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  1. Sep 2025
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      Reply to the reviewers

      Manuscript number: RC-2025-02879 Corresponding author(s): Matteo Allegretti; Alia dos Santos

      1. General Statements

      In this study, we investigated the effects of paclitaxel on both healthy and cancerous cells, focusing on alterations in nuclear architecture. Our novel findings show that:

      • Paclitaxel-induced microtubule reorganisation during interphase alters the perinuclear distribution of actin and vimentin. The formation of extensive microtubule bundles, in paclitaxel or following GFP-Tau overexpression, coincides with nuclear shape deformation, loss of regulation of nuclear envelope spacing, and alteration of the nuclear lamina.

      • Paclitaxel treatment reduces Lamin A/C protein levels via a SUN2-dependent mechanism. SUN2, which links the lamina to the cytoskeleton, undergoes ubiquitination and consequent degradation following paclitaxel exposure.

      • Lamin A/C expression, frequently dysregulated in cancer cells, is a key determinant of cellular sensitivity to, and recovery from, paclitaxel treatment.

      Collectively, our data support a model in which paclitaxel disrupts nuclear architecture through two mechanisms: (i) aberrant nuclear-cytoskeletal coupling during interphase, and (ii) multimicronucleation following defective mitotic exit. This represents an additional mode of action for paclitaxel beyond its well-established mechanism of mitotic arrest.

      We thank the reviewers for their time and constructive feedback. We have carefully considered all comments and have carried out a full revision. The updated manuscript now includes additional data showing:

      • Overexpression of microtubule-associated protein Tau causes similar nuclear aberration phenotypes to paclitaxel. This supports our hypothesis that increased microtubule bundling directly leads to nuclear disruption in paclitaxel during interphase.

      • Paclitaxel's effects on nuclear shape and Lamin A/C and SUN2 expression levels occur independently of cell division.

      • Reduced levels of Lamin A/C and SUN2 upon paclitaxel treatment occur at the protein level via ubiquitination of SUN2.

      • The effects of paclitaxel on the nucleus are conserved in breast cancer cells.

      Full Revision

      We have also edited our text and added further detail to clarify points raised by the reviewers. We believe that our revised manuscript is overall more complete, solid and compelling thanks to the reviewers' comments.

      1. Point-by-point description of the revisions

      Reviewer #1 Evidence, reproducibility and clarity

      This description of the down-regulation of the expression of lamin A/C upon treatment with paclitaxel and its sensitivity to SUN2 is quite interesting but still somehow preliminary. It is unclear whether this effect involves the regulation of gene expression, or of the stability of the proteins. How SUN2 mediates this effect is still unknown.

      We thank the reviewer for this valuable comment. To elucidate the mechanism behind the decrease in Lamin A/C and SUN2 levels, we have now performed several additional experiments. First, we performed RT-qPCR to quantify mRNA levels of these genes, relative to the housekeeping gene GAPDH (Supplementary Figure 3B and O). The levels of SUN2 and LMNA mRNA remained the same between control and paclitaxel-treated cells, indicating that this effect instead occurs at the protein level. We have also tested post-translational modifications as a potential regulatory mechanism for Lamin A/C and SUN2. In addition to the phosphorylation of Ser404 which we had already tested (Supplementary Figure 3C), we have now included additional Phos-tag gel and Western blotting data showing that the overall phosphorylation status of Lamin A/C is not affected by paclitaxel (Supplementary Figure 3E and F). We also pulled-down Lamin A/C from cell lysates and then Western blotted for polyubiquitin and acetyl-lysine, which showed that the ubiquitination and acetylation states of Lamin A/C are also not affected by paclitaxel (Supplementary Figure 3G-I). However, Western blots for polyubiquitin of SUN2 pulled down from cell lysates showed that paclitaxel treatment results in significant SUN2 ubiquitination (Figure 3M and N). Therefore, we propose that the downregulation of SUN2 following paclitaxel treatment occurs by ubiquitin-mediated proteolysis.

      The roles of free tubulins and polymerized microtubules, and thus the potential role of paclitaxel, need to be uncovered.

      We addressed this important point by using an alternative method to stabilise/bundle microtubules in interphase, namely by overexpressing GFP-Tau, as suggested by reviewer 2. Following GFP- Tau overexpression, large microtubule bundles were observed throughout the cytoplasm (Figure 4A), and this resulted in a significant decrease in nuclear solidity (Figure 4B). Furthermore, in cells where microtubule bundles extensively contacted the nucleus, the nuclear lamina became unevenly distributed and appeared patchy (Figure 4C). This supports our hypothesis that the aberrations to nuclear shape and Lamin A/C localisation in paclitaxel-treated cells are due to the presence of microtubules bundles surrounding the nucleus.

      The doses of paclitaxel at which occur the effects described in the paper are not fully consistent with all the conclusions. Most experiments have been done at 5 nM. However, at this dose the effect of lamin A/C over or down expression on the growth (differences in the slopes of the curves in Figure 4A) are not fully convincing and not fully consistent with the clear effect on viability as well (in addition, duration of treatments before assessing vialbility are not specified). At 1 nM, cell growth is reduced and the rescuing effect of lamin over-expression is much more clear (Fig 4A), and the nucleus deformation clear (Fig 2A) but this dose has no effect on lamin A/C expression (Fig 3C), which questions how lamins impact nucleus shape and cell survival. Cytoskeleton reorganisation in these conditions is not described although it could clarify the respective role of force production (suggested in figure 1) and nuclei resistance (shown in figure 2) in paclitaxel sensitivity.

      We thank the reviewer for raising this important point. We have addressed this by conducting additional repeats for the cell confluency measurements to increase the statistical power of our experiments (Figure 5A). Our data now show that GFP-lamin A/C had a statistically significant effect on rescuing cell growth at both 1 nM and 5 nM paclitaxel, while Lamin A/C knockdown exacerbated the inhibition of cell growth at 5 nM paclitaxel but not 1 nM paclitaxel (Figure 5A). In addition, we note that the duration of paclitaxel treatment before assessing viability was specified in the figure legend: "Bar graph comparing cell viability between wild-type (red), GFP-Lamin A/C overexpression (green), and Lamin A/C knockdown (blue) cells following 20 h incubation in 0, 1, 5, or 10 nM paclitaxel." We also repeated cell viability analysis after 48 h incubation in paclitaxel instead of 20 h to allow for a longer time for differences to take effect (Figure 5B).

      We also added figures showing the cytoskeletal reorganisation at both 1 and 10 nM in addition to 0 and 5 nM (Supplementary Figure 1A) showing that microtubule bundling and condensation of actin into puncta correlated with increased paclitaxel concentration. Vimentin colocalised well with microtubules at all concentrations.

      We have also included in our results section further clarification for the use of 5nM paclitaxel in this study. The new section reads as follows: "Experiments were performed at 5 nM paclitaxel (with additional experiments to determine dose relationships at 1 and 10 nM) because this aligns with previous studies7,14,24. Furthermore, previous analysis of patient plasma reveals that typical concentrations are within the low nanomolar range8, and concentrations of 5-10 nM are required in cell culture to reach the same intracellular concentrations observed in vivo in patient tumours9. This aligns with in vitro cytotoxic studies of paclitaxel in eight human tumour cell lines which show that paclitaxel's IC50 ranges between 2.5 and 7.5 nM41."

      Finally, although the absence of role of mitotic arrest is clear from the data, the defective reorganisation of the nucleus after mitosis still suggest that the effect of paclitaxel is not independent of mitosis.

      We thank the reviewer for pointing out the need for clarification in the wording of our manuscript. We have reworded the title and relevant sections of our abstract, introduction, and discussion to make it clearer that the effects of paclitaxel on the nucleus are due to a combination of aberrant nuclear cytoskeletal coupling during interphase and multimicronucleation following mitotic slippage. We have also added additional data in support of the effect of paclitaxel on nuclear architecture during interphase. For this, we used serum-starved cells (which divide only very slowly such that the majority of cells do not pass through mitosis during the 16 h incubation in paclitaxel [Supplementary Figure 2D]). Our new data confirmed that paclitaxel's effects on nuclear solidity, and Lamin A/C and SUN2 proteins levels can occur independently of cell division (Figure 2C; Figure 3H-J). Finally, when we overexpressed GFP-Tau (as discussed above) we observed similar aberrations to nuclear solidity and Lamin A/C localisation. This indicates that these effects occur due to microtubule bundling in interphase, especially as in our study GFP-Tau did not lead to multimicronucleation or appear to affect mitosis (Figure 4).

      Below are the main changes to the text regarding the interphase effect of paclitaxel:

      • Title: "Paclitaxel compromises nuclear integrity in interphase through SUN2-mediated cytoskeletal coupling"

      • Abstract: "Overall, our data supports nuclear architecture disruption, caused by both aberrant nuclear-cytoskeletal coupling during interphase and exit from defective mitosis, as an additional mechanism for paclitaxel beyond mitotic arrest."

      • Introduction: "Here we propose that cancer cells have increased vulnerability to paclitaxel both during interphase and following aberrant mitosis due to pre-existing defects in their NE and nuclear lamina."

      • Discussion: "Overall, our work builds on previous studies investigating loss of nuclear integrity as an anti-cancer mechanism of paclitaxel separate from mitotic arrest14,20,21. We propose that cancer cells show increased sensitivity to nuclear deformation induced by aberrant nuclear-cytoskeletal coupling and multimicronucleation following mitotic slippage. Therefore, we conclude that paclitaxel functions in interphase as well as mitosis, elucidating how slowly growing tumours are targeted."

      minor: a more thorough introduction of known data about dose response of cells in culture and in vivo would help understanding the range of concentrations used in this study.

