons.”

(8) Some Protocols are missing. For example, in Figure 4, many stimulus combinations were used to test behavior. How were stimuli of different modalities applied to the animals? Further details need to be provided in the protocols.

We thank the Reviewer for identifying this oversight. The methods section of our original submission detailed most of the stimulus combinations but omitted the opto + mechano combination (4F). We updated our methods to correct these omissions.

(9) It might be helpful if the authors could provide a sample video for each behavior to clarify how they were each defined.

Our manuscript includes a table with a detailed description of the behaviors (Table S2), and we added two annotated videos that show representative behavioral responses to optogenetic nociceptor or epidermis stimulation.

(10) A supplementary summary table of genotypes might be helpful for the reader.

Experimental genotypes are provided in the figure legends, and a detailed list of all alleles used in the study as well as their source is provided in supplemental table S1.

Reviewer #2 (Public Review):

Summary.

The authors provide compelling evidence that stimulation of epidermal cells in Drosophila larvae results in the stimulation of sensory neurons that evoke a variety of behavioral responses. Further, the authors demonstrate that epidermal cells are inherently mechanoresponsive and implicate a role for store-operated calcium entry (mediated by Stim and Orai) in the communication to sensory neurons.

Strengths.

The study represents a significant advance in our understanding of mechanosensation. Multiple strengths are noted. First, the genetic analyses presented in the paper are thorough with appropriate consideration to potential confounds. Second, behavioral studies are complemented by sophisticated optogenetics and imaging studies. Third, identification of roles for store-operated calcium entry is intriguing. Lastly, conservation of these pathways in vertebrates raise the possibility that the described axis is also functional in vertebrates.

Weaknesses.

The study has a few conceptual weaknesses that are arguably minor. The involvement of store-operated calcium entry implicates ER calcium store release. Whether mechanical stimulation evokes ER calcium release in epidermal cells and how this might come about (e.g., which ER calcium channels, roles for calcium-induced calcium release etc.) remains unaddressed. On a related note, the kinetics of store-operated calcium entry is very distinct from that required for SV release. The link between SOC and epidermal cells-neuron transmission is not reconciled. Finally, it is not clear how optogenetic stimulation of epidermal cells results in the activation of SOC.

(1) The involvement of store-operated calcium entry implicates ER calcium store release. Whether mechanical stimulation evokes ER calcium release in epidermal cells and how this might come about (e.g., which ER calcium channels, roles for calcium-induced calcium release etc.) remains unaddressed.

Our studies suggest that mechanically evoked responses in epidermal cells involve both ER calcium release and store-operated calcium entry. Notably, we show that depletion of ER calcium stores before mechanical stimulation, by treating with thapsigargin, reduces (but does not eliminate) mechanically evoked calcium responses in fly epidermal cells (Fig. 6C-6F). Likewise, fly epidermal cells and human keratinocytes both exhibit mechanically evoked calcium responses in the absence of extracellular calcium (10mM EGTA to chelate all free calcium ions). These data support a model whereby mechanical stimuli trigger calcium release from ER stores and influx. Indeed, several cell types have been shown to display mechanically evoked release of calcium from stores. For example, mechanical stimulation of enteroendocrine cells of the gut epithelium results in both calcium release from ER stores and calcium influx across the plasma membrane (Knutson et al., 2023). Similar to our findings, Knutson et al found that depleting stores decreased mechanically evoked calcium signals by over 70% in these gut epithelial stores. In our revised manuscript we have more clearly emphasized these points.

We agree with the reviewer that deciphering the mechanisms by which mechanical stimuli promote ER calcium release and subsequent store-operated calcium entry is an exciting topic to explore. One potential mechanism is the activation of a mechanosensitive receptor that promotes calcium release from the ER via calcium-induced calcium release or IP3 production, as has been proposed for enteroendocrine cells. A recent paper demonstrated that the ER itself is mechanosensitive and that mechanical stimuli promotes calcium release via the opening of calcium-permeable ion channels in the ER membrane (Song et al., 2024). Determining the relative contributions of store-operated calcium entry and ER calcium release and deciphering their underlying mechanisms will require a thorough investigation of ER calcium channels and receptors, thus we believe this would be beyond the scope of the present manuscript and merits publication on its own. However, we now include this in our discussion as an exciting new direction we aim to pursue.

(2) The kinetics of store-operated calcium entry is very distinct from that required for SV release. The link between SOC and epidermal cells-neuron transmission is not reconciled.

The Reviewer raises an interesting point regarding the mode of epidermal cell-neuronal communication. We demonstrated a requirement for dynamin-dependent vesicle release from epidermal cells in mechanical sensitization. However, the nature of the vesicular pool, the mode and kinetics of release, and the type of neuromodulator released remain to be characterized. Hence, it’s not clear that kinetics of synaptic vesicle release is an appropriate comparison. Our studies do demonstrate that behavioral responses to optogenetic epidermal stimulation are relatively slow – on the order of seconds – which is not incompatible with the kinetics of store-operated calcium entry. Furthermore, the primary functional output we define for epidermal mechanosensory responses, mechanical nociceptive sensitization, is apparent 10 sec following the stimulus and persists for minutes in our behavior assays. Consistent with this model, studies of the mammalian touch dome have shown that touch-sensitive Merkel cells secrete neurotransmitters to modulate neurons and promote sustained action potential firing on a similar timescale. Likewise, mechanically evoked ER calcium-release promotes sustained secretion of serotonin from enterochromaffin cells.

(3) It is not clear how optogenetic stimulation of epidermal cells results in the activation of SOC.

We appreciate the opportunity to clarify our results. We demonstrate that optogenetic epidermal stimulation elicits behavioral responses in larvae and calcium responses in somatosensory neurons, but we do not claim that optogenetic epidermal stimulation elicits SOC. Our optogenetic studies demonstrate the capacity for epidermal stimulation to modulate somatosensory function, but we characterize contributions of SOC only to mechanical stimuli which are more physiologically relevant. However, it is worth noting that CsChrimson is a calcium-permeable channel, suggesting that an increase in intracellular calcium may trigger epidermal-evoked neuronal responses and behaviors during optogenetic stimulation.

References

Hwang, RY, Zhong, L, Xu, Y, Johnson, T, Zhang, F, Deisseroth, K, and Tracey, WD (2007). Nociceptive neurons protect Drosophila larvae from parasitoid wasps. Curr Biol 17, 2105–2116.

Knutson, KR, Whiteman, ST, Alcaino, C, Mercado-Perez, A, Finholm, I, Serlin, HK, Bellampalli, SS, Linden, DR, Farrugia, G, and Beyder, A (2023). Intestinal enteroendocrine cells rely on ryanodine and IP3 calcium store receptors for mechanotransduction. J Physiol 601, 287–305.

Maksimovic, S, Nakatani, M, Baba, Y, Nelson, AM, Marshall, KL, Wellnitz, SA, Firozi, P, Woo, S-H, Ranade, S, Patapoutian, A, et al. (2014). Epidermal Merkel cells are mechanosensory cells that tune mammalian touch receptors. Nature 509, 617–621.

Mikesell, AR, Isaeva, O, Moehring, F, Sadler, KE, Menzel, AD, and Stucky, CL (2022). Keratinocyte PIEZO1 modulates cutaneous mechanosensation. Elife 11, e65987.

Song, Y, Zhao, Z, Xu, L, Huang, P, Gao, J, Li, J, Wang, X, Zhou, Y, Wang, J, Zhao, W, et al. (2024). Using an ER-specific optogenetic mechanostimulator to understand the mechanosensitivity of the endoplasmic reticulum. Dev Cell 59, 1396-1409.e5.

Woo, S-H, Ranade, S, Weyer, AD, Dubin, AE, Baba, Y, Qiu, Z, Petrus, M, Miyamoto, T, Reddy, K, Lumpkin, EA, et al. (2014). Piezo2 is required for Merkel-cell mechanotransduction. Nature 509, 622–626.

es podria veure millor aquesta taula? no es veu massa bé. Si li fas un as.tibble(regicor)? o pots fer trampes i fer-li un gt de fons jeje

Ho canviaria per una altra frase tipo formula specification o algo així

Faltaria llistar les condicions del model o ho diràs de veu? Independència Normalitat del residus Homogeneïtat de les variàncies

Aquí és important que diguis que no hi ha jerarquia entre les variables. X sobre Y ó Y sobre X ho llegirem igual.

I també està pensada per raons linials i que si no ho són la rho no és interpretable (feu el gràfic). Els podries mostrar el quartet de Anscombe's

Trauria el punt o faria una entradeta dels punts de sota per deixar clar que el concepte és aquest.

T'has passat... ;)... si tens, com els passa als genetistes, una matriu de 1000x1000 o més per algun lloc has de començar a descartar... no ens agrada, però és realista. Trauria la frase, però comentaria de veu les greus limitacions.

Per lligar-ho amb el títol de la presentació aquí podries anunciar o definir els GLM i explicar-ne 4 tipus en funció de la variable dependent i la seva distribució. I dir que n'hi ha més.

Els Cox no són GLM per tant ni els mencionaria.

para hacer lo que dice Cristian, en el cambio anterior de poner el lung en esta diapo, puedes poner head(lung, 3 o 4)

called model1 / named model1 o si es una instrucción: name it model2

called model1 / named model1 o si es una instrucción: name it model2

Potser faria un tip general pel punt 4 que fos: use the survfit object. Valorar aleshores si treure aquest o no.

Afegiria que transformesin a factor les variables que tinguin ordre, o algo així. Perquè sino no se'ls hi ocurrirà.

Reviewer #3 (Public review):

Summary:

Here the authors describe the role of mORs in synaptic glutamate release from substance P and cholinergic neurons in the medial habenula to the interpeduncular nucleus (IPN) circuit in adult mice. They show that mOR activation reduces evoked glutamate release from substance P neurons yet increases evoked glutamate release and Ach release from cholinergic neurons. Unlike glutamate release, Ach release is only detected when potassium channels are blocked with 4-AP or dendrotoxin, implicating Kv1.2. The authors also report a previously unidentified glutamatergic input to IPR mediated from SP neurons and describe the developmental timing of mOR-facilitation in adolescent mice.

Strengths:

(1) The experiments provide new insight into the role of mORs in controlling evoked glutamate release in a circuit with high levels of mORs and established roles in relevant behaviors.

(2) The experimental design is generally rigorous, and the results are clear-cut. The conclusions are largely supported by the data.

(3) The findings will be of interest to those working in the field.

Weaknesses:

(1) The mechanistic underpinnings of the most interesting results are not pursued. For example, the experiments do not provide new insight into the differential effects of evoked and spontaneous glutamate/Ach release by Gi/o coupled mORs, nor the differential threshold for glutamate versus Ach release.

(2) The significance of the ratio of AMPA versus nACh EPSCs shown in Figure 6 is unclear since nAChR EPSCs measured in the K+ channel blockers are compared to AMPA EPSCs in control (presumably 4-AP would also increase AMPA EPSCs).

(3) The authors note that blocking Kv1 channels typically enhances transmitter release by slowing action potential repolarization. The idea that Kv1 channels serve as a brake for Ach release in this system would be strengthened by showing that these channels are the target of neuromodulators or that they contribute to activity-dependent regulation that allows the brake to be released.

Realiza aqui as tuas anotações sobre o diagnósticos das DTM.

Obrigada. Filipa Ricardo

to ensure that someone else can

O texto pode ser útil para uso no tcc para contextualizar. Esse quadro se agrava quando há o reconhecimento de que a violência em meio escolar, sobretudo nas escolas da rede pública, não é fenômeno recente, tendeu a se agravar egerou,inclusive,umsignificativoceticismoquantoàspossíveissoluções.Poroutrolado,verifica-se,também,queessetipodeproble-manãoé privilégiodepaísespobres,poisatinge,deformasdiversas,paísesdesenvolvidos

Unidad básica de la actividad refleja integrada, que consta de un órgano sensorial, una neurona aferente, una o más sinapsis dentro de una estación de integración central, una neurona eferente y un efector.

We must not 'challenge' God, for He cannot be challenged. WE must not question His mercy, we must not think that we are above sin and that we are not sinners. Because who could survive if we were judged based off our sins alone, how could any of us make it if it were not for our Redeemer, the Lord Himself?

DOI: 10.1016/j.xcrm.2025.102036

Resource: (BioLegend Cat# 502505, RRID:AB_315230)

Curator: @scibot

SciCrunch record: RRID:AB_315230

What is this?

DOI: 10.1016/j.neuron.2025.03.004

Resource: (BioLegend Cat# 829901, RRID:AB_2564934)

Curator: @scibot

SciCrunch record: RRID:AB_2564934

What is this?

DOI: 10.1016/j.jcmgh.2025.101511

Resource: (Cell Signaling Technology Cat# 14896, RRID:AB_2798639)

Curator: @scibot

SciCrunch record: RRID:AB_2798639

What is this?

Rabelais has vague historical background, but what is well known from his lifetime was his transition from a religious lifestyle to the studies of medicine and humanities (Coutinho 4). This shift in mindset reflects heavy on Rabelais work as he pokes fun at religious symbols and uses biblical events in what was considered "poor taste" at the time. This is one of the numerous reasons Rabelais' novels were considered "forbidden books" at the time in frace (Coutinho 4). Gargantua gives thanks to God for liquor and uses Jesus' betrayal in a satirical form of gratitude.

Coutinho, Léo, Carlos Henrique Ferreira Camargo, and Hélio Afonso Ghizoni Teive. “François Rabelais and His Dystonic Giants.” Arquivos de neuro-psiquiatria 82.7 (2024): 1–2. Web.

While this may sound unique, you can see the heritage in this passage with a biblical passage. This saying appears to be derived from an inspiration of the book of Ephesians. This would entail another contrast in the irony Rabelais wishes to display to the reader. What is a reference in Ephesians to awake to the Jesus - instead he is singing to those around him - reference the idea of free interpretation of biblical verses and being guided by the spirit to speak such things rather than the catholic church. The paragraph before and after dive deeper into the protestant ideology that stride father from the catholic ideology that with protestants, they can interpret and illustrates scriptural and theology losely rather than strict interpretation like at the time during reformation in the 16th century in france. This makes the most sense as well in the idea that Reinian is a german name and what was at the time eastern frankia was heavily interactive with - the lutheran church spread into france quickly and rapidly.

https://en.wikipedia.org/wiki/Lutheranism https://www.houseofnames.com/reinmann-family-crest https://www.biblegateway.com/passage/?search=Ephesians%205%3A13-15&version=KJ21

The name of this song, beati quorum was the perfect choice for the song before battle, "o lord be not in thine anger" (MyMemory). This choice of song is just another example of the constant contrasting ideas between what the author chooses vs. what makes sense. It makes sense to ask the Lord to not be angry before battle, but I feel as if unlike Gargantua it wouldn't be a song to put someone to sleep, but rather continue with the anxiety present before the battle. Results for Beati quorum via, translation from Latin to English. Beati quorum via, in English with contextual examples. (n.d.). https://mymemory.translated.net/en/Latin/English/beati-quorum-via%2C

When I read this part of the text, I felt quite shocked because of the level of detail the human anatomy was mentioned. Of course, my shock then subsided because I remembered that Rabelais was a doctor. I've read that Rabelais actually did use some of his writing as a way to make some more known about the privilege of medicine only to the elite. Simply taking some time to add the names of human anatomy rather than describing, for example, "the top of the head" (coronal joint) or "right temple/side of the head" (crotaphic artery, with a little bit of research). This definitely further reinforces his position as both a writer and someone with expertise in medicine and the human body.

This, I think, also applies to the whole "vital urge" aspect of the writing because it is anatomical and gruesome towards the end of the passage. Also, this is simply the effect of panic and adrenaline, a human response.

Anderson, J. “The Francois Rabelais school of medicine.” BMJ (Clinical research ed.) vol. 323,7327 (2001): 1456-7. doi:10.1136/bmj.323.7327.1456

A partir de 2028 (terei só 56 anos) a quantidade é 105.

Possivelmente irei atingir 105 pontos em 2039 (com 67 anos), 67+38,58= 105,48

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

Reviewer #1 (Evidence, reproducibility, and clarity)

The manuscript by Song et al presents evidence to show that the predicted cysteine protease type 6 secretion system (T6SS) effector Cpe1 inhibits target cell growth by cleaving type II DNA Topoisomerases GyrB and ParE. The authors determined the structure of the protein complex formed by Cpe1 and its immunity protein Cpi1, which allowed them to reveal the mechanism of inhibition. Moreover, the authors identified type II DNA topoisomerases GyrB and ParE as the targets of Cpe1. Overall, the major conclusions were well supported by experimental data of high quality. The findings have expanded our appreciation of the mechanism utilized by T6SS effectors to inhibit target cell growth.

We thank the reviewer for their positive remarks and valuable suggestions to improve this manuscript.

Major comments

To better establish that GyrB and ParE are the sole targets of Cpe1, the authors should express the GG mutant in target cells and determine whether these cells become resistant to Cpe1-mediated killing (inhibition). They can also determine whether co-expression of the cleavage resistant mutants suppresses the toxicity of Cpe1.

We appreciate the reviewer’s suggestion to investigate additional substrates of Cpe1 beyond GyrB and ParE, which may not have been fully captured in our crosslinking-mass spectrometry experiments due to technical limitations or low protein abundance. To address this topic, we generated target cells heterologously expressing cleavage-resistant GyrB and ParE variants (GyrBΔG102 and ParEΔG98) that are not susceptible to Cpe1, as described in our original manuscript (Figures 3h, i). We performed both Cpe1 expression assay and competition assay to assess if expression of the cleavage-resistant variants suppresses Cpe1 toxicity (Author Response Figures 1a, b). However, we did not observe a substantial protective effect. While this outcome could suggest that GyrB and ParE are not the sole targets of Cpe1, alternative explanations are also plausible. In the Cpe1 expression assay, high levels of Cpe1 could still act on endogenous wild-type GyrB and ParE, and although we attempted to increase variant expression, precise quantification remains challenging. In the competition assay, highly active Cpe1 may have continued to target wild-type substrates throughout the experiment, potentially masking any protective effect. Additionally, reduced activity of the mutant proteins could contribute to the observed results. Finally, deletion of the global repressor H-NS in the Cpe1-producing E. coli strain may have induced other interbacterial competition mechanisms1, leading to growth inhibition independently of Cpe1. Addressing these questions comprehensively would require a more systematic investigation under a wider range of conditions. We consider this an important avenue for future studies.

Results in Figure 7 clearly show that Cpi1 is capable of displacing ParE from Cpe1 due to higher affinity. Yet, the "competitive inhibition model" described in the last result section does not completely match what is really happening in Cpe1-mediated interbacterial competition. If Cpi1 is in the target cell, it would more likely engage the incoming Cpe1 before it can interact with ParE or GyrB, so competition does not occur in this scenario. Similarly, in the predatory cells expressing Cpe1 and Cpi1, these two proteins will form a stably protein complex, and no competition with the target will occur. The authors should reconsider their model.

We thank the reviewer for their comments and appreciate the opportunity to clarify this point. First, we believe the reviewer is referring to Figure 5 rather than Figure 7. In our model, the primary role of immunity proteins in interbacterial competition is to neutralize cognate toxins and prevent self- or kin-intoxication. These immunity proteins exhibit high specificity and strong binding affinity toward their associated toxins, ensuring effective protection2. In predatory cells, immunity proteins are typically co-expressed with their corresponding toxins, likely enabling immediate suppression upon translation. During kin competition, immunity proteins can protect cells even after foreign toxins engage their substrates.

Our results demonstrate that Cpi1 binds Cpe1 with higher affinity than its substrates and can displace them from pre-formed Cpe1-substrate complexes (Figures 5b-f). This aligns with the established function of immunity proteins in interbacterial competition and provides a mechanistic basis for how they confer protection, even when toxins have initially engaged their targets2. We acknowledge the reviewer’s point that in both scenarios—whether in the recipient cell or the toxin-producing cell—Cpe1 may first encounter Cpi1. However, our model underscores that Cpi1 not only binds at the substrate site but also exhibits superior affinity for Cpe1, ensuring robust protection against Cpe1-mediated toxicity.

Minor comments

"Intoxication" was used throughout the text numerous times to describe the activity of Cpe1. Looking in the Marriam-Webster dictionary, "Intoxication" means "a condition of being drunk". This word should be replaced with "toxicity" or some other terms in this line.

We thank the reviewer for this comment. We acknowledge that the term "intoxication" is commonly associated with alcohol consumption, yet the Merriam-Webster dictionary also defines it as "an abnormal state that is essentially a poisoning" (https://www.merriam-webster.com/dictionary/intoxication). This definition aligns with its well-established usage in the field of interbacterial competition to describe the effects of interbacterial toxins during antagonism3-5, which we have adopted in our manuscript. However, we appreciate the reviewer’s concern and remain open to revising the terminology if deemed necessary for clarity.

Lines 46-48, references on contact-dependent killings by these systems mentioned should cited. Ref. 9 cited does NOT cover the information at all.

We thank the reviewer for this comment. We have revised the citation and now reference studies that specifically describe contact-dependent killing systems in the relevant sentences (Lines 45–____50)

"characterizations" should be "characterization".

We have now modified the sentence as requested (Line 69)

Line 229 "Cpe1-Bpa monomers" should be " apo Cpe1-Bpa". The results cannot distinguish whether these bands are monomers or multimers.

We appreciate the reviewer’s careful assessment of our manuscript. The results in Line 233 (Figure 3c) show the enrichment of His-tagged proteins, including crosslinked complexes and overproduced Cpe1-Bpa. Based on the molecular weight marker, the Cpe1-Bpa bands appear between 10–15 kDa, consistent with the molecular weight of Cpe1 monomers (Figure 3a). Therefore, we have labeled this band as “Cpe1-Bpa monomers” and maintained this terminology throughout the text. This designation aligns with previous studies utilizing site-specific crosslinking via Bpa incorporation6,7

Line 283, was the mutation deletion? Substitution was used I think.

We thank the reviewer for highlighting this point. The GyrB and ParE mutants used to confirm the cleavage sites were deletion mutants, with a single glycine removed from the predicted double-glycine motifs. We have now revised the text for clarity (Lines 285–290)

Lines 439-444 the discussion should be extended to include other bacterial toxins that target type II DNA topoisomerases (e.g. PMID: 26299961 and PMID: 26814232).

We appreciate the reviewer’s suggestion. The studies referenced (PMID: 26299961 and PMID: 26814232) describe FicT toxin with adenylyl transferase activity that target and post-translationally modify GyrB and ParE at their ATPase domains, highlighting a potential hotspot for topoisomerase inhibition. We have now incorporated an additional paragraph in the Discussion section to describe these findings (Lines 424–439).

Reviewer #1 (Significance)

The authors determined the structure of the protein complex formed by Cpe1 and its immunity protein Cpi1, which allowed them to reveal the mechanism of inhibition. Moreover, the authors identified type II DNA topoisomerases GyrB and ParE as the targets of Cpe1. Overall, the major conclusions were well supported by experimental data of high quality. The findings have expanded our appreciation of the mechanism utilized by T6SS effectors to inhibit target cell growth.

We sincerely thank the reviewer for their positive comments and for the suggestions to improve our manuscript.

Reviewer #2 (Evidence, reproducibility, and clarity)

The manuscript, titled "An Interbacterial Cysteine Protease Toxin Inhibits Cell Growth by Targeting Type II DNA Topoisomerases GyrB and ParE", describes how an effector family was identified and characterized as a papain-like cysteine protease (PLCP) that negatively impacts bacterial growth in the absence of its co-encoded immunity protein. This thorough report includes (1) bioinformatic analysis of prevalence, finding this PLCP effector encoded in many gram-negative bacteria, (2) confirming conservation of catalytic active site via structural (crystallographic) analysis, as well as visualizing contacts with the immunity protein, (3) validation of results using growth studies combined with mutagenesis, (4) using a cell-based cross-linking method to pull out potential targets, which were subsequently identified via mass spectrometry, (5) validation of these results using in vitro protease assays with purified (potential) substrates, including verification of the motif recognized on the substrate(s), and cell-based phenotype analyses, and finally, (6) demonstrating competition between immunity protein and ParE substrate using an in vitro pull-down approach. Overall, this is a strong body of work with compelling conclusions that are well supported by multiple experimental approaches.

We appreciate the reviewer for their positive comments regarding our original submission.

Major comments

The claims made based on the presented results are well supported, including that this PLCP effector toxin is widespread, is neutralized in a competitive mechanism by its immunity partner, and that it effectively cleaves both GyrB and ParE (subunits of bacterial type II topoisomerases) at a conserved motif, resulting in suppression of bacterial cell growth via mis-regulating chromosome segregation. No additional experiments are needed to further validate these results, and the authors are commended on the cell-based and in vitro studies to deduce very specific mechanisms and structural details.

We appreciate the reviewer’s positive feedback.

Minor comments

While the writing and data presentation are extremely clear, in general I recommend the authors indicate the level(s) of replication for experiments. Figure legends generally note that mean values with standard deviations are shown, but I did not find where the number of replicates (and independent versus technical) were listed.

We appreciate the reviewer’s suggestion. We have now revised the manuscript to specify the levels of replication (independent vs. technical) for each experiment in the figure legends, particularly in Figures 2 and 3.

The figures are very clear, but in many instances the addition of PLCP toxin is indicated as "before" and "after"; while a modest change, I recommend altering this to some type of "-" and "+" type nomenclature rather than a time-based notation (especially as presumably both samples were treated identically, just with or without protease).

We thank the reviewer for this helpful comment. In Figures 3 and Supplementary Figures 5, 9, we used "before" and "after" to indicate the time points for in vitro cleavage assays verifying Cpe1 cleavage. To minimize variations between reactions, the catalytic mutant Cpe1tox (Cpe1toxC362A) was used as a comparison rather than a reaction without Cpe1tox. In these assays, duplicate reaction mixtures were prepared: one was denatured immediately after preparation ("before" reaction) to serve as a baseline, while the other was incubated to allow enzymatic activity ("after" reaction). This labeling clarifies the comparison between initial and processed samples. We believe this approach clearly distinguishes the effects of Cpe1 activity and provides a reliable basis for assessing proteolysis in our assays.

I also suggest quantifying the intensities of the gel images presented in Figure 5c, d (for example, Cpe1 intensity as a ratio to that of the ParE ATPase domain), to make the interpretation even more evident.

We thank the reviewer for the valuable suggestion to quantify the signal intensities of the gel images presented in Figures 5c, d. We have now included the quantification results in Supplementary Figures 9e, f and have updated the respective text in the manuscript (Lines 826-828 and 1066-1087).

Crystallographic structure: the PDB report notes some higher-than-expected RZR (RSRZ) scores; I interpret this to mean that there was strain around the catalytic site of one of the two toxins in the asymmetric unit, or that this copy was less well ordered. The RZR outliers likely arise from non-optimal weighting for geometric restraints. While no figures of electron density are presented, these modest outliers are not expected to alter the conclusions reached in the current work. One point of interest that is not addressed, however, is if any variance between the two complexes in the asymmetric unit are noted? A passage compares the current toxins to others in the larger subfamily and notes a rotation of a side chain is needed to superpose (Line 159). Can the authors please clarify around which bond this rotation is needed, and if both copies in the asymmetric unit are in the same orientation at this site?

We appreciate the reviewer’s insightful comments.

1. We have provided the electron density map for the RSR-Z outlier residues along with the model (Author response Figure 2a). These outlier residues are located at the loop regions of a molecule within the asymmetric unit in the crystal (Chain B). As a result, the electron density for their side chains appears to be noisier compared to residues in the well-folded regions, leading to higher RSR-Z scores. Notably, when we superimposed the models of two complexes within the asymmetric unit, the calculated RMSD value was 0.402 Å (Author response Figure 2b), indicating that the two models are structurally very similar and that these residues are properly assigned. Therefore, the RSR-Z outliers do not significantly impact the overall structure.
2. Here, we provide a zoomed-in view of Figure 2d, highlighting the superimposed crystal structures of Cpe1 and the closely related PLCPs, ComA and LahT (Author response Figure 2c). As shown, the side chain of the catalytic cysteine residue in ComA adopts a different orientation, positioning it slightly farther from the homologous residues in Cpe1 and LahT. However, since the backbone and catalytic pockets remain structurally intact, we believe that this deviation arises due to results from crystal packing effects rather than an inherent functional distinction. We have now modified the main text (Lines 159-166) to clarify this and prevent any potential misinterpretation.

Reviewer #2 (Significance)

Bacteria encode numerous effectors to successfully compete in natural environments or to mediate virulence; these effectors are typically associated with type VI secretion system machinery or referred to as contact dependent inhibition systems. The current work has identified a sub-family of papain-like cysteine protease effectors that are unique by targeting type II topoisomerases. Among the actionable findings is the identification of both the specific site of interaction with the topo substrates, as well as the specific motif recognized for cleavage. This should enable the field to move forward probing for this activity with other toxins and substrates. The insights provided by the competitive neutralization mechanism also stand out as an important contribution that can be more broadly applied. Within the literature, few effector targets are identified, making the current study stand out as impactful by the well-executed experiments that directly support the conclusions.

While the current study has strong elements of novelty and is complete, it also nicely sets up future studies for remaining open questions. For example, does the nucleotide-bound status of the ATPase domain, or other catalytic intermediate, impact the susceptibility of topoisomerases to cleavage? Is this identified motif found in other ATPase domains? Is the negative supercoiling activity unique to gyrase also impacted, or is the phenotypic mechanism of cell toxicity reliant only on chromosome segregation? What types of kinetic parameters do this class of toxins demonstrate, and does sequence variability alter this? These ideas are a testament to the intriguing study as presented, capturing the readers' curiosity for additional details that are clearly beyond the scope of the current work.

I anticipate this work will be of interest to the broad field of microbiologists that study interbacterial communication as well as pathogenic mechanisms. While the research is largely fundamental in nature, it is wide in scope with applications to many gram-negative bacteria that inhabit a myriad of niches. The work will also be of interest to specialists in topoisomerases, as the list of toxins that target these essential enzymes is growing and the therapeutic utility of topoisomerase inhibition remains vital. My interest lies in the latter, in toxin-mediated inhibition of topoisomerase enzymes as a means to alter bacterial cell growth. While I have strong expertise in structural biology, I am lacking in expertise for mass spectrometry. I note this because this method was used for the identification of the target substrate.

We appreciate the reviewer’s insightful discussion and interest in our study. We agree that further investigations are crucial to address the open questions posed, and we have initiated work on some of these avenues.

For example, considering Cpe1's specificity for the ATPase domain of GyrB and ParE, we have begun examining whether Cpe1 targets other ATPase domains by searching for the consensus sequence or double glycine motifs in the sequences of ATPase domains beyond GyrB and ParE. Among the 42 E. coli ATPase domains identified by the PEC database8, we found several with double glycine residues. However, none contained the exact LHAGGKF consensus sequence identified in GyrB and ParE, which are targeted by Cpe1 (Author Response Figure 3). These findings suggest that Cpe1 is less likely to target other ATPase domains. Nonetheless, due to Cpe1’s potential tolerance of certain variations within the consensus sequence, we cannot draw a definitive conclusion without further investigation into the cleavage sites.

Another critical open question is the impact of Cpe1-mediated cleavage on the function of GyrB and ParE. To address this topic, we have begun investigating if Cpe1 cleavage affects the ATPase activity of these proteins. As expected, our biochemical analysis has demonstrated a significant decrease in ATP hydrolysis in the presence of active Cpe1tox, but not in the presence of the catalytic mutant Cpe1toxC362A (Author response Figures 4a, b). These results confirm that the ATP-dependent activities of both GyrB and ParE are disrupted following Cpe1 cleavage9. Previous work on FicT toxin that inhibits GyrB and ParE ATPase activity through post-translational modification found that ATP-dependent activities such as DNA supercoiling, relaxation, and decatenation were inhibited10,11. Interestingly, GyrB’s relaxation of negative supercoiled DNA, which does not require ATP, was also affected to some extent. This outcome raises the question as to whether Cpe1-cleaved GyrB results in similar downstream defects. Investigating this possibility would provide valuable insights into Cpe1’s mode of action, although we feel doing so is beyond the scope of the current study. Consequently, we view this as an important area for future research.

Finally, regarding the potential applications of Cpe1, we are interested in further investigating its enzymatic specificity and properties. In this study, we analyzed the binding kinetics between Cpe1 and its substrate (Figure 5f) and currently we are endeavoring to characterize the kinetics of Cpe1-mediated proteolysis. To better probe hydrolytic dynamics, we plan to utilize a substrate with a reporting group (such as a chromogenic or fluorogenic leaving group) to monitor cleavage over time. We could achieve this by designing a recombinant substrate based on our knowledge of Cpe1’s native substrates (GyrB and ParE) and the target sequence (“LHAGGKF”). Alternatively, a secondary reaction leading to colorimetric changes could be employed for detection. We consider this an exciting research direction and an important next step for this study.

Overall, we are grateful for the reviewer’s recognition of the novelty and importance of our work in advancing the understanding of interbacterial toxins and their inhibitory effects on topoisomerases. We plan to further investigate the consequences of Cpe1 cleavage on GyrB and ParE and to explore Cpe1 kinetics and its mechanistic actions in more detail. This will not only deepen our understanding of bacterial toxin-mediated inhibition but may also provide critical insights into strategies for targeting type II DNA topoisomerases. The reviewer’s insightful feedback has proven invaluable in shaping our ongoing and future research directions.

Reviewer #3 (Evidence, reproducibility, and clarity)

Bacterial warfare in microbial communities has become illuminated by recent discoveries on molecular weapons that allow contact-dependent injection of bacterial toxins between competitors. Among the best characterized systems are the type VI secretion system (T6SS) or the contact-dependent inhibition (CDI) system (i.e. some of the T5SSs). These systems are delivering a plethora of toxins with various biochemical activities and a broad range of targets. In recent years many such toxins have been characterized and their relevance in pointing at appropriate drug targets is increasing.

In this study the authors built on a previously published association of a family of proteins, papain-like cysteine proteases (PLCPs), with their delivery by T6SS or CDI into target bacterial cells. Whereas this observation is not particularly novel, the findings that this set of proteins, that the authors called now Cpe1, can specifically target bacterial proteins such as ParE and GyrB, so that it affects chromosome partitioning and cell division, is groundbreaking. The authors are clearly demonstrating that Cpe1 cleaves their target proteins at double glycine recognition site which is in line with previous characterization of such proteases when fused to a particular category of ABC transporters. Even more remarkably they can show using biochemical approaches that Cpi1 is a cognate immunity for CpeI, preventing its activity, not by interfering with the catalytic site, but instead with the substrate binding site. The mechanism of competitive inhibition between immunity and substrate is also substantiated by biochemical data.

We sincerely appreciate the reviewer’s interest in and support of our study.

Major comments

• This is a very well conducted study which combines bacterial genetics and phenotypes with excellent biochemical evidence.

We thank the reviewer for their positive comments.

• There are 8 targets identified for Cpe1 and yet only two are cleaved by the enzyme. It is intriguing that FtsZ is one identified target by the pull down but not confirmed for cleavage. The authors rules this as false positive but the cell division defect associated with Cpe1 activity would be consistent here. Are there any double glycine in FtsZ that could be identified as cleavage site? Is it possible that slightly different incubation conditions may promote degradation of FtsZ?

We appreciate the reviewer’s thoughtful comment regarding FtsZ as a potential substrate of Cpe1. This was indeed an intriguing possibility, especially given the cell division defects observed following Cpe1 intoxication. Early on in the project, we also identified FtsZ as a Cpe1 interactor in our proteomic crosslinking assays, which further fueled the hypothesis that FtsZ might be a target.

To explore this possibility, first we examined the FtsZ protein sequence for potential Cpe1 cleavage sites and identified several double glycine motifs (Author response Figure 5a). However, none of these motifs matched the consensus sequence identified in GyrB and ParE, which is LHAGGKF, a sequence that we have shown to be critical for Cpe1 cleavage activity. In an effort to better understand if FtsZ could still be cleaved by Cpe1, we conducted additional cleavage assays under various conditions (Author response Figure 5b). We tested different incubation temperatures, including increasing the temperature to 37 °C, and extended the reaction time to overnight. However, we did not observe any cleavage of FtsZ under these conditions. Given that FtsZ undergoes significant conformational changes upon binding to GTP12, we also considered the possibility that the GTP-bound form of FtsZ might be cleaved by Cpe1. However, even under those conditions, no significant cleavage of FtsZ was detected (Author response Figure 5b). Based on these results, we do not have any evidence to support that FtsZ is a target of Cpe1. The observed cell division defects are more likely a secondary effect resulting from the cleavage of GyrB and ParE, direct targets of Cpe1 that are crucial for chromosome segregation.

• Could it be structurally predicted whether the GG of ParE or GyrB is fitted into the catalytic site of Cpe1.

We appreciate the reviewer’s insightful question regarding the structural prediction of the GG motif of ParE and GyrB fitting into the catalytic site of Cpe1. To address this possibility, we used Alphafold 3 to predict the interaction structure between Cpe1 and its substrates13. The resulting model of Cpe1 interacting with the ATPase domain of GyrB (GyrBATPase) is shown in Supplementary Figure 9c. As illustrated, the loop of the GyrB ATPase domain containing the consensus targeting sequence (“LHAGGKF”) fits into the catalytic site of Cpe1, with the GG motif positioned closest to the catalytic cysteine residue, which likely facilitates hydrolysis. We also attempted to model the interaction between Cpe1 and the ATPase domain of ParE. However, confidence for this model was lower (ipTM = 0.74, pTM = 0.71), possibly due to Alphafold’s preference for certain protein configurations. To gain a more accurate understanding of how Cpe1 binds and recognizes its substrates, we are currently working on co-crystallizing Cpe1tox with GyrB and ParE. This long-term project aims to provide precise structural insights into the Cpe1-substrate interaction and further elucidate the mechanism of cleavage.

Minor comments

• The authors described a family of proteases, PLPCs, and characterized one here called Cpe1. Not clear whether this is a generic name or one specific protein from one particular bacterial species. Indeed, it is unclear from which bacterial strain the Cpe1 protein studied here originates.

We thank the reviewer for this comment and apologize for the lack of clarity. To provide better context, we have now revised the manuscript (Lines 136-137 and 141-145) to clearly state that the Cpe1 protein characterized in this study originates from E. coli strain ATCC 11775.

• It may be worth to emphasize that the Cpe1 domain is found in all possible configurations as T6SS cargo and that is to be linked to VgrG, PAAR or Rhs.

Thank you for this suggestion. We have revised the manuscript accordingly to emphasize this point (Lines 106-109).

• Line 49 the authors could indicate that the Esx system is also known as type VII secretion system (T7SS).

Thank you for this suggestion. We have revised the manuscript accordingly (Line 48-50).

• Line 113 it may be better to use Proteobacteria instead of Pseudomonadota

We have revised the manuscript (Lines 114-115) as suggested by the reviewer. It is important to note that following the recent decision by the International Committee on Systematics of Prokaryotes (ICSP) to amend the International Code of Nomenclature of Prokaryotes (ICNP) and formally recognize "phylum" under official nomenclature rules14,15, the taxonomy database used in our analysis has adopted the updated nomenclature. To ensure consistency, we followed this updated nomenclature throughout the original manuscript.

Reviewer #3 (Significance)

This is an excellent piece of work. The characterization of Cpe1 might look poorly novel at the start when compared to previous studies. Yet the findings go crescendo by characterizing original mechanisms of action of the cognate immunity, and by identifying the molecular target of Cpe1. This is providing real conceptual advance in the T6SS field and not just reporting yet another T6SS toxin.

As a T6SS expert I genuinely feel that these findings are groundbreaking and could be targeted to broad audience since the possible implications of these observations for future antimicrobial drugs discovery or therapeutic approaches is highly relevant.

We sincerely appreciate the reviewer’s positive remarks and support of our study.

References

1. Ishihama, A., and Shimada, T. (2021). Hierarchy of transcription factor network in Escherichia coli K-12: H-NS-mediated silencing and Anti-silencing by global regulators. FEMS Microbiol Rev 45. 10.1093/femsre/fuab032.
2. Hersch, S.J., Manera, K., and Dong, T.G. (2020). Defending against the Type Six Secretion System: beyond Immunity Genes. Cell Rep 33, 108259. 10.1016/j.celrep.2020.108259.
3. Russell, A.B., Singh, P., Brittnacher, M., Bui, N.K., Hood, R.D., Carl, M.A., Agnello, D.M., Schwarz, S., Goodlett, D.R., Vollmer, W., and Mougous, J.D. (2012). A widespread bacterial type VI secretion effector superfamily identified using a heuristic approach. Cell Host Microbe 11, 538-549. 10.1016/j.chom.2012.04.007.
4. Jana, B., Fridman, C.M., Bosis, E., and Salomon, D. (2019). A modular effector with a DNase domain and a marker for T6SS substrates. Nat Commun 10, 3595. 10.1038/s41467-019-11546-6.
5. Halvorsen, T.M., Schroeder, K.A., Jones, A.M., Hammarlof, D., Low, D.A., Koskiniemi, S., and Hayes, C.S. (2024). Contact-dependent growth inhibition (CDI) systems deploy a large family of polymorphic ionophoric toxins for inter-bacterial competition. PLoS Genet 20, e1011494. 10.1371/journal.pgen.1011494.
6. Nguyen, T.T., Sabat, G., and Sussman, M.R. (2018). In vivo cross-linking supports a head-to-tail mechanism for regulation of the plant plasma membrane P-type H(+)-ATPase. J Biol Chem 293, 17095-17106. 10.1074/jbc.RA118.003528.
7. Liu, Y., Yu, J., Wang, M., Zeng, Q., Fu, X., and Chang, Z. (2021). A high-throughput genetically directed protein crosslinking analysis reveals the physiological relevance of the ATP synthase 'inserted' state. FEBS J 288, 2989-3009. 10.1111/febs.15616.
8. Yamazaki, Y., Niki, H., and Kato, J. (2008). Profiling of Escherichia coli Chromosome database. Methods Mol Biol 416, 385-389. 10.1007/978-1-59745-321-9_26.
9. Reece, R.J., and Maxwell, A. (1991). DNA gyrase: structure and function. Crit Rev Biochem Mol Biol 26, 335-375. 10.3109/10409239109114072.
10. Harms, A., Stanger, F.V., Scheu, P.D., de Jong, I.G., Goepfert, A., Glatter, T., Gerdes, K., Schirmer, T., and Dehio, C. (2015). Adenylylation of Gyrase and Topo IV by FicT Toxins Disrupts Bacterial DNA Topology. Cell Rep 12, 1497-1507. 10.1016/j.celrep.2015.07.056.
11. Lu, C., Nakayasu, E.S., Zhang, L.Q., and Luo, Z.Q. (2016). Identification of Fic-1 as an enzyme that inhibits bacterial DNA replication by AMPylating GyrB, promoting filament formation. Sci Signal 9, ra11. 10.1126/scisignal.aad0446.
12. Matsui, T., Han, X., Yu, J., Yao, M., and Tanaka, I. (2014). Structural change in FtsZ Induced by intermolecular interactions between bound GTP and the T7 loop. J Biol Chem 289, 3501-3509. 10.1074/jbc.M113.514901.
13. Abramson, J., Adler, J., Dunger, J., Evans, R., Green, T., Pritzel, A., Ronneberger, O., Willmore, L., Ballard, A.J., Bambrick, J., et al. (2024). Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature 630, 493-500. 10.1038/s41586-024-07487-w.
14. Oren, A., Arahal, D.R., Rossello-Mora, R., Sutcliffe, I.C., and Moore, E.R.B. (2021). Emendation of Rules 5b, 8, 15 and 22 of the International Code of Nomenclature of Prokaryotes to include the rank of phylum. Int J Syst Evol Microbiol 71. 10.1099/ijsem.0.004851.
15. Oren, A., and Garrity, G.M. (2021). Valid publication of the names of forty-two phyla of prokaryotes. Int J Syst Evol Microbiol 71. 10.1099/ijsem.0.005056.

I loved the start of this poem, this brings me back to a time in my own life, where I was drinking. Much like what I would write about in my own journal. The stanza's "tis not that wine is wanting," "yet here I sit in sorrow" "my troubles have been many" these are brilliant!

I like this, for my own personal reason. I can relate to the feeling of wanting to see someone again, with a strong desire. Life seems hard until you see them again, once you see them, nothing can bring you pain. It's like the philosophical expression "Love is a drug," which I agree and feel it is the strongest drug, once we find acceptance from a person or group we are like addicts and soldiers at the very same time.

A similar experience to reading this might be listening to a song by Fantasia.

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 #1

Evidence, reproducibility and clarity

Title: Hypoxia impedes differentiation of cranial neural crest cells into derivatives relevant for craniofacial development

Synopsis: Cleft lip w/ or w/o cleft palate is the second-most common birth defect worldwide. Defects are often traceable to cranial neural crest cells through genetics or environmental factors. Schmid and coauthors focus on the environmental factor of hypoxia and investigate the effects of hypoxic conditions on the ability of CNCCs to differentiate and migrate. They performed RNA-seq analysis with qRT-PCR validation for specific markers and show that hypoxia appears to repress differentiation without markedly affecting proliferation. Hypoxic conditions did not demonstrated significant perturbations in cell proliferation; however, chondrocyte, osteoblast, and smooth muscle differentiation was significantly reduced for cell lines cultured under hypoxia. Bulk RNA-seq and PCA revealed dysregulation of genes implicated in cytoskeletal integrity (such as actin γ-2), neural crest cell migration (hedgehog co-receptor brother of CDO) and amino acid metabolism (cysteine dioxygenase), which Schmid and colleagues termed OFC risk genes.

Major comments

• The authors performed qRT-PCR validation for markers of differentiation and hypoxia, with a major absence of VEGF and HIF1a. The paper would be strengthened by mention of these factors, especially by qRT-PCR or Western blot.
• Please provide justification of selection 0.5% as their hypoxic condition or perhaps repeat experiments in a less extreme environment to see if their conclusions still hold true.
• standard immunohistochemistry or histology of differentiated cells would strengthen the authors' claims of reduced differentiation under hypoxic conditions, e.g., Alcian blue, alk-phos or Alizarin red, and smooth muscle actin or other indicator.
• The authors identify a few genes that appear down-regulated in all three differentiation conditions. If it is within the scope of the study, it would strengthen the claim of these genes' function to show the effect of knock-down or knock-out for validation.
• Another major critique lies in the initial claim that proliferation of O9-1 cells is not significantly impacted by hypoxia. In figures 1E-H, photograms of the cells cultured 24 -72 hours and quantifications of live vs dead cells are shown as evidence for this argument. However, the increased density of cells in normoxic conditions may be a confounding variable in this assay. It would be interesting for the researchers to assess the percent of dead vs alive cells between normoxic and hypoxic conditions when the plates reach equivalent densities.
• At end of Fig 1 section authors attempt to tie phenotypes observed in a cell line in vitro to the complex biological processes. They are not comparable and in vivo models would be better suited for these types of comparisons.
• Fig 2: if qRT-PCR did not show statistically different results between experimental and control groups why move on to bulk RNA seq?
• Fig 5: hypoxia this intense is going to affect broad range of biological processes and genes. Finding a few genes that are affected in extreme hypoxia that are also risk genes is highly unlikely. How can the authors be assured that these overlaps are actually significant and not just by chance?
• Would appreciate discussion on how examination of neural crest is relevant for OFC, as most animal models of OFC demonstrate the pathogenesis in embryonic epithelium or periderm, not in the neural crest. Defects in neural crest are associated with other congenital craniofacial anomalies such as craniosynostosis or complex (Tessier) clefts, not the typical orofacial cleft. Please revise rationale of study, interpretation of data and Discussion to specifically state how neural crest cells are involved in the pathogenesis of orofacial cleft.

Minor comments

• The author should replace "Final proof" in the introduction with "further evidence supporting."
• Authors are inconsistent when referring to Figures- sometimes they capitalize (i.e. 1J) and other times they leave lower case (i.e. 1i). Needs to be consistent throughout. Figures are not numbered
• In figures authors would sometimes list 21% O2 first then 0.5% O2 or vice versa. (i.e. Fig on page 21 panels I, J, K). Needs to be consistent.
• Figures on pages 28, 29, 30 panel J and page 31 panel F: there is no legend on what the scale/measurement is for the difference in expression level other than it ranges from -1 to +3.
• Will the authors please comment on the one normoxic sample in Figure 1I that did not cluster with the others? Did this meet the standards to merit exclusion as an outlier?
• The authors refer to DEG as deregulated genes; while not strictly incorrect, the more standard usage is "differentially expressed genes." Please address.

Significance

This work on neural crest cells and hypoxia are biologically and clinically significant.

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 #1

Evidence, reproducibility and clarity

Title: Hypoxia impedes differentiation of cranial neural crest cells into derivatives relevant for craniofacial development

Synopsis: Cleft lip w/ or w/o cleft palate is the second-most common birth defect worldwide. Defects are often traceable to cranial neural crest cells through genetics or environmental factors. Schmid and coauthors focus on the environmental factor of hypoxia and investigate the effects of hypoxic conditions on the ability of CNCCs to differentiate and migrate. They performed RNA-seq analysis with qRT-PCR validation for specific markers and show that hypoxia appears to repress differentiation without markedly affecting proliferation. Hypoxic conditions did not demonstrated significant perturbations in cell proliferation; however, chondrocyte, osteoblast, and smooth muscle differentiation was significantly reduced for cell lines cultured under hypoxia. Bulk RNA-seq and PCA revealed dysregulation of genes implicated in cytoskeletal integrity (such as actin γ-2), neural crest cell migration (hedgehog co-receptor brother of CDO) and amino acid metabolism (cysteine dioxygenase), which Schmid and colleagues termed OFC risk genes.

Major comments

• The authors performed qRT-PCR validation for markers of differentiation and hypoxia, with a major absence of VEGF and HIF1a. The paper would be strengthened by mention of these factors, especially by qRT-PCR or Western blot.
• Please provide justification of selection 0.5% as their hypoxic condition or perhaps repeat experiments in a less extreme environment to see if their conclusions still hold true.
• standard immunohistochemistry or histology of differentiated cells would strengthen the authors' claims of reduced differentiation under hypoxic conditions, e.g., Alcian blue, alk-phos or Alizarin red, and smooth muscle actin or other indicator.
• The authors identify a few genes that appear down-regulated in all three differentiation conditions. If it is within the scope of the study, it would strengthen the claim of these genes' function to show the effect of knock-down or knock-out for validation.
• Another major critique lies in the initial claim that proliferation of O9-1 cells is not significantly impacted by hypoxia. In figures 1E-H, photograms of the cells cultured 24 -72 hours and quantifications of live vs dead cells are shown as evidence for this argument. However, the increased density of cells in normoxic conditions may be a confounding variable in this assay. It would be interesting for the researchers to assess the percent of dead vs alive cells between normoxic and hypoxic conditions when the plates reach equivalent densities.
• At end of Fig 1 section authors attempt to tie phenotypes observed in a cell line in vitro to the complex biological processes. They are not comparable and in vivo models would be better suited for these types of comparisons.
• Fig 2: if qRT-PCR did not show statistically different results between experimental and control groups why move on to bulk RNA seq?
• Fig 5: hypoxia this intense is going to affect broad range of biological processes and genes. Finding a few genes that are affected in extreme hypoxia that are also risk genes is highly unlikely. How can the authors be assured that these overlaps are actually significant and not just by chance?
• Would appreciate discussion on how examination of neural crest is relevant for OFC, as most animal models of OFC demonstrate the pathogenesis in embryonic epithelium or periderm, not in the neural crest. Defects in neural crest are associated with other congenital craniofacial anomalies such as craniosynostosis or complex (Tessier) clefts, not the typical orofacial cleft. Please revise rationale of study, interpretation of data and Discussion to specifically state how neural crest cells are involved in the pathogenesis of orofacial cleft.

Minor comments

• The author should replace "Final proof" in the introduction with "further evidence supporting."
• Authors are inconsistent when referring to Figures- sometimes they capitalize (i.e. 1J) and other times they leave lower case (i.e. 1i). Needs to be consistent throughout. Figures are not numbered
• In figures authors would sometimes list 21% O2 first then 0.5% O2 or vice versa. (i.e. Fig on page 21 panels I, J, K). Needs to be consistent.
• Figures on pages 28, 29, 30 panel J and page 31 panel F: there is no legend on what the scale/measurement is for the difference in expression level other than it ranges from -1 to +3.
• Will the authors please comment on the one normoxic sample in Figure 1I that did not cluster with the others? Did this meet the standards to merit exclusion as an outlier?
• The authors refer to DEG as deregulated genes; while not strictly incorrect, the more standard usage is "differentially expressed genes." Please address.

Significance

This work on neural crest cells and hypoxia are biologically and clinically significant.

To menu prosím upravme na "O nás" "naše značky" "lidé" "kontakt"

upravme i v hlavní menu

Este capítulo proporciona una guía detallada para seleccionar el método de investigación adecuado según los objetivos y el alcance previstos, asegurando coherencia entre la metodología y los resultados esperado

Mi investigación busca comprender el uso que se le da al color en la narrativa visual de cierta pelicula, por lo que necesito recolectar y analizar información tanto del tema del color como de la historia y esto se puede lograr con este trabajo de gabinete y claro una investigación cualitativa.

He de asimilar que mi tema busca un análisis, que permita poder entender la función de escenarios y su aporte narrativo, pero como es bien sabido, el estudio de animación que me he de basar es Studio Ghibli, un estudio de origen japonés, por ende es de entenderse, que la manera de acceder a información que me permita colaborar la manera en que estos escenarios se diseñan y crean es por medio de libros (ya sea físicos o digitales), que me narren lo que en otro continente sucede.

Mi investigación se basara en el estudio de casos, debido estudiará un pequeño caso, y como es que funcionan los elementos de este, siendo así que el análisis será solo de este entorno específico, además que se complementara con el análisis de documentos, información y entrevistas de los creativos, lo que hace que se haga un análisis completo de toda la información y documentos para lograr los objetivos de esta.

Aunque al principio no podía relacionar mi investigación con alguno de los métodos, es este aspecto esencial en el estudio de caso que me acercara más al resultado que quiero al tener dos casos en el cual su propósito descriptivo daría un veredicto final a la comparación que realizaré con los conciertos del cantante Louis Tomlinson y cómo se presenta su identidad por medio de los efectos especiales.

Considera que para mi investigación un enfoque cualitativo me ayudaría más ya que las principales herramientas que voy a usar en mi análisis son la descripción y a comparación de los dos eventos para obtener una respuesta respecto a la identidad constante del artista y su música con el diseño de sus efectos especiales.

Las familias que considero que son la más adecuadas para mi tema son la investigación cualitativa y el trabajo de gabinete. Mi investigación se enfoca en identificar las representaciones visuales más emblemáticas de Alicia en el país de las maravillas desde la época victoriana hasta principios del siglo XXI y para logarlo se necesitan consultar fuentes ya existentes, como fuentes documentales y bibliográficas. Este no requiere precisamente un trabajo de campo, sino lo contrario, ya que, a partir de algunos ejemplos encontrados en las fuentes primarias, se ira profundizando el tema.

no ho havia vist mai :O

one independent factor o poden ser més?

Estas herramientas de trabajo colaborativo son algo desconocidas, pero al entrar en actividad con ellas también surgen preguntas como por ejemplo si hacen o asemejan las aplicaciones mas conocidas como word -- si son compatibles.

¿puedo hacer estas anotaciones con cualquier lectura?

pull yourself up by your bootstraps narrative

idea that humans have complete dominion over nature an must exercise it; rooted in Biblical ideas no doubt

Algunas personas cambiaron las fechas límite...

o

Las fechas límite cambiaron...

Alguien comenzó la intervención militar...

o

La intervención militar comenzó...

vincula o crédito ao pagamento na etapa anterior

se não tiver como consultar os pagamentos recolhe e depois o comite devolve em 3 dias

hoje na compensação o contribuinte que escolhe, com a reforma será ordem cronologica

Por exemplo, Comprei e paguei em dinheiro, não vai ter split, eu que pago o imposto

the. O per no repetir tant, "whether there are differences in age between groups"

Falten subindex i a O i a E.

Això pot ser posar-ho com un resource no? O sino aquí però diferenciat de la resta?

També és poden fer amb %'s o densitat. Potser diria:

Histograms are constructed by binning the data and counting the number of observations in each bin, or the percentage or density:

Això no pot anar aquí. Et proposo el següent ordre: Standard deviation Range Interquartile range ... i aleshores aprofites per explicar què és un percentil de veu o en una slide extra. Què et sembla?

The location where cached data is stored—either in main memory or on disk—has significant implications for system performance and reliability. Storing cached data in main memory offers faster access times compared to disk storage, making it an attractive option for systems that require high performance. However, main memory is volatile, meaning that cached data may be lost in the event of a crash, which introduces reliability concerns. In contrast, disk caches are non-volatile and ensure that cached data is preserved even during system failures, but they are slower due to the inherent latency of disk I/O. Hybrid solutions, such as using main memory for high-speed access and disk for persistent storage, combine the advantages of both. The choice between memory and disk caching often depends on the system's resource constraints and performance requirements, with main memory being more favorable for high-performance, low-latency systems and disk being preferred for reliability and persistence.

The cluster-based DFS model is designed for large-scale, data-intensive applications that require high fault tolerance, scalability, and efficient data access. This model is exemplified by systems like the Google File System (GFS) and Hadoop Distributed File System (HDFS). In this architecture, clients interact with a master metadata server that manages the file system’s structure and location of data, which is distributed across multiple data servers. Each data server stores chunks of files, and these chunks are replicated across servers to ensure data redundancy and faster access. The cluster-based model excels in environments with massive amounts of data because it allows for parallel reading and writing of file chunks across multiple servers, improving throughput and system efficiency. The master server maintains metadata, such as file locations and directory structure, but clients can directly interact with the data servers for file access, reducing the load on the metadata server. This model is widely used in big data applications, where scalability and fault tolerance are paramount.

When a user requests access to a remote file in a distributed file system (DFS), the system first locates the server that holds the file using the naming system. Then, the file data must be transferred to the user. One common way to do this is through a remote-service method, where the user's request is sent to the server, the server accesses the file, and the result is sent back. This is similar to how local file systems access data from a disk. Remote Procedure Call (RPC) is often used to handle these interactions. To improve performance, caching is used—not just to reduce disk access like in regular systems, but also to cut down network traffic in DFS. This helps speed up file access and reduce delays.

I/O virtualization presents significant challenges due to the diversity of hardware devices and performance considerations. VMMs offer different approaches, such as dedicating devices to specific guests or abstracting hardware access through virtualized device drivers. Direct device access enhances performance but limits sharing, while shared access requires careful VMM management to ensure security and efficiency. Networking in virtualized environments involves complex IP address management and virtual switching to connect guests efficiently. Firewalls and network address translation (NAT) mechanisms provide security and isolation. This section highlights how VMMs handle I/O operations to balance performance, flexibility, and security in virtualized environments.

Paravirtualization modifies guest operating systems to improve performance by reducing the overhead of full virtualization. Instead of simulating hardware, the VMM provides an optimized interface that guests interact with. Xen pioneered this approach, introducing efficient device I/O using shared memory buffers and hypercalls for memory management. Paravirtualized guests require modifications but achieve better efficiency. Over time, Xen adapted to hardware-assisted virtualization, reducing reliance on paravirtualization. However, some hypervisors, especially Type 0, still use paravirtualization techniques to optimize system performance and reduce resource overhead.

Type 1 hypervisors, also known as "bare-metal" hypervisors, function as specialized operating systems that manage guest VMs directly on hardware. They run in kernel mode, handling CPU scheduling, memory management, and I/O operations while offering APIs for external tools. They improve data-center efficiency by consolidating workloads, enabling live migration, and simplifying backup and replication. However, their complexity and cost, especially for commercial options like VMware ESX, pose challenges. Some general-purpose operating systems, such as Red Hat Enterprise Linux, incorporate Type 1 hypervisors, treating VMs like processes but providing special execution handling.