      As mentioned above, we have now included additional information in our Results section to clarify our paclitaxel dose range: "Experiments were performed at 5 nM paclitaxel (with additional experiments to determine dose relationships at 1 and 10 nM) because this aligns with previous studies7,14,24. Furthermore, previous analysis of patient plasma reveals that typical concentrations are within the low nanomolar range8, and concentrations of 5-10 nM are required in cell culture to reach the same intracellular concentrations observed in vivo in patient tumours9. This aligns with in vitro cytotoxic studies of paclitaxel in eight human tumour cell lines which show that paclitaxel's IC50 ranges between 2.5 and 7.5 nM41."

      Significance

      In this manuscript, Hale and colleagues describe the effect of paclitaxel on nucleus deformation and cell survival. They showed that 5nM of paclitaxel induces nucleus fragmentation, cytoskeleton reorganisation, reduced expression of LaminA/C and SUN2, and reduced cell growth and viability. They also showed that these effects could be at least partly compensated by the over-expression of lamin A/C. As fairly acknowledged by the authors, the induction of nuclear deformation in paclitaxel-treated cells, and the increased sensitivity to paclitaxel of cells expressing low level of lamin A/C are not novel (reference #14). Here the authors provided more details on the cytoskeleton changes and nuclear membrane deformation upon paclitaxel treatment. The effect of lamin A/C over and down expression on cell growth and survival are not fully convincing, as further discussed below. The most novel part is the observation that paclitaxel can induce the down-regulation of the expression of lamin A/C and that this effect is mediated by SUN2.

      We appreciate the reviewer's summary and thank them for their time. We believe our comprehensive revisions have addressed all comments, strengthening the manuscript and making it more robust and compelling.

      Reviewer #2 Evidence, reproducibility and clarity This study investigates the effects of the chemotherapeutic drug paclitaxel on nuclear-cytoskeletal coupling during interphase, claiming a novel mechanism for its anti-cancer activity. The study uses hTERT-immortalized human fibroblasts. After paclitaxel exposure, a suite of state- of-the-art imaging modalities visualizes changes in the cytoskeleton and nuclear architecture. These include STORM imaging and a large number of FIB-SEM tomograms.

      We thank the reviewer for the summary and for highlighting our efforts in using the latest imaging technical advances.

      Major comments:

      The authors make a major claim that in addition to the somewhat well-described mechanism of paclitaxel on mitosis, they have discovered 'an alternative, poorly characterised mechanism in interphase'.

      However, none of the data proves that the effects shown are independent of mitosis. To the contrary, measurements are presented 48 hours after paclitaxel treatment starts, after which it can be assumed that 100% of cells have completed at least one mitotic event. The appearance of micronuclei evidences this, as discussed by the authors shortly. It looks like most of the results shown are based on botched mitosis or, more specifically, errors on nuclear assembly upon exit from mitosis rather than a specific effect of paclitaxel on interphase. The readouts the authors show just happen to be measurements while the cells are in interphase.

      Alternative hypotheses are missing throughout the manuscript, and so are critical controls and interpretations.

      We thank the reviewer for highlighting the lack of clarity in our wording. We have revised the title, abstract and relevant sections of the introduction and discussion to clarify our message that the effects of paclitaxel on the nucleus arise from a combination of aberrant nuclear-cytoskeletal coupling during interphase and multimicronucleation following exit from defective mitosis. We have also included additional data where we used slow-dividing, serum-starved cells (under these conditions, the majority of cells do not undergo mitosis during the 16 h incubation in paclitaxel [Supplementary Figure 2D]). Our new data show that even in these cells there is a clear effect of paclitaxel on nuclear solidity, and Lamin A/C and SUN2 protein levels, further supporting our hypothesis that these phenotypes can occur independently of cell division (Figure 2C; Figure 3H-J). Furthermore, we performed additional experiments where we used overexpression of GFP-Tau as an alternative method of stabilising microtubules in interphase and observed similar aberrations to nuclear solidity and Lamin A/C localisation. As GFP-Tau overexpression did not lead to micronucleation or appear to affect mitosis, these data support the hypothesis that nuclear aberrations occur due to microtubule bundling in interphase (Figure 4). We discuss these experiments in more detail below. Finally, we have reworded the introduction to better introduce alternative hypotheses and mechanisms for paclitaxel's activity.

      The authors claim that 'Previously, the anti-cancer activity of paclitaxel was thought to rely mostly on the activation of the mitotic checkpoint through disruption of microtubule dynamics, ultimately resulting in apoptosis.' The authors may have overlooked much of the existing literature on the topic, including many recent manuscripts from Xiang-Xi Xu's and another lab.

      We would like to note that the paper from Xiang-Xi Xu's lab (Smith et al, 2021) was cited in our original manuscript (reference 14 in both the original and revised manuscripts). We have now also included additional review articles from the Xiang-Xi Xu lab (PMID:36368286 20 and PMID: 35048083 21). Furthermore, we have clarified the wording in both the introduction and discussion to better reflect the current understanding of paclitaxel's mechanism and alternative hypotheses.

      The data, e.g. in Figure 1, does not hold up to the first alternative hypothesis, e.g. that paclitaxel stabilizes microtubules and that excessive mechanical bundling of microtubules induces major changes to cell shape and mechanical stress on the nucleus. Even the simplest controls for this effect (the application of an alternative MT stabilizing drug or the overexpression of an MT stabilizer, e.g., tau).

      We thank the reviewer for suggesting this control experiment using the microtubule stabiliser Tau. We have now included these experiments in the revised version of the manuscript (Figure 4). The overexpression of GFP-Tau supports our hypothesis that cytoskeletal reorganisation in paclitaxel exerts mechanical stress on the nucleus during interphase, resulting in nuclear deformation and aberrations to the nuclear lamina. In particular, GFP-Tau overexpression resulted in large microtubule bundles throughout the cytoplasm (Figure 4A). Notably, in cells where these bundles extensively contacted the nucleus, we observed a significant decrease in nuclear solidity (Figure 4B) accompanied by changes in nuclear lamina organisation, including a patchy lamina phenotype, similar to that induced by paclitaxel (Figure 4C).

      The focus on nuclear lamina seems somewhat arbitrary and adjacent to previously published work by other groups. What would happen if the authors stained for focal adhesion markers? There would probably be a major change in number and distribution. Would the authors conclude that paclitaxel exerts a specific effect on focal adhesions? Or would the conclusion be that microtubule stabilization and the following mechanical disruption induce pleiotropic effects in cells? Which effects are significant for paclitaxel function on cancer cells?

      We thank the reviewer for raising important points regarding the specificity of paclitaxel's effects. We agree that microtubule stabilisation can induce myriad cellular changes, including alterations to focal adhesions and other cytoskeletal components. Our focus on Lamin A/C and nuclear morphology is grounded both in the established clinical relevance of nuclear mechanics in cancer and builds on mechanistic work from other groups.

      Lamin A/C expression is commonly altered in cancer, and nuclear morphology is frequently used in cancer diagnosis35. Lamin A/C also plays a crucial role in regulating nuclear mechanics32 and, importantly, determines cell sensitivity to paclitaxel14. However, the mechanism by which Lamin A/C determines sensitivity of cancer cells to paclitaxel is unclear.

      Our data are consistent with Lamin A/C being a determinant of paclitaxel survival sensitivity. We also provide evidence that paclitaxel itself reduces Lamin A/C protein levels and disrupts its organisation at the nuclear envelope. We directly link these effects to microtubule bundling around the nucleus and degradation of force-sensing LINC component SUN2, highlighting the importance of nuclear architecture and mechanics to overall cellular function. Furthermore, we show that recovery from paclitaxel treatment depends on Lamin A/C expression levels. This has clinical relevance, as unlike cancer cells, healthy tissue with non-aberrant lamina would be able to selectively recover from paclitaxel treatment.

      Minor comments:

      While I understand the difficulty of the experiments and the effort the authors have put into producing FIB-SEM tomograms, I am not sure they are helping their study or adding anything beyond the light microscopy images. Some of the images may even be in the way, such as supplementary Figure 6, which lacks in quality, controls, and interpretation. Do I see a lot of mitochondria in that slice?

      We agree with the reviewer that Supplementary Figure 6 does not add significant value to the manuscript and thank the reviewer for pointing this out. We have removed it from the manuscript accordingly.

      I may have overlooked it, but has the number of cells from which lamellae have been produced been stated?

      We thank the reviewer for pointing out the missing information. For our cryo-ET experiments, we collected data from 9 lamellae from paclitaxel-treated cells and 6 lamellae from control cells, with each lamella derived from a single cell. This information has now been added to the figure legend (Figure 2F).

      Significance

      The significance of studying the effect of paclitaxel, the most successful chemotherapy drug, should be broad and of interest to basic researchers and clinicians.

      As outlined above, I believe that major concerns about the design and interpretation of the study hamper its significance and advancements.

      We appreciate the reviewer's concerns and have performed major revisions to strengthen the significance of our study. Specifically, we conducted two key sets of experiments to validate our original conclusions: serum starvation to control for the effects of cell division, and overexpression of the microtubule stabiliser Tau to demonstrate that paclitaxel can affect the nucleus via its microtubule bundling activity in interphase.

      By elucidating the mechanistic link between microtubule stabilisation and nuclear-cytoskeletal coupling, our findings contribute to our understanding of paclitaxel's multifaceted actions in cancer cells.

      My areas of expertise could be broadly defined as Cell Biology, Cytoskeleton, Microtubules, and Structural Biology.

      Reviewer #3 Evidence, reproducibility and clarity The manuscript presents interesting new ideas for the mechanism of an old drug, taxol, which has been studied for the last 40 years.

      We thank the reviewer for the positive feedback.

      Although similar ideas are published, which may be suitable to be cited? • Paclitaxel resistance related to nuclear envelope structural sturdiness. Smith ER, Wang JQ, Yang DH, Xu XX. Drug Resist Updat. 2022 Dec;65:100881. doi: 10.1016/j.drup.2022.100881. Epub 2022 Oct 15. PMID: 36368286 Review. • Breaking malignant nuclei as a non-mitotic mechanism of taxol/paclitaxel. Smith ER, Xu XX. J Cancer Biol. 2021;2(4):86-93. doi: 10.46439/cancerbiology.2.031. PMID: 35048083 Free PMC article.

      We thank the reviewer for bringing to our attention these important review articles. In our initial manuscript, we only cited the original paper (14, also reference 14 in the original manuscript). We have now included citations to the suggested publications (20,21).

      We would also like to emphasise how our manuscript distinguishes itself from the work of Smith et al.14,20,21:

      • Cell-type focus: In their study 14, Smith et al. examined the effect of paclitaxel on malignant ovarian cancer cells and proposed that paclitaxel's effects on the nucleus are limited to cancer cells. However, our data extends these findings by demonstrating paclitaxel's effects in both cancerous and non-cancerous backgrounds.

      • Cytoskeletal reorganisation: Smith et al. show reorganisation of microtubules in paclitaxel-treated cells14. Our data show re-organisation of other cytoskeletal components, including F-actin and vimentin.

      • Multimicronucleation: Smith et al. propose that paclitaxel-induced multimicronucleation occurs independently of cell division14. Although we observe progressive nuclear abnormalities during interphase over the course of paclitaxel treatment, our data do not support this conclusion; we find that multimicronucleation occurs only following mitosis.

      • Direct link between microtubule bundling and nuclear aberrations: We show that nuclear aberrations caused by paclitaxel during interphase (distinct from multimicronucleation) are directly linked to microtubule bundling around the nucleus, suggesting they result from mechanical disruption and altered force propagation.

      • Lamin A/C regulation: Consistent with Smith et al.14, we show that Lamin A/C depletion leads to increased sensitivity to paclitaxel treatment. However, we further demonstrate that paclitaxel itself leads to reduced levels of Lamin A/C and that this effect occurs independently of mitosis and is mediated via force-sensing LINC component SUN2. Upon SUN2 knockdown, Lamin A/C levels are no longer affected by paclitaxel treatment.

      • Recovery: Finally, our work reveals that cells expressing low levels of Lamin A/C recover less efficiently after paclitaxel removal. This might help explain how cancer cells could be more susceptible to paclitaxel.

      Only one cell line was used in all the experiments? "Human telomerase reverse transcriptase (hTERT) immortalised human fibroblasts" ? The cells used are not very relevant to cancer cells (carcinomas) that are treated with paclitaxel. It is not clear if the observations and conclusions will be able to be generalized to cancer cells.

      We thank the reviewer for this comment. Our initial study aimed to understand the effects of paclitaxel on nuclear architecture in non-aberrant backgrounds. To show that the observed effects of paclitaxel are also applicable to cancer cells, we have now repeated our main experiments using MDA-MB-231 human breast cancer cells (Supplementary Figure 1B; Supplementary Figure 3P-T). Similar to our findings in human fibroblasts, paclitaxel treatment of MDA-MB-231 led to cytoskeletal reorganisation (Supplementary Figure 1B), a decrease in nuclear solidity (Supplementary Figure 3P), aberrant (patchy) localisation of Lamin A/C (Supplementary Figure 3Q), and a reduction in Lamin A/C and SUN2 levels (Supplementary Figure 3R-T).

      "Fig. 1. (B) STORM imaging of α-tubulin immunofluorescence in cells fixed after 16 h incubation in control media or 5 nM paclitaxel. Lower panels show α-tubulin clusters generated with HDBSCAN analysis. Scale bars = 10 μm." It needs explanation of what is meaning of the different color lines in the lower panels, just different filaments?

      We have added further detail to the figure legend for clarification: "Lower panels show α-tubulin clusters generated with HDBSCAN analysis. Different colours distinguish individual α-tubulin clusters, representing individual microtubule filaments or filament bundles."

      Generally, the figures need additional description to be clear.

      We have added further clarification and detail to our figure legends.

      "Figure 3 - Paclitaxel results in aberrations to the nuclear lamina." The sentence seems not to be well constructed. "Paclitaxel treatment causes ..."?

      We changed this sentence to: "Figure 3 - Paclitaxel treatment results in aberrant organisation of the nuclear lamina and decreased Lamin A/C levels via SUN2."

      Lamin A and C levels are different in different images (Fig. 3B, H): some Lamin A is higher, and sometime Lamin C is higher? This may possibly due to culture condition or subtle difference in sample handling?.

      We thank the reviewer for pointing this out and we agree that the ratio of Lamin A to Lamin C can vary with culture conditions. To confirm that paclitaxel treatment reduces total Lamin A/C levels regardless of this ratio, we repeated the Western blot analysis in three additional biological replicates using cells in which Lamin C levels exceeded Lamin A levels. These experiments confirmed a comparable decrease in total Lamin A/C levels. Figure 3B and 3C have been updated accordingly.

      Also, the effect on Lamin A/C and SUN2 levels are not significant of robust.

      Decreased Lamin A/C and SUN2 levels following paclitaxel treatment were consistently seen across three or more biological repeats (Figure 3B-C), and this could be replicated in a different cell type (MDA-MB-231) (Supplementary Figure 3R-T). Furthermore, Western blotting results are consistent with the patchy Lamin A/C distribution observed using confocal and STORM following paclitaxel treatment (Figure 3A; Supplementary Figure 3A), where Lamin A/C appears to be absent from discrete areas of the lamina.

      Any mechanisms are speculated for the reason for the reduction?

      We have now included additional data which aims to shed light on the mechanism behind the decrease in Lamin A/C and SUN2 levels following paclitaxel treatment. We found that SUN2 is selectively degraded during paclitaxel treatment. Immunoprecipitation of SUN2 followed by Western blotting against Polyubiquitin C showed increased SUN2 ubiquitination in paclitaxel (Figure 3M and N). Furthermore, in our original manuscript, we showed that Lamina A/C levels remained unaltered during paclitaxel treatment in cells where SUN2 had been knocked down. We propose that changes in microtubule organisation affect force propagation to Lamin A/C specifically via SUN2 and that this leads to Lamina A/C removal and depletion. Future work will be needed to fully understand this mechanism.

      In addition to the findings described above, we report no significant changes in mRNA levels for LMNA or SUN2 in paclitaxel (Supplementary Figure 3B and O). Phos-tag gels followed by Western blotting analysis for Lamin A/C also did not detect changes to the overall phosphorylation status of Lamin A/C due to paclitaxel treatment. This is in agreement with our initial data showing no changes to Lamin A/C Ser 404 phosphorylation levels (Supplementary Figure 3E and F). Finally, Lamin A/C immunoprecipitation experiments followed by Western blotting for Polyubiquitin C and acetyl-lysine showed no significant changes in the ubiquitination and acetylation state of Lamin A/C in paclitaxel-treated cells (Supplementary Figure 3G-I).

      Also, the about 50% reduction in protein level is difficult to be convincing as an explanation of nuclear disruption.

      The nuclear lamina and LINC complex proteins play a critical role in regulating nuclear integrity, stiffness and mechanical responsiveness to external forces28,31-33,54,75, as well as in maintaining the nuclear intermembrane distance69,74. In particular, SUN-domain proteins physically bridge the nuclear lamina to the cytoskeleton through interactions with Nesprins, thereby preserving the perinuclear space distance30,69,74. Mutations in Lamins have been shown to disrupt chromatin organization, alter gene expression, and compromise nuclear structural integrity, and experiments with LMNA knockout cells reveal that nuclear mechanical fragility is closely coupled to nuclear deformation47. Furthermore, nuclear-cytoskeletal coupling is essential during processes such as cell migration, where cells undergo stretching and compression of the nucleus; weakening or loss of the lamina in such cases compromises cell movement47,73. In our work, we show that alterations to nuclear Lamin A/C and SUN2 by paclitaxel treatment coincide with nuclear deformations (Figure 2A-D, F, G; Figure 3A-D, F, G; Supplementary Figure 3A, P-T) and that these deformations are reversible following paclitaxel removal (Supplementary Figure 4B-D). Our experiments also demonstrate that Lamin A/C expression levels significantly influence cell growth, cell viability, and cell recovery in paclitaxel (Figure 5). Therefore, drawing on current literature and our results, we propose that, during interphase, paclitaxel induces severe nuclear aberrations through the combined effects of: i) increased cytoskeletal forces on the NE caused by microtubule bundling; ii) loss of ~50% Lamin A/C and SUN2; iii) reorganisation of nucleo-cytoskeletal components.

      Significance

      The manuscript presents interesting new ideas for the mechanism of an old drug, taxol, which has been studied for the last 40 years.

      The data may be improved to provide stronger support.

      Additional cell lines (of cancer or epithelial origin) may be repeated to confirm the generality of the observation and conclusions.?

      We thank the reviewer for the feedback and valuable suggestions. In response, we have included experiments using human breast cancer cell line MDA-MB-231 to further corroborate our findings and interpretations. We believe these additions have improved the clarity, robustness and impact of our manuscript, and we are grateful for the reviewer's contributions to its improvement.

    1. Week 14: PEER-TO-PEER WRITING (Thursday, December 4, 2025)

      peer-to-peer writing

      If only we could have that in an atuonomous setting that would be the right counterballance

      Gives a good target date

    1. schwannomas vestibulares,

      Un schwannoma vestibular, o neurinoma del acústico, es un tumor no canceroso que se origina en las células de Schwann que recubren el nervio del equilibrio (nervio vestibular) y del oído interno al cerebro. Suelen ser de crecimiento lento y afectan generalmente a un solo oído, causando síntomas como pérdida de audición, zumbidos (tinnitus) y pérdida de equilibrio.