Type 0 hypervisors are hardware-based and load guest operating systems at boot time, enabling efficient execution. They partition resources such as CPUs, memory, and I/O devices to guests, simplifying implementation. However, I/O sharing is a challenge, requiring management by the hypervisor or a control partition. Some type 0 hypervisors support dynamic resource reallocation, allowing guests to adjust CPU and memory use. This section also explains how type 0 hypervisors enable nested virtualization, where guests act as VMMs themselves. These hypervisors are distinct because they provide close-to-hardware execution with minimal overhead.

Prueba

• Informativo nº 843
• 18 de março de 2025.CORTE ESPECIAL
• Compartilhe:
• Processo: REsp 2.072.206-SP, Rel. Ministro Ricardo Villas Bôas Cueva, Corte Especial, por maioria, julgado em 13/2/2025, DJEN 12/3/2025.

Ramo do Direito DIREITO PROCESSUAL CIVIL, DIREITO EMPRESARIAL

TemaPaz, Justiça e Instituições Eficazes <br /> Desconsideração da personalidade jurídica. Natureza jurídica de demanda incidental. Existência de litigiosidade. Improcedência do pedido. Não inclusão do sócio (ou da empresa) no polo passivo. Honorários advocatícios de sucumbência. Cabimento. Superação da jurisprudência dominante.

Destaque - O indeferimento do pedido de desconsideração da personalidade jurídica, tendo como resultado a não inclusão do sócio (ou da empresa) no polo passivo da lide, dá ensejo à fixação de verba honorária em favor do advogado de quem foi indevidamente chamado a litigar em juízo.

Informações do Inteiro Teor - Cinge-se a controvérsia em definir se é possível a fixação de honorários advocatícios na hipótese de rejeição do pedido formulado em incidente de desconsideração da personalidade jurídica.

• Há julgados do Superior Tribunal de Justiça, inclusive já na vigência do CPC/2015, afirmando a impossibilidade de condenação em honorários advocatícios nos incidentes processuais, ressalvadas situações excepcionais.

• Com idêntica linha de raciocínio, a Terceira Turma desta Corte debruçou-se sobre o assunto e concluiu, por maioria, que, tratando-se de incidente de desconsideração da personalidade jurídica, "(...) o descabimento da condenação nos ônus sucumbenciais decorre da ausência de previsão legal excepcional, sendo irrelevante se apurar quem deu causa ou foi sucumbente no julgamento final do incidente."(REsp 1.845.536/SC, Rel. Ministra Nancy Andrighi, Rel. para acórdão Ministro Marco Aurélio Bellizze, Terceira Turma, DJe de 9/6/2020).

• Todavia, no julgamento do REsp 1.925.959/SP, o Ministro Paulo de Tarso Sanseverino propôs ao Órgão Colegiado nova reflexão a respeito da matéria, trazendo relevantes aspectos que levaram o Órgão Julgador a meditar mais profundamente sobre a questão, estando o primeiro deles consubstanciado no fato de que o incidente de desconsideração da personalidade jurídica foi incluído no capítulo das intervenções de terceiros, a exigir a análise do tema sob esse específico enfoque.

• Com efeito, o que se busca com a instauração do incidente é a formação de um litisconsórcio, com ampliação subjetiva da lide, para que no polo passivo da relação jurídica litigiosa passem a figurar terceiros, que assim são considerados até o momento em que são regularmente cientificados da intenção de serem incluídos na lide como responsáveis por dívidas que não contraíram.

• Tal pretensão pode ser exercitada na petição inicial, conforme faculdade conferida pelo art. 134, § 2º, do CPC/2015, ou em outras fases do processo, sendo mais comum a hipótese em que o pedido de desconsideração é formulado já na fase de cumprimento de sentença ou na própria execução.

• Sob esse prisma, e considerando a efetiva existência de uma pretensão resistida, manifestada contra terceiro(s) que até então não figurava(m) como parte, entende-se que a improcedência do pedido formulado no incidente, tendo como resultado a não inclusão do sócio (ou da empresa) no polo passivo da lide - situação que se equipara à sua exclusão quando indicado desde o princípio para integrar a relação processual -, mesmo que sem a ampliação do objeto litigioso, dará ensejo à fixação de verba honorária em favor do advogado de quem foi indevidamente chamado a litigar em juízo, como vem entendendo a doutrina.

• Em suma, com base no princípio hermenêutico segundo o qual onde há a mesma razão, aplica-se o mesmo direito - ubi eadem ratio ibi eadem jus -, entende-se que pode ser aplicada ao caso a mesma orientação adotada para a hipótese de extinção parcial do processo em virtude da exclusão de litisconsorte passivo, que dá ensejo à condenação do autor ao pagamento de honorários advocatícios sucumbenciais em favor do advogado do excluído.

• Como já havia advertido a eminente Ministra Nancy Andrighi em voto proferido no julgamento do REsp nº 1.845.536/SC, "o incidente de desconsideração da personalidade jurídica tem natureza semelhante à de um procedimento comum e autônomo, capaz de alterar substancialmente o rumo da ação principal, monitória, em fase de cumprimento de sentença, porquanto poderia acarretar a inclusão ou a exclusão da sócia recorrida do alcance dos efeitos da execução forçada promovida em juízo. Nessas circunstâncias, portanto, a despeito de não haver previsão expressa no art. 85, § 1º, do CPC/15, a parte que requer a desconsideração e não obtêm êxito em seu propósito deveria, em tese, arcar com os ônus referentes à sucumbência. Isso porque há, no julgamento ocorrido na vigência do CPC/15, inegável decisão parcial de mérito por meio de decisão interlocutória, porquanto permanece em curso o processo quanto à pessoa jurídica que originariamente ocupa o polo passivo da demanda".

• Nesse mesmo julgado, contudo, a eminente Ministra Nancy Andrighi defendeu a aplicação do princípio da causalidade para impedir que a parte exequente fosse responsabilizada pelo pagamento de encargos que se fizeram necessários na busca de seu direito de crédito, ao ressaltar que "mesmo que não estejam presentes os requisitos autorizadores da desconsideração, afrontaria à equidade impor ao credor, que sequer consegue a satisfação de seu crédito, a responsabilidade pelo pagamento de honorários em favor do advogado da parte que, além de não ter encerrado corretamente sua empresa, ainda sairia vitoriosa da lide, fazendo jus à verba honorária em prol de sua defesa.".

• Nesse específico ponto, todavia, ao menos no âmbito da Terceira Turma, prevaleceu entendimento em sentido contrário, visto que a desconsideração da personalidade jurídica é medida excepcional, de modo que, sendo invocada fora das hipóteses estritamente previstas em lei, os encargos da sucumbência devem ser imputados a quem se utilizou indevidamente do instituto.

• Já em caso de deferimento do pedido de desconsideração (direta ou inversa), com o efetivo redirecionamento da demanda contra o sócio ou a pessoa jurídica, conforme o caso, o eventual sucumbimento destes somente poderá ser aferido ao final, a depender do juízo de procedência ou improcedência da pretensão contra eles direcionada.

• Ressalta-se, por fim, que a definição dos critérios de fixação dos honorários advocatícios na hipótese de improcedência do pedido formulado em incidente de desconsideração da personalidade jurídica é matéria que pode exigir maiores esforços no futuro, mas não foi devolvida a esta Corte no presente recurso especial.

• Informativo nº 842
• Processo: REsp 1.909.271-PR, Rel. Ministro Ricardo Villas Bôas Cueva, Terceira Turma, por unanimidade, julgado em 11/2/2025, DJEN 14/2/2025.

Ramo do Direito DIREITO PROCESSUAL CIVIL

TemaPaz, Justiça e Instituições Eficazes <br /> Apresentação espontânea do réu. Fase postulatória. Momento anterior ao exame prévio da petição inicial e à audiência de conciliação ou mediação. Deflagração do prazo para oferecimento da contestação. Inaplicabilidade. Aplicação das regras dos incisos I e II do art. 335 do CPC/2015.

Destaque - A apresentação do réu no instante inicial da fase postulatória, em momento anterior à decisão do magistrado a respeito do recebimento da inicial e da designação de audiência de conciliação ou mediação, não deflagra automaticamente o prazo para o oferecimento de contestação, o qual será contabilizado nos termos dos incisos I e II do art. 335 do CPC/2015.

Informações do Inteiro Teor - A controvérsia resume-se em definir se a apresentação do réu no instante inicial da fase postulatória, em momento anterior à decisão do magistrado a respeito do recebimento da inicial e da designação de audiência de conciliação ou mediação deflagra, automaticamente, o prazo para o oferecimento de contestação, nos termos do art. 239, § 1º, última parte, do Código de Processo Civil (CPC/2015).

• Entre as novas diretrizes trazidas pelo atual Código de Processo Civil está a previsão de que a solução consensual dos conflitos deve ser, sempre que possível, promovida pelo Estado e estimulada pelos partícipes da relação jurídica processual. Nessa linha, o primeiro passo para a autocomposição passou a ser dado logo no início da marcha processual e antes mesmo da apresentação da defesa do réu, com a marcação de audiência específica que só pode ser dispensada em virtude de sua manifesta inutilidade.

• Por esse motivo, a citação, que, na vigência do diploma processual de 1973, era definida como o ato pelo qual se chama a juízo o réu a fim de que se defenda, conforme previa o art. 213 do código revogado, passou a ser conceituada, no art. 238 do atual CPC, como o ato pelo qual são convocados o réu, o executado ou o interessado para integrar a relação processual.

• No procedimento comum para os direitos disponíveis, em regra, a citação do réu para integrar a relação processual conterá a sua convocação para manifestar o seu interesse em participar da audiência de mediação e conciliação do art. 340 do CPC/2015, não envolvendo necessariamente, portanto, a apresentação imediata da defesa.

• Nesse sentido, a indevida falta ou a nulidade de citação é irregularidade grave que ostenta a natureza de vício transrescisório, mas que também pode ser suprida ainda durante a tramitação da ação pelo comparecimento espontâneo do réu, o qual, nos termos do § 1º do art. 239 do diploma processual vigente, tem o efeito de providenciar-lhe a condição de parte, passando ele a se sujeitar aos efeitos do processo, tal qual houvesse ocorrido a citação válida.

• Conforme jurisprudência do STJ, "o comparecimento espontâneo do réu supre a citação quando é atingida a finalidade do ato, qual seja, informar a parte, de modo inequívoco, acerca da demanda ajuizada contra si e de suas respectivas consequências, a fim de viabilizar o exercício do seu direito de defesa" (REsp 1.698.821/RJ, Rel. Ministro Ricardo Villas Bôas Cueva, Terceira Turma, julgado em 6/2/2018, DJe de 15/2/2018).

• Todavia, a previsão final § 1º art. 239 do CPC/2015, segundo a qual o prazo para apresentação de contestação flui a partir da data do comparecimento espontâneo, somente tem aplicabilidade lógica e sistemática na hipótese em que o réu se apresenta ao processo em estado avançado do procedimento, notadamente após a decretação da sua revelia.

• À luz dessas considerações, constata-se que a determinação legal segundo a qual o prazo para a apresentação da contestação tem início imediato na data do comparecimento espontâneo, somente tem aplicação, no procedimento comum relacionado a ações que versem sobre direitos disponíveis, se a apresentação do réu aos autos ocorrer em momento mais adiantado do procedimento, notadamente após a decretação da sua revelia.

• Por outro lado, na hipótese em que a apresentação do réu ocorre ainda no momento inicial da fase postulatória, o prazo para a apresentação da contestação será contabilizado nos termos dos incisos I e II do art. 335 do CPC/2015, solução que homenageia o devido processo legal e a boa-fé, na vertente da proteção da expectativa legítima, no sentido de que o termo inicial será a data: I - da audiência de conciliação ou de mediação, ou da última sessão de conciliação, quando qualquer parte não comparecer ou, comparecendo, não houver autocomposição; II - do protocolo do pedido de cancelamento da audiência de conciliação ou de mediação apresentado pelo réu, quando ocorrer a hipótese do art. 334, § 4º, inciso I".

esto es un comercial, no un tip o sugerencia, lo pondría como blockquote

Author response:

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

Reviewer #1 (Public review):

Summary:

In this manuscript, De La Forest Divonne et al. build a repertory of hemocytes from adult Pacific oysters combining scRNAseq data with cytologic and biochemical analyses. Three categories of hemocytes were described previously in this species (i.e. blast, hyalinocyte, and granulocytes). Based on scRNAseq data, the authors identified 7 hemocyte clusters presenting distinct transcriptional signatures. Using Kegg pathway enrichment and RBGOA, the authors determined the main molecular features of the clusters. In parallel, using cytologic markers, the authors classified 7 populations of hemocytes (i.e. ML, H, BBL, ABL, SGC, BGC, and VC) presenting distinct sizes, nucleus sizes, acidophilic/basophilic, presence of pseudopods, cytoplasm/nucleus ratio and presence of granules. Then, the authors compared the phenotypic features with potential transcriptional signatures seen in the scRNAseq. The hemocytes were separated in a density gradient to enrich for specific subpopulations. The cell composition of each cell fraction was determined using cytologic markers and the cell fractions were analysed by quantitative PCR targeting major cluster markers (two per cluster). With this approach, the authors could assign cluster 7 to VC, cluster 2 to H, and cluster 3 to SGC. The other clusters did not show a clear association with this experimental approach. Using phagocytic assays, ROS, and copper monitoring, the authors showed that ML and SGC are phagocytic, ML produces ROS, and SGC and BGC accumulate copper. Then with the density gradient/qPCR approach, the authors identified the populations expressing anti-microbial peptides (ABL, BBL, and H). At last, the authors used Monocle to predict differentiation trajectories for each subgroup of hemocytes using cluster 4 as the progenitor subpopulation.

The manuscript provides a comprehensive characterisation of the diversity of circulating immune cells found in Pacific oysters.

Strengths:

The combination of the two approaches offers a more integrative view.

Hemocytes represent a very plastic cell population that has key roles in homeostatic and challenged conditions. Grasping the molecular features of these cells at the single-cell level will help understand their biology.

This type of study may help elucidate the diversification of immune cells in comparative studies and evolutionary immunology.

Weaknesses:

The study should be more cautious about the conclusions, include further analyses, and inscribe the work in a more general framework.

Reviewer #1 (Recommendations for the authors):

The manuscript provides a comprehensive characterisation of the diversity of circulating immune cells found in Pacific oysters.

Major comments:

(1) The introduction would benefit from a clear description of what is known about immune cell development and diversity in this model. The bibliography on the three subtypes origins and properties (i.e. blast, hyalinocyte, and granulocytes) should be described in the introduction.

We thank Reviewer #1 for their valuable comments, which have allowed us to further improve our manuscript. We have enriched the introduction with the following addition (line 79 to 82):

“Blast-like cells are considered as undifferentiated hemocyte types (20), hyalinocytes (21) seem to be more involved in wound repair, and granulocytes, more implicated in immune surveillance. The latter are considered as the main immunocompetent hemocyte types (22).”

(2) The authors mentioned a previous scRNAseq dataset produced in another oyster species. They should compare the two datasets to show the robustness of the molecular signatures determined in the present study. In addition, the authors do not mention markers identified in the literature that could be relevant to characterize the clusters (e.g. inflammatory pathway PMID: 29751033, proliferative markers PMID: 36591234/ PMID: 29317231, granulocyte markers PMID: 30633961 ... list not exhaustive). Overall, the comparison of this manuscript dataset and the available literature is too partial

We appreciate the reviewer’s suggestion to compare our dataset with previously published scRNAseq data and to integrate markers from the literature. Below, we address these points in detail.

The transcription factors involved in hematopoiesis, such as Tal1, Sox, Runx, and GATA, are highly conserved across metazoans. These markers were identified in our dataset, consistent with findings in other species (1–3), including the previously mentioned scRNA-seq dataset in C. hongkongensis (4). However, defining robust and specific markers for distinct hemocyte types remains an ambitious goal that requires validation across diverse biological contexts - work that is beyond the scope of the present study. Additionally, meaningful comparisons between datasets are constrained by differences in annotation frameworks and the absence of a standardized system for defining hemocyte subtypes. These limitations underscore the need for harmonization efforts to facilitate robust cross-study comparisons. Nonetheless, our dataset provides a strong foundation for future comparative analyses once such standardization is achieved.

In response to the reviewer’s comment, we have added a paragraph to the discussion (lines 747 - 760) detailing that we identified conserved transcription factor markers in C. gigas and C. hongkongensis.

(3) The authors sequenced 3000 cells without providing more comprehensive information/rationale on the analysed population. What is the number of hemocytes found in an adult? What proportion of the whole hemocyte population does this analysis represent? Does it include the tissue-interacting hemocytes? Also, what is the rationale for choosing that specific stage?

We thank the reviewer for their insightful questions regarding the analyzed hemocyte population.

Adult 18-month-old Crassostrea gigas contain approximately 1 million circulating hemocytes per mL of hemolymph, with an average of 1 mL of hemolymph per individual. Thus, this represents approximately 1 million circulating hemocytes per oyster. For our scRNA-seq analysis, we sampled 3,000 hemocytes, which corresponds to 0.3% of the total circulating hemocyte population.

The number of cells processed was optimized to minimize the occurrence of doublets during scRNAseq. Following 10x Genomics Chromium guidelines, we loaded 4,950 cells to successfully recover a target of 3,000 cells, with a doublet rate of 2.4%, well below the target threshold of 2.5%. This information has been added on line 125 of the document. The target was 3,000 cells, and as reported in Supplementary Table S1, the estimated number of cells after STAR-solo alignment was 2,937. This ensures the reliability and accuracy of single-cell transcriptomic data.

We selected 18-month-old oysters for two key reasons: (i) to facilitate hemolymph collection, as hemocyte counts are more stable and sufficient at this stage, enabling us to collect enough cells for all planned experiments, including functional and cytological analyses; and (ii) to use oysters that are not susceptible to OsHV-1 μVar herpesvirus, which predominantly affects younger animals. This ensured that the hemocyte populations analyzed were not influenced by viral infections or related immune responses.

Our study focused on circulating hemocytes collected from hemolymph, which does not include tissue-interacting hemocytes. While these cells may represent an additional population of interest, they fall outside the scope of our current investigation.

By carefully selecting the animal stage and optimizing cell sampling, we ensured that the scRNA-seq dataset provides a robust representation of circulating hemocyte diversity while maintaining high data quality.

(4) For the GO term enrichment analysis, the authors included all genes presenting a cluster enrichment above L2FC>0.25. This seems extremely low to find distinct functions for each cluster. The risk is to call "cluster specific GO term" GO terms for which the genes are poorly enriched in the cluster. For the most important GO term mentioned in the text, the authors should show the expression levels of the genes (with DotPlot similar to Fig1D) to illustrate the specificity of the GO term. At last, the GO enrichment scores were apparently calculated using the whole genome as background. The analysis, aiming at finding differences between hemocyte subgroups, should use the genes detected in the dataset as background.

We appreciate the reviewer's concerns regarding the threshold used for GO term enrichment analysis and the choice of background genes. Below, we provide clarification on these points.

For nuanced comparisons, such as those between activation states of the same cell type, lower thresholds for log2FC (e.g., ≥0.25) are commonly used to detect subtle regulatory shifts. In single-cell RNA sequencing (scRNA-seq) analyses, it is typical to use a log2FC threshold between 0.25 and 0.5 to ensure that biologically relevant, yet subtle, changes are captured. For our analysis, this threshold was chosen to maintain sensitivity to such shifts, particularly given the diversity and functional specialization of hemocyte clusters.

To address the reviewer's suggestion, we will include DotPlot representations (similar to Fig. 1D) for the most significant GO terms highlighted in the text. This will illustrate the expression levels of the associated genes across clusters and demonstrate their specificity to the identified GO terms.

Regarding the background used in the GO enrichment analysis, we employed the Rank Based Gene Ontology Analysis (RBGOA) approach, which explicitly states in its documentation: "It is important to have the latter two tables representing the whole genome (or transcriptome) — at least the portion that was measured — rather than some select group of genes since the test relies on comparing the behavior of individual GO categories to the whole." Our analysis was conducted in agreement with these initial recommendations, ensuring that the results are consistent with the methodology outlined for RBGOA.

(5) The authors reannotated the genes of C. gigas to reach 73.1% annotation. What are the levels of annotations found prior to the reannotation? What do the scores/scale bars from the RBGOA analysis mean in Figures 2B-D?

Thank you for your comment. The original annotation for C. gigas was based on the work of Penaloza et al. (5), which provided GO annotations for 18,750 out of 30,724 genes, corresponding to 61% annotation. Following our reannotation efforts, we were able to increase the annotation coverage to 73.1%, enhancing the resolution of downstream analyses. In response to the reviewer’s comment, we have updated the results section (line 211 and 216) to explicitly include the original annotation coverage of 61% from the work of Penaloza et al., followed by details on our newly achieved annotation percentage of 73.1%.

Thank you for pointing this out. We apologize for the oversight regarding the scale bar in Figures 2BD. The colors in the original figure correspond to a z-score calculated from the gene ratio, which was not clearly explained and may have caused confusion. In the revised version of the manuscript, we propose a new representation to facilitate understanding and improve the clarity of the data presentation (Figure 2B).

(6) The authors describe first the result of the Kegg enrichment analysis and then of the RBGOA. To gain fluidity, I would suggest merging the results of both Kegg and RBGOA for each cluster.

Thank you for the suggestion. To enhance the fluidity of the results section, we have redesigned the KEGG/RBGOA figure (see figure 2A and 2B) to present the results for each cluster in an integrated manner. This revised approach aims to provide a clearer and more cohesive representation of the findings.

(7) The authors make correlations between gradient fraction containing multiple hemocyte populations and qPCR expression levels of cluster-specific markers to associated cytologic features with specific clusters. If feasible, I would recommend validating the association of several markers with hemocyte subgroups using in situ hybridisation or immunolabelling.

Cytological identification of hemocytes in our study relies on MCDH staining, which provides detailed morphological and cytological information. Unfortunately, the fixation methods required for in situ hybridization (ISH) or immunolabeling are not compatible with those used for MCDH staining. We attempted to combine these approaches but found that the fixation protocols necessary for ISH or immunolabeling compromised the quality of the cytological features observed with MCDH staining. Consequently, such validation was not feasible within the constraints of our experimental setup.

(8) Anti-microbial peptides are mentioned as enriched in agranular cells based on the gradient/qPCR analysis (Figure 6). Are these AMPs regulated by inflammatory pathways? Are any inflammatory pathways enriched in any scRNAseq cluster? In addition, without validating the data by directly labelling AMP in the different populations, it seems hard to conclude that AMP are expressed only by agranular cells.

In oysters, two families of antimicrobial peptides/proteins appear to be transcriptionally regulated in hemocytes in response to an infection. The first is that of Cg-BigDefs (6). A 2020 article indicates that the expression of CgBigDef1 is regulated by CgRel, an ortholog of the NFkB transcription factor, which also control the expression of the proinflammatory cytokine CgIL17 (7). Cg-BPI is induced in response to infection but its regulatory pathways remain unknown (8). The last well characterized family of antimicrobial peptides is Cg-Defs. It exhibits constitutive expression in hemocytes.

In our scRNA-seq analysis, CgRel (G12420) shows an increased expression in cluster 5, with a log2FC of 0.4 (equivalent to a 1.32-fold change or 32% higher expression compared to other clusters). Cluster 5 corresponds to blast-like cells, which are transcriptionally distinct and predominantly found in fractions 1, 2, and 3. These same fractions exhibit the highest CgBigDef expression, as demonstrated by qPCR.

From our qPCR results, we see no expression of the three AMP families in cell-sorted granular cells while the cell-sorted agranular cells are positive for the three AMP families, even for inducible ones. Still, we agree that labelling of cell sorted hemocyte populations would reinforce our data. We now specify in the text that further staining would be necessary to confirm these transcriptomic results (Discussion, lines 695 to 296).

(9) The authors should play down some statements concerning cluster identity. In the absence of a true lineage tracing approach, it is possible that those clusters represent states rather than true cell subtypes. Immune cells are very plastic in nature and able to adapt to the environment, even in conditions that are considered homeostatic.

We appreciate the reviewer’s insightful comment regarding the plasticity of immune cells and the potential for clusters to represent states rather than distinct cell subtypes. We agree that, in the absence of a lineage tracing approach, definitive classification of clusters as fixed subtypes is challenging. Immune cells, including those in invertebrates, are known for their high degree of plasticity and adaptability to environmental cues.

In response to the reviewer’s comment, we have revised the Discussion section to include a statement clarifying that these clusters may represent dynamic states rather than fixed subtypes, thereby acknowledging the plasticity of immune cells (lines 766 to 770).

(10) Related to the above issue, there is no indication of stem cells being present in the cell population. Is there any possibility to look for proliferative or progenitor markers? In homeostatic and in challenged conditions (for example Zymosan treatment)? This would provide some hints into the cellular pathways involved in the response. Perhaps determining the number/fraction of phagocytic cells in challenged conditions would help as well, in the absence of time-lapse assays.

Thank you for highlighting the possibility of stem cells or progenitor markers in our hemocyte populations. In our current analysis, we did not detect any known stem cell or proliferative markers, nor evidence of a clearly defined hematopoiesis site in the hemolymph. Indeed, previous work suggests that oyster hematopoiesis may occur in tissues such as the gills, implying that stem or progenitor cells might not circulate in the hemolymph under homeostatic conditions. Consequently, it is plausible that our observation of no proliferative cell populations partly reflects their absence in hemolymph, especially in naïve (unstimulated) oysters. To conclusively identify potential progenitor cells and their proliferative activity, further approaches involving deliberate perturbation of hemocyte homeostasis - such as immunological challenge (e.g., Zymosan treatment) combined with lineagetracing or proliferation assays - would be necessary. These future investigations would not only clarify whether proliferative cells emerge in the hemolymph in response to environmental or pathological stimuli but also help elucidate the broader cellular pathways underlying oyster immune responses.

In response to the reviewer’s comment, we have revised the Discussion (lines 742 to 745) and added : “Nevertheless, we did not detect any canonical stem or progenitor cell populations in our dataset, underscoring the need for future investigations - potentially involving immunological challenges and lineage-tracing assays - to clarify whether proliferative cells circulate in the hemolymph or instead reside primarily in tissue compartments.”

(11) Could the authors discuss the phagocytic hemocytes in light of scavenger receptor expression?

We thank the reviewer for this insightful question. Our study identifies macrophage-like cells and small granule cells as the principal phagocytes in Crassostrea gigas, capable of robust pathogen engulfment. Transcriptomic data reveal that these cell types express markers associated with endocytosis and immune defense pathways, such as CLEC and LACC24, which are integral to their phagocytic functionality.

Interestingly, our single-cell RNA sequencing analysis indicates that cluster 3, corresponding to small granule cells, expresses the scavenger receptor cysteine-rich (SRCR) gene G3876, annotated as an Low-density lipoprotein receptor-related protein with a Log2 fold change (Log2FC) of 0.77. This finding directly links small granule cells to scavenger receptor-mediated functions, supporting their role as professional phagocytes. Scavenger receptors, including SRCR proteins, are known for their ability to bind and internalize diverse ligands, including pathogens, and their presence in small granule cells highlights a potential mechanism for pathogen recognition and clearance.

Additionally, scavenger receptors are significantly expanded in oysters, as shown in Wang et al. (9). These receptors exhibit dynamic upregulation in hemocytes upon pathogen exposure, particularly following stimulation with pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS). This evidence suggests that SRCR proteins, including the one identified in our study, play a pivotal role in the phagocytic activities of hemocytes by facilitating pathogen recognition and internalization.

We propose to add this paragraph (lines 610 to 618) in the Discussion : “Interestingly, our scRNA-seq analysis indicates that SGC (cluster 3) expresses the scavenger receptor cysteine-rich (SRCR) gene G3876, annotated as an Low-density lipoprotein receptor-related protein with a Log2 fold change (Log2FC) of 0.77 linking them to scavenger receptor-mediated pathogen recognition and clearance. This aligns with findings by Wang et al. (9), who demonstrated significant expansion and dynamic regulation of SRCR genes in response to pathogen-associated molecular patterns. “

(12) I am not convinced by the added value of the lineage analysis and the manuscript could stand without it. There is no experimental validation to substantiate the filiation between the clusters. In addition, rooting the lineage to cluster 4 is poorly justified (enrichment in the ribosomal transcript). Cluster 6 is also enriched in ribosomal transcripts and this enrichment can be caused by the low threshold used for the selection of cluster-specific genes (L2FC >0.25). At last, cluster 4 > VC and cluster 4 >SGC belong to the same lineage according to Figure 7 FH.

We thank the reviewer for their detailed comments regarding the lineage analysis. We acknowledge the limitations in experimentally validating the proposed filiation between clusters, as hemocytes in Crassostrea gigas cannot currently be cultivated ex-vivo, and we lack the ability to isolate cells specifically from cluster 4 for further functional assays. Consequently, our lineage analysis is based solely on transcriptomic data and pseudo-time trajectory analysis.

Hematopoietic stem cells (HSCs) are a population of stem cells that are largely cell-cycle-quiescent (G0 phase) with low biosynthetic activity. Upon stimulation and stress HScs undergo proliferation and differentiation and produce all lineages of hemocytes.

Ribosomal proteins play a multifaceted role in preserving the balance between stem cell quiescence and activation. By ensuring precise regulation of protein synthesis, they allow stem cells to maintain their undifferentiated state while remaining poised for activation when needed. Furthermore, ribosomal proteins contribute to the cellular stress response, safeguarding stem cells from oxidative damage and other stressors that could compromise their functionality. Importantly, ribosomal biogenesis and the dynamic assembly of ribosomes provide a regulatory mechanism that fine-tunes the transition from self-renewal to differentiation, a critical feature of hematopoietic stem cells (HSCs) and other stem cell types. These mechanisms collectively highlight the indispensable role of ribosomal proteins in stem cell biology, underscoring their relevance to our study's findings.

In vertebrate, the maintenance of hematopoietic stem cells (HSCs) and hematopoietic homeostasis is widely acknowledged to rely on the proper regulation of ribosome function and protein synthesis (10). This process necessitates the coordinated expression of numerous genes, including genes that encode ribosomal proteins (RP genes) and those involved in regulating ribosome biogenesis and protein translation. Disruptions or mutations in these critical genes are associated with the development of congenital disorders (11). Among these, Rpl22 (found in cluster 4 with a Log2FC of 1.59) has been shown to play a pivotal role in HSC maintenance by balancing ribosomal protein paralog activity, which is critical for the emergence and function of HSCs (12).