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      I wonder what the differences of play in different cultures is/ Historically each culture has had some form of play or the other. So I wonder what similarities and differences the different forms of play in different cultures had. Every culture is somewhat different, but could play be something common among them all?

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      Usually when thinking of play I notice that I more over look at it from a psychological or scientific perspective rather then a scientific one. I find it interesting that this study looks at it from a more cultural lens, as I never really saw play as related to culture. I am looking forward to learning more about the exact relationship between play and culture.

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    5. ºှ ှ !ှ ှ ှ ှ !ှ &E1ှ &೸Dzှ !ှ &ှ & ှ&EJ 1ှ ME ှ ှ ှ &EJှ J!J+ှ Jှ & ှ Eှ 1ှ ှ Eှ &J&Jှ Eှ ှ ှ  J&Jှ ű¤

      Considering how ancient people also played things like boardgame, the argument sounds convincing.

    6. ˆqìlˆŠœmì Š½ì ·ÌŠÈqì œŠÈq ̧c¥¥Üì gq½Šmqì ˆŠ¦½qœzì ӊȈì mqœŠ„ˆÈì È ̧c©½¶² ̧Éqmìjqܲ©mì ˆŠ¦½q¥yì È²ì ½Ìl‰ì c©ì qÚÉq©Éì ȈcÉì ˆqì _œ¦²½Éì jv¥ŠqÏq½ì ˆqìclÈÌ_œœÜ슽ì½Ìlˆìc©mì ½Ìlˆì _ìɈŠ©„ӊȈ²ÌÈì ˆ²ÓqÏq ̧ìӈ²œœÜ윲½Š©‚ìl²©½lŠ²Ì½©q½½ì ²yì ² ̧mŠ©c ̧Üì ̧qcœŠÉÜ

      Is it fair to say that play is a means of escape from reality?

    7. ý ှ ¹ှ ¹ှ ‹ှ ှ $ ှ $ှ ¹ှ‹ှ  ‹ ှ કှ ‹‹& ှ ¹ ှ ¹‹ ¹‹ ှ Ǿ¹ ှ  ှ ‹ှ ƚp !ȥ ¹ှZှ ‹ှ ¹ှ ¹ ှ $ှ$ှ ¹ှ $¹¡íှ

      I didn't understand what this meant so I looked it up; apparently "warp and woof" refers to the fundamental components of weaving, and as an idiom it refers to the "essential foundation or base" of a structure. So, basically what he's saying is that repetition and alternation are essential components on which play is built.

    8. ˷Fှ=ှ =I Iှ  ှ =ှ = Iှ  Ưှ ှ =# ှ Fှ c ှcှc#ှ Fှ $=Icှ  ̈Ɵ= ှ =$ှ Àှ

      Interesting. Sometimes I am tired and don't feel like going to basketball practice or training. But I do go, because I feel like I have to, and in general I do like the sport, just not all the time. According to this argument, on the when I don't feel like going, I am not actually playing?

    9. u ှ Fှှ c#ှ =$ှ ှ# ̈ ှ c= ှ  ှ ̈== ှ I ှ  ှ Ŏ nှ

      This thought makes me think of the way watching a dog or another animal run around highlights its muscles and athleticism. I've never thought about it this way, but physical play does, in a way, display the body in it's most active and beautiful form.

    1. Art. 19.
      • Informativo nº 860
      • 2 de setembro de 2025.
      • PRIMEIRA TURMA
      • Processo: REsp 2.023.326-SC, Rel. Ministro Paulo Sérgio Domingues, Primeira Turma, por unanimidade, julgado em 5/8/2025, DJEN 19/8/2025.

      Ramo do Direito DIREITO TRIBUTÁRIO

      TemaPaz, Justiça e Instituições Eficazes <br /> Execução fiscal. Reconhecimento da procedência do pedido. Honorários advocatícios sucumbenciais. Art. 19, caput e § 1º, da Lei 10.522/2002. Interpretação sistemática. Dispensa de condenação.

      Destaque - Sempre que houver desistência nos moldes da Lei n. 10.522/2002, a Fazenda Nacional estará <u>exonerada</u> do pagamento de honorários advocatícios sucumbenciais.

      Informações do Inteiro Teor - A controvérsia refere-se à condenação da Fazenda Nacional ao pagamento de honorários advocatícios, mesmo após o reconhecimento da procedência do pedido pela União.

      • A Lei n. 10.522/2002 estabelece, em seu art. 19, a dispensa da Procuradoria-Geral da Fazenda Nacional de "contestar, de oferecer contrarrazões e de interpor recursos", bem como a autoriza a "desistir de recursos já interpostos", nas matérias ali elencadas. Trata-se de situações em que o Estado reconhece a insubsistência da dívida objeto de cobrança.

      • O inciso I do § 1º do art. 19 da Lei 10.522/2002 prevê, nas matérias tratadas nesse artigo, a possibilidade de a Fazenda Nacional não ser condenada ao pagamento de honorários advocatícios quando reconhecer a procedência do pedido, ao ser citada para apresentar resposta, nos embargos à execução fiscal e nas exceções de pré executividade.

      • A leitura desses dispositivos evidencia que a norma tem caráter autorizativo, sendo dirigida à atuação profissional dos procuradores da Fazenda Nacional. Visa orientar a conduta da PGFN no reconhecimento da procedência do pedido ou na desistência da execução fiscal, quando a própria Administração Tributária reconhece a existência de fundamentos jurídicos relevantes para tanto - como nos casos em que a controvérsia versa acerca de tema objeto de parecer, vigente e aprovado, pela PGFN, ou esteja amparada em súmula ou parecer do Advogado-Geral da União favorável ao pleito do particular (incisos II e IV do art. 19 da Lei n. 10.522/2002).

      • O termo "nas matérias de que trata este artigo", presente no § 1º, deve ser compreendido à luz do conjunto normativo da lei, de modo que, sempre que houver desistência nos moldes da Lei n. 10.522/2002, haverá a exoneração da Fazenda Nacional ao pagamento de honorários advocatícios.

      • A eventual imposição de ônus à Fazenda Nacional, quando ela atua em hipóteses legitimamente autorizadas pela própria lei, poderia gerar efeito contrário ao pretendido pelo legislador, estimulando a litigância porque, a toda evidência, desistir não faria sentido.

      • Por fim, em se tratando de norma interna autorizativa que regula a atuação da PGFN, não cabe ao Poder Judiciário exercer sindicabilidade sobre os fundamentos que levaram à desistência, mas apenas reconhecer o não cabimento da condenação em honorários sempre que a desistência for realizada nos termos da Lei n. 10.522/2002.

    1. III
      • Informativo nº 860
      • 2 de setembro de 2025.
      • PRIMEIRA TURMA
      • Processo: AgInt no REsp 2.109.509-RS, Rel. Ministro Sérgio Kukina, Primeira Turma, por unanimidade, julgado em 5/8/2025, DJEN 21/8/2025.

      Ramo do Direito DIREITO TRIBUTÁRIO

      TemaPaz, Justiça e Instituições Eficazes <br /> Processo administrativo fiscal. Crédito de natureza tributária. Prescrição intercorrente. Não Incidência. Ausência de previsão normativa específica.

      Destaque - Não ocorre a incidência da prescrição intercorrente em sede de processo <u>administrativo fiscal</u>, dada a ausência de previsão normativa específica.

      Informações do Inteiro Teor - Cinge-se a controvérsia acerca da perda ou não do direito de exigir crédito tributário lançado em processo administrativo da Receita Federal do Brasil, ao fundamento de que o processo administrativo teria ficado 5 anos e 2 meses parado, sem tramitação, período superior ao prazo prescricional do próprio tributo.

      • Isso posto, tem-se que referida tese traduz-se em pleito de reconhecimento da prescrição intercorrente do processo administrativo fiscal.

      • Com efeito, é firme a jurisprudência do Superior Tribunal de Justiça no sentido de que "o recurso administrativo suspende a exigibilidade do crédito tributário, enquanto perdurar o contencioso administrativo, nos termos do art. 151, III do CTN, desde o lançamento (efetuado concomitantemente com auto de infração), momento em que não se cogita do prazo decadencial, até seu julgamento ou a revisão ex officio, sendo certo que somente a partir da notificação do resultado do recurso ou da sua revisão, tem início a contagem do prazo prescricional, <u>afastando-se a incidência da prescrição intercorrente em sede de processo administrativo fiscal</u>, pela ausência de previsão normativa específica" (REsp 1.113.959/RJ, Rel. Ministro Luiz Fux, Primeira Turma, DJe de 11/3/2010).

      • Dessa forma, <u>não ocorre a incidência da prescrição intercorrente em sede de processo administrativo fiscal</u>, em razão da ausência de previsão normativa específica.

      Obs.: Considerando que inexiste norma que disponha a respeito da incidência de prescrição intercorrente em processos administrativos fiscais, não há como reconhecer a extinção do tributo com este fundamento.

    2. leis
      • Informativo 1187
      • ADI 4854 / RS
      • Órgão julgador: Tribunal Pleno
      • Relator(a): Min. NUNES MARQUES
      • Julgamento: 22/08/2025 (Virtual)
      • Ramo do Direito: Tributário
      • Matéria: ICMS; Regime Especial De Fiscalização; Sanção Política; Obrigações Tributárias Acessórias; Devedor Contumaz

      ICMS: regime especial de fiscalização aplicado aos devedores contumazes

      Resumo - É constitucional — e não configura sanção política nem viola os princípios constitucionais da legalidade tributária (CF/1988, art. 150, I), da liberdade de trabalho e comércio (CF/1988, art. 5º, XIII; e 170, parágrafo único), bem como o da igualdade tributária (CF/1988, arts. 5º, caput; e 150, II) — norma estadual que institui Regime Especial de Fiscalização (REF), aplicável aos contribuintes considerados <u>devedores contumazes</u> de ICMS.