Regarding the justification for rooting the lineage to cluster 4, our decision was informed by the enrichment of ribosomal transcripts and functional annotations suggesting a role in translation and cell proliferation, consistent with a precursor-like state. The use of a log2 fold-change (L2FC) threshold of >0.25, while conservative, allowed us to include subtle but meaningful transcriptional shifts essential for resolving lineage transitions.

Finally, the lineage progression from cluster 4 to vesicular cells (VC), macrophage-like cells (ML), and ultimately small granule cells (SGC) is supported by trajectory analysis (Figure 7FH), which consistently places VC and ML as intermediates in the differentiation process toward SGC. Although experimental validation is currently not feasible, these findings provide a conceptual framework for future investigations when cell isolation and functional validation tools become available.

(13) The figures containing heatmaps (Figure 7, Figure 2, Figure S10) or too many subpanels (Figure S5) and Table S5 are hardly readable.

Thank you for highlighting the issues related to the clarity of the heatmaps (Figures 2, 7, and S10), the multi-panel figure (Figure S5), and Table S5. In response to your feedback, we have revised all of these elements to enhance readability and comprehension. Specifically, we increased font sizes, optimized color scales, and reorganized the layout of the subpanels to emphasize the key findings. We also updated Table S5 to ensure that the data are presented in a clear and easily interpretable format.

We trust that these modifications address the concerns raised and improve the overall clarity of the figures and table.

(14) A number of single-cell analyses are now available in different species and the authors allude to similar pathways/transcription factors being involved. Perhaps the authors could expand on this in the discussion section.

Transcription factors involved in hematopoiesis, such as Tal1, Runx and GATA, are highly conserved across metazoans. Consistent with findings in other species, our dataset identifies these markers, reinforcing the evolutionary conservation of these pathways. Furthermore, these markers are also reported in the previous scRNA-seq dataset for C. hongkongensis (4), supporting the robustness of our molecular signatures. However, defining specific and robust markers for distinct hemocyte types remains an ambitious task, requiring additional validation in diverse biological and experimental contexts. This validation is beyond the scope of the present study.

In addition, meaningful comparisons between scRNA-seq datasets are constrained by differences in annotation frameworks and the absence of standardized definitions for hemocyte subtypes. Harmonizing these datasets to enable robust cross-species comparisons is a critical challenge for future studies. Nonetheless, the insights provided by our dataset establish a strong foundation for such comparative analyses when these standardization efforts are realized.

In crayfish (1), 16 transcriptomic clusters were identified corresponding to three hemocyte types, with markers such as integrin prominently expressed in hyalinocytes, consistent with our identification of integrin-related genes in hemocytes. In shrimp (1), 11 transcriptomic clusters were described, with markers of hemocytes in immune-activated states, that we observed also in our dataset. For Anopheles gambiae (2), 8 transcriptomic clusters were identified, including clusters with high ribosomal activity, analogous to those we described in our study. Finally, in Bombyx mori (3), 20 transcriptomic clusters were reported, corresponding to five cytological hemocyte types. Transcription factors such as bHLH, myc, and runt were identified in granulocytes and oenocytoid, showing parallels with markers identified in our dataset.

Despite these similarities, cross-species comparisons are hindered by variability in genome availability and annotation quality, which complicates the precise identification and functional characterization of genes across datasets. Notably, we did not detect pro-phenoloxidase genes in our dataset, unlike shrimp and crayfish, suggesting potential species-specific differences in immune mechanisms.

Regarding the previously published C. hongkongensis scRNA-seq dataset (4), we observe overlap in markers such as runx and GATA. However, direct comparisons remain limited due to differences in dataset annotations and definitions of hemocyte subtypes. This underscores the need for standardized frameworks to facilitate cross-study comparisons. While we emphasize that robust cross-species validation was beyond the scope of this study, our findings contribute valuable insights into the molecular signatures of oyster hemocytes and provide a framework for future comparative research.

We have expanded our discussion to include comparisons with available scRNAseq data from other invertebrate specie (lines 747 to 760)

Minor comments:

(1) Figure 2A-D: to increase the readability of the figure, the authors should display only the GO terms mentioned in the text and keep the full list in supplementary data.

To enhance the fluidity of the results section, we have redesigned the KEGG/RBGOA figure to present the results for each cluster in an integrated manner (See figure 2A and 2B).

(2) Line 223: the authors mention that cluster 1 is characterized by its morphology without providing an explanation or evidence.

We have revised the description of Cluster 1 to remove references to morphology, ensuring consistency with the data presented at this stage of the manuscript (lines 227 to 229) : ”Cluster 1, comprising 27.6 % of cells, is characterized by GO-terms related to myosin complex, lamellipodium, membrane and actin cytoskeleton remodelling, as well as phosphotransferase activity.”

(3) Line 306: the authors mentioned expression levels and associated them with Log2FC, which represents an enrichment, not the level of expression.

Thank you for pointing this out. We agree that log2FC represents enrichment rather than absolute expression levels. We have revised the text in the manuscript to clarify this distinction (line 309). The corrected text now states that log2FC reflects the degree of enrichment or depletion of a gene in a specific cluster relative to others, rather than its absolute expression level.

(4) Figure 4B: the figure shows the distribution of all hemocytes subgroups for each fraction. To better appreciate the distribution of the subgroups in the different fractions, it would be good to have the number of cells of each subtype in the fractions.

We thank the reviewer for their suggestion to include the number of cells of each subtype in the fractions. While we do not have the exact total number of cells per fraction, we systematically performed hemocyte counts for each fraction as part of our methodology. These counts provide a robust estimation of hemocyte distributions across fractions.

Including these counts in the figure could be an alternative approach; however, we believe it would not significantly enhance the interpretability of the data, as the focus of this analysis is on the relative proportions of hemocyte subtypes rather than absolute numbers. The current representation provides a clear and concise overview of subtype distribution patterns, which aligns with the goals of the study.

Nevertheless, if the reviewer considers it essential, we are open to integrating the hemocyte counts into the figure or supplementing the information in the text or supplementary materials to provide additional context.

(5) Line 487-488: the authors mentioned that monocle 3 can deduce the differentiation pathway from the mRNA splice variant. I did not find this information in the publication associated with the statement.

Thank you for pointing this out. We acknowledge the inaccuracy in our statement regarding Monocle3's capabilities. Monocle3 does not deduce differentiation pathways based on mRNA splice variants, as was erroneously suggested in the manuscript. Instead, Monocle3 performs trajectory inference using gene expression profiles. It calculates distances between cells based on their transcriptomic profiles, where cells with similar profiles are positioned closer together, and those with distinct profiles are farther apart. This method enables the construction of potential differentiation trajectories by identifying paths between transcriptionally related cells.

We revise the text in the manuscript to accurately describe this process and remove the incorrect reference to mRNA splice variants (lines 495 to 497).

(6) Figures 6C-H display heatmaps with two columns representing the beginning and the end of the lineage predicted. It would be more talkative to show the whole path presented in Figure S10.

Thank you for pointing out that Figures 7C–H currently only show the beginning and end of the predicted lineage, limiting the clarity of the intermediate stages. In response to your suggestion, we have revised these figures to include the full trajectory as presented in Figure S10, ensuring that the intermediate transitions are more clearly visualized. We believe these modifications offer a more comprehensive overview of the entire lineage and enhance the interpretability of our results.

Bibliography:

(1) F. Xin, X. Zhang, Hallmarks of crustacean immune hemocytes at single-cell resolution. Front. Immunol. 14 (2023).

(2) H. Kwon, M. Mohammed, O. Franzén, J. Ankarklev, R. C. Smith, Single-cell analysis of mosquito hemocytes identifies signatures of immune cell subtypes and cell differentiation. eLife 10, e66192 (2021).

(3) M. Feng, L. Swevers, J. Sun, Hemocyte Clusters Defined by scRNA-Seq in Bombyx mori: In Silico Analysis of Predicted Marker Genes and Implications for Potential Functional Roles. Front. Immunol. 13 (2022).

(4) J. Meng, G. Zhang, W.-X. Wang, Functional heterogeneity of immune defenses in molluscan oysters Crassostrea hongkongensis revealed by high-throughput single-cell transcriptome. Fish & Shellfish Immunology 120, 202–213 (2022).

(5) C. Peñaloza, A. P. Gutierrez, L. Eöry, S. Wang, X. Guo, A. L. Archibald, T. P. Bean, R. D. Houston, A chromosome-level genome assembly for the Pacific oyster Crassostrea gigas. GigaScience 10, giab020 (2021).

(6) R. D. Rosa, A. Santini, J. Fievet, P. Bulet, D. Destoumieux-Garzón, E. Bachère, Big Defensins, a Diverse Family of Antimicrobial Peptides That Follows Different Patterns of Expression in Hemocytes of the Oyster Crassostrea gigas. PLOS ONE 6, e25594 (2011).

(7) Y. Li, J. Sun, Y. Zhang, M. Wang, L. Wang, L. Song, CgRel involved in antibacterial immunity by regulating the production of CgIL17s and CgBigDef1 in the Pacific oyster Crassostrea gigas. Fish & Shellfish Immunology 97, 474–482 (2020).

(8) Evidence of a bactericidal permeability increasing protein in an invertebrate, the Crassostrea gigas Cg-BPI | PNAS. https://www.pnas.org/doi/abs/10.1073/pnas.0702281104.

(9) L. Wang, H. Zhang, M. Wang, Z. Zhou, W. Wang, R. Liu, M. Huang, C. Yang, L. Qiu, L. Song, The transcriptomic expression of pattern recognition receptors: Insight into molecular recognition of various invading pathogens in Oyster Crassostrea gigas. Developmental & Comparative Immunology 91, 1–7 (2019).

(10) R. A. J. Signer, J. A. Magee, A. Salic, S. J. Morrison, Haematopoietic stem cells require a highly regulated protein synthesis rate. Nature 509, 49–54 (2014).

(11) A. Narla, B. L. Ebert, Ribosomopathies: human disorders of ribosome dysfunction. Blood 115, 3196–3205 (2010).

(12) Y. Zhang, A.-C. E. Duc, S. Rao, X.-L. Sun, A. N. Bilbee, M. Rhodes, Q. Li, D. J. Kappes, J. Rhodes, D. L. Wiest, Control of Hematopoietic Stem Cell Emergence by Antagonistic Functions of Ribosomal Protein Paralogs. Developmental Cell 24, 411–425 (2013).

Reviewer #2 (Public review):

Summary:

This work provides a comprehensive understanding of cellular immunity in bivalves. To precisely describe the hemocytes of the oyster C. gigas, the authors morphologically characterized seven distinct cell groups, which they then correlated with single-cell RNA sequencing analysis, also resulting in seven transcriptional profiles. They employed multiple strategies to establish relationships between each morphotype and the scRNAseq profile. The authors correlated the presence of marker genes from each cluster identified in scRNAseq with hemolymph fractions enriched for different hemocyte morphotypes. This approach allowed them to correlate three of the seven cell types, namely hyalinocytes (H), small granule cells (SGC), and vesicular cells (VC). A macrophage-like (ML) cell type was correlated through the expression of macrophage-specific genes and its capacity to produce reactive oxygen species. Three other cell types correspond to blast-like cells, including an immature blast cell type from which distinct hematopoietic lineages originate to give rise to H, SGC, VC, and ML cells. Additionally, ML cells and SGCs demonstrated phagocytic properties, with SGCs also involved in metal homeostasis. On the other hand, H cells, nongranular cells, and blast cells expressed antimicrobial peptides. This study thus provides a complete landscape of oyster hemocytes with functional validation linked to immune activities. This resource will be valuable for studying the impact of bacterial or viral infections in oysters.

Strengths:

The main strength of this study lies in its comprehensive and integrative approach, combining single-cell RNA sequencing, cytological analysis, cell fractionation, and functional assays to provide a robust characterization of hemocyte populations in Crassostrea gigas.

(1) The innovative use of marker genes, quantifying their expression within specific cell fractions, allows for precise annotation of different cellular clusters, bridging the gap between morphological observations and transcriptional profiles.

(2) The study provides detailed insights into the immune functions of different hemocyte types, including the identification of professional phagocytes, ROS-producing cells, and cells expressing antimicrobial peptides.

(3) The identification and analysis of transcription factors specific to different hemocyte types and lineages offer crucial insights into cell fate determination and differentiation processes in oyster immune cells.

(4) The authors significantly advance the understanding of oyster immune cell diversity by identifying and characterizing seven distinct hemocyte transcriptomic clusters and morphotypes.

These strengths collectively make this study a significant contribution to the field of invertebrate immunology, providing a comprehensive framework for understanding oyster hemocyte diversity and function.

Weaknesses:

(1) The authors performed scRNAseq/lineage analysis and cytological analysis on oysters from two different sources. The methodology of the study raises concerns about the consistency of the sample and the variability of the results. The specific post-processing of hemocytes for scRNAseq, such as cell filtering, might also affect cell populations or gene expression profiles. It's unclear if the seven hemocyte types and their proportions were consistent across both samples. This inconsistency may affect the correlation between morphological and transcriptomic data.

We thank the reviewer for highlighting the importance of sample consistency and potential variability, and we acknowledge the need for clarification regarding the use of oysters from two different sources.

Oysters from La Tremblade (known pathogen-free in standardized conditions) were used to establish the hemocyte transcriptomic atlas through scRNA-seq and for cytological analyses. Oysters from the Thau Lagoon (Bouzigues) were used for cytological, functional, and fractionation experiments. These oysters were sampled during non-epidemic periods and monitored under Ifremer’s microbiological surveillance to ensure pathogen free status.

The cytological results (hemocytograms) presented in Figure 3 and Supplementary Figure S3 were derived from Thau Lagoon oysters. To clarify, we updated The Table 3 in Figure 3 and Supplementary Figure S3 to explicitly display hemocyte counts for oysters from both La Tremblade and Thau Lagoon. These data confirm consistent proportions of hemocyte types across both sources, with no significant differences (p > 0.05).

Hemocyte isolation and filtering protocols were rigorously optimized to preserve cell viability and morphology during scRNA-seq library preparation. Viability assays and cytological evaluations confirmed that these procedures did not significantly alter hemocyte populations or their proportions. Sample processing times were minimized to ensure that the scRNA-seq results accurately reflect the native state of the hemolymph.

Taken together, our results confirm that variability between oyster sources or methodological processes did not compromise our findings. This ensures that the correlations between morphological and transcriptomic data are reliable and robust.

(2) The authors claim to use pathogen-free adult oysters (lines 95 and 119), but no supporting data is provided. It's unclear if the oysters were tested for bacterial and viral contaminations, particularly Vibrio and OsHV-1 μVar herpesvirus.

The oysters used in this study were sourced from two distinct origins. First, the animals (18 months old) utilized for scRNA-seq and cytological analyses were obtained from the Ifremer controlled farm located in La Tremblade, France (GPS coordinates: 45.7981624714465, -1.150171788447683). This facility exclusively produces standardized oysters bred in controlled conditions with filtered seawater, entirely isolated from environmental known pathogens. The oysters from this source are certified “pathogen-free” upon arrival at the laboratory, following Ifremer's stringent quality control protocols. We have replaced the term 'pathogen-free' with 'known pathogen-free’ (line 123) to accurately reflect the animals' true status.

Second, for the fractionation experiments and functional tests, oysters were either sourced from the aforementioned Ifremer farm or from a producer located in the Thau Lagoon, France (GPS coordinates: 43.44265228308842, 3.6359883059292057). The Thau Lagoon is subject to comprehensive environmental and microbiological surveillance by the Ifremer monitoring network and the regional veterinary laboratory. For these experiments, we specifically selected oysters aged 18 months - an age associated with reduced susceptibility to OsHV-1 μVar herpesvirus - and ensured that sampling occurred outside of any detected epidemic periods. Furthermore, prior to experimentation, hemocyte samples from all oysters were examined. Oysters showing signs of contamination or exhibiting abnormal hemocyte profiles were excluded from the study.

These measures ensured that the oysters used in this work were of high health status and minimized the likelihood of bacterial or viral contamination, including Vibrio and OsHV-1 μVar.

(3) The KEGG and Gene Ontology analyses, while informative, are very descriptive and lack interpretation. The use of heatmaps with dendrograms for grouping cell clusters and GO terms is not discussed in the results, missing an opportunity to explore cell-type relationships. The changing order of cell clusters across panels B, C, and D in Figure 2 makes it challenging to correlate with panel A and to compare across different GO term categories. The dendrograms suggest proximity between certain clusters (e.g., 4 and 1) across different GO term types, implying similarity in cell processes, but this is not discussed. Grouping GO terms as in Figure 2A, rather than by dendrogram, might provide a clearer visualization of main pathways. Lastly, a more integrated discussion linking GO term and KEGG pathway analyses could offer a more comprehensive view of cell type characteristics. The presentation of scRNAseq results lacks depth in interpretation, particularly regarding the potential roles of different cell types based on their transcriptional profiles and marker genes. Additionally, some figures (2B, C, D, and 7C to H) suffer from information overload and small size, further hampering readability and interpretation.

Thank you for your valuable suggestions regarding the presentation and interpretation of our KEGG and Gene Ontology (GO) analyses. In response, we revised Figure 2 to enhance clarity and provide deeper insights into cell-type relationships and biological processes.

The revised figure 2 reorganizes GO term analysis into a more intuitive layout, grouping related biological processes and pathways in a structured manner. This approach replaces the dendrogram organization and provides a clearer visualization of key pathways for each cell cluster.

(4) The pseudotime analysis presented in the study provides modest additional information to what is already manifest from the clustering and UMAP visualization. The central and intermediate transcriptomic profile of cluster 4 relative to other clusters is apparent from the UMAP and the expression of shared marker genes across clusters (as shown in Figure 1D). The statement by the authors that 'the two types of professional phagocytes belong to the same granular cell lineage' (lines 594-596) should be formulated with more caution. While the pseudotime trajectory links macrophage-like (ML) and small granule-like (SGC) cells, this doesn't definitively establish a direct lineage relationship. Such trajectories can result from similarities in gene expression induced by factors other than lineage relationships, such as responses to environmental stimuli or cell cycle states. To conclusively establish this lineage relationship, additional experiments like cell lineage tracing would be necessary, if such tools are available for C. gigas.

We appreciate the reviewer’s detailed feedback on the pseudotime analysis and its interpretation. While we acknowledge that the clustering and UMAP visualization provide valuable insights, the pseudotime analysis offers a complementary approach by highlighting significantly expressed genes, including key transcription factors, that might otherwise be overlooked in differential expression analysis based solely on Log2FC between clusters. In our study, the pseudotime analysis revealed transcription factors known to play crucial roles in hemocyte differentiation, providing additional depth to our understanding of hemocyte lineage relationships and functional specialization.

Regarding the statement on lines 594 - 596, we agree that the evidence provided by pseudotime trajectories does not definitively establish a direct lineage relationship between macrophage-like (ML) and small granule-like (SGC) cells. Instead, these trajectories suggest potential developmental connections that warrant further investigation. We propose the following revised sentence (lines 616 to 618) :

"The pseudotime trajectory linking macrophage-like (ML) and small granule-like (SGC) cells suggests a potential developmental relationship within the granular cell lineage; however, this hypothesis requires further validation."

We also concur with the reviewer that additional experiments, such as cell lineage tracing, would be necessary to definitively establish this relationship. Unfortunately, the long-term cultivation of hemocytes in C. gigas is currently not feasible. However, we are planning to develop FACS-based approaches to separate the seven hemocyte subtypes, which will allow us to refine their ontology and explore their potential lineage relationships more precisely.

(6) Given the mention of herpesvirus as a major oyster pathogen, the lack of discussion on genes associated with antiviral immunity is a notable omission. While KEGG pathway analysis associated herpesvirus with cluster 1, the specific genes involved are not elaborated upon.

Thank you for your valuable observation regarding the lack of discussion on genes associated with antiviral immunity, particularly in the context of herpes virus infection. The KEGG pathway analysis indeed identified a weak signature associated with herpesvirus in Cluster 1, primarily involving genes encoding beta integrins. In humans, beta integrins have been described as receptors facilitating herpesvirus entry (1). However, in the case of naive oysters used in this study, the KEGG signature was subtle, likely reflecting the absence of active viral infection. Additionally, beta integrins are multifunctional molecules that also play critical roles in processes such as cell adhesion, a function attributed to hyalinocytes, as highlighted in our results.

Given the naive status of the oysters and the weak antiviral signature observed, we chose not to discuss these findings in detail in this study. However, ongoing work in our laboratory aims to further investigate the specific hemocyte populations targeted by OsHV-1, which may shed light on the role of integrins in antiviral immunity in oysters.

We hope this clarifies our approach and the context of the KEGG findings. Thank you for bringing this important perspective to our attention.

(7) The discussion misses an opportunity for comparative analysis with related species. Specifically, a comparison of gene markers and cell populations with Crassostrea hongkongensis, could highlight similarities and differences across systems.

In response to the reviewer’s comment, we have added a comparative analysis between C. hongkongensis and C. gigas hemocyte populations, situating our findings within the broader context of invertebrate immune cell diversity and specialization (lines 747 to 760)

Reviewer #2 (Recommendations for the authors):

(1) Lines 92-93: The authors should add references associated with transcriptomic studies of C. gigas hemocytes.

Thank you for pointing this out. In the revised manuscript, we have added references to previous transcriptomic studies of C. gigas hemocytes (line 83).

(2) Line 121 and 127: The authors should clarify whether 3,000 represents the number of cells loaded or their target for analysis.

The number of cells processed was optimized to minimize the occurrence of doublets during scRNAseq. Following 10x Genomics Chromium guidelines, we loaded 4,950 cells to successfully recover a target of 3,000 cells, with a doublet rate of 2.4%, well below the target threshold of 2.5%. This information has been added on line 125 of the document. The target was 3,000 cells, and as reported in Supplementary Table S1, the estimated number of cells after STAR-solo alignment was 2,937. This ensures the reliability and accuracy of single-cell transcriptomic data.

(3) Line 129: "Supp. Table 1" in the text and "Supp. Table S1" in the figure title should be edited.

The inconsistency between "Supp. Table 1" in the text and "Supp. Table S1" in the figure title has been corrected for uniformity throughout the manuscript (line 134).

(4) Line 138-139: The authors should clarify that the heatmap displays the top 10 positively enriched marker genes for each cluster, as identified by Seurat's differential expression analysis. It is important to note that the analysis does not explicitly show under-represented transcripts, but rather highlights the contrast between cluster-specific overexpressed genes and their lower expression in other clusters.

We have clarified that the heatmap displays the top 10 positively enriched marker genes for each cluster, as identified by Seurat's differential expression analysis, and that the analysis highlights cluster-specific overexpressed genes rather than explicitly showing under-represented transcripts (lines 143 - 145).

(5) Figure 1: The authors should consider improving or potentially removing Figure 1C. The gene IDs are not readable due to their small size, which significantly reduces the informative value of the figure. In addition, the data presented in this heatmap is largely redundant with the more informative and readable dot plot in Figure 1D, which shows both expression levels and the percentage of cells expressing each gene.

Thank you for your suggestion regarding Figure 1C. In the revised manuscript, we have removed the original panel C from the main figure and transferred it to Supplementary Figure S1K, which improves readability while retaining the relevant data. We have also renumbered the remaining panels for clarity, with the former panel D now designated as panel C. We believe these adjustments address the reviewer’s concerns and streamline the presentation of the data.

(6) Table 1: The authors should clarify in the legend the statistical significance criteria (adjusted p-value) for the genes listed.

As requested, we have added the adjusted p-value threshold (adj. p-value < 0.05) to the legend of Table 1.

(7) Line 188: The authors should align the text description of the KEGG pathways in cluster 7 with Figure 2A, describing Wnt signaling pathway and clarifying the terminology "endosome pathway" to ensure consistency.

In the revised text, we have aligned our description with Figure 2A by explicitly mentioning the Wnt signaling pathway in cluster 7 (lines 193 to 194).

The endo-lysosomal pathway encompasses a series of membrane-bound compartments and trafficking events responsible for the uptake of macromolecules from the extracellular environment, their subsequent sorting in endosomes, and eventual degradation in lysosomes. This pathway is tightly regulated, ensuring not only the breakdown of macromolecules but also the recycling of membrane components and signaling receptors essential for maintaining cellular homeostasis (2). In our study, the KEGG signatures of cluster 7 highlight the involvement of the endo-lysosomal pathway.

(8) Line 223: The authors should revise the description of cluster 1, avoiding references to morphology at this point in the manuscript, as no morphological data has been presented yet.

We have revised the description of Cluster 1 to remove references to morphology, ensuring consistency with the data presented at this stage of the manuscript (lines 227 to 229) : ”Cluster 1, comprising 27.6 % of cells, is characterized by GO-terms related to myosin complex, lamellipodium, membrane and actin cytoskeleton remodelling, as well as phosphotransferase activity.”

(9) Figure 2: The authors should revise Figure 2 to improve the clarity. For Figure 2A, they should address the redundancy in the "Global and overview maps" category by removing overlapping pathways such as carbon metabolism and biosynthesis of amino acids, which are likely represented in more specific metabolic categories (glycolysis, pentose). They could consider grouping similar pathways together, such as combining "Amino acid metabolism" with "Metabolism of other amino acids," and separating metabolic pathways from cellular processes for easier interpretation. They should also address the surprising absence of certain expected pathways like lipid metabolism, nucleotide metabolism, and cofactor/vitamin metabolism, as well as cellular processes such as cell growth and chromatin modeling. Even if these pathways are not enriched in specific clusters, mentioning their absence could provide valuable context for the reader.

In the revised version of the manuscript, we propose a new representation to facilitate understanding and improve the clarity of the data presentation.

(10) For Figures 2B, C, and D, the authors should significantly increase the font size of text and numbers, ensuring readability at 100% scale in PDF format. They could also add labels directly on each graph to clearly indicate the type of GO terms represented, (Biological Process, Cellular Component, or Molecular Function).

In the revised version of the manuscript, we propose a new representation to facilitate understanding and improve the clarity of the data presentation.

(11) Line 247-250: The authors should revise their description of cell types to follow the same order as presented in Figure 3A.

We have revised the description of cell types in the manuscript to follow the same order as presented in Figure 3A, as requested.

(12) Line 265-266: The authors should develop the significance of the nucleo-cytoplasmic ratio in hemocyte morphology and identification.

We thank the editor for bringing this to our attention and apologize for the discrepancy between the terminology used in the text and the results presented in Figure 3. The text refers to the nuclear-tocytoplasmic ratio (N/C), while the figure mistakenly displays the inverse ratio, cytoplasmic-to-nuclear ratio (C/N). We recognize that this inversion may cause confusion and will ensure consistency between the text and the figure.

To address this, we propose correcting the figure legend and labels in Figure 3 to align with the terminology used in the text (N/C ratio). This will prevent confusion and maintain clarity throughout the manuscript.

The nuclear-to-cytoplasmic (N:C) ratio, also known as the nucleus:cytoplasm ratio or N/C ratio, is a well-established measurement in cell biology that reflects the relative size of the nucleus to the cytoplasm. This ratio is frequently used as a morphologic feature in the diagnosis of atypia and malignancy in human cells, underscoring its diagnostic value. In the context of our study, we use the N:C ratio to provide a more precise and quantitative description of hemocyte types in Crassostrea gigas. Specifically, the N:C ratio allows us to distinguish between different hemocyte morphotypes, such as blasts and granular cells, and to enrich the characterization of their functional specialization. This quantitative measure supports the morphological classification and enhances the reproducibility and clarity of hemocyte identification.

(13) Line 286-294: The authors should review and correct the legend for Figure 3. It seems that the description of results related to Figure 3C has been mistakenly inserted into the legend.

We thank the reviewer for pointing out this issue with the legend of Figure 3. The description of results related to Figure 3C has now been removed from the legend. The revised legend focuses solely on the figure elements, improving clarity and consistency. We believe this adjustment addresses the reviewer's comment effectively.

(14) Figure 3: The authors should revise the legend for Figure 3A to provide more detailed and explicit descriptions of the "Size, shape and particularities" of the ML, SGC, BGC, and VC hemocyte types.

We thank the reviewer for their insightful suggestion to provide more explicit descriptions in the legend for Figure 3A. We have revised the legend to include detailed explanations of the "Size, shape, and particularities" for the ML, SGC, BGC, and VC hemocyte types. Specifically, we have clarified that size refers to the average granule diameter, shape describes the morphology of the granules (e.g., spherical or elongated), and particularities highlight distinguishing features such as granule color or fluorescence properties observed under specific staining or imaging conditions. We believe this updated legend provides the level of detail requested and enhances the clarity of the figure (lines 294 - 297).

(15) Figure 4: The authors should clarify the method used for calculating relative gene expression in Figure 4A and Figure 6. They should explicitly state in the figure legend that the expression was normalized to the Cg-rps6 reference gene, as mentioned in line 835. The authors should also provide details on the calculation method used (e.g., 2-ΔCt method) and confirm whether the reference gene was expressed at similar levels across all clusters.

We thank the reviewer for pointing out the need for additional clarity regarding the calculation of relative gene expression in Figures 4A and 6. To address this, we have revised the legends for both figures to explicitly state that gene expression levels were normalized to the reference gene Cg-rps6 and calculated using the 2^-ΔCt method. We have also confirmed that Cg-rps6 was stably expressed across all hemocyte clusters and explicitly mentioned this in the revised legends. These changes ensure greater transparency and address the reviewer’s concerns (lines 342 to 346).

(16) The authors could consider removing or modifying Figure 4B, as it appears to be redundant with Figure 3C. Both figures show the average percentage of each hemocyte type in the seven Percoll gradient fractions.

We thank the reviewer for highlighting potential redundancy between Figures 3C and 4B. While both figures present the distribution of hemocyte types across Percoll gradient fractions, Figure 4B serves a distinct and critical purpose in the manuscript. Specifically, it provides the numerical data necessary to understand the correlations shown in Figure 4A, where we analyze the relationship between gene expression levels and the distribution of hemocyte types. These detailed percentages are essential for interpreting the statistical robustness and biological relevance of the correlation matrix, which could not be derived solely from the qualitative visualization in Figure 3C.

(17) Figure 5: The authors should address the redundancy between Figure S7B and Figure 5B, as they appear to present the same data. In Figure S7B, "SGC" is incorrectly abbreviated as "G".

In the revised version of the manuscript, we addressed the redundancy between the two figures and we corrected the incorrectly abbreviated SGC.

(18) Line 412: The authors should correct the typographical error, changing "Pecoll" to "Percoll".

In the revised version of the manuscript, we correct this typographical error (line 417).

(19) Line 417: The statement about the inhibitor apocynin likely refers to Figure 5D, not Figure 5C.

In the revised version of the manuscript, we have corrected this reference error to accurately refer to Figure 5D (line 422).

(20) Line 441-444: The authors should provide references to support their annotation of cluster 1 as macrophage-like cells based on macrophage-specific genes. These references should cite established literature on known macrophage gene markers, particularly in bivalves or related species if available. They need to clarify whether specific gene markers exist for each of the hemocyte morphotypes they have identified. If such markers are known from previous studies, they should be mentioned and referenced.

We propose to modify lines 446 to 449 to address the reviewer's concerns. Cluster 1, which we have termed "macrophage-like" due to its pronounced phagocytic activity and reactive oxygen species (ROS) production, is enriched in Angiopoietin-1 receptor expression (Table 1). Angiopoietin receptors belong to the Tie receptor family, which is expressed in a subset of macrophages known as Tie2-expressing monocytes (TEMs) in humans (3–5). While our analysis reveals a strong overexpression of the Angiopoietin-1 receptor, we acknowledge that this receptor is not an exclusive marker for macrophages.

In bivalves, including oysters, no definitive molecular markers have been established for macrophagelike cells as they are defined functionally in this study. Consequently, the identification of such cells relies on their functional characteristics rather than strict marker expression. To clarify, we propose the following revision to the sentence:

Furthermore, this cluster expresses macrophage-related genes, including the macrophage-expressed gene 1 protein (G30226) (Supp. Data S1), along with maturation factors for dual oxidase, an enzyme involved in peroxide formation (Supp. Fig. S8), supporting its designation as macrophage-like based on functional characteristics.

(21) Figure 7: For Figures 7C to 7H, the authors should increase the font size of gene names and descriptions to ensure legibility in both printed versions and digital formats. To simplify these figures, the authors could consider displaying less differentially expressed genes for each lineage, along with the top genes for each differentiation pathway. If detailed gene information is crucial, they could move the full list to a supplementary table and reference it in the figure legend. Regarding Figure 7I, the authors should reorder the transcription factor genes by cluster and specificity to improve visualization and interpretation, like in Figure 1D.

Thank you for these valuable suggestions regarding Figure 7. We have revised Figures 7C–H to ensure improved readability. Furthermore, we have simplified these panels by highlighting fewer differentially expressed genes for each lineage. In Figure 7I, we have reordered the transcription factor genes by cluster and specificity, following a layout similar to Figure 1D, to facilitate clearer visualization and interpretation of the data.

(22) Line 490: The authors should provide more precise references to the specific GO terms and figure panels they are discussing.

To address this comment, we have revised the sentence and provided additional information in the text to clearly indicate where the corresponding figure panels can be found in the manuscript (line 499)

(23) Line 510: The authors state that "5 cell lineages could be defined," but the subsequent text and Figure 7C to H actually present 6 distinct lineages.

We have corrected in the manuscript. 6 lineages could be defined (line 521).

(24) Line 534: The authors should consider further investigating the pluripotent potential of cluster 4 cells by exploring known or potential stem cell markers in their scRNAseq data.

Thank you for highlighting the possibility of pluripotent potential of cluster 4. In our current analysis, we did not detect any known stem cell or proliferative markers, nor evidence of a clearly defined hematopoiesis site in the hemolymph. Indeed, previous work suggests that oyster hematopoiesis may occur in tissues such as the gills, implying that stem or progenitor cells might not circulate in the hemolymph under homeostatic conditions. Consequently, it is plausible that our observation of no proliferative cell populations partly reflects their absence in hemolymph, especially in naïve (unstimulated) oysters. To conclusively identify potential progenitor cells and their proliferative activity, further approaches involving deliberate perturbation of hemocyte homeostasis - such as immunological challenge (e.g., Zymosan treatment) combined with lineage-tracing or proliferation assays - would be necessary. These future investigations would not only clarify whether proliferative cells emerge in the hemolymph in response to environmental or pathological stimuli but also help elucidate the broader cellular pathways underlying oyster immune responses.

In response to the reviewer’s comment, we have revised the Discussion (lines 695 to 696) and added : “Nevertheless, we did not detect any canonical stem or progenitor cell populations in our dataset, underscoring the need for future investigations - potentially involving immunological challenges and lineage-tracing assays - to clarify whether proliferative cells circulate in the hemolymph or instead reside primarily in tissue compartments.”

(25) Figure S10: The authors should significantly improve the readability of Figure S10 by increasing the font size. Currently, the small font size makes it impossible for readers to discern the information presented.

Thank you for highlighting the readability concerns regarding Figure S10. In response to your comment, we have increased the overall size and font of the figure, ensuring that all labels and legends are clearly legible in both printed and digital formats. We believe these adjustments will allow readers to more easily interpret the information presented.

(26) Line 896: The authors should correct the typographical error on line 896 by deleting the additional bracket.

In the revised version of the manuscript, we correct this typographical error.

(27) Figure S12: The authors should address the absence of any reference to Figure S12 in the main text of the manuscript.

The reference to Supp. Figure S12 has been corrected. It was a referencing error between Supp. Figure S11(in the discussion, line 670) and Supp. Figure S12.

Bibliography:

(1) G. Campadelli-Fiume, D. Collins-McMillen, T. Gianni, A. D. Yurochko, Integrins as Herpesvirus Receptors and Mediators of the Host Signalosome. Annual Review of Virology 3, 215–236 (2016).

(2) J. P. Luzio, P. R. Pryor, N. A. Bright, Lysosomes: fusion and function. Nat Rev Mol Cell Biol 8, 622–632 (2007).

(3) A. S. Harney, E. N. Arwert, D. Entenberg, Y. Wang, P. Guo, B.-Z. Qian, M. H. Oktay, J. W. Pollard, J. G. Jones, J. S. Condeelis, Real-Time Imaging Reveals Local, Transient Vascular Permeability, and Tumor Cell Intravasation Stimulated by TIE2hi Macrophage-Derived VEGFA. Cancer Discov 5, 932–943 (2015).

(4) M. De Palma, R. Mazzieri, L. S. Politi, F. Pucci, E. Zonari, G. Sitia, S. Mazzoleni, D. Moi, M. A. Venneri, S. Indraccolo, A. Falini, L. G. Guidotti, R. Galli, L. Naldini, Tumor-targeted interferon-alpha delivery by Tie2-expressing monocytes inhibits tumor growth and metastasis. Cancer Cell 14, 299–311 (2008).

(5) M. De Palma, M. A. Venneri, R. Galli, L. Sergi Sergi, L. S. Politi, M. Sampaolesi, L. Naldini, Tie2 identifies a hematopoietic lineage of proangiogenic monocytes required for tumor vessel formation and a mesenchymal population of pericyte progenitors. Cancer Cell 8, 211–226 (2005).

Reviewer #3 (Public review):

The paper addresses pivotal questions concerning the multifaceted functions of oyster hemocytes by integrating single-cell RNA sequencing (scRNA-seq) data with analyses of cell morphology, transcriptional profiles, and immune functions. In addition to investigating granulocyte cells, the study delves into the potential roles of blast and hyalinocyte cells. A key discovery highlighted in this research is the identification of cell types engaged in antimicrobial activities, encompassing processes such as phagocytosis, intracellular copper accumulation, oxidative bursts, and antimicrobial peptide synthesis.

A particularly intriguing aspect of the study lies in the exploration of hemocyte lineages, warranting further investigation, such as employing scRNA-seq on embryos at various developmental stages.

In the opinion of this reviewer, the discussion should compare and contrast the transcriptome characteristics of hemocytes, particularly granule cells, across the three species of bivalves, aligning with the published scRNA-seq studies in this field to elucidate the uniformities and variances in bivalve hemocytes.

Reviewer #3 (Recommendations for the authors):

Minor Concerns:

(1) In the context of C. gigas, the notable expansion of stress and immune-related genes in its genome stands out. It is anticipated that the article will discuss the expression patterns of classical immune-related genes like TLR and RLR across different cell clusters.

We appreciate the reviewer's interest in the expression patterns of classical immune-related genes, such as Toll-like receptors (TLRs) and RIG-I-like receptors (RLRs), across different cell clusters in Crassostrea gigas. In our single-cell RNA sequencing (scRNA-seq) analysis, we did not detect significant expression of TLR or RLR genes. This absence can be attributed to several factors. First, technical limitations of scRNA-seq: The droplet-based scRNA-seq technology employed in our study captures only a fraction of the transcripts present in each cell approximately 10–20% (https://kb.10xgenomics.com/hc/en-us/articles/360001539051-What-fraction-of-mRNA-transcriptsare-captured-per-cell). This inherent limitation often results in the underrepresentation of genes with low expression levels. Consequently, TLRs and RLRs, which may be expressed at low levels in certain hemocytes, could be undetected due to this capture inefficiency. TLRs are typically expressed at low basal levels under resting conditions and are upregulated in response to specific stimuli or pathogenic challenges (1, 2). Given that our study analyzed hemocytes in their basal state, the expression levels of these receptors may have been below the detection threshold of the scRNA-seq platform. Furthermore, as highlighted by De Lorgeril et al. (3) the expression of these immune receptors varies depending on the resistance of the oyster. This variability further underscores the dynamic and context-dependent nature of TLR and RLR expression

To comprehensively assess the expression patterns of TLRs and RLRs across different hemocyte clusters, future studies could incorporate targeted enrichment strategies, such as bulk RNA-seq or single-cell technologies with higher capture efficiencies. Additionally, analyzing hemocytes under stimulated conditions or comparing oysters with varying levels of resistance could provide insights into the inducible and context-specific expression of these immune receptors.

(2) Clarification is needed in lines 265-266 regarding the nucleo-cytoplasmic ratio (N/C) terminology to prevent confusion, considering the discrepancy with the results presented in Figure 3.

We thank the editor for bringing this to our attention and apologize for the discrepancy between the terminology used in the text and the results presented in Figure 3. The text refers to the nuclear-tocytoplasmic ratio (N/C), while the figure mistakenly displays the inverse ratio, cytoplasmic-to-nuclear ratio (C/N). We recognize that this inversion may cause confusion and will ensure consistency between the text and the figure.

To address this, we propose correcting the figure legend and labels in Figure 3 to align with the terminology used in the text (N/C ratio). This will prevent confusion and maintain clarity throughout the manuscript.

(3) The selection of cluster 4 as the root for pseudotime analysis based on high ribosomal protein expression raises questions. It would be beneficial to elaborate on the inclusion of other genes, such as cell cycle or mitotic-related genes, to validate the pseudotime analysis outcomes.

We appreciate the reviewer’s insightful comment on the significance of ribosomal proteins in stem cell maintenance.

Hematopoietic stem cells (HSCs) are a population of stem cells that are largely cell-cycle-quiescent (G0 phase) with low biosynthetic activity. Upon stimulation and stress HScs undergo proliferation and differentiation and produce all lineages of hemocytes.

Ribosomal proteins play a multifaceted role in preserving the balance between stem cell quiescence and activation. By ensuring precise regulation of protein synthesis, they allow stem cells to maintain their undifferentiated state while remaining poised for activation when needed. Furthermore, ribosomal proteins contribute to the cellular stress response, safeguarding stem cells from oxidative damage and other stressors that could compromise their functionality. Importantly, ribosomal biogenesis and the dynamic assembly of ribosomes provide a regulatory mechanism that fine-tunes the transition from self-renewal to differentiation, a critical feature of hematopoietic stem cells (HSCs) and other stem cell types. These mechanisms collectively highlight the indispensable role of ribosomal proteins in stem cell biology, underscoring their relevance to our study's findings.

In vertebrate, the maintenance of hematopoietic stem cells (HSCs) and hematopoietic homeostasis is widely acknowledged to rely on the proper regulation of ribosome function and protein synthesis (4). This process necessitates the coordinated expression of numerous genes, including genes that encode ribosomal proteins (RP genes) and those involved in regulating ribosome biogenesis and protein translation. Disruptions or mutations in these critical genes are associated with the development of congenital disorders (5). Among these, Rpl22 (found in cluster 4 with a Log2FC of 1.59) has been shown to play a pivotal role in HSC maintenance by balancing ribosomal protein paralog activity, which is critical for the emergence and function of HSCs (6).

(4) What is the resolution of the cell clustering employed in the study? Given that cluster 1 potentially encompasses two distinct cell types, Macrophage-Like and Big Granule cells, further sub-clustering efforts and correlation analyses between cluster markers and cell morphologies could aid in their differentiation.

Thank you for your inquiry regarding the resolution of our cell clustering. As described in the Materials and Methods section, we used the Seurat FindClusters function with a resolution parameter of r = 0.1 for the scRNA-seq dataset. We performed sub-clustering within Cluster 1, resulting in four distinct subclusters. However, despite analyzing various specific markers, we did not identify any marker uniquely associated with the Big Granule Cell (BGC) morphology. Notably, LACC24 specifically marks a subset of cells within Cluster 1, as shown in Supplementary Figure S8, although this gene alone was insufficient to definitively distinguish a distinct BGC population.

(5) Line 78's statement regarding the primary identification of three hemocyte cell types in C. gigas-blast, hyalinocyte, and granulocyte cells would benefit from including references to substantiate this claim.

We thank Reviewer #1 for their valuable comments, which have allowed us to further improve our manuscript. We have enriched the introduction with the following addition (lines 79 to 82):

“Blast-like cells are considered undifferentiated hemocyte types (Donaghy et al., 2010), hyalinocytes appear to play a key role in wound repair (de la Ballina et al., 2020), and granulocytes are primarily involved in immune surveillance. Among these, granulocytes are regarded as the main immunocompetent hemocyte type (Wang et al., 2017).”

Conclusion:

The authors largely achieved their primary objective of providing a comprehensive characterization of oyster immune cells. They successfully integrated multiple approaches to identify and describe distinct hemocyte types. The correlation of these cell types with specific immune functions represents a significant advancement in understanding oyster immunity. However, certain aspects of their objectives have not been fully achieved. The lineage relationships proposed on the basis of pseudotime analysis, while interesting, require further experimental validation. The potential of antiviral defense mechanisms, an important aspect of oyster immunity, has not been discussed in depth.

This study is likely to have a significant impact on the field of invertebrate immunology, particularly in bivalve research. It provides a new standard for comprehensive immune cell characterization in invertebrates. The identification of specific markers for different hemocyte types will facilitate future research on oyster immunity. The proposed model of hemocyte lineages, while requiring further validation, offers a framework for studying hematopoiesis in bivalves.

Bibliography:

(1) J. Chen, J. Lin, F. Yu, Z. Zhong, Q. Liang, H. Pang, S. Wu, Transcriptome analysis reveals the function of TLR4-MyD88 pathway in immune response of Crassostrea hongkongensis against Vibrio Parahemolyticus. Aquaculture Reports 25, 101253 (2022).

(2) Y. Zhang, X. He, F. Yu, Z. Xiang, J. Li, K. L. Thorpe, Z. Yu, Characteristic and Functional Analysis of Toll-like Receptors (TLRs) in the lophotrocozoan, Crassostrea gigas, Reveals Ancient Origin of TLR-Mediated Innate Immunity. PLOS ONE 8, e76464 (2013).

(3) J. de Lorgeril, B. Petton, A. Lucasson, V. Perez, P.-L. Stenger, L. Dégremont, C. Montagnani, J.M. Escoubas, P. Haffner, J.-F. Allienne, M. Leroy, F. Lagarde, J. Vidal-Dupiol, Y. Gueguen, G.

Mitta, Differential basal expression of immune genes confers Crassostrea gigas resistance to Pacific oyster mortality syndrome. BMC Genomics 21, 63 (2020).

(4) R. A. J. Signer, J. A. Magee, A. Salic, S. J. Morrison, Haematopoietic stem cells require a highly regulated protein synthesis rate. Nature 509, 49–54 (2014).

(5) A. Narla, B. L. Ebert, Ribosomopathies: human disorders of ribosome dysfunction. Blood 115, 3196–3205 (2010).

(6) Y. Zhang, A.-C. E. Duc, S. Rao, X.-L. Sun, A. N. Bilbee, M. Rhodes, Q. Li, D. J. Kappes, J. Rhodes, D. L. Wiest, Control of Hematopoietic Stem Cell Emergence by Antagonistic Functions of Ribosomal Protein Paralogs. Developmental Cell 24, 411–425 (2013).

有时候我们不想让 Git 来自动添加、甚至显示 untracted，就可以使用 .gitignore 文件来，在里面声明 pattern 来让 Git 忽略。

需要被忽略的文件通常是日志文件、或者是由 build 系统产生的文件。

Reviewer #2 (Public review):

Summary:

In this study, the authors investigate the molecular mechanism behind kinesin-1's coordinated movement along microtubules, with a focus on how ATP binding, hydrolysis, and microtubule attachment/detachment are regulated in the leading and trailing heads. Using pre-steady state kinetics and single-molecule assays, they show that the neck linker's conformation modulates nucleotide affinity and detachment rates in each head differently, establishing an asynchronous chemo-mechanical cycle that prevents simultaneous detachment. Supported by cryo-EM structural data, their findings suggest that strain-induced conformational changes in the nucleotide-binding pockets are crucial for kinesin's hand-over-hand movement, presenting a detailed kinetic model of its stepping mechanism. The manuscript is well-crafted, technically rigorous, and should be of significant interest to cell biology and cytoskeletal motor researchers.

Significance:

All conclusions are well-supported by the provided data. The findings address a critical gap in our understanding of how kinesin's two motor domains coordinate their movements, offering insights into the molecular basis of its stepping mechanism. This work should be of significant interest to the cytoskeletal research community.

Comments on latest version:

The authors have satisfactorily addressed my comments, although I recommend the addition of the following reference:

Lu Rao, Jan O. Wirth, Jessica Matthias, and Arne Gennerich. 2025. A Two-Heads-Bound State Drives KIF1A Superprocessivity. bioRxiv 2025.01.14.632505

This paper provides conclusive evidence that kinesin-1 predominantly adopts a one-head-bound state at limiting ATP concentrations and remains in this state for a significant portion of its enzymatic cycle even at saturating ATP. This limits its processivity compared to KIF1A, which predominantly adopts a two-heads-bound state under saturating ATP conditions. These findings directly support the authors' conclusion that trailing head dissociation is favored over leading head detachment.

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

Revision Plan (Response to Reviewers)

1. General Statements [optional]

Response: We are pleased the reviewers appreciate the power of this novel proteomics methodology that allowed us to uncover new depths on the complexity of the ribosome ubiquitination code in response to stress. We also appreciate that the reviewers think that this is a “very timely” study and “interesting to a broad audience” that can change the models of translation control currently adopted in the field. Characterizing complex cellular processes is critical to advance scientific knowledge and our work is the first of its kind using targeted proteomics methods to unveil the integrated complexity of ribosome ubiquitin signals in eukaryotic systems. We also appreciate the fairness of the comments received and below we offer a comprehensive revision plan substantially addressing the main points raised by the reviewers. According to the reviewers’ suggestions, we will also expand our studies to two additional E3 ligases (Mag2 and Not4) known to ubiquitinate ribosomes, which will create an even more complete perspective of ubiquitin roles in translation regulation.

2. Description of the planned revisions

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

The authors present a potentially powerful proteomics platform using parallel reaction monitoring (PRM) to quantitatively profile ribosomal protein (RP) ubiquitylation, with a focus on yeast under hydrogen peroxide (H₂O₂) stress. This approach robustly identifies both known and novel RP modifications, including basal ubiquitylation events previously undetected, and identifies Hel2-dependent mechanisms. The data support the conclusion that RPs are regulated by a multifaceted ubiquitin code, establishing a good foundation for the study.

However, the study's focus shifts in a manner that introduces several limitations. Following the rigorous PRM-based analyses, the reliance on Western blotting without replication or quantification (e.g., single-experiment data in Figs. 3-5) significantly weakens the evidence. Experimental design becomes inconsistent, with variable combinations of stressors (H₂O₂, MMS, 4-NQO) and genetic backgrounds (WT, hel2Δ, rad6Δ) that preclude systematic comparisons. For instance, Fig. 3C/E and Fig. 4 omit critical controls (e.g., MMS in Fig. 4, rad6Δ in Fig. 3E), while Fig. 5 conflates distinct variables by comparing H₂O₂-treated rad6Δ with MMS-treated hel2Δ-a design that obscures causal relationships. Furthermore, Fig. 3F highlights that 4-NQO and MMS elicit divergent responses in hel2Δ, undermining the rationale for using these stressors interchangeably. These inconsistencies culminate in a fragmented narrative; attempts to link ISR activation or ribosome stalling to RP ubiquitylation become impossible, leaving the primary takeaway as "stress responses are complex" rather than advancing mechanistic insight.

        __Response: __We appreciate the evaluation of our work and that the power of our proteomics method established a good foundation for the study. We also understand the reviewer’s concerns and we will detail below a plan to enhance quantification and increase systematic comparisons. The experiments presented here were conducted with biological replicates, but in several instances, we focused on presence and absence of bands, or their pattern (mono vs poly-ub) because of the semi-quantitative nature of immunoblots. We will revise the figures and present their quantification and statistical analyses. In additional, we did not intend to use these stressors interchangeably, but instead, to use select conditions to highlight the complexity the stress response. In particular, we followed up with H2O2 *versus* 4-NQO because both chemicals are considered sources of oxidative stress. Even though it is unfeasible to compare every single stress condition in every strain background, in the revised version, we will include additional controls to increase the cohesion of the narrative, and expand the comparison between MMS, H2O2, and 4-NQO, as suggested. Details below.

To strengthen the work, the following revisions are essential:

R1.1. Repeat and quantify immunoblots: All Western blotting data require biological replicates and statistical analysis to support claims.

        __Response: __As requested, we will display quantification and statistical analysis of the suggested and new immunoblots that will be conducted during the revision period.

R1.3. Remove non-parallel comparisons: The mRNA expression analysis in Fig. 5, which compares dissimilar conditions (e.g., rad6Δ + H₂O₂ vs. hel2Δ + MMS), should be omitted or redesigned to enable direct, strain- and stressor-matched contrasts.

        __Response: __We will follow the reviewers’ suggestion and redesign the analysis to increase consistency and prioritize data under identical conditions. To increase confidence in the mRNA data analysis, we intend to perform follow up experiments and analyze protein abundance of *ARG proteins* and *CTT1 *under different conditions. The remaining data using non-parallel comparisons will be moved to supplemental material and de-emphasized in the final version of the manuscript.

R1.4. Standardize experimental variables: Restructure the study to maintain identical genetic backgrounds and stressors across all figures, enabling systematic interrogation of enzyme- or stress-specific effects on the ubiquitin code.

        __Response: __To ensure a better comparison across strains and conditions, we will re-run several experiments and focus on our main stress conditions. Specifically:

• 3D: We plan to re-run this experiment and include MMS

• 3E: We plan to perform the same panel of experiments in rad6D ,and display WT data as main figure.

• 4A-B: We plan to perform translation output (HPG incorporation) experiments with MMS as suggested

• 4C: We plan to re-run blots for p-eIF2a under MMS for improved comparison.

Reviewer #1 (Significance (Required)):

The authors present a potentially powerful proteomics platform using parallel reaction monitoring (PRM) to quantitatively profile ribosomal protein (RP) ubiquitylation, with a focus on yeast under hydrogen peroxide (H₂O₂) stress. This approach robustly identifies both known and novel RP modifications, including basal ubiquitylation events previously undetected, and identifies Hel2-dependent mechanisms. The data support the conclusion that RPs are regulated by a multifaceted ubiquitin code, establishing a good foundation for the study.

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

In this manuscript the authors use a new target proteomics approach to quantify site-specific ubiquitin modification across the ribosome before and after oxidative stress. Then they validate their findings following in particular ubiquitination of Rps20 and Rps3 and extend their analysis to different forms of oxidative stress. Finally they question the relevance of two known actors of ribosome ubiquitination, Hel2 and Rad6. It is not easy to summarize the observations because in fact the major finding is that the patterns of ribosome ubiquitination occur in a stresser and enyzme specific manner (even when considering only oxidative stress). However, the complexity revealed by this study is very relevant for the field, because it underlies that the ubiquitination code of ribosomes is not easy to interpret with regard to translation dynamics and responses to stress or players involved. It suggests that some of the models that have generally been adopted probably need to be amended or completed. I am not a proteomics expert, so I cannot comment on the validity of the new proteomics approach, of whether the methods are appropriately described to reproduce the experiments. However, for the follow up experiments, the results following Rps20 and Rps3 ubiquitination are well performed, nicely controlled and are appropriately interpreted.

Maybe what one can regret is that the authors have limited their analysis to the study of Hel2 and Rad6, and not included other enyzmes that have already been associated with regulation of ribosome ubiquitination, to get a more complete picture. It may not take that much time to test more mutants, but of course there is the risk that rather than enable to make a working model it might make things even more complex.

        __Response: __We value the positive evaluation of our work. We also appreciate the notion that it meaningfully expands the knowledge on the complexity of the ribosome ubiquitination code, challenges the current models of translation control, and conducted well-performed, and nicely controlled experiments. To address the main concern of the reviewer, we will expand our work by studying two additional enzymes involved in ribosome ubiquitination (Mag2 and Not4) and provide a more comprehensive picture of this integrated system. Specifically, we will generate yeast strains deleted for *MAG2* and *NOT4*, and evaluate their impact in ribosome ubiquitination under our main conditions of stress. We will investigate the role of these additional E3s in translation output (HPG incorporation), and in inducing the integrated stress response via phosphorylated eIF2α and Gcn4 expression. Additional follow up experiments will be performed according to our initial results.

Reviewer #2 (Significance (Required)):

In recent years, regulation of translation elongation dynamics has emerged as a much more relevant site of control of gene expression that previously envisonned. The ribosome has emerged as a hub for control of stress responses. Therefore this study is certainly very timely and interesting for a broad audience. However, it does fall short of giving any simple picture, and maybe the only point one can question is whether it is interesting to publish a manuscript that concludes that regulation is complicated, without really being able to provide any kind of suggestive model.

My feeling is nevertheless that it will impact how scientists in the field design their experiments and what they will conclude. It will certainly also drive new experiments and approaches, and lead to investigations on how all the different players in regulation of ribosome modification talk to each other and signal to signaling pathways.

        __Response: __We appreciate the comments and the balanced view that studies like ours will still be impactful and contribute to a number of fields in multiple and meaningful ways. With the new experiments proposed here, and used of additional mutants and strains, we intend to propose and provide a more unified model that explain this complex and dynamic relationship.

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

Recent studies have shown that the ubiquitination of uS3 (Rps3) is crucial for the quality control of nonfunctional rRNA, specifically in the process known as 18S noncoding RNA degradation (NRD). Additionally, the ubiquitination of uS10 (Rps20) plays a significant role in ribosome-associated quality control (RQC). However, the dynamics of ribosome ubiquitination in response to oxidative stress are not yet fully understood.

In this study, the authors developed a targeted proteomics method to quantify the dynamics of ribosome ubiquitination in response to oxidative stress, both relatively and stoichiometrically. They identified 11 ribosomal sites that exhibited increased ubiquitin modification after exposure to hydrogen peroxide (H2O2). This included two known targets: uS10 and uS3 (of Hel2), which recognize collided ribosomes and initiate the processes of 18S NRD and translation quality control (RQC). Using isotope-labeled peptides, the researchers demonstrated that these modifications are non-stoichiometric and display significant variability among different peptides.

Furthermore, the authors explored how specific enzymes in the ubiquitin system affect these modifications and their impact on global translation regulation. They found that uS3 (Rps3) and uS10 (Rps20) were modified differently by various stressors, which in turn influenced the Integrated Stress Response (ISR). The authors suggest that different types of stressors alter the pattern of ubiquitinated ribosomes, with Rad6 and Hel2 potentially competing for specific subpopulations of ribosomes.

Overall, this study emphasizes the complexity of the ubiquitin ribosomal code. However, further experiments are necessary to validate these findings before publication.

Major Comments:

I consider the additional experiments essential to support the claims of the paper.

R3.1. To understand the roles of ribosome ubiquitination at the specific sites, the authors must perform stressor-specific suppression of global translation, as demonstrated in Figures 4 and 5. This should include the uS10-K6R/K8R and uS3-K212R mutants.

        __Response: __We understand the importance of the suggested experiment. We have already requested and kindly received strains expressing these mutations, which will reduce the time required to successfully address this point. We will perform our translation and ISR assays such as the one referred by the reviewer in Figs. 4A-C and 5E, and results will determine the role of individual ribosome ubiquitination sites in translation control.

R3.2. It is crucial to ensure that experiments are adequately replicated and that statistical analysis is thorough, with precise quantification. For a more accurate comparison between wild-type (WT) and Hel2 deletion mutants regarding ribosome ubiquitination, the authors should quantify the ubiquitinated ribosomes in both WT and Hel2 mutants under stress conditions. This quantification should be conducted on the same blot, using diluted control samples. Similarly, in Figures 3F and 4C, for an accurate comparison between WT and Hel2 or Rad6 deletion mutants, the authors should quantify the ubiquitinated ribosomes across these conditions. Again, this quantification should be performed on the same blot with the dilution of control samples.

        __Response: __As was also requested by reviewer 1 and discussed above (point R1.1), we will conduct quantification and display statistical analyses for our immunoblots. In addition, we will re-run the aforementioned experiments to improve quantification following the reviewers’ request (same gel & diluted control samples).

Reviewer #3 (Significance (Required)):

• General assessment:

Recent studies reveal that the ubiquitination of uS3 (Rps3) is essential for the quality control of nonfunctional rRNA (18S NRD), while the ubiquitination of uS10 (Rps20) plays a crucial role in ribosome-associated quality control (RQC). However, the dynamics of ribosome ubiquitination in response to oxidative stress remain unclear.