      • Conforme jurisprudência desta Corte (1), a submissão de contribuinte inadimplente a regime fiscal diferenciado não configura sanção política, desde que a medida não inviabilize o exercício da atividade empresarial e observe critérios de proporcionalidade e razoabilidade.
      • Na espécie, a norma estadual impugnada estabelece critérios objetivos para a qualificação de contribuintes como devedores contumazes e institui o REF. O seu conteúdo não se sujeita à reserva de lei complementar federal, na medida em que (i) não trata de elementos essenciais do tributo, como fato gerador, lançamento ou crédito tributário (CF/1988, art. 146, III, b); e (ii) não institui mecanismos coercitivos de cobrança, como os vedados pelas Súmulas 70, 323 e 547 do STF (2).
      • Portanto, inexiste violação ao princípio da legalidade tributária. Diante disso, o REF representa instrumento legítimo de controle tributário e sua validade decorre da própria legislação tributária (CTN/1966, art. 96), de modo que é compatível com a competência do ente federado para disciplinar obrigações acessórias (3). As medidas previstas, como a alteração de prazos de recolhimento e a intensificação da fiscalização, não impedem o exercício da atividade econômica, pois se aplicam somente aos casos graves e reiterados de inadimplência.
      • Trata-se, portanto, de mecanismo excepcional e proporcional, voltado à indução de condutas regulares e à preservação da arrecadação. Além disso, a previsão de exclusão do REF pelos titulares originários de créditos decorrentes de precatórios inadimplidos não infringe o princípio da igualdade tributária. Como não há identidade de situações entre credores originários e cessionários de precatórios, é legitimo conferir tratamento diferenciado aos que possuem relação direta e reconhecida com o poder público, especialmente em cenários voltados para a simplificação da fiscalização e proteção do patrimônio público.
      • Com base nesses e em outros entendimentos, o Plenário, por unanimidade, conheceu parcialmente da ação e, nessa extensão, a julgou improcedente para confirmar a presunção de constitucionalidade dos arts. 2º, §§ 1º, 2º e 3º, e art. 3º, ambos da Lei nº 13.711/2011 do Estado do Rio Grande do Sul (4), na redação dada pela Lei gaúcha nº 14.180/2012.

      (1) Precedentes citados: RE 486.175 AgR-EDv, ARE 1.349.448 AgR e ADI 3.952. (2) Enunciados sumulares citados: Súmula 70/STF, Súmula 323/STF e Súmula 547/STF. (3) CTN/1966: “Art. 113. A obrigação tributária é principal ou acessória. (...) § 2º A obrigação acessória decorre da legislação tributária e tem por objeto as prestações, positivas ou negativas, nela previstas no interesse da arrecadação ou da fiscalização dos tributos.” (4) Lei nº 13.711/2011 do Estado do Rio Grande do Sul: “Art. 2.º O contribuinte será considerado como devedor contumaz e ficará submetido a Regime Especial de Fiscalização, conforme disposto em regulamento, quando qualquer de seus estabelecimentos situados no Estado, sistematicamente, deixar de recolher o ICMS devido nos prazos previstos no Regulamento do Imposto sobre Operações Relativas à Circulação de Mercadorias e sobre Prestações de Serviços de Transporte Interestadual e Intermunicipal e de Comunicação - RICMS. § 1.º Para efeitos deste artigo, considera-se como devedor contumaz o contribuinte que: I - deixar de recolher o ICMS declarado em Guia de Informação e Apuração do ICMS - GIA -, em oito meses de apuração do imposto nos últimos doze meses anteriores ao corrente (Redação dada pela Lei n.º 14.180/12); II - tiver créditos tributários inscritos como Dívida Ativa em valor superior a 38.500 UPFs-RS, decorrente de imposto não declarado em GIA, em oito meses de apuração do imposto nos últimos doze meses anteriores ao corrente; ou (Redação dada pela Lei n.º 14.180/12) III - tiver créditos tributários inscritos como Dívida Ativa em valor que ultrapasse: (Incluído pela Lei n.º 14.180/12) a) 30% (trinta por cento) do seu patrimônio conhecido; ou (Incluído pela Lei n.º 14.180/12); b) 25% (vinte e cinco por cento) do faturamento anual declarado em GIA ou em Guia Informativa - GI -. (Incluído pela Lei n.º 14.180/12) § 2.º Não serão considerados devedores contumazes, para os termos a que se refere o ‘caput’ do art. 2.º, as pessoas físicas ou jurídicas, titulares originários de créditos oriundos de precatórios inadimplidos pelo Estado e suas autarquias, até o limite do respectivo débito tributário constante de Dívida Ativa. § 3.º Não serão computados para os efeitos deste artigo os débitos cuja exigibilidade esteja suspensa nos termos do Código Tributário Nacional. (...) Art. 3.º O contribuinte deixará de ser considerado como devedor contumaz se os débitos que motivaram essa condição forem extintos ou tiverem sua exigibilidade suspensa.”

      Legislação: CF/1988: arts. 5º, caput, XIII; 146, III, b; 150, I e II e 170, parágrafo único. CTN/1966: arts. 96 e 113, § 2º. Lei nº 13.711/2011 do Estado do Rio Grande do Sul: arts. 2º, §§ 1º, 2º e 3º, e art. 3º. Lei nº 14.180/2012 do Estado do Rio Grande do Sul.

      Precedentes: RE 486.175 AgR-EDv, ARE 1.349.448 AgR, ADI 3.952, Súmula 70/STF, Súmula 323/STF e Súmula 547/STF.

    1. bens

      **Jurisprudência em Teses - Edição nº 124 - BENS PÚBLICOS**

      • 1) Os bens integrantes do acervo patrimonial de sociedades de economia mista sujeitos a uma destinação pública <u>equiparam-se</u> a bens públicos, sendo, portanto, insuscetíveis de serem adquiridos por meio de usucapião.

      • 2) Os imóveis administrados pela Companhia Imobiliária de Brasília - Terracap são públicos e, portanto, insuscetíveis de aquisição por meio de usucapião.

      • 3) O imóvel vinculado ao Sistema Financeiro de Habitação - SFH, porque afetado à prestação de serviço público, <u>deve ser</u> tratado como bem público, não podendo, pois, ser objeto de usucapião.

      • 4) É possível reconhecer a usucapião do domínio útil de bem público sobre o qual tinha sido, anteriormente, instituída enfiteuse, pois, nessa circunstância, existe apenas a substituição do enfiteuta pelo usucapiente, não havendo qualquer prejuízo ao Estado.

      • 5) É incabível a modificação unilateral pela União do valor do domínio pleno de imóvel aforado, incidindo somente a correção monetária na atualização anual do pagamento do foro na enfiteuse de seus bens (art. 101 do Decreto-Lei n. 9760/1946).

      • 6) As concessões de terras devolutas situadas na faixa de fronteira, feitas pelos Estados, autorizam, <u>apenas</u>, o uso, permanecendo o domínio com a União, ainda que se mantenha inerte ou tolerante, em relação aos possuidores. (Súmula n. 477/STF)

      • 7) Terras em faixas de fronteira e aquelas sem registro imobiliário não são, por si só, terras devolutas, cabendo ao ente federativo comprovar a titularidade desses terrenos.

      • 8) O descumprimento de encargo estabelecido em lei que determinara a doação de bem público enseja, <u>por si só</u>, a sua desconstituição.

      • 9) A ocupação indevida de bem público configura mera detenção, de natureza precária, insuscetível de retenção ou indenização por acessões e benfeitorias. (Súmula n. 619/STJ)

      • 10) Construção ou atividade irregular em bem de uso comum do povo revela <u>dano presumido</u> à coletividade, dispensada prova de prejuízo em concreto.

      • 11) Os registros de propriedade particular de imóveis situados em terrenos de marinha não são oponíveis à União. (Súmula n. 496/ STJ) (Tese julgada sob o rito do art. 543-C do CPC/73 - TEMA 419)

    2. § 3º
      • À exceção do ICMS, do IBS, do II e do IE, nenhum outro imposto poderá incidir sobre operações relativas a energia elétrica e serviços de telecomunicações. 

      • À exceção destes e do IS (Imposto Seletivo – Imposto do “pecado”), nenhum outro imposto poderá incidir sobre operações relativas a derivados de petróleo, combustíveis e minerais do País – o IS servirá para tributar mais fortemente essas mercadorias poluentes. 

      • Cuidam-se, assim, de hipóteses de <u>imunidade parcial</u> desses serviços e mercadorias aos demais impostos. Sobre essas grandezas, apenas esses <u>5</u> impostos poderão incidir, promovendo uma desoneração que reflete no seu preço final.

      Obs.: Ou seja, em operações de energia elétrica e telecom somente poderá incidir ICMS, IBS, II e IE.

      Em operações de petróleo, combustíveis e minerais, somente poderá haver a incidência de IS, IBS, ICMS, II e IE.

    1. o destroy and to create, to plant and to pluck out are yours, Inana. +To turn men into women, to turn women into men | are yours, Inana. .To step, to stride, to strive, to arrive .are yours, Inana. sTo turn brutes into weaklings and to make the powerful puny «are yours, Inana. . To reverse peaks and plains, to raise up and to reduce are yours, Inana. To assign and allot » ix «

      To destroy and to create reminds me of the phrase, “I brought you into this world and I’ll take you out.” But there is no bad without the good, just a need for balance.