• Advance:

In this study, the authors developed a targeted proteomics method to quantify ribosome ubiquitination dynamics in response to oxidative stress, both relatively and stoichiometrically. By utilizing isotope-labeled peptides, they demonstrated that these modifications are non-stoichiometric and exhibit significant variability across different peptides. They identified 11 ribosomal sites that showed increased ubiquitin modification following H2O2 exposure, including two known targets of Hel2, which recognize collided ribosomes and induce translation quality control (RQC).

• Audience: This information will be of interest to a specialized audience in the fields of translation, ribosome function, quality control, ubiquitination, and proteostasis.

• The field: Translation, ribosome function, quality control, ubiquitination, and proteostasis.

__ Response:__ We appreciate that our work will be valuable to a number of fields in protein dynamics and that our method advances the field by measuring ribosome ubiquitination relatively and stoichiometrically in response to stress.

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

Response: All requested changes require experiments and data analyses, and a complete revision plan is delineated above in section #2.

• *

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

• *

R1.2. Leverage the PRM platform: Apply the established quantitative proteomics approach to validate or extend findings in Fig. 3 (e.g., RAD6-dependent ubiquitylation), ensuring methodological consistency.

        __Response: __Although we understand the interest on the proposed result for consistency, this is the only requested experiment that we do not intend to conduct. Because of the lack of overall ubiquitination of ribosomal proteins in *rad6**D* in response to H2O2 (e.g., Silva et al., 2015, Simoes et al., 2022), we believe that this PRM experiment in unlikely to produce meaningful insight on the ubiquitination code. In this context, we expected that sites regulated by Hel2 will be the ones largely modified in rad6*D *and we followed up on them via immunoblot. Moreover, this experiment would not be time or cost-effective, and resources and efforts could be used to strengthen other important areas of the manuscript, such as including the E3’s Mag2 and Not4 into our work.

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

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Referee #1

Evidence, reproducibility and clarity

The authors present a potentially powerful proteomics platform using parallel reaction monitoring (PRM) to quantitatively profile ribosomal protein (RP) ubiquitylation, with a focus on yeast under hydrogen peroxide (H₂O₂) stress. This approach robustly identifies both known and novel RP modifications, including basal ubiquitylation events previously undetected, and identifies Hel2-dependent mechanisms. The data support the conclusion that RPs are regulated by a multifaceted ubiquitin code, establishing a good foundation for the study.

However, the study's focus shifts in a manner that introduces several limitations. Following the rigorous PRM-based analyses, the reliance on Western blotting without replication or quantification (e.g., single-experiment data in Figs. 3-5) significantly weakens the evidence. Experimental design becomes inconsistent, with variable combinations of stressors (H₂O₂, MMS, 4-NQO) and genetic backgrounds (WT, hel2Δ, rad6Δ) that preclude systematic comparisons. For instance, Fig. 3C/E and Fig. 4 omit critical controls (e.g., MMS in Fig. 4, rad6Δ in Fig. 3E), while Fig. 5 conflates distinct variables by comparing H₂O₂-treated rad6Δ with MMS-treated hel2Δ-a design that obscures causal relationships. Furthermore, Fig. 3F highlights that 4-NQO and MMS elicit divergent responses in hel2Δ, undermining the rationale for using these stressors interchangeably. These inconsistencies culminate in a fragmented narrative; attempts to link ISR activation or ribosome stalling to RP ubiquitylation become impossible, leaving the primary takeaway as "stress responses are complex" rather than advancing mechanistic insight.

To strengthen the work, the following revisions are essential:

1. Repeat and quantify immunoblots: All Western blotting data require biological replicates and statistical analysis to support claims.
2. Leverage the PRM platform: Apply the established quantitative proteomics approach to validate or extend findings in Fig. 3 (e.g., RAD6-dependent ubiquitylation), ensuring methodological consistency.
3. Remove non-parallel comparisons: The mRNA expression analysis in Fig. 5, which compares dissimilar conditions (e.g., rad6Δ + H₂O₂ vs. hel2Δ + MMS), should be omitted or redesigned to enable direct, strain- and stressor-matched contrasts.
4. Standardize experimental variables: Restructure the study to maintain identical genetic backgrounds and stressors across all figures, enabling systematic interrogation of enzyme- or stress-specific effects on the ubiquitin code.

Significance

The authors present a potentially powerful proteomics platform using parallel reaction monitoring (PRM) to quantitatively profile ribosomal protein (RP) ubiquitylation, with a focus on yeast under hydrogen peroxide (H₂O₂) stress. This approach robustly identifies both known and novel RP modifications, including basal ubiquitylation events previously undetected, and identifies Hel2-dependent mechanisms. The data support the conclusion that RPs are regulated by a multifaceted ubiquitin code, establishing a good foundation for the study.

Significa que, durante el ejercicio, tu cuerpo está quemando entre un 40% y un 50% más de energía que la que usaría si estuvieras en reposo (sin hacer nada).

PROTEINAS, CARBOHIDRATOS o GRASAS, que tu cuerpo usa para CONSTRUIR cosas como tus músculos, piel, o incluso energía.

Los ALIMENTOS que comes, como PAN, FRUTAS o CARNE, que tu cuerpo ROMPE en pedacitos más pequeños, como azúcar o ácidos grasos, para conseguir energía.

Hypothes.is annotation will be lost in this process of editing metadata, so as he says to make sure annotation are kept.

2012 was a while ago, especially in technology terms, but the majority of the content in this position statement is still relevant and applicable to current day. Some technologies are named or referred to as "cutting edge" that are now just standard technology. That's the biggest marker of age. Otherwise, it's still good information.

• 15 Mas, se tardar, para que saibas como convém andar na casa de Deus, que é a igreja do Deus vivo, a coluna e firmeza da verdade.
• 20 Mas vocês têm uma unção que procede do Santo, e todos vocês têm conhecimento.
• 21 Não lhes escrevo porque não conhecem a verdade, mas porque vocês a conhecem e porque nenhuma mentira procede da verdade. 27 Quanto a vocês, a unção que receberam dele permanece em vocês, e não precisam que alguém os ensine; mas, como a unção dele recebida, que é verdadeira e não falsa, os ensina acerca de todas as coisas, permaneçam nele como ele os ensinou.
• 13 Mas, quando vier aquele, o Espírito de verdade, ele vos guiará em toda a verdade; porque não falará de si mesmo, mas dirá tudo o que tiver ouvido, e vos anunciará o que há de vir. 14 Ele me glorificará, porque há de receber do que é meu, e vo-lo há de anunciar.
• 10 mas Deus o revelou a nós por meio do Espírito. O Espírito sonda todas as coisas, até mesmo as coisas mais profundas de Deus. 11 Pois, quem dentre os homens conhece as coisas do homem, a não ser o espírito do homem que nele está? Da mesma forma, ninguém conhece as coisas de Deus, a não ser o Espírito de Deus. 12 Nós, porém, não recebemos o espírito do mundo, mas o Espírito procedente de Deus, para que entendamos as coisas que Deus nos tem dado gratuitamente.

• Excelência - É incontestável a qualidade moral, ética e filosófica das Escrituras.
• Eficácia - como regra de fé e prática, sempre resulta em uma igreja saudável e fiel.
• Estilo - A multiplicidade de estilos, mas em harmonia.
• Harmonia - todos os livros do cânon apontam para Deus e o plano de redenção em Cristo de forma harmônica, seja de forma direta, ou por meio de pontes.
• Escopo - o seu propósito não é contradicente ou temporal.

Não foi a igreja que outorgou as Escrituras ao povo, mas sim, o contrário, como vemos, especialmente nas epístolas, é Escritura que estabelece o quê e como é uma igreja. Todavia, o bom uso e conhecimento da E. é capaz de mover uma sociedade inteira ao seu apreço. Isso porquê é à Igreja que cabe a docência, e onde podemos estudar e aprender em comunidade acerca dela.

• Toda a Escritura é divinamente inspirada, e proveitosa para ensinar, para repreender, para corrigir, para instruir em justiça;
• Se recebemos o testemunho dos homens, o testemunho de Deus é maior; porque o testemunho de Deus é este, que de seu Filho testificou.
• Por isso também damos, sem cessar, graças a Deus, pois, havendo recebido de nós a palavra da pregação de Deus, a recebestes, não como palavra de homens, mas (segundo é, na verdade), como palavra de Deus, a qual também opera em vós, os que crestes.

Por que cremos que a Bíblia deve ser crida e obedecida? A autoria das Escrituras é o que confere a ela o status de regra de fé e prática. Declaramos na primeira seção que o Senhor escolheu revelar-se dessa forma, que é superior ao senso do divino advindo da natureza.

• Estamos estudando o cap. mais longo da CFW. E que difere, em ordem, do CMW. Por que essa troca de ordem foi feita? Qual vocês creem ser a necessidade de tanto esmero na discussão do tópico das Escrituras?

• Nas seções 3 e 4, falamos sobre a composição canônica. Como sabemos quais livros fazem parte do cânon e quais não podem fazer? Quais são alguns dos critérios para que um livro tenha sido aceito como canônico?

SUM-161 DEPÓSITO. CONDENAÇÃO A PAGAMENTO EM PECÚNIA (mantida) - Res. 121/2003, DJ 19, 20 e 21.11.2003 - Se não há condenação a pagamento em pecúnia, descabe o depósito de que tratam os §§ 1º e 2º do art. 899 da CLT (ex-Prejulgado nº 39).

SUM-245 - DEPÓSITO RECURSAL. PRAZO (mantida) - Res. 121/2003, DJ 19, 20 e 21.11.2003 - O depósito recursal deve ser feito e comprovado no prazo alusivo ao recurso. A interposição antecipada deste não prejudica a dilação legal.

OJ-SDI2-69 FUNGIBILIDADE RECURSAL. INDEFERIMENTO LIMINAR DE AÇÃO RESCISÓRIA OU MANDADO DE SE-GURANÇA. RECURSO PARA O TST. RECEBIMENTO CO-MO AGRAVO REGIMENTAL E DEVOLUÇÃO DOS AUTOS AO TRT (inserida em 20.09.2000) - Recurso ordinário interposto contra despacho monocrático indeferitório da petição inicial de ação rescisória ou de mandado de segurança pode, pelo princípio de fungibilidade recursal, ser recebido como agravo regimental. Hipótese de não conhecimento do recurso pelo TST e devolução dos autos ao TRT, para que aprecie o apelo como agravo regimental.

Conforme Instrução Normativa n° 39 do TST, não cabe, no processo do trabalho, o disposto no art. 1.010, § 3º do CPC. Isto é, o juízo do primeiro grau deve efetuar juízo de admissibilidade dos recursos. Eventual indeferimento sujeita-se à interposição de Agravo de Instrumento.

Nessa linha: - Logo, no âmbito trabalhista, o juízo de primeiro grau deve examinar os pressupostos recursais do recurso ordinário, podendo, com isso, negar seguimento a esse apelo.

• O prazo do agravo de instrumento, como mencionado, é de oito dias, de acordo com a previsão do art. 897, caput, da CLT.838

• Diferentemente do processo civil, o agravo de instrumento, no âmbito da Justiça do Trabalho, não é cabível em face de outras decisões interlocutórias (arroladas no art. 1.015 do CPC), mas apenas no caso acima destacado. (GARCIA, Gustavo Filipe B. Curso de Direito Processual do Trabalho - 12ª Edição 2024. 12. ed. Rio de Janeiro: Saraiva Jur, 2024. E-book. p.296. ISBN 9788553622825. Acesso em: 21 mar. 2025.)

Tema 17

TST-IRR-0000239-55.2011.5.02.0319

Cumulação de Adicionais de Periculosidade e de Insalubridade amparados em fatos geradores distintos e autônomos.

Tema 17 - Tese firmada

• CUMULAÇÃO DE ADICIONAIS DE PERICULOSIDADE E DE INSALUBRIDADE AMPARADOS EM FATOS GERADORES DISTINTOS E AUTÔNOMOS. O art. 193, § 2º, da CLT foi recepcionado pela Constituição Federal e veda a cumulação dos adicionais de insalubridade e de periculosidade, ainda que decorrentes de fatos geradores distintos e autônomos.

Súmula 510 do STF

Praticado o ato por autoridade, no exercício de competência delegada, contra ela cabe o mandado de segurança ou a medida judicial.

O STF admitiu a possibilidade de habeas corpus coletivo.

O habeas corpus se presta a salvaguardar a liberdade. Assim, se o bem jurídico ofendido é o direito de ir e vir, quer pessoal, quer de um grupo determinado de pessoas, o instrumento processual para resgatá-lo é o habeas corpus, individual ou coletivo.

A ideia de admitir a existência de habeas corpus coletivo está de acordo com a tradição jurídica nacional de conferir a maior amplitude possível ao remédio heroico (doutrina brasileira do habeas corpus).

Apesar de não haver uma previsão expressa no ordenamento jurídico, existem dois dispositivos legais que, indiretamente, revelam a possibilidade de habeas corpus coletivo. Trata-se do art. 654, § 2º e do art. 580, ambos do CPP.

O art. 654, § 2º estabelece que compete aos juízes e tribunais expedir ordem de habeas corpus de ofício. O art. 580 do CPP, por sua vez, permite que a ordem concedida em determinado habeas corpus seja estendida para todos que se encontram na mesma situação.

Assim, conclui-se que os juízes ou Tribunais podem estender para todos que se encontrem na mesma situação a ordem de habeas corpus concedida individualmente em favor de uma pessoa.

Existem mais de 100 milhões de processos no Poder Judiciário, a cargo de pouco mais de 16 mil juízes, exigindo do STF que prestigie remédios processuais de natureza coletiva com o objetivo de emprestar a máxima eficácia ao mandamento constitucional da razoável duração do processo e ao princípio universal da efetividade da prestação jurisdicional.

Diante da inexistência de regramento legal, o STF entendeu que se deve aplicar, por analogia, o art. 12 da Lei nº 13.300/2016, que trata sobre os legitimados para propor mandado de injunção coletivo.

Assim, possuem legitimidade para impetrar habeas corpus coletivo:

1) o Ministério Público;

2) o partido político com representação no Congresso Nacional;

3) a organização sindical, entidade de classe ou associação legalmente constituída e em funcionamento há pelo menos 1 (um) ano;

4) a Defensoria Pública.

STF. 2ª Turma.HC 143641/SP. Rel. Min. Ricardo Lewandowski, julgado em 20/2/2018 (Info 891).

Não é condição para o cabimento de ação popular a demonstração de prejuízo material aos cofres públicos, dado que o art 5°, LXXIII, da CF estabelece que qualquer cidadão é parte legítima para propor ação popular e impugnar, ainda que separadamente, ato lesivo ao patrimônio material, MORAL, cultural ou histórico do Estado ou de entidade de que ele participe (STF, Tese 836 de Repercussão Geral)

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Reviewer #1 (Public review):

In this study, Tiang et al. explore the role of ubiquitination of non-structural protein 16 (nsp16) in the SARS-CoV-2 life cycle. nsp16, in conjunction with nsp10, performs the final step of viral mRNA capping through its 2'-O-methylase activity. This modification allows the virus to evade host immune responses and protects its mRNA from degradation. The authors demonstrate that nsp16 undergoes ubiquitination and subsequent degradation by the host E3 ubiquitin ligases UBR5 and MARCHF7 via the ubiquitin-proteasome system (UPS). Specifically, UBR5 and MARCHF7 mediate nsp16 degradation through K48- and K27-linked ubiquitination, respectively. Notably, degradation of nsp16 by either UBR5 or MARCHF7 operates independently, with both mechanisms effectively inhibiting SARS-CoV-2 replication in vitro and in vivo. Furthermore, UBR5 and MARCHF7 exhibit broad-spectrum antiviral activity by targeting nsp16 variants from various SARS-CoV-2 strains. This research advances our understanding of how nsp16 ubiquitination impacts viral replication and highlights potential targets for developing broadly effective antiviral therapies.

Strengths:

The proposed study is of significant interest to the virology community because it aims to elucidate the biological role of ubiquitination in coronavirus proteins and its impact on the viral life cycle. Understanding these mechanisms will address broadly applicable questions about coronavirus biology and enhance our overall knowledge of ubiquitination's diverse functions in cell biology. Employing in vivo studies is a strength.

Weaknesses:

Minor comments:<br /> Figure 5A- The authors should ensure that the figure is properly labeled to clearly distinguish between the IP (Immunoprecipitation) panel and the input panel.

Author response:

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

Reviewer #1 (Public review):

Major comments:

(1) In Figure 1 the authors could reference and use NSP8 (PMID: 38275298) and Nucleocapsid (PMID: 37185839) in their experiments as positive controls.

Thank you for your suggestion! In Figure 1A, during our screening of SARS-CoV-2 nsp proteins regulated by MG132, we confirmed that nsp8 can also be restored by MG132. This finding indicates that nsp8 is degraded via the proteasome pathway and can therefore serve as a positive control for the experiment. It has been reported that nsp8 undergoes degradation via the ubiquitin-proteasome pathway following its ubiquitination mediated by TRIM22. We have added the description at line 115 in the manuscript.

(2) The data indicating that NSP16 is ubiquitinated come from overexpression systems, and it is possible that NSP16 ubiquitination only occurs in expression contexts, not during coronavirus infection. If NSP16 ubiquitination can't be measured in the context of infection, it is unclear how we can make any conclusions. The authors need to demonstrate the ubiquitination of NSP16 in the context of viral infection.

We greatly appreciate the reviewer's suggestion and have incorporated the corresponding experimental results. As shown in Figure 5A, co-IP experiments using an endogenous nsp16 antibody were conducted following infection with the SARS-CoV-2 Wuhan strain. These experiments confirmed that the nsp16 protein encoded by the virus undergoes ubiquitination in infected cells. This finding highlights the ubiquitination of nsp16 within a biological context, thereby supporting our conclusions in expression contexts.

(3) In Figure 4, adding controls will strengthen the authors' conclusion.

a) Is it possible to observe ubiquitination of NSP16 by transfecting in NSP16-FLAG tagged, immunoprecipitate NSP16, run a western blot, and probe for endogenous ubiquitin?

b) Can the authors please include an empty vector control as well as WT ubiquitin in these panels for comparison?

c) In addition, why are the Ubiquitination patterns different in the IP panels of D and E vs B?? Without an empty vector control, it is challenging to conclude what the background is.

Thank you for your valuable suggestions! We have made the following changes and additions in response to your comments:

a) We have conducted the experiments as per the reviewer's suggestion. Figure 3B shows the result. Co-IP experiments were performed, and endogenous ubiquitination of nsp16 was observed using the endogenous ubiquitin antibody.

b) We apologize for previously focusing solely on presenting multiple ubiquitin mutants on a single panel of nsp16 IP without considering the inclusion of an empty vector control and WT ubiquitin. The experiment has been redesigned and conducted, and the results are now presented in Figures 3E and 3F.

c) The differences in the ubiquitination patterns observed between the IP panels in Figures 3E and 3F compared to 3C may be due to varying plasmids, differences in antibody and depth of exposure. To address this, we have standardized the plasmids in the figure and included an empty vector control as a negative control to clarify the background signal.

(4) Overexpression of the ubiquitin mutants may have an indirect effect on protein homeostasis. The authors can also utilize linkage-specific antibodies in their studies to elucidate the ubiquitin linkage associated with NSP16 ubiquitination. K63-linkage Specific Polyubiquitin (D7A11) Rabbit mAb, 5621S, and K48-linkage Specific Polyubiquitin (D9D5) Rabbit mAb, 8081S from Cell Signaling Technologies?

We greatly appreciate the reviewer's excellent suggestion! Using linkage-specific antibodies to elucidate the ubiquitin linkage associated with nsp16 ubiquitination would indeed provide more direct evidence. However, due to the long lead time for obtaining these antibodies, we plan to conduct further verification in future experiments.

(5) The authors discussed the subcellular localization of overexpressed NSP16- showing the localization of NSP16 in the context of viral infection would strengthen the study. If this is challenging, can the authors express NSP16 along with the co-factor NSP10 and examine its subcellular localization?

Thank you for your suggestion! During viral infection, we observed the ubiquitination of the nsp16 protein through co-IP experiments, indicating that the presence of nsp10 does not influence the regulation of nsp16 ubiquitination by MARCHF7 or UBR5 (Figure 5A). Therefore, we believe that investigating the co-localization of nsp10 and nsp16 would not provide additional value to our results. Additionally, through a literature review, we found studies that have already examined the localization of nsp10 and nsp16 following viral infection. These studies revealed that nsp10 was located in the cytoplasm, while nsp16 can be detected in both the nucleus and cytoplasm (PMID: 33080218; PMID: 34452352). This observation is consistent with the localization of nsp16 that we observed in our overexpression experiments.

(6) a) In Figure 3A, the authors should note that the interaction of NPS16 appears weak with UBR5. The authors should confirm that the interaction of NSP16 and the E3 ligases is relevant in the context of viral infection.

b) In Figure 3B, the scale bars should be labeled in at least one panel, as well as in the legend.

c) The authors discussed nuclear localization of MARCHF7, UBR5, and NSP16, therefore a control with a nuclear stain should be included in this figure to enhance the study.

d) Some panels look overexposed while others are blurry which decreases the robustness of the interaction as the authors stated in line 191. To strengthen the results of Figure 3, consider GST purification and in vitro, cell-free binding assays to confirm a direct interaction between nsp16 and the E3 ligases

Thank you for the reviewer’s thoughtful suggestions! We have made the following changes and adjustments based on your recommendations:

a) On the interaction between nsp16 and UBR5:

The interaction between nsp16 and UBR5 appears to be weak, possibly due to the large size of the UBR5 protein (300 kDa). As a result, there are challenges in presenting the experimental results, including difficulties in both expression and protein level detection. To further confirm the relevance of the interaction between nsp16 and the E3 ligases in the context of viral infection, we have performed experiments, and the results are presented in Figure 5A.

b) On scale bars:

The issue regarding the scale bars in Figure 4 has been addressed, and we have now included them in the figure legend for clarity (Line 885).

c) On nuclear localization control:

For the localization of MARCHF7, UBR5, and nsp16 in Figure 4C, given that both MARCHF7 and UBR5 are tagged with CFP, DAPI staining would result in spectral overlap. However, we conducted co-localization experiments for MARCHF7 or UBR5 with nsp16 in Figure 4—figure supplements 1E and 1F, where DAPI staining was included to illustrate the localization of these three proteins. Our experiments showed that while these proteins are present in both the nucleus and cytoplasm, they are predominantly localized in the cytoplasm.

d) On validation of direct interaction:

We attempted GST purification and in vitro cell-free binding assays to verify the direct interaction between nsp16 and the E3 ligases. However, UBR5 and MARCHF7 are both large proteins, with UBR5 being particularly large, which significantly increased the difficulty of purification. Additionally, we faced challenges in purifying nsp16, as the purified nsp16 protein tended to aggregate. We will continue to optimize purification techniques and conditions in future experiments.

We appreciate your valuable comments, which have greatly contributed to improving our experiments and conclusions.

.

(7) To confirm the knockdown of the E3 ligases by siRNA, the authors should use western blotting to show the presence/absence/decrease of the protein levels in addition to mRNA levels by RT-PCR. The authors have the lysates, and they have shown that the antibodies for MARCHF7 and UBR5 work therefore including this throughout the manuscript to help substantiate the authors' conclusions.

Thank you for the reviewer’s valuable suggestion! We have validated the knockdown efficiency at the protein level for the experiments involving siRNA knockdown. Corresponding Western blot images are now included in the relevant experiments to substantiate our conclusions, in addition to the RT-PCR data, including Figures 2, 4 and 5.

(8) In the overexpression studies of the E3 ligases with viral infection in Figure 5, the authors should include the catalytic mutants for the E3 ligases with the nsp16 gradient experiment. This would strengthen the conclusion of the studies.

Thank you for the reviewer’s suggestion! We have conducted the relevant experiments based on your recommendation, and the corresponding data are presented in the Figure 6—figure supplements 2A-H. These results strengthen the conclusions of our study.

(9) Figure 5: For C and F, for a better comparison of the efficacy against the 2 strains, the authors should use the same scale. This could benefit from a kinetics experiment.

Thank you for the reviewer’s suggestion! We have made revisions in Figures 5E and 5H in responses to your recommendation.

(10) Is there a synergistic effect of double E3 knockdown on viral replication?

Thank you for the reviewer’s question! In Figures 5—figure supplement 1A-B, we conducted experiments by individually and simultaneously knocking down MARCHF7 or UBR5, followed by infection with viral SARS-CoV-2 transmissible virus-like particles. The results revealed that simultaneous knockdown further enhances viral replication, demonstrating a synergistic effect.

(11) In lines 98-100 the authors state "This dual targeting by MARCHF7 and UBR5 impairs the 2'-O-MTase activity of nsp16, blocking the conversion of cap-0 to cap-1 at the 5 'end of viral RNA, ultimately exhibiting potent antiviral activity against SARS-CoV-2". The authors did not examine the 2'-O-MTase activity of nsp16. The authors should rephrase this or provide the data if this experiment was done.

Thank you for the reviewer’s valuable suggestion! Based on your comment, we have revised the ambiguous wording located in lines 100-104.

(12) In the discussion, the authors reported that elucidating a specific lysine residue (s) that is ubiquitinated was challenging and stated that they generated multiple mutants including truncated mutants, and wrote "data not shown". The authors need to include this data as supplementary.

Thank you for the reviewer’s suggestion! Based on your comment, we have included the data regarding the specific lysine residue(s) that is ubiquitinated, along with the truncated mutants, as supplementary data (Appendix-figure S2).

(13) In Figure 7, the authors showed a copy number of SARS CoV-2 E in lung tissue. The authors should show viral titers using either the plaque assay or the TCID50 assay.

Thank you for the reviewer’s suggestion! Based on your comment, we measured the TCID50 of the virus in the lung tissue homogenates, and the results are presented in Figure 7D.

Minor comments:

(1) Line 76: while many E3 ubiquitin ligases directly recognize and bind to their target substrates, cullin-RING ligases directly bind an adaptor, which binds a substrate receptor and/or the substrate directly, while the RING-box protein binds a different surface of the cullin and is also not directly interacting with substrate.

Thank you for the reviewer’s valuable suggestion! Based on your comment, we have revised the ambiguous wording in line 76.

(2) Line 161: having introduced the suggestion that NSP16 is ubiquitinated by these ligases, consider moving Figure 4 to the Figure 3 spot.

Based on your comment, we have rearranged the order of the figures and moved Figure 4 to the Figure 3 spot.

(3) Figure 2: Can the authors please do +/- MG132 for each siRNA? It is possible that the lanes where we don't see NSP16 were because there was no NSP16 expressed, OR it was degraded, MG132 would confirm one or the other.

Thank you for the reviewer’s suggestion! Based on your comment, we have redesigned the experiment and included the MG132 treatment for each siRNA. The results are presented in Figure 2A.

(4) Line 165: The authors write "As confirmed by MS, both Myc-tagged MARCHF7 and endogenous UBR5 interact with nsp16, as seen in the Co-IP experiment" should be the reverse, MS suggests NSP16-E3 interaction, the co-ip confirms this.

Based on your comment, we have revised the wording in line 183 to ensure accuracy. MS suggests the interaction between nsp16 and the E3 ligases, while the Co-IP experiment confirms this interaction.

(5) Line 178: the cited paper doesn't clearly show NSP16 nuclear localization, nor do the authors of said paper claim that they found it there. It is cytoplasmic. Additionally, said paper used overexpression, and it is unclear if NSP16 is nuclear in the context of viral infection.

Thank you for the reviewer’s suggestion! The referenced paper states, "As can be seen in the Supplementary Fig. S2, the viral proteins are either cytoplasmic (NSP2, NSP3C, NSP4, NSP8, Spike, M, N, ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8, ORF9b, and ORF10) or both nuclear and cytoplasmic (NSP1, NSP3N, NSP5, NSP6, NSP7, NSP9, NSP10, NSP12, NSP13, NSP14, NSP15, NSP16, E, and ORF9a)," indicating that nsp16 is localized in both the nucleus and cytoplasm. Upon reviewing the literature, we found that the paper (PMID: 33080218) reports the distribution of nsp16 protein following viral infection. The results indicate that nsp16 is present in both the nucleus and cytoplasm, although the authors of the referenced paper claim that ns16 was located in the nucleus.

(6) Line 197: in addition to the 7 lysine residues, ubiquitin can also form linear N-terminal linkages.

Thank you for the reviewer’s suggestion! Linear N-terminal ubiquitination, with its distinct linkage and substrate recognition mechanism, is typically mediated by a complex consisting of the E3 ubiquitin ligases HOIL-1 and HOIP, and differs from classical ubiquitination. Therefore, this type of ubiquitin chain was not investigated in our experiments.

(7) Line 202: Authors state "Interestingly, all single-lysine Ub mutants promoted nsp16 ubiquitylation to varying degrees, indicating a complex polyubiquitin chain structure on nsp16 potentially regulated by multiple E3 ligases". However, not all the mutants. K33 isn't supported by the blot.

Thank you for pointing that out! Indeed, we made an error in our description. The K33 mutant did not promote nsp16 ubiquitylation, and we have corrected this in the manuscript accordingly in line 173.

(8) Line 204: consider including "E2-E3 ligase pairs" for RING ligases the E2 determines the linkage type see: Cell Research (2016) 26:423-440.

Thank you for your suggestion! We have included the term "E2-E3 ligase pairs" in the article in line 176.

(9) Line 235: The authors used the real virus, the inclusion of the BLS2 virus here is extraneous, it doesn't add anything. The authors can consider removing it.

Thank you for your suggestion! In our experiments, we performed simultaneous knockdown of two E3 ligases, so we believe this data is relevant and should not be removed.

(10) Line 238: Authors state: "led to a significant increase in SARS-CoV-2 levels compared to the control group". What is meant by "levels?"

Thank you for your careful reading. We have updated "levels" to "replication" as suggested to clarify the meaning in line 237.

(11) Line 245: increased titers. This could be improved for specificity by saying, 1-log increase for example.

Thank you for the reviewer's valuable suggestions. We have made the necessary changes and specified "increased titers" as a "1-log increase" in lines 249 and 261.

(12) Line 249: in Figure 5H again, the authors are showing relative mRNA levels. Ideally should show protein levels by western blot.

Thank you for the reviewer's suggestion! We have performed protein-level detection of the knockdown efficiency for the samples, and the bands have been placed in the corresponding positions in Figure 5I.

(13) Line 259: "strongly linked to their ability to modulate..." This appears to be an overextension of the data. The data show nsp16 levels can compensate for E3 overexpression, but not that the E3 ligases are modulating this activity. We can infer this from previous experiments. Perhaps increasing the NSP12 levels would also have the same effect as they don't show that this is specific to NSP16. What about a catalytically dead E3?

Thank you for the reviewer's thoughtful suggestion. We have revised the wording accordingly and designed the viral-related experiments with E3 enzyme activity mutants in Figure 6 supplement 2.

(14) Figure 6: In panel H the MW for UBR5 is incorrect, should be around 300kDa.

Thank you for the reviewer's detailed suggestions. We have made the necessary revisions in Figure 6H.

(15) Line 267: "suggesting a more conserved sequence". What are the authors referring to? More conserved than what? This section would benefit from a discussion of which residues are mutated. Are they potential Ub sites, which could point to differential degradation by the E3s as due to more ubiquitination? Or rather to more efficient interaction with the E3? Is this conserved in related CoVs: original SARS and MERS, for instance?

Thank you for the reviewer’s detailed suggestions. In this context, by “conservation,” we refer to the relative conservation of nsp16 proteins across different subtypes of the Omicron variant. We found that most of the mutation sites contained only 1 to 2 mutations. Additionally, we have constructed and validated multiple-mutant nsp16 proteins, which are still degraded by MARCHF7 or UBR5. Given the ongoing prevalence of the Omicron variant, we aim to explore the broad-spectrum degradation and antiviral effects of these two E3 ligases. While it would be ideal if these experiments could aid in identifying the ubiquitination sites, we have not yet identified any mutant forms that escape degradation. We also compared the nsp16 proteins of several other coronaviruses (such as human coronaviruses 229E, HKU1, MERS-CoV, NL63, OC43, and SARS-CoV-1), and found that these viruses' nsp16 proteins are not highly conserved. As a result, we have not further investigated whether MARCHF7 or UBR5 regulate the nsp16 proteins of these viruses.

(16) Line 347: 2C of what virus?

Thank you for the reviewer’s careful reading. We have made the necessary additions to address this point in line 357.

(17) Line 890: "Scale bars, 25 mm". Should it be 25nm?

Thank you for your feedback! I realized there was an error in the unit labeling, and I have corrected the relevant sections in line 904. I appreciate your careful reading.

Reviewer #2 (Recommendations for the authors):

(1) In Figure 6, the authors found that increasing amounts of nsp16 restored the replication of SARS-CoV-2 in the presence of MARCHF7 or UBR5. The authors better discuss the possibility that nsp16 may stimulate viral replication regardless of these E3 ligases, or provide evidence to further clarify this.

Thank you for your thoughtful suggestion! Given the strong functionality of nsp16 itself, your consideration is very comprehensive. In Figure 6—figure supplement 2A–H, we conducted transfection experiments with E3 activity-deficient proteins and reintroduced nsp16. The results showed that, in the absence of active MARCHF7 or UBR5 antiviral function, overexpression of nsp16 did not promote viral replication, although the RNA levels of the M protein slightly increased. Therefore, in our experiments, excess nsp16 did not significantly stimulate viral replication.

(2) In Figure 7, the in vivo data supports the function of both E3 ligases to reduce viral infectivity. Is it possible that tail vein injection of naked plasmid DNA may stimulate the innate immune system, e.g., induce IFN as a DNA vaccine, which may contribute to the inhibitory effect? The authors are suggested to discuss or address it.

Upon reviewing the relevant literature, we found that the hydrodynamic gene delivery (HGD) method using naked DNA is both highly efficient and associated with a low risk of triggering immune responses or oncogenesis. Studies have shown that HGD only weakly activates host immunity (reference: 37111597), which is less of a concern compared to other gene delivery methods. Although some studies have reported strong immune responses following the injection of naked DNA (e.g., Otc cDNA) in human trials, it is noteworthy that no such responses were observed in 17 other participants. This suggests that the immune reactions observed in some cases may be due to individual variability or limitations in animal models, which may not fully translate to human trials.

Based on these findings, we believe that the antiviral effects observed in our study are primarily attributable to the intrinsic properties and functions of the E3 ligases.  Furthermore, it has been reported that mice and non-human primates exhibit significantly greater resistance to innate immune activation compared to humans. This highlights the challenges in translating these findings into effective antiviral therapeutics and underscores the need for further research in this area. We have incorporated the requested discussion into the manuscript in lines 393-410.

(3) The authors shall include some of the key data in supplementary figures in the main text, such as the study on UBR5 and MARCHF7 mediate broad-spectrum degradation of nsp16 variants and SARS-CoV-2 infection decreases UBR5 and MARCHF7 expression, which make it easier for readers to follow.

Thank you for your valuable suggestion regarding the organization of our manuscript. In response to your feedback, we have moved the study on nsp16 variants to the Figure 6—figure supplement 3. Additionally, the data showing changes in UBR5 and MARCHF7 levels following viral infection have been added as supplementary data in Figure 6—figure supplement 4.

(4) The diagrammatic sketches in Figures 1E, S1A and B, 7A, and 8 had low resolutions. Please change them to higher resolutions. Moreover, please state the licensing rights of these diagrammatic sketches.

Thank you for your detailed review! In response to your comment, we have improved the resolution of Figures 1E, S1A and B, 7A, and 8. Additionally, we have specified the drawing tools and source websites in the figure legends (lines 794, 813, 999, and 1013). And we have obtained the necessary licenses for each diagram.

Figure 1E: Created in BioRender. Li, Z. (2025) https://BioRender.com/h43f612

Figure S1B: Created in BioRender. Li, Z. (2025) https://BioRender.com/b98t559

Figure 7A: Created in BioRender. Li, Z. (2025) https://BioRender.com/e76g512

Figure 8: Created in BioRender. Li, Z. (2025) https://BioRender.com/o84p897

(5) The authors suggested that both UBR5 and MARCHF7 had a function in triggering the degradation of NSP16, however, the expression of UBR5 but not MARCHF7 was shown to be associated with the severity of clinical symptoms. Further, why did the host evolve 2 kinds of E3 ligases to adjust only 1 viral target? Please discuss them.

Thank you for your insightful comments. We acknowledge that the limited number of patients with varying degrees of illness in our study could potentially mask some of the observed phenomena. Additionally, individual variability may also play a significant role, which highlights the challenges in translating findings from animal models to human trials.

Regarding the presence of two E3 ligases targeting the same substrate, we view this as part of an evolutionary arms race between the host and the virus. Viruses evolve mechanisms to counteract the host’s antiviral responses, while the host, in turn, develops multiple pathways and strategies to combat viral infection. This dynamic may explain why multiple E3 ligases regulate the levels of the same factor, reflecting the host’s complex and redundant antiviral defense mechanisms. We have incorporated the requested discussion into the manuscript in lines 359-362.

(6) Please standardize the symbol size of the bar charts in the same figure, just like in Figures 1D and 5.

Thank you for your constructive suggestion. We have standardized the symbol sizes of the bar charts in the figure as per your recommendation, ensuring consistency across all panels.

(7) The use of English could be improved.

Thank you for your feedback regarding the language. We have carefully reviewed the manuscript and made revisions to improve the clarity and fluency of the English.

Reviewer #3 (Recommendations for the authors):

Major points:

(1) In Figure 1: The expression level of NSP6, 10, 11, and 12 is weak. Include a higher exposure blot (right next to these blots marking as higher exposure) to show the expression of these plasmids. Here, the NSP12 plasmid has no expression, so it is difficult to conclude the effect of MG132 from this blot. It will be appropriate to show the molecular weight of each gene fragment since some of the plasmids have multiple bands. Verify the densitometric analysis, the NSP4 (+/- MG132) blot, and the densitometric analysis do not correlate. Figure 1B: It is recommended to include appropriate control (media only) for NH4Cl. The DMSO control serves well for the drugs, not for Ammonium Chloride. In Figure 1C, how did the authors arrive at the 15-hour time point? The correlation does not appear as the authors claim. Where is the 15-hour sampling time point for MG132 or CHX chase? The experimental approach to screen the E2/E3 Ub ligase is appreciated.

Thank you for your valuable feedback! Regarding your questions, we have made the following revisions:

On the expression of nsp6, nsp10, nsp11, and nsp12 in Figure 1:

We have replaced the blots for nsp10, nsp11, and nsp12 with higher exposure blots. However, due to the strong expression of NSP14, we were unable to generate a higher exposure blot for nsp6. Based on the current exposure, it is clear that nsp6 is not regulated by the proteasome. Additionally, in the high-exposure blot for nsp12, we were able to observe its expression and found that this protein is weakly regulated by MG132. Following your suggestion, we have labeled the molecular weights of the proteins in the figure.

On the densitometric analysis of nsp4 protein:

We recalculated the densitometric analysis for nsp4 and found no issues. Although the band intensities do not show large changes, the relative fold changes appear more pronounced because we normalized the data using GAPDH as an internal control. We have added detailed description in the figure legend.

On the NH4Cl control:

In this experiment, ammonium chloride was dissolved in DMSO. We reviewed the solubility data and found that ammonium chloride has a solubility of 50 mg/ml in DMSO, which is sufficient to reach the concentrations used in our experiment. While the solubility is higher in water, we believe that DMSO is an appropriate solvent for this compound in our context.

On the 15-hour time point in Figure 1C:

Regarding the 15-hour time point mentioned in Figure 1C, we did not collect samples at that time. We performed semi-quantitative analysis of protein levels at different time points using ImageJ and estimated the half-life time point based on the half-life calculation formula. Thank you for your suggestion; we will clarify this in the figure legend.

Once again, thank you for your thoughtful review and constructive suggestions. We have made the necessary revisions and improvements to the figures based on your feedback.

(2) In Figure 2: I do not find a reason to include DMSO control in the siRNAs for E2/E3 Ub. Please justify why it is necessary. It is requested to include WB for the siRNA-treated samples. It is strongly recommended to show the WB data for siRNA-treated samples because you are showing siRNA treatment of MARCHF7 in shUBR5 cells and vice versa. However, if antibodies for corresponding targets are not available, qPCR can be shown in graphical representation in supplementary data indicating the siRNA target region and qPCR target. Show a graphical representation of domains/ deleted regions of MARCHF7 and UBR5.

Thank you for your valuable feedback! We have addressed your concerns as follows:

On the inclusion of the DMSO control group:

The DMSO group was initially included as a control for the MG132-treated group. By comparing with the MG132 group, we aimed to observe whether nsp16 levels were restored by MG132 treatment. Additionally, in siRNA knockdown experiments, the DMSO group was included to compare nsp16 protein levels after knockdown with those in the NC group, as well as to assess differences in nsp16 restoration between MG132 treatment and factor knockdown. However, we acknowledge some issues in the control design. To address this, we have redesigned and conducted the experiments with improved controls (Figure 2A).

On validating knockdown efficiency:

We have included Western blot data for UBR5 and MARCHF7 knockdown efficiencies. For other factors where specific antibodies were unavailable, we followed your suggestion and provided graphical representations in the Appendix-figure S1, illustrating the siRNA target regions and qPCR target sites to confirm knockdown specificity and efficiency.

(3) In Figure 4 A: Write details on how this IP was done. What was the transfection time of this plasmid? Is the transfection time different from that of NSP16 in Figure 1A which shows a significant degradation of NSP16? Please discuss this in detail. It is recommended that this IP be done in +/- MG132. Since you have used siRNA and performed an IP, It is recommended to repeat the IP (with +/- MG132) using the MARCHF7 and UBR5 plasmids

Thank you for your detailed review and suggestions! We have addressed your concerns as follows:

On the specific protocol for the co-IP in Figure 3A:

The detailed protocol for the immunoprecipitation (IP) experiment is as follows: on day 1, cells were plated, and on day 2, we co-transfected nsp16 and Ub expression plasmids. After 32 hours of transfection, we treated the cells with MG132 for 16 hours, then harvested the cells for IP. We included MG132 treatment in all ubiquitination IP experiments because, without MG132, nsp16 would be degraded, preventing us from observing changes in ubiquitination levels. We apologize for not clearly labeling this in the figure, and we have made the necessary modifications.

On the use of MG132 and NSP16 degradation:

Following your suggestion, we have clarified the use of MG132 in the IP experiments, which differs from the degradation of nsp16 shown in Figure 1A. In Figure 1A, we show the degradation of nsp16 in the absence of MG132 treatment.

On the overexpression of UBR5 and MARCHF7:

The effect of overexpressing UBR5 or MARCHF7 on ubiquitination has been validated in Figure 4 supplement 2. In these experiments, we explored the effect of UBR5 activity domain inactivation on nsp16 ubiquitination, as well as the effect of MARCHF7 truncation on nsp16 ubiquitination modification. In these experiments, overexpression of the wild-type E3 ligases was also included, and the results yielded the same conclusions as those from the E3 knockdown experiments, thereby validating the robustness of our findings.

(4) In Figure 4C: Appropriate controls are missing. The authors claim NSP16 is ubiquitinated and degraded by UBR5 and MARCHF7 via K27 and K48 chains. There is no NSP16 Only control. We cannot compare the NSP16 without an NSP16 transfection. I will suggest the authors repeat these individual controls in both the presence and absence of MG132.

Thank you for your careful review and valuable suggestion! In response to your comment, we have redesigned the experiment and added a control group without nsp16 transfection. We have repeated the validation in the presence of MG132. Without MG132 treatment, nsp16 is degraded, leading to very low protein levels, making it difficult to observe the phenomenon. We have updated the figure accordingly and made the necessary adjustments based on your suggestion (Figure 3E-F).

(5) In my opinion, the Figure 8 needs modification. It is requested to show the levels of strand-specific viral mRNA under UBR5 and MARCHF7 knock-down in +/- of MG312. This figure should also be supported by WB indicating the level of NSP16 (capping activity) and any of the viral proteins. This may validate that if the capping activity is lost, viral translation is affected and hence there is a reduction in virus titre. Alternatively, the figure can be modified by putting a sub-heading box over 7mGppA-RNA section and marking it as a future direction/ hypothesis.

Thank you for your thorough and thoughtful review! Regarding the modification of Figure 8, we completely agree with your suggestion. Currently, examining the impact of viral RNA cap modification is technically challenging for us. Therefore, we have followed your advice and marked the investigation of how nsp16 degradation affects viral RNA cap structures as a future direction/hypothesis in the schematic of Figure 8. This revision helps provide direction for future experiments and enhances the clarity of the figure. Thank you for your thoughtful consideration and valuable suggestion!

Minor points:

(1) Figure 2A: Align NSP16 Blot to actin.

Thank you for your constructive feedback! We have redesigned the experiment and included an MG132 treatment group in Figure 2A. Consequently, the figure has been revised comprehensively, and the nsp16 blot has been aligned with tubulin.

(2) Figure 2C: It is recommended to properly align the lanes where the pLKO and shRNA labelling are overlapping.

Thank you for your thoughtful suggestion! We have revised Figure 2C based on your recommendation to ensure that the pLKO and shRNA labeling no longer overlap. We sincerely apologize for any confusion this may have caused and appreciate your understanding and support.

(3) Just a curious question, what happens if we silence both UBR5 and MARCHF7 and check for virus titre? This is an additional work, but if the authors do not agree, it is ok.

Thank you for your valuable suggestion! Regarding your question about silencing both UBR5 and MARCHF7, we indeed attempted to generate knockout cell lines, but unfortunately, we were not successful at this stage. We plan to explore alternative methods to establish stable knockout cell lines in our future experiments. Meanwhile, as shown in Figure 5 supplement 1, we have performed experiments where both UBR5 and MARCHF7 were knocked down simultaneously, followed by infection with virus-like particles. The results indicate that dual knockdown further enhances viral replication. These findings may partially address your question. Thank you again for your insightful suggestion!

las clases entienden o los objetos entiende? Creo que soy yo la que no entiendo ;)

Me parece interesante la discusión de la vez pasada en torno al cuestionamiento a la noción de objeto, que se presentaba en el video de Anjana Vakil, que incluso más que verbo lo pensábamos como la emergencia o inter/intra acción de esos "objetos".

Esta es la definición de esto según wikipedia: es en computación aquel que puede representar valores de lógica binaria, esto es 2 valores, que normalmente representan falso o verdadero.

Aquí sería también importante una gráfica, así solo con texto me cuesta imaginarlo.

Esta página la he reescrito varias veces en la medida en que distintos talleres avanzan y luego de estar en la variante de Fossil de otro proyecto, llamado Holónica, finalmente está en esta última iteración de la Grafoscopedia.

Intenta dar una mirada conceptual de alto nivel, mientras prepara el terreno para el lugar donde explicaremos la sintaxis más en detalle. La idea es llegar a que podamos hacer ejercicios introductorios de para ver cómo estos conceptos o semántica encarnan en dicha sintaxis.

Valdría la pena profundizar más el concepto de conjunto porque siento que aquí no es muy claro. O al menos no para mi :(

Ejemplo de como mentalizarme para magnetizar mi mente

Este es un compromiso a seguir fielmente para convertirse en un mejor ser humano, consta de 5 puntos interesantes.

• Teses: 1. É constitucional a norma estadual que assegura, no âmbito da educação superior: (i) a livre criação e a auto-organização de centros e diretórios acadêmicos, (ii) seu funcionamento no espaço físico da faculdade, (iii) a livre circulação das ideias por eles produzidas, (iv) o acesso dos seus membros às salas de aula e (v) a participação em órgãos colegiados, em observância aos mandamentos constitucionais da liberdade de associação (CF/1988, art. 5º, XVII), da promoção de uma educação plena e capacitadora para o exercício da cidadania (CF/1988, art. 205) e da gestão democrática da educação (CF/1988, art. 206, VI).

• 2. Entretanto, a norma não se aplica às instituições federais e particulares de ensino superior, em vista de integrarem o sistema federal (arts. 209 e 211, CF c/c os arts. 16 e 17 da Lei 9.394/1996).

[ADI 3.757, rel. min. Dias Toffoli, j. 17-10-2018, P, DJE de 27-4-2020.]

• Os estados não possuem competência para credenciar e autorizar o funcionamento de instituição de ensino superior oriunda de outra unidade da federação para atuar em seu território. (...) À luz do que dispõe o art. 10 da Lei de Diretrizes Básicas da Educação, compete aos Estados organizar, manter e desenvolver os órgãos e instituições oficiais dos seus sistemas de ensino. A única forma prevista em lei para que uma instituição de ensino superior estadual ofereça cursos em outro Estado é a modalidade de ensino à distância, na forma do art. 80 da Lei 9.394/1996, o que requer credenciamento por parte da União.

[ACO 1.197, rel. min. Edson Fachin, j. 25-4-2023, P, DJE de 8-5-2023.]

• A interdependência e complementariedade das noções de Estado Laico e Liberdade de Crença e de Culto são premissas básicas para a interpretação do ensino religioso de matrícula facultativa previsto na Constituição Federal, pois a matéria alcança a própria liberdade de expressão de pensamento sob a luz da tolerância e diversidade de opiniões. (...) A singularidade da previsão constitucional de ensino religioso, de matrícula facultativa, observado o binômio Laicidade do Estado (CF, art. 19, I)/Consagração da Liberdade religiosa (CF, art. 5º, VI), implica regulamentação integral do cumprimento do preceito constitucional previsto no artigo 210, §1º, autorizando à rede pública o oferecimento, em igualdade de condições (CF, art. 5º, caput), de ensino confessional das diversas crenças.

• A Constituição Federal garante aos alunos, que expressa e voluntariamente se matriculem, o pleno exercício de seu direito subjetivo ao ensino religioso como disciplina dos horários normais das escolas públicas de ensino fundamental, ministrada de acordo com os princípios de sua confissão religiosa e baseada nos dogmas da fé, inconfundível com outros ramos do conhecimento científico, como história, filosofia ou ciência das religiões.

• O binômio Laicidade do Estado/Consagração da Liberdade religiosa está presente na medida em que o texto constitucional (a) expressamente garante a voluntariedade da matrícula para o ensino religioso, consagrando, inclusive o dever do Estado de absoluto respeito aos agnósticos e ateus; (b) implicitamente impede que o Poder Público crie de modo artificial seu próprio ensino religioso, com um determinado conteúdo estatal para a disciplina; bem como proíbe o favorecimento ou hierarquização de interpretações bíblicas e religiosas de um ou mais grupos em detrimento dos demais.

• Ação direta julgada improcedente, declarando-se a constitucionalidade dos artigos 33, caput e §§ 1º e 2º, da Lei 9.394/1996, e do art. 11, § 1º, do Acordo entre o Governo da República Federativa do Brasil e a Santa Sé, relativo ao Estatuto Jurídico da Igreja Católica no Brasil, e afirmando-se a constitucionalidade do ensino religioso confessional como disciplina facultativa dos horários normais das escolas públicas de ensino fundamental.

[ADI 4.439, red. do ac. min. Alexandre de Moraes, j. 27-9-2017, P, DJE de 21-6-2018.]

EMENTA AGRAVO INTERNO EM MANDADO DE SEGURANÇA. AMPLA DEFESA E CONTRADITÓRIO EM PROCESSO ADMINISTRATIVO. PROFESSORES SOB REGIME DE DEDICAÇÃO EXCLUSIVA DA UNIVERSIDADE FEDERAL DE MINAS GERAIS. POSSIBILIDADE DE ALOCAÇÃO DOS PROFISSIONAIS EM ATIVIDADES DE COORDENAÇÃO DE TURMAS EM CURSOS DE PÓS-GRADUAÇÃO LATO SENSU.

1. É necessária a observância do contraditório e da ampla defesa em sede de processo administrativo que tramita no Tribunal de Contas da União, se da decisão resultar invalidação de ato que afete a esfera jurídica de quem o expediu.

2. O princípio da autonomia universitária (CF, art. 207) e diversas normas infraconstitucionais (Leis n. 9.394/1996 e 12.772/2012, além de resoluções do Conselho Nacional de Educação e da entidade educacional) conferem à Universidade Federal de Minas Gerais (UFMG) autoridade para gerir suas atividades de ensino, pesquisa e extensão.

3. Os professores contratados sob regime de dedicação exclusiva que desempenhem atividades regulares de ensino na UFMG podem exercer a coordenação de turmas de pós-graduação lato sensu (especialização, aperfeiçoamento e outros).

4. Agravo interno desprovido.

(MS 27800 AgR, Relator(a): NUNES MARQUES, Segunda Turma, julgado em 02-07-2022, PROCESSO ELETRÔNICO DJe-153 DIVULG 02-08-2022 PUBLIC 03-08-2022)

Ementa: DIREITO CONSTITUCIONAL E ADMINISTRATIVO. ATOS DE NOMEAÇÃO, PRETÉRITOS E FUTUROS, DE REITORES E VICE-REITORES DE UNIVERSIDADES FEDERAIS PELO PRESIDENTE DA REPÚBLICA A PARTIR DE LISTA TRÍPLICE. ATO COMPLEXO PREVISTO NA LEGISLAÇÃO. EXERCÍCIO DE DISCRICIONARIEDADE MITIGADA PELO CHEFE DO PODER EXECUTIVO. ABSOLUTO CUMPRIMENTO AO PROCEDIMENTO E FORMA ESTABELECIDOS EM LEI. RESPEITO AO PROCEDIMENTO DE CONSULTA REALIZADO PELAS UNIVERSIDADES FEDERAIS, CONDICIONANTES DE TÍTULO E CARGO E OBRIGATORIEDADE DE ESCOLHA DE UM DOS NOMES QUE FIGUREM NA LISTA TRÍPLICE. INEXISTÊNCIA DE OFENSA À AUTONOMIA UNIVERSITÁRIA (ART. 207, CF) E AOS PRINCÍPIOS DA GESTÃO DEMOCRÁTICA DO ENSINO (ART. 206, VI, CF), DO REPUBLICANISMO (ART. 1º, CAPUT) E DO PLURALISMO POLÍTICO (ART. 1º, V). AUSÊNCIA DE FUMUS BONI IURIS. MEDIDA CAUTELAR INDEFERIDA.

1. A autonomia científica, didática e administrativa das universidades federais, prevista no art. 207 da Constituição Federal, concretiza-se pelas deliberações colegiadas tomadas por força dos arts. 53, 54, 55 e 56 da Lei 9.394/1996. A escolha de seu dirigente máximo pelo Chefe do Poder Executivo, a partir de lista tríplice, com atribuições eminentemente executivas, não prejudica ou perturba o exercício da autonomia universitária, não significando ato de fiscalização ou interferência na escolha ou execução de políticas próprias da instituição, escolhidas por decisão colegiada e participativa de seus integrantes.

2. A Constituição Federal e legislação complementar preveem, para instituições essenciais ao equilíbrio democrático, como Tribunais Superiores, o Ministério Público e a Defensoria Pública, escolha de integrantes ou dirigentes máximos a partir de ato discricionário do Presidente da República, com ou sem formação de lista tríplice pelos pares. Tal previsão não afasta ou prejudica a autonomia institucional, administrativa e jurídica de tais entes face ao Poder Executivo, pois fundado na legitimação política da escolha pelo titular eleito democraticamente.

3. Sendo a escolha determinada a partir de lista tríplice, não se justifica a imposição de escolha no nome mais votado, sob pena de total inutilidade da votação e de restrição absoluta à discricionariedade mitigada concedida ao Chefe do Poder Executivo.

4. Ausência dos requisitos necessários para deferimento da medida cautelar, uma vez que se trata de exceção ao princípio segundo o qual os atos normativos são presumidamente constitucionais.

5. Desnecessidade de deferimento parcial do pleito cautelar para a fixação de balizas já previstas na Lei 5.540/1968, com a redação dada pela Lei 9.192/1995, e que continua em vigor. 6. Medida liminar indeferida.

(ADPF 759 MC-Ref, Relator(a): EDSON FACHIN, Relator(a) p/ Acórdão: ALEXANDRE DE MORAES, Tribunal Pleno, julgado em 08-02-2021, PROCESSO ELETRÔNICO DJe-071 DIVULG 14-04-2021 PUBLIC 15-04-2021)

EMENTA: AÇÃO DIRETA DE INCONSTITUCIONALIDADE MANEJADA CONTRA O ART. 1º DA LEI FEDERAL 11.301/2006, QUE ACRESCENTOU O § 2º AO ART. 67 DA LEI 9.394/1996. CARREIRA DE MAGISTÉRIO. APOSENTADORIA ESPECIAL PARA OS EXERCENTES DE FUNÇÕES DE DIREÇÃO, COORDENAÇÃO E ASSESSORAMENTO PEDAGÓGICO. ALEGADA OFENSA AOS ARTS. 40, § 5º, E 201, § 8º, DA CONSTITUIÇÃO FEDERAL. INOCORRÊNCIA. AÇÃO JULGADA PARCIALMENTE PROCEDENTE, COM INTERPRETAÇÃO CONFORME.

I - A função de magistério não se circunscreve apenas ao trabalho em sala de aula, abrangendo também a preparação de aulas, a correção de provas, o atendimento aos pais e alunos, a coordenação e o assessoramento pedagógico e, ainda, a direção de unidade escolar.

II - As funções de direção, coordenação e assessoramento pedagógico integram a carreira do magistério, desde que exercidos, em estabelecimentos de ensino básico, por professores de carreira, excluídos os especialistas em educação, fazendo jus aqueles que as desempenham ao regime especial de aposentadoria estabelecido nos arts. 40, § 5º, e 201, § 8º, da Constituição Federal.

III - Ação direta julgada parcialmente procedente, com interpretação conforme, nos termos supra.

(ADI 3772, Relator(a): CARLOS BRITTO, Relator(a) p/ Acórdão: RICARDO LEWANDOWSKI, Tribunal Pleno, julgado em 29-10-2008, DJe-059 DIVULG 26-03-2009)

Ementa: CONSTITUCIONAL E ADMINISTRATIVO. RECURSO EXTRAORDINÁRIO. APOSENTADORIA ESPECIAL DOS PROFESSORES (CONSTITUIÇÃO, ART. 40, § 5º). CONTAGEM DE TEMPO EXERCIDO DENTRO DA ESCOLA, MAS FORA DA SALA DE AULA.

1. Revela especial relevância, na forma do art. 102, § 3º, da Constituição, a questão acerca do cômputo do tempo de serviço prestado por professor na escola em funções diversas da docência para fins de concessão da aposentadoria especial prevista no art. 40, § 5º, da Constituição.

2. Reafirma-se a jurisprudência dominante desta Corte nos termos da seguinte tese de repercussão geral: Para a concessão da aposentadoria especial de que trata o art. 40, § 5º, da Constituição, conta-se o tempo de efetivo exercício, pelo professor, da docência e das atividades de direção de unidade escolar e de coordenação e assessoramento pedagógico, desde que em estabelecimentos de educação infantil ou de ensino fundamental e médio.

3. Repercussão geral da matéria reconhecida, nos termos do art. 1.035 do CPC. Jurisprudência do SUPREMO TRIBUNAL FEDERAL reafirmada, nos termos do art. 323-A do Regimento Interno. (RE 1039644 RG, Relator(a): ALEXANDRE DE MORAES, Tribunal Pleno, julgado em 12-10-2017, PROCESSO ELETRÔNICO REPERCUSSÃO GERAL - MÉRITO DJe-257 DIVULG 10-11-2017 PUBLIC 13-11-2017)

Não inclui o magistrado nesta fase, apenas depois para a homologação.

DOI: 10.1016/j.xcrm.2025.102020

Resource: (BioLegend Cat# 320907, RRID:AB_493196)

Curator: @scibot

SciCrunch record: RRID:AB_493196

What is this?