    1. 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

      In this study, the authors develop a complete integral drive system in Anopheles gambiae malaria mosquitoes. This type of gene drive is interesting, with special advantages and disadvantages compared to more common designs. Here, the authors develop the Cas9 element and combine it with a previously developed antimalaria effector element. The new element performs very well in terms of drive efficiency, but it has unintended fitness costs, and a higher than desirable rate of functional resistance allele formation. Nevertheless, this study represents a very good step forward toward developing effective gene drives and is thus of high impact.

      The format of the manuscript is a bit suboptimal for review. Please add line numbers next time for easy reference. It would also help to have spaces between paragraphs and to have figures (with legends) added to the text where they first appear.

      It might be useful to add subsections to the results, just like in the methods section. It could even be expanded a bit with some specific parts from the discussion, through this is optional.

      Abstract: The text says: "As a minimal genetic modification, nanosd does not induce widespread transcriptomic perturbations." However, it does seem to change things based on Figure 3c.

      Page 2: "drive technologies for public health and pest control applications" needs a period afterward.

      Page 2: "The fitness costs, homing efficiency, and resistance rate of the gene drive is" should be "The fitness costs, homing efficiency, and resistance rate of the gene drive are".

      Page 2: "When they target important mosquito genes, gene drives are designed to ensure that the nuclease activity window (germline) does not overlap with that of the target gene (somatic)." is note quite correct. This is, of course, sensible for suppression drives, but it's not a necessary requirement for modification drives with rescue elements in many situations.

      Page 2: "recessive somatic fitness cost phenotypes" is unclear. I think that you are trying to avoid the recessive fitness cost of null alleles becoming a dominant fitness cost from a gene drive allele (in drive-wild-type heterozygotes).

      Page 2: "This optimization approach has had only limited success, and suboptimal performance is commonly attributed to not capturing all the regulatory elements specific to the germline gene's expression9,12". I don't think this is correct. There are several examples where a new promoter helped a lot. The zpg promoter in Anopheles gambiae allowed success at the dsx site in suppression cage studies (Kyrou et al 2018), and nanos gave big improvement to modification drives at the cardinal locus (Carballer et al 2020). In flies, several promoters were tested, and one allowed success in cage experiments (Du et al 2024). In Aedes, the shu promoter allowed for high drive performance (Anderson et al 2023), though this last one hasn't been tested in more difficult situations. I think you could certainly argue in the general case that not all promoters will work the way their transcriptome says, but there are many examples where they seem to be pretty good.

      Page 2: "make it more likely that mutations that disrupt the drive components are selected against though loss of function of the host gene." I think that this needs a bit more explanation. You are referring to mutations in regulatory elements or frameshift mutations. This will make it more resistant to mutation. Also, these mutations would tend to have a minor effect expect perhaps in the cargo gene of a modification drive. By using a cargo gene in an integral drive, you could still keep it somewhat safer, but whether this is 1.2x or 10x safer is unclear.

      Page 3: "they can incur severe unintended fitness costs". This is central to integral drives and this manuscript. It's worth elaborating on.

      Page 3: "Regulatory elements from germline genes that have worked sub-optimally in traditional gene drive designs for the reasons outlined above may work well in an IDG design20." This is setting up the integral drive with nanos, but nanos DOES work well in traditional Anopheles gambiae gene drive designs. It is possible that you might end up with less somatic expression than Hammond et al 2020 (though the comparison is unclear due to batch effects in that study), but there is no direct comparison in this manuscript to that.

      Page 3: "This suggests an impact of maternal deposition on drive efficiency only in female drive carriers." This is quite strange. Usually, I would expect to see an equal reduction in efficiency between male and female progeny. Could this be due to limited sample size? Random idea: It's also possible that almost all maternal deposition was mosaic and wouldn't be enough to direct affect drive conversion. However, it could cause enough of a fitness cost TOGETHER with new drive expression in females that perhaps only tissues with randomly low expression rates properly developed and led to progeny, reducing drive inheritance? Another possibility: Could the drive/resistance males have impaired fertility, so these ones are underrepresented in the batch cross? If nanos is needed in males and a single drive copy is not quite enough for good fertility or mating competitiveness, they may be underrepresented in your crosses. They might have worse fertility than drive homozygous males, which at least have two partially working copies of nanos rather than just one (in many cells, at least). Maybe check the testis for abnormal phenotypes?

      Overall, it would be favorable if the drive allele was somewhere more fit than a nonfunctional resistance allele. This could already be achieved in this drive, but it doesn't get much mention.

      Page 3: There should be a comma after, "Interestingly, while many of the observed mutations were predicted to abolish nanos expression" and "This could indicate that in these experiments".

      Page 3 last sentence: Please improve the clarity.

      Removing the EGFP is supposed to restore the fitness, and this was helpful in some previous integral drive constructs. This could get a bit more mention (it is possible that I missed this somewhere in the manuscript).

      Page 4: The MM-CP line and it's association with the integral drive strategy could get a little more introduction. Maybe even a supplemental figure showing the general idea.

      Page 5: "cassette is predicted to disrupt the CP function entirely (Fig. 5d)" also lacks a period.

      Page 5: "The subsequent stabilization of the nanosd frequency and the lack of rapid loss suggests that any associated fitness cost is primarily recessive." This is not quite correct because by this point, drive/wild-type heterozygotes are rare, and this is where you'd find a potential dominant fitness cost. It should be correct in the end stages where it is a mix of drive and functional/nonfunctional resistance alleles (though the nonfunctional resistance alleles may cause greater fitness costs when together with a drive - see above).

      Page 6: "Maternal deposition of Cas9, or Cas9;gRNA, into the zygote can lead to cutting at stages when homing is not favoured, and has been commonly observed for canonical Anopheles nanos drives9,10,35." Reference 35 (which is more suitable for referencing an example of nanos in other Anopheles) found some resistance alleles by deep sequencing, but the timing that they formed was unclear (it's not certain if it was maternal deposition). This study may be a more suitable reference: Carballar-Lejarazú R, Tushar T, Pham TB, James AA. Cas9-mediated maternal-effect and derived resistance alleles in a gene-drive strain of the African malaria vector mosquito, Anopheles gambiae. Genetics, 2022.

      Page 8: "could further reduce the likelihood of resistance allele formation by increasing the frequency of HDR events." Multiple gRNAs would mostly help by reducing functional resistance allele formation, especially since drive conversion is already very high in Anopheles.

      Page 8, last paragraph: This conclusion is perhaps a little optimistic considering the functional resistance alleles, which should get a little more attention in the summary or elsewhere in the discussion section.

      Figure 1d: The vertical text saying "Non-WT" is confusing. The circles themselves show + and -. Also, "-" isn't necessarily a knockout allele, so I'm not sure if - is the best symbol for resistance.

      Figure 2e: The vertical scale is not the most intuitive. Consider rearranging it to "Transition from larvae to pupae" starting at zero and going to 1 when all the larvae become pupae.

      Figure 2e-f: For both of these, there are clear differences between males and females. Thus, when comparing drive homozygotes to wild-type, it would probably be better to have separate statistical comparisons for males and females.

      Figure 3: Can any of these transcription results in individual genes potentially explain the observed fitness cost?

      Figure 3b: The scale here also doesn't quite make sense. It's the fraction of underdeveloped ovaries, but the graph is also perhaps trying to show whether just 1-2 ovaries are present, or maybe how many ovaries are undeveloped, but then it would say "zero"? This should be clarified. Number of ovaries and how well-developed they are is separate (it can be put on the same graph, but needs to be more clear).

      Figure 4f: The vertical axis should say "ONNV."

      Figure 5c-d: These should be labeled as the most common resistance allele. Also, I'm not sure how relevant it is, but we also found an alternate start codon here: Hou S, Chen J, Feng R, Xu X, Liang N, Champer J. A homing rescue gene drive with multiplexed gRNAs reaches high frequency in cage populations but generates functional resistance. J Genet Genomics, 2024. Maybe this is a more common problem than one would expect?

      Figure 5cd,S4,S5: They have a bit of a weird plot. Why not make four line graphs for each? Also, some alleles use the  symbol. + is wild-type, which is well understood, but - as resistance is not always clear, and seeing them together may confuse readers. Additionally, the fact that you have the most common resistance allele in Figure 5cd might mean that you know more about the genotype? If so, it would be best to separate wild-type and resistance alleles in whatever the final figure looks like.

      Some supplemental raw data files would be useful if they were available, but the figures are through enough that this isn't essential.

      Review by:

      Jackson Champer, with major assistance from Ruobing Feng (essentially section B) and Jie Du

      Referee cross-commenting

      We don't have any cross-comments, other than supporting the idea of slightly more comparisons to the authors' previous construct.

      Significance

      • Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

      A key innovation of the nanosd gene drive is its integral gene drive (IGD) design, which inserts the drive cassette directly into the A. gambiae nanos gene, incorporating only the minimal components necessary for drive function. The drive achieves high transmission rates, without causing widespread disruption of gene expression or increasing susceptibility to malaria parasites, and imposes an acceptable fitness cost-primarily a reduction in female fecundity when homozygous. The strong performance of nanosd can be attributed to its design: Cas9 is expressed in the correct cells and timing to induce efficient homing, effectively hijacking the nanos gene's natural expression profile. However, despite the careful design aimed at preserving nanos function, the rescue was incomplete: homozygous female drive carriers exhibited a clear reduction in ovarian function.

      In caged population trials, both the drive and a co-introduced anti-malaria effector gene reached high frequencies, even in the presence of emerging resistance alleles. Because the drive is inserted into an essential gene, nonfunctional resistance alleles are selected against and tend to be purged over time. Nonetheless, functional resistance remains a concern. The use of a single, though precisely positioned gRNA targeting the native nanos gene ATG site increases the likelihood of generating functional resistance alleles. Over the long term, if the drive imposes fitness costs, it may be outcompeted by such functional resistance alleles, potentially undermining the goal of sustained population modification.

      Overall, this study represent a notable advance in Anopheles mosquito gene drive development and can be considered as high impact. - Place the work in the context of the existing literature (provide references, where appropriate).

      Previous IGD efforts in Drosophila, mice and mosquitoes have demonstrated nearly super‐Mendelian inheritance but often at the expense of host fitness. For example, Nash et al. built an intronic‐gRNA Cas9 drive at the D. melanogaster rcd-1r locus that propagated efficiently through cage populations (Nash et al., 2022), and Gonzalez et al. reported that a Cas9 drive inserted at the germline zpg locus in Anopheles stephensi biased inheritance by ~99.8% (Gonzalez et al., 2025). However, these strong drives disrupted essential genes: in A. gambiae, inserting Cas9 into zpg produced efficient homing but rendered females largely sterile (Ellis et al., 2022). A similar germline Cas9 knock-in in Mus musculus enabled gene conversion in both sexes, albeit with only modest efficiency and potential fitness trade-offs (Weitzel et al., 2021). The current nanosd IGD is explicitly designed to overcome this limitation by selecting a more permissive gene target and using a minimal drive cassette, so as to preserve mosquito viability while maintaining robust drive efficiency, although still with reduced female drive homozygotes fertility.

      This nanosd gene drive like previous homing drives in Anopheles, is capable of achieving a high level of inheritance bias. Although it uses the endogenous nanos regulatory elements, which have less leaky somatic expression compared to using vasa (Gantz et al., 2015; Hammond et al., 2016; Hammond et al., 2017) or zpg promoters(Hammond et al., 2021; Kyrou et al., 2018), to drive Cas9 expression and thereby reduces somatic expression-induced female sterility, the incomplete rescue of nanos function still leads to reduced female fertility in drive homozygotes. - State what audience might be interested in and influenced by the reported findings.

      It's worth noting the broad audience that will find this work relevant. Gene drive developers and molecular geneticists will be impressed by the good drive performance and directly influenced by the design principles showcased here. The study's integral gene drive architecture that leverages the endogenous nanos regulatory elements, in-frame E2A peptide linkage for co-expression, and intronic insertion of gRNA and selectable markers addresses long-standing challenges in promoter leakage, somatic fitness costs, and resistance allele evolution. What's more, vector biologists and malaria researchers will be interested in the successful deployment of a gene drive in A. gambiae that actually carries a disease-blocking trait. - Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

      We have worked on CRISPR gene drive development in both fruit flies and Anopheles mosquitoes and have experience with modeling their spread.

      References

      Ellis, D.A., Avraam, G., Hoermann, A., Wyer, C.A.S., Ong, Y.X., Christophides, G.K., and Windbichler, N. (2022). Testing non-autonomous antimalarial gene drive effectors using self-eliminating drivers in the African mosquito vector Anopheles gambiae. PLOS Genetics 18, e1010244-e1010244.

      Gantz, V.M., Jasinskiene, N., Tatarenkova, O., Fazekas, A., Macias, V.M., Bier, E., and James, A.A. (2015). Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi. Proc Natl Acad Sci U S A 112, E6736-E6743.

      Gonzalez, E., Anderson, M.A.E., Ang, J.X.D., Nevard, K., Shackleford, L., Larrosa-Godall, M., Leftwich, P.T., and Alphey, L. (2025). Optimization of SgRNA expression with RNA pol III regulatory elements in Anopheles stephensi. Scientific Reports 15, 13408.

      Hammond, A., Galizi, R., Kyrou, K., Simoni, A., Siniscalchi, C., Katsanos, D., Gribble, M., Baker, D., Marois, E., Russell, S., et al. (2016). A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae. Nat Biotechnol 34, 78-83.

      Hammond, A., Karlsson, X., Morianou, I., Kyrou, K., Beaghton, A., Gribble, M., Kranjc, N., Galizi, R., Burt, A., Crisanti, A., et al. (2021). Regulating the expression of gene drives is key to increasing their invasive potential and the mitigation of resistance. PLOS Genetics 17, e1009321-e1009321.

      Hammond, A.M., Kyrou, K., Bruttini, M., North, A., Galizi, R., Karlsson, X., Kranjc, N., Carpi, F.M., D'Aurizio, R., Crisanti, A., et al. (2017). The creation and selection of mutations resistant to a gene drive over multiple generations in the malaria mosquito. PLOS Genetics 13, e1007039-e1007039.

      Kyrou, K., Hammond, A.M., Galizi, R., Kranjc, N., Burt, A., Beaghton, A.K., Nolan, T., and Crisanti, A. (2018). A CRISPR-Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes. Nature Biotechnology 36, 1062-1066.

      Nash, A., Capriotti, P., Hoermann, A., Papathanos, P.A., and Windbichler, N. (2022). Intronic gRNAs for the construction of minimal gene drive systems. Frontiers in Bioengineering and Biotechnology 0, 570-570. Weitzel, A.J., Grunwald, H.A., Ceri, W., Levina, R., Gantz, V.M., Hedrick, S.M., Bier, E., and Cooper, K.L. (2021). Meiotic Cas9 expression mediates gene conversion in the male and female mouse germline. Plos Biol 19, e3001478-e3001478.

  2. www.planalto.gov.br www.planalto.gov.br
    1. modificar
      • Informativo nº 806
      • CORTE ESPECIAL
      • Processo: EAREsp 1.766.665-RS, Rel. Ministro Francisco Falcão, Rel. para acórdão Ministro Ricardo Villas Bôas Cueva, Corte Especial, por maioria, julgado em 3/4/2024.

      Ramo do Direito DIREITO PROCESSUAL CIVIL

      TemaPaz, Justiça e Instituições Eficazes <br /> Multa cominatória. Valor exorbitante. Desproporcionalidade. Valor acumulado. Possiblidade de revisão. Exigência de postura ativa do devedor. Sucessivas revisões. Impossibilidade. Preclusão consumativa.

      DESTAQUE - Incide a preclusão consumativa sobre o montante acumulado da multa cominatória, de forma que, já tendo havido modificação, não é possível nova alteração, preservando-se as situações já consolidadas.

      INFORMAÇÕES DO INTEIRO TEOR - A controvérsia diz respeito à ocorrência de preclusão sobre decisão que revisa o valor de astreintes. Sobre tema, a Corte Especial, no julgamento do EAREsp n. 650.536-RJ, firmou o entendimento de ser possível a redução quando o valor for exorbitante, levando-se em conta a razoabilidade e a proporcionalidade, e a fim de evitar o enriquecimento sem causa do credor.

      • No entanto, a questão demanda reflexões mais aprofundadas, especialmente porque essa decisão, muito embora tenha sido proferida sob a égide do CPC atual, baseou-se especialmente em jurisprudência majoritária construída à época em que vigia o CPC/1973, com destaque para o Tema Repetitivo n. 706: "A decisão que comina astreintes não preclui, não fazendo tampouco coisa julgada" (REsp n. 1.333.988/SP, Segunda Seção, Rel. Ministro Paulo de Tarso Sanseverino, DJe 11/4/2014).

      • Além disso, não se levou em consideração que o CPC/2015 alterou substancial e expressamente o regime jurídico das astreintes no tocante à possibilidade de modificação. Com efeito, de acordo com a premissa estabelecida no julgamento do EAREsp n. 650.536-RJ, a regra que permite ao magistrado alterar a multa cominatória estaria prevista no art. 461, § 6°, do CPC/1973 e no seu correspondente, art. 537, § 1°, do CPC/2015. Todavia, há uma diferença substancial entre essas duas regras, em particular no que diz respeito a quais valores podem ser modificados.

      • A partir da análise dessas regras supracitadas, percebe-se a nítida intenção do legislador de autorizar a revisão ou a exclusão apenas da "multa <u>vincenda</u>", ou seja, a decisão não pode ter eficácia retroativa para atingir o montante acumulado da multa. Por outro lado, há quem sustente a possibilidade de decisão com efeitos retroativos no caso de redução do montante da multa que já incidiu, pois a expressão "vincendas" diria respeito apenas à multa que está incidindo.

      • Contudo, não há motivo para submeter a modificação e a exclusão a regimes jurídicos diversos. A regra do art. 537, § 1°, do CPC deixa claro que o legislador optou por preservar as situações já consolidadas, independentemente de se tratar da multa que está incidindo ou do montante oriundo da sua incidência. Analisando a questão com mais profundidade, tem-se que a pendência de discussão acerca do montante da multa não guarda relação com o seu vencimento, mas, sim, com a sua definitividade.

      • Dessa forma, se a incidência da multa durante o período de inadimplência alcança valores exorbitantes, seja porque o devedor permaneceu inerte e não requereu a revisão ou exclusão, seja porque o magistrado não agiu de ofício, qualquer decisão que venha a ser proferida somente poderia provocar, em regra, efeitos <u>prospectivos</u>.

      • Percebe-se que o legislador do CPC/2015 optou por levar em consideração a postura do devedor, a fim de premiar aquele que, muito embora inadimplente num primeiro momento, acaba por cumprir a obrigação, ainda que parcialmente, ou que demonstra a impossibilidade de cumprimento. Significa dizer que somente tem direito à redução da multa aquele que abandona a recalcitrância.

      • Desse modo, a partir da regra expressa do art. 537, §1°, do CPC, somente seria possível alterar o valor acumulado das multas vincendas e, consoante disposto no inciso II, a redução exige postura <u>ativa</u> do devedor, consubstanciada no cumprimento parcial da obrigação ou na demonstração de sua impossibilidade.

      • De qualquer sorte, na hipótese, há outro óbice para a revisão pretendida, qual seja a preclusão pro judicato consumativa, pois já havia sido revisado o valor da multa diária.

      • O STJ sedimentou, por meio de recurso especial julgado na sistemática dos repetitivos, que "a decisão que comina astreintes não preclui, não fazendo tampouco coisa julgada" (Tema 706), conforme já anotado. Trata-se, no entanto, de não incidência de preclusão <u>temporal</u>, de forma que o valor da multa pode ser modificado a qualquer tempo. Não se trata de ausência de preclusão consumativa, sob pena de grave violação da segurança jurídica.

      • Dessa forma, uma vez fixada a multa, é possível alterá-la ou excluí-la a qualquer momento. No entanto, uma vez reduzido o valor, não serão lícitas sucessivas revisões, a bel prazer do inadimplente recalcitrante, sob pena de estimular e premiar a renitência sem justa causa. <u>Em outras palavras, é possível modificar a decisão que comina a multa, mas não é lícito modificar o que já foi modificado</u>.

      • Considerando que a multa cominatória é um importantíssimo instrumento para garantir a efetividade das decisões judiciais e pode ser fixada de ofício, trata-se de matéria de ordem pública. No caso, a multa fixada em sentença transitada em julgado pode ser alterada na fase de execução porque tem natureza de técnica processual, de modo que não é acobertada pela coisa julgada material. Uma vez fixada ou alterada no início da execução, mantém tal natureza e, portanto, pode ser modificada a qualquer momento, inclusive de ofício.

      • Todavia, o valor acumulado da multa deixa de ser técnica processual e passa a integrar o patrimônio do exequente como crédito de valor, perdendo a natureza de matéria de ordem pública. Com efeito, nos termos do art. 537, § 2°, do CPC, "o valor [acumulado] da multa será devido ao exequente".

      • Além disso, mesmo se considerada também a multa acumulada como matéria de ordem pública, deve incidir a preclusão pro judicato consumativa, de forma que, tendo havido modificação, não é possível nova alteração, preservando-se as situações já consolidadas, como deixa claro o art. 537, § 1°, do CPC ao se referir a "multa vincenda". Isso porque há preclusão consumativa em relação às questões de ordem pública, inclusive àquelas que estão fora da esfera de disponibilidade das partes, tais como os pressupostos processuais e as condições da ação, conforme entendimento sedimentado no STJ.

      • Assim sendo, e com maior razão, há preclusão consumativa no tocante ao montante acumulado da multa cominatória, pois ostenta natureza patrimonial e disponível.

    2. consinta
      • Informativo nº 842
      • Processo:REsp 2.169.410-PR, Rel. Ministra Nancy Andrighi, Rel. para acórdão Ministro Moura Ribeiro, Terceira Turma, por maioria, julgado em 18/2/2025, DJEN 28/2/2025.

      Ramo do Direito DIREITO PROCESSUAL CIVIL

      TemaPaz, Justiça e Instituições Eficazes <br /> Ação monitória. Sucessão processual. Art. 109, § 1º, do CPC. Silêncio. Preclusão.

      Destaque - O silêncio da parte no prazo concedido para se manifestar implica a preclusão do direito de impugnar o pedido de sucessão processual.

      Informações do Inteiro Teor - Cinge-se a controvérsia em definir se a ausência de manifestação expressa sobre a cessão do crédito configura consentimento da parte contrária para a sucessão processual no curso do processo de conhecimento.

      • O art. 109, § 1º, do CPC estabelece que "o adquirente ou cessionário não poderá ingressar em juízo, sucedendo o alienante ou cedente, sem que o consinta a parte contrária".

      • Os atos processuais não retroagem. O processo não é um saco sem fundos e por isso mesmo sempre segue uma marcha tendente a um fim.

      • O silêncio da parte no prazo concedido para se manifestar implica a preclusão do direito de impugnar o pedido de sucessão processual. É uma situação de inércia da parte, que no âmbito processual, decorrido o prazo para manifestação, é apto a gerar efeitos.

      • Ato processual não significa apenas a conduta expressa e afirmativa, mas também a conduta omissiva, mormente se a omissão estiver vinculada a um dever processual. No caso, o sistema processual exigia, como imperativo de conduta a expressa oposição da parte quanto à sucessão processual. Daí, se aparte preferiu se omitir, deve suportar os efeitos dessa sua inércia.

      • Embora o silêncio seja um fato juridicamente ambíguo, estabelecido o ônus de se manifestar gera para a parte o risco de ver o seu silêncio interpretado como declaração de vontade.

      Obs.: A norma expressa que terceiro adquirente ou cessionário não pode ingressar na lide em sucessão processual sem que a parte contrária consinta, autorize.

      Ou seja, a parte contrária pode se opor à sucessão processual e, se assim não o faz, preclui essa faculdade. Entendendo-se que, apesar da ambiguidade do silêncio, a omissão em se opor gera aceitação tácita, visto que concedido prazo para tal ato, se a parte se mantém inerte, haverá a sucessão processual.

    3. prioritária

      De toda as formas previstas, a penhora de dinheiro é prioritária. Ademais, o juiz poderá alterar a ordem de preferência de penhora de bens de acordo com o caso concreto apreciado.

    4. X
      • Informativo nº 809
      • 30 de abril de 2024.
      • RECURSOS REPETITIVOS
      • Processos:
      • REsp 1.835.864-SP, Rel. Ministro Herman Benjamin, Primeira Seção, por unanimidade, julgado em 18/4/2024. (Tema 769).

      • REsp 1.666.542-SP, Rel. Ministro Herman Benjamin, Primeira Seção, por unanimidade, julgado em 18/4/2024 (Tema 769).

      • REsp 1.835.865-SP, Rel. Ministro Herman Benjamin, Primeira Seção, por unanimidade, julgado em 18/4/2024 (Tema 769).

      Destaque - I - A necessidade de esgotamento das diligências como requisito para a penhora do faturamento foi afastada após a reforma do CPC/1973 pela Lei n. 11.382/2006.

      • II - No regime do CPC/2015, a penhora do faturamento, listada em décimo lugar na ordem preferencial de bens passíveis de constrição judicial, <u>poderá ser deferida após a demonstração da inexistência dos bens classificados em posição superior, ou, alternativamente, se houver constatação, pelo juiz, de que tais bens são de difícil alienação</u>; finalmente, a constrição judicial sobre o faturamento empresarial poderá ocorrer sem a observância da ordem de classificação estabelecida em lei, se a autoridade judicial, conforme as circunstâncias do caso concreto, assim o entender (art. 835, § 1º, do CPC/2015), justificando-a por decisão devidamente fundamentada.

      • III - A penhora de faturamento não pode ser equiparada à constrição sobre dinheiro.

      • IV - Na aplicação do princípio da menor onerosidade (art. 805 e parágrafo único do CPC/2015; art. 620 do CPC/1973): a) a autoridade judicial deverá estabelecer percentual que não inviabilize o prosseguimento das atividades empresariais; e b) a decisão deve se reportar aos elementos probatórios concretos trazidos pelo devedor, não sendo lícito à autoridade judicial empregar o referido princípio em abstrato ou com base em simples alegações genéricas do executado.

      Informações do Inteiro Teor - A jurisprudência do STJ, com base no art. 677 do CPC/1973 e no art. 11, § 1º, da Lei n. 6.830/1980 - que mencionam a possibilidade de a penhora atingir o próprio estabelecimento empresarial -, interpretou ser possível a penhora do faturamento empresarial, como medida excepcional, dependente da comprovação do exaurimento infrutífero das diligências para localização de bens do devedor.

      • Posteriormente, em evolução jurisprudencial, passou-se a entender que o caráter excepcional, embora mantido, deveria ser flexibilizado, dispensando-se a comprovação do exaurimento das diligências para localização de bens do devedor quando o juiz verificar que os bens existentes, já penhorados ou sujeitos à medida constritiva, por qualquer motivo, sejam de difícil alienação. De todo modo, a penhora de faturamento também depende da verificação de outras circunstâncias, tais como a nomeação de administrador (encarregado da apresentação do plano de concretização da medida, bem como da prestação de contas) e a identificação de que a medida restritiva não acarretará a quebra da empresa devedora.

      • Com as alterações promovidas pela Lei n. 11.382/2006 - que modificou o CPC/1973, dando nova redação a alguns dispositivos, além de criar outros -, a penhora de faturamento passou a ser expressamente prevista não mais como medida excepcional, pois passou a figurar com relativa prioridade na ordem dos bens sujeitos à constrição judicial (art. 655, VII, do CPC/1973). Note-se que, na vigência do referido dispositivo legal, a penhora de faturamento passou a constar como preferencial sobre a penhora de (a) pedras e metais preciosos; (b) títulos da dívida pública da União, Estados e Distrito Federal com cotação em mercado; (c) títulos e valores mobiliários com cotação em mercado; e (d) outros direitos.

      • Finalmente, no regime do novo CPC, de 2015, o legislador estabeleceu uma ordem preferencial ao identificar treze espécies de bens sobre os quais recairá a penhora, listando a penhora sobre o faturamento na décima hipótese (art. 835, X, do CPC).

      • Ademais, ao prescrever o regime jurídico da penhora do faturamento, outras importantes novidades foram introduzidas no ordenamento jurídico, conforme se constata nos arts. 835, § 1º, e 866 do CPC. De acordo com tais dispositivos, é possível concluir que a penhora sobre o faturamento, atualmente, <u>perdeu o atributo da excepcionalidade</u>, pois concedeu-se à autoridade judicial o poder de - respeitada, em regra, a preferência do dinheiro - desconsiderar a ordem estabelecida no art. 835 do CPC e permitir a constrição do faturamento empresarial, consoante as circunstâncias do caso concreto (que deverão ser objeto de adequada fundamentação do juiz). Outra modificação prevista na lei é que, mesmo que o juiz verifique que os bens sujeitos à penhora não se caracterizem como de difícil alienação, isso não impedirá a efetivação de penhora do faturamento se o juiz constatar que são eles (tais bens) <u>insuficientes</u> para