DOI: 10.1016/j.ebiom.2025.105628

Resource: (BD Biosciences Cat# 563172, RRID:AB_2744297)

Curator: @scibot

SciCrunch record: RRID:AB_2744297

What is this?

sanık ve aynı zamanda başvurucu döktürmüş

Great point

This section depicts the root of Melville's disdain for publishers and critics. As we have discussed, he was very particular about his style and hard on his own writing making it clear how any changes could be viewed as abominable. Furthermore, when it is his writing being criticized, it would be frustrating to know it had been altered in his own name. This idea is reflected in Pierre's struggles to find his footing as a writer when everyone in the world wants something different but he simply wants to write for himself and share that with the world.

Author response:

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

We appreciate that both reviewers found our findings significant and recognized the strength of the presented data in demonstrating the potential value of ASO-mediated Emc10 expression modulation for treating 22q11.2DS. We are grateful for the reviewers' valuable input and constructive suggestions, which we believe have significantly strengthened our manuscript. Below, we address the main points and concerns, followed by our point-by-point responses:

Evaluation of ASO-Mediated Emc10 Reduction: We appreciate the feedback and the opportunity to clarify this point. While we agree that ASO-mediated reduction of Emc10 should ideally be evaluated at both the mRNA and protein levels, we would like to emphasize that this was indeed performed in our study. Specifically, we conducted both qRT-PCR and Western Blot (WB) assays on the same animal cohort, focusing on the left and right hippocampus (rather than the PFC) following ASO injection (see Figure S11C and D). We prioritized the hippocampus for the WB assay because our primary behavioral assays and observed phenotypes in this study are strongly hippocampus-centric. This approach reflects our aim to investigate Emc10's role in the brain regions most relevant to the observed phenotypes. We hope this clarification addresses the reviewer’s concerns. While protein-level analysis would ideally complement RNA measurements, the Emc10 antibodies available were suboptimal in specificity and sensitivity, requiring substantial optimization. Additionally, challenges in obtaining sufficient high-quality protein from small regions like the hippocampus limited the use of protein detection as a standalone method. We plan to refine antibody protocols or explore alternative methods in future work. Notably, in all instances where we performed parallel protein and RNA measurements in both, mouse brain tissue and human cell lines, there was excellent concordance between the datasets, strongly suggesting that mRNA levels are a reliable indicator of Emc10 protein levels in our model.

ASO Neuronal Uptake: While ASO uptake by neurons in the brain can vary considerably depending on factors such as ASO chemistry, delivery method, target brain region, and cell type, our targeted delivery approach, ASO design optimization, and ASO screening strategy were specifically tailored to achieve uniform and efficient uptake across hippocampal and cortical regions, in both neurons and glia. The figures included in our manuscript at both low and high magnification (see Figure S14A) clearly display the extensive (over 97%) overlap of ASO-positive cells (green signal) with cells expressing the neuronal marker NeuN (red signal). While quantifying ASO-positive cells in different brain regions could add value, the robust diffusion of ASO into neurons and glia is effectively demonstrated in the current figures and indirectly supported by the robust downregulation of Emc10 in ASO-treated animals as shown by qRT-PCR assays of hippocampal and cortical brain regions.

Transcriptomic Data in Mutant EMC10 NGN2-iNs: Reduction in EMC10 levels is not expected to directly affect transcription or to broadly reorganize the differential gene expression profile of the Q6/Q5 patient/control NGN2-iN lines. Accordingly, our transcriptional profiling was not designed to assess the direct impact of EMC10 deficiency on gene expression but rather to serve as an indirect measure of cellular pathways affected by the reduction in EMC10 levels in the patient Q6 line. We aimed to identify genes and related functional pathways differentially expressed between the Q6/Q5 patient/control lines, where these expression differences are either abolished or significantly attenuated in Q6/EMC10^HET or Q6/EMC10^HOM NGN2-iNs.

Statistical Analysis: We have meticulously reviewed all statistical analyses in the manuscript to ensure their appropriateness and adherence to established practices. For Figure S2, we acknowledge that the statistical details were not fully specified in the figure legend, though they are provided for each miRNA in Supplemental Table S2. In the revised manuscript, we ensured that the statistical methods and corresponding values are clearly indicated for each comparison.

We are confident that the revisions outlined above, along with the point-by-point responses provided below, will significantly strengthen our manuscript and address all the concerns raised by the reviewers. We would like to express our sincere thanks to the reviewers for their valuable feedback and constructive suggestions.

Reviewer #1 (Recommendations For The Authors):

My comments here are generally limited to minor comments that reflect possible small additions or edits to the manuscript:

(1) Panel 1A is very small. Please consider making that bigger as space permits.

We have increased the panel size of Figure 1A in the revised manuscript to improve its visibility and clarity.

(2) Are you able to identify the dot that represents EMC10 in panel 1C? I understand that EMC10 is represented in Supplementary Figure 4A.

We appreciate the reviewer's observation. In Figure 1C, the volcano plot depicts differentially expressed miRNAs in the Q5/Q6 neuronal samples, as identified through miRNA-sequencing. Since EMC10, as a protein-coding gene and a downstream target of miRNA dysregulation, is not included in this analysis. However, as the reviewer correctly notes, EMC10 gene expression is represented in the volcano plot in Supplementary Figure 4A, which displays differentially expressed genes identified through bulk RNA-seq analysis of the same neuronal samples. To avoid any confusion, we have clarified the title of Figure 1C to emphasize that it represents miRNA expression changes.

(3) With regard to studies using iPSC. Some of the studies are executed across multiple distinct pairs and some are only done in a single pair. Overall, while results are coherent and often complimentary, would it be valuable for the authors to comment on experiments where studies in multiple pairs seemed particularly important, or others wherein it was less important?

We thank the reviewer for this insightful question regarding our use of multiple versus single hiPSC pairs. Our investigation began with the Q5/Q6 sibling (dizygotic twin) pair, which shares the most similar genetic background. This minimized the impact of confounding genetic factors and provided a robust foundation for testing our hypothesis that EMC10 upregulation, driven by miRNA dysregulation, is a key consequence of the 22q11.2 deletion in human neurons, thus validating our previous findings from the Df(16)A^+/- mouse model (Stark et al., 2008; Xu et al., 2013). To ensure the generalizability of our findings, we incorporated additional hiPSC lines from another sibling pair as well as a case/control pair, demonstrating that EMC10 upregulation is a consistent feature of 22q11.2DS. Subsequently, we focused on the well-matched Q5/Q6 pair for detailed morphological, functional, and genetic rescue experiments. This approach allowed us to perform in-depth studies while controlling for potential genetic confounders. By using both multiple and single hiPSC pairs, we balanced the need for generalizable findings with the practical considerations of conducting technically complex and resource-intensive experiments. This strategy enabled us to provide both broad and detailed insights into the mechanisms underlying 22q11.2DS. We have modified the introductory paragraph of the Results section to better highlight this issue.

(4) While the majority of the experiments seem sufficiently powered to test the hypothesis in question in the iPSC studies, Figure 2B raises the question if the study replicates here were underpowered, and perhaps the authors might consider mentioning this, although this is a very minor comment.

We thank the reviewer for raising this point. We acknowledge that the statistical power to detect a significant difference in pre-miR-485 levels in Figure 2B may be limited due to the relatively small sample size and the inherent variability in hiPSC-derived neuronal cultures. However, it is important to emphasize that the functional impact of miRNAs is primarily mediated by their mature transcript forms. Our miRNA-seq data (Supplementary Table 2 and Figure S2) did not show significant alterations in the levels of mature miR-485-5p or miR-485-3p. This finding aligns with the reported expression pattern of miR-485 in hiPSC-derived neurons, where relatively low levels are observed in early neuronal development, with increased expression occurring in older, more mature neurons (Soutschek et al. 2023; https://ethz-ins.org/igNeuronsTimeCourse/ database from the Institute of Neurogenomics, ETH Zurich). This database provides a valuable resource for examining gene expression dynamics during human neuronal differentiation. Given that our hiPSC-derived neurons were analyzed at a relatively early developmental stage (DIV8 for these experiments), it is likely that miR-485 expression had not yet reached levels sufficient to reveal significant differences. While we acknowledge the potential limitation in statistical power for detecting subtle changes in pre-miR-485 levels, the combined evidence suggests that miR-485 may not be a significant contributor to the observed phenotypes at this developmental stage.

A paragraph has been added in the corresponding Results section to address this issue.

(5) There are a few situations where the authors could help out the reader a little bit by providing more labels on the figures directly. For example: in Figure 2, there are expression levels, over-expression, and inhibition of miRNA but the X-axis is named with similar labels for the miRNAs in question for each of these distinct experiments. If the authors want to help the reader, they may consider labeling these panels with a descriptive title to reflect the experiment being done or use more descriptive terms in the X-axis panels. Again, this is minor. Similarly, in Figure 5, it might be helpful for the authors to help out the reader again with more labels on the panels, such as in Figures 5B, 5C, and 5D. Would they consider labeling these panels, HPC, PFC, SSC with the brain location as they did in Figure 4?

We thank the reviewer for these helpful suggestions to improve the clarity of our figures. We have implemented the proposed changes. In Figure 2C-E, we have added specific titles to the panels to clearly distinguish between the different experimental conditions such as miRNA overexpression and inhibition. Similarly, in Figure 5, we labeled panels 5B, 5C, and 5D with the brain regions analyzed (HPC, PFC, SSC) to match the labeling used in Figure 4. We believe these revisions enhance the readability and overall interpretability of the figures, making it easier for readers to follow the experiments and results.

(6) Figure 3: There is some overshoot of the data in EMC10 homozygous null, in panel 3E, and also, overshoot of the het in panel 3H. Would there be value in the authors commenting on the potential basis for this in the discussion? Some issues are minor, such as the lack of electrophysiological analysis of circuits in vivo or in ex vivo slices that may further support the proposed rescue.

The reviewer correctly highlights the observation in Figures 3E and 3H, where the number of branch points in the Q6/EMC10^HOM line exceeds wildtype levels and the calcium response in the Q6/EMC10^HET and Q6/EMC10^HOM lines surpasses that of the control. This overshoot is indeed intriguing and warrants discussion. EMC10 is part of the ER Membrane Complex (EMC), which plays a critical role in the proper folding and localization of various membrane proteins, including neurotransmitter receptors and ion channels such as voltage-gated calcium channels (Chitwood et al., 2018; Shurtleff et al., 2018; Chitwood and Hegde, 2019). In the context of the 22q11.2 deletion, EMC10 dysregulation may disrupt the proper localization of these proteins at the synapse, affecting both dendritic morphology and calcium signaling. The precise basis of this overshoot remains unclear. The overshoot may result from a dosage-sensitive inhibitory effect of Emc10, where both reduced and increased expression alter normal neuronal processes, with excessive responses potentially triggered upon gene restoration by the mutant system’s adaptation to dysfunction, leading to altered receptor sensitivity or signaling dynamics. This underscores the critical importance of precise Emc10 expression for proper neuronal development and function, in line with previous findings suggesting that EMC10 plays an auxiliary or modulatory role in EMC function. A short comment on the potential basis for this overshoot has been added in the corresponding Results section of the manuscript. Regardless of the underlying mechanisms, these findings emphasize the importance of precise titration of ASO constructs, rigorous gene dosage controls, and thorough analysis of context-specific responses to ensure both efficacy and safety in clinical applications.

We also agree with the reviewer that electrophysiological studies, particularly in the 22q11.2 deletion mouse model, would provide valuable insights into the impact of EMC10 modulation by ASOs on neuronal activity and circuit function at the in vivo and ex vivo levels. Incorporating such experiments into future studies will allow us to assess synaptic transmission and plasticity, contributing to a more comprehensive understanding of the therapeutic potential of ASO-mediated EMC10 modulation in 22q11.2DS.

(7) Did the authors take out the behavior studies further than 9 weeks? Would the authors consider commenting on what they speculate might be the duration of the treatment effect? For both mice and definitely humans.

We thank the reviewer for raising the important question regarding the duration of the ASO treatment effect, which is crucial for translating our findings into clinically relevant therapies. While behavioral studies beyond 9 weeks were not conducted in this study, our in vivo experiments and findings from prior publications (detailed below) enable an informed speculative assessment.

We utilized 2'-O-methoxyethyl (2'-MOE) modified ASOs, known for their enhanced binding affinity, nuclease resistance, and increased metabolic stability. In our in vivo post-injection screening of ASOs (Figure S13C), we predicted that Emc10 expression levels return to normal WT levels (~T100%) approximately 26 weeks post-treatment in Emc10^ASO (#1466182) treated mice. This prediction is supported by our Emc10 expression profiles across various brain regions, which demonstrate robust repression of Emc10 lasting up to 10 weeks post-administration (Figure 6D-F). While these findings suggest that the treatment effect in our model could extend significantly beyond 10 weeks following a single ASO injection, further empirical validation is required through extended follow-up studies. Encouragingly, long-term effects of 2'-MOE ASOs have been observed in other neurological disorders (Kordasiewicz et al., 2012; Scoles et al., 2017; Finkel et al., 2017; Darras et al., 2019). However, factors such as ASO distribution, target cell turnover, and disease-specific pathophysiology could influence the duration of the effect. To address these uncertainties, we have added a paragraph in the Discussion section emphasizing the need for additional studies, including extended follow-up periods and eventual clinical trials, to determine the specific duration of effect for our Emc10^ASO constructs in treating 22q11.2DS.

Reviewer #2 (Recommendations For The Authors):

(1) It is acknowledged that the iPSC-derived cells in Figure 1 are no longer progenitors, but differentiation markers for astrocytes and glia are also needed in Figure 1b to establish that equal rates of differentiation have occurred across genotypes.

We thank the reviewer for raising this important point about ensuring equal rates of differentiation across genotypes. As the reviewer notes, we employed a well-established protocol for directed differentiation of hiPSCs into cortical neurons using a combination of small molecule inhibitors, as previously described by Qi et al. (2017). This protocol has been extensively validated and is known to robustly generate cortical neurons while actively suppressing glial differentiation, as evidenced by the lack of upregulation of glial markers such as GFAP, AQP4, or OLIG2 in the original study. Given the established neuronal specificity of this protocol and our focus on neuronal phenotypes, we prioritized the confirmation of successful neuronal differentiation using the established neuronal markers TUJ1 and TBR1. Therefore, additional markers for astrocytes and glia are not included in this figure, as we did not expect significant glial differentiation under these conditions. A sentence has been added in the corresponding Results section to address this issue.

(2) For the RNA-seq experiments outlined in Figures 3J and K, a more comprehensive analysis is needed of the genes disrupted in the parental Q6 line relative to the het and homo lines. What percent are rescued, unaffected, vs uniquely disrupted?

Reduction in EMC10 levels is not expected to directly affect transcription or broadly reorganize the gene expression profile of the Q6/Q5 NGN2-iN lines. Our transcriptional profiling was not designed to assess the direct impact of EMC10 deficiency on gene expression but rather to measure the cellular pathways affected by reduced EMC10 in the patient Q6 line. We identified genes differentially expressed between the Q6 (patient) and Q5 (control) lines, whose expression differences were either abolished or significantly attenuated ("rescued") in the Q6/EMC10^HET or Q6/EMC10^HOM lines. In the Q6/EMC10^HET line, 237 DEGs (6%) were rescued, while in the Q6/EMC10^HOM line, 382 DEGs (11%) were rescued. Importantly, further analysis revealed 103 shared rescued DEGs in these lines, which was statistically significant (enrichment factor = 1.7; p < 0.0001, based on a hypergeometric test). We added a new figure panel (Figure 3L) to visualize the significant overlap of rescued DEGs from the Q6/EMC10^HET and Q6/EMC10^HOM lines. This overlap suggests these genes play a critical role in biological pathways impacted by EMC10 levels, particularly in nervous system development, as indicated by our functional annotation analysis. We also performed protein-protein interaction (PPI) network analysis to explore the functional relationships among these 103 shared DEGs (Figure S8). Future studies will further investigate these gene sets to gain deeper insights into the molecular mechanisms underlying 22q11.2DS and the role of EMC10.

(3) The authors claim that 50% EMC10 loss in adult mice is safe and should be toned down. EMC10 knockout mice have motor, anxiety, and social phenotypes. It would be unique amongst highly dosage-sensitive genes (MeCP2, CDKL5, TCF4, FMR1, etc.) for there to only be a neurodevelopmental component. In all those cases, and others, the effects of over and under-expression are reversible into adulthood. Establishing the range in adults is critical to establishing therapeutic utility. Absent a detailed examination of non-cognitive phenotypes, this claim cannot be made.

The reviewer raises an important point about the potential effects of EMC10 reduction in adult mice and the need to establish a safe therapeutic window by evaluating both cognitive and non-cognitive phenotypes. We agree that such a comprehensive evaluation is critical for assessing the safety and translational potential of Emc10-targeting therapies. While the International Mouse Genotyping Consortium reported motor and anxiety phenotypes in homozygous Emc10 knockout mice, these data are unpublished and based on a relatively small number of animals. Furthermore, in our previous work (Diamantopoulou et al., 2017), we demonstrated that complete Emc10 loss does not impair cognition or social behavior, as assessed by prepulse inhibition (PPI), working memory (WM), and social memory (SM) assays (see Figure 3A-D; Diamantopoulou et al., 2017). Additionally, heterozygous Emc10 mice, which exhibit a ~50% reduction in Emc10 expression similar to that achieved with our ASO treatment, showed no evidence of motor deficits or anxiety-like behavior. Specifically, Emc10^+/- mice displayed locomotor activity comparable to WT mice in the open field (OF) test (Figure S4A, Diamantopoulou et al., 2017). Moreover, genetic normalization of Emc10 expression in Df(16)A^+/- mice demonstrated no signs of anxiety-like behavior, as assessed by the OF test (Figure S4A) and elevated plus maze (EPM) (Figure S4B; Diamantopoulou et al., 2017). To further support these findings, we have added new data to the current manuscript (see Figure S10J) showing that TAM treatment-mediated restoration of Emc10 levels in the brain of adult Df(16)A^+/- mice did not affect the time that mutant mice spent in the center area of the OF (Fig. S10J), suggesting that Emc10 reduction does not influence anxiety-related behavior. These results suggest that a 50% reduction in EMC10 expression is unlikely to result in motor or anxiety-like phenotypes in adult mice. Finally, as noted in the manuscript, in addition to prior findings from animal models, a substantial number of relatively rare LoF variants or potentially damaging missense variants have been identified in the human EMC10 gene among likely healthy individuals in gnomAD, a database largely devoid of individuals known to be affected by severe neurodevelopmental disorders (NDDs).

Nevertheless, the Discussion has been revised to underscore the importance of establishing a more detailed safety profile, including non-cognitive phenotypes, to fully validate the therapeutic potential of Emc10-targeting approaches. It also highlights the need for future studies to expand on these evaluations, addressing this critical aspect and laying a stronger foundation for advancing these findings into clinical drug development

(4) Supplemental Figure 10: The protein validation of Emc10 knockout following tamoxifen injection needs to be validated in all brain regions, not just the PFC. This is particularly important as the rest of the paper focuses on HPC-mediated phenotypes.

First, we want to emphasize that we conducted both qRT-PCR and WB assays on the same animal cohort, specifically examining the left and right hippocampus following ASO injection (see Figure S11C and D). This approach is crucial, given the central role of hippocampus in the phenotypes investigated in our ASO-mediated Emc10 knockdown experiments.

The reviewer raises an important point regarding the validation of EMC10 reduction at the protein level across all relevant brain regions using the Emc10 conditional knockout strain. We agree that such validation would ideally confirm the efficacy of our tamoxifen-induced knockout model comprehensively. However, we hope the reviewer appreciates that obtaining sufficient high-quality protein for WB analysis from smaller brain regions like the hippocampus poses a significant technical challenge. This difficulty is further compounded by the need to reserve the same samples for qRT-PCR to ensure consistency between mRNA and protein measurements. Importantly, our data from ASO-mediated Emc10 knockdown experiments (Figures S11C-D) demonstrate a clear and consistent correlation between reductions in Emc10 mRNA and protein levels in both the left and right hippocampus. Furthermore, in our constitutive Emc10-knockout mouse model (Diamantopoulou et al., 2017; see Figure S1A-B), we observed a strong agreement between mRNA and protein levels, supporting the reliability of mRNA data as a proxy for EMC10 protein levels in our experiments. Importantly, in all instances where we performed parallel protein and RNA measurements in human cell lines, there was excellent concordance between the datasets. Thus, while we acknowledge the limitations of relying primarily on mRNA data, we are confident that the Emc10 mRNA expression data in Figure S10 accurately reflect protein-level changes across brain regions in our conditional knockout model. To address this concern more fully in the future, we are working to refine antibody detection and optimize our protein extraction protocols to enable more routine and precise protein-level validation across smaller brain regions. We appreciate the reviewer’s feedback and will continue to refine our methodologies to strengthen the robustness of our findings.

(5) Figure 3: 1 way ANOVA would be more appropriate to analyze the data in B-G than t-tests.

We appreciate the suggestion of the reviewer. As mentioned above, we carefully selected statistical tests appropriate for each analysis. For Figure 3B-G, we chose to use pairwise t-tests to address specific hypotheses regarding the disease phenotype and rescue effects. This approach is consistent with prior experimental studies in the field, including our own (e.g., Xu et al., 2013; Figure 7H-I). Importantly, most of our t-tests yielded highly significant results (p < 0.001 or p < 0.01), reinforcing the robustness of our findings.

(6) Figure 5-6: Protein data is needed to complement the mRNA knockdown data.

We agree with the reviewer on the importance of protein-level validation to complement the mRNA knockdown data. As mentioned in our response to Reviewer’s Comment (4), in all instances where we performed parallel protein and RNA measurements, either in mouse brain or human cell lines, we observed excellent concordance between the datasets. This supports the reliability of our mRNA data as a proxy for protein changes. Nevertheless, we acknowledge the value of including protein validation in future experiments and will consider incorporating it to further strengthen our findings.

(7) The use of additional phenotypic measures is applauded in Figure 6, however, to appropriately interpret the data more is needed. Shao et al 2021 (Figure S9) show data from the International Mouse Genotyping Consortium claiming EMC10 KO mice have gait, activity, and anxiety phenotypes. All of these parameters could impact the SM assay and the y-maze assay. Changes in SM interaction time could be linked to anxiety or motor impairments, but interpreted as cognitive deficits because these symptoms were not assessed. At a minimum, discussion is needed about this limitation, as well as the inclusion of distance explored in the SM and Y-maze assays.

We thank the reviewer for their insightful comment regarding the potential influence of locomotor, gait, or anxiety phenotypes on the observed deficits in the SM and Y-maze assays. The behavioral phenotypes reported for Emc10 knockout mice by the International Mouse Genotyping Consortium (https://www.mousephenotype.org/data/genes/MGI:1916933) were limited to homozygous female mice and based on a small sample size (4–6 females) compared to a larger WT control group. Moreover, these data are unpublished and thus challenging to evaluate fully. Importantly, no abnormal behaviors were reported for Emc10 heterozygous knockout mice in these datasets. Additionally, the claim by Shao et al. (2021) regarding cognitive impairments in Emc10 knockout mice based on our previous work (Diamantopoulou et al., 2017) is inaccurate.

Our analysis of both the constitutive Emc10 knockout model (Diamantopoulou et al., 2017) and the current conditional Emc10 heterozygous knockout model consistently demonstrates that Emc10 reduction does not affect locomotor activity or anxiety-like behavior. In our earlier characterization of constitutive heterozygous Emc10 knockout mice (Emc10^+/-), we observed no signs of anxiety-like behavior or motor impairments in OF assays (see Figure 2A-B and Figure S4A, Diamantopoulou et al., 2017). Similarly, results from Df(16)A^+/- mice with genetically normalized Emc10 expression [Df(16)A^+/-; Emc10^+/-] also showed no indications of anxiety-like behavior or locomotor changes in the OF and EPM assays (see Figure S4A-B, Diamantopoulou et al., 2017). Consistent with these findings, our current data from Df(16)A^+/- mice with conditional Emc10 reduction in the brain show no significant differences in locomotor activity and anxiety-related measures as assessed by OF assays (Figure S10J). Furthermore, total arm entries in Y-maze assays conducted in Df(16)A^+/- mice treated with Emc10 ASOs were comparable to controls (Figures S14C and G-H), providing additional support for the conclusion that locomotor activity remains unaffected in these models.

We further appreciate the reviewer’s suggestion that changes in social interaction time during the SM assay could be influenced by anxiety or motor impairments. However, we consider this scenario unlikely in our model. Interaction times during the first trial of the SM assay, which measures general social interest, are comparable between Df(16)A^+/- mice with reduced Emc10 expression (either genetically or through ASO treatment) and WT controls (see Figures 4E, 5E, and S10G). These findings indicate that our mouse models do not exhibit inherent difficulties in initiating social interaction, as might be expected if motor impairments or heightened anxiety were present. Reduced social interaction is commonly used as a behavioral marker for anxiety in rodent studies (reviewed by Bailey and Crawley, Anxiety-Related Behaviors in Mice, 2009). “Anxious” mice typically exhibit decreased social interaction, spending less time engaging with other mice compared to non-anxious counterparts. However, the specific deficit we observe in the second trial of the SM assay—when mice are reintroduced to a familiar juvenile—is indicative of impaired social recognition memory, as previously documented for Df(16)A^+/- mice (Piskorowski et al., 2016; Donegan et al., 2020). This deficit is distinct from the general social avoidance typically associated with heightened anxiety.

Based on our comprehensive assessment of locomotor activity, anxiety-related behaviors, and social interaction, we conclude that the observed rescue of social memory and spatial memory deficits in mice with reduced Emc10 expression is most likely due to improved cognitive function rather than alterations in motor or anxiety-related domains.

(8) For ASO optimization experiments, it is not sufficient to claim robust uptake. A quantitative measure is needed using the PO antibody showing what percentage of cells were positive for the ASO. Since the contention is that only Emc10 in excitatory neurons is important, it would be helpful if this also included a breakdown of ASO uptake in excitatory and inhibitory neurons and astrocytes.

We thank the reviewer for highlighting the importance of quantifying ASO uptake and assessing cell-type specificity. To address this, we have added new data to the panel, as shown in the high-magnification images in Figure S14A. These images provide evidence that a large majority of NeuN-positive neurons exhibit a strong ASO signal. Specifically, we observed widespread ASO uptake (green) that extensively colocalized with the neuronal marker NeuN (red) in both the hippocampus and prefrontal cortex. Quantitative analysis of this overlap indicates that over 97% of NeuN-positive neurons were ASO-positive, demonstrating efficient neuronal uptake. This robust neuronal uptake aligns with the significant normalization of Emc10 levels and the behavioral improvements observed in ASO-treated Df(16)A^+/- mice, further supporting the functional efficacy of our approach in modulating Emc10 expression within the relevant neuronal populations. Overall, the observed ASO uptake in neurons, as demonstrated by IHC, combined with RNA assays and the behavioral improvements in treated mice, strongly supports the efficacy of our approach in targeting Emc10 expression in the intended neuronal populations.

(9) An interpretation is needed in Figure S3 as to why ~50% of the pathways increased are also present on the decreased list. Ie. G1/transition, viral reproductive process, pos regulator of cell stress, etc. 4/10 GO terms are present in both increased and decreased groups in A and 7/10 in B.

We thank the reviewer for pointing out the overlap between pathways enriched in both the upregulated and downregulated miRNA groups in Figure S3. This overlap likely reflects the complex nature of miRNA regulation, where individual miRNAs can target multiple genes within a pathway, and single genes can be regulated by multiple miRNAs, sometimes with opposing effects (reviewed in Bartel, 2009; Bartel, 2018). For example, in the “G1/S transition” pathway, upregulated miRNAs such as miR-92a-3p, miR-92b-3p, and miR-34a-5p may promote the transition by targeting cell cycle regulators like FBXW7, CDKN1C, and CDK6 (Zhou et al., 2015; Zhao et al., 2021; Oda et al., 2024). Conversely, downregulated miRNAs such as miR-143-3p and miR-200b are known to suppress the transition by targeting genes such as HK2 and GATA-4 (Zhou et al., 2015; Yao et al., 2013). Our analysis identified overlapping predicted target genes for both upregulated and downregulated miRNAs, supporting the notion that many genes are subject to complex regulation by multiple miRNAs with potentially synergistic or antagonistic effects. Thus, the enrichment of certain GO terms in both groups likely reflects this intricate interplay of miRNA-mediated gene regulation. Future investigations focusing on specific miRNA-target interactions within these pathways will be critical to fully elucidate the underlying mechanisms and better understand the functional consequences of these opposing regulatory effects.

Minor Concerns:

(1) Define SM before using it.

We have defined the SM assay in the main text upon its first mention, where we describe the assay and its relevance to cognitive function (see page 11 of the revised manuscript).

(2) Statistics have been run in Figure S2, but not presented. The text only states that the differences between groups are significant. Please add in.

We have revised the legend of Figure S2 to include the specific statistical test used (students t-tests) and the corresponding p-values.

(3) The switch from ASO1 to ASO2 between Figures 5 and 6 needs more discussion. Why were new ASOs generated when ASO1 worked?

We thank the reviewer for their question regarding the transition from Emc10^ASO1 to Emc10^ASO2 between Figure 4 and Figures 5-6. Emc10^ASO1 served as our initial proof-of-concept ASO construct, successfully demonstrating the feasibility of inhibiting Emc10 mRNA expression and providing evidence for behavioral rescue in our mouse model. As outlined in the manuscript, Emc10^ASO2 targets a different region of the Emc10 transcript (intron 1, Figure 5A) compared to Emc10^ASO1 (intron 2, Figure 4A). This distinction provides an additional layer of validation for our targeting strategy and ensures specificity in modulating Emc10 expression. In addition, Emc10^ASO1 exhibited limited distribution in the brain, primarily targeting the hippocampus with weaker inhibition of Emc10 in other regions such as the cortex (Figure 4C, right panel). Emc10^ASO2 overcame this limitation and achieve broader brain distribution, as demonstrated by the qRT-PCR data in Figure 5C. Given that 22q11.2DS can affect multiple brain regions and cognitive domains beyond the hippocampus, achieving broader distribution of the ASO is critical for a more comprehensive assessment of therapeutic potential.

(4) Page 3: Define "LoF"

We have defined Loss-of-Function (LoF) in the main text where it is first mentioned in the Introduction, where we discuss the potential of using LoF mutations to devise therapeutic interventions (see page 3 of the revised manuscript).

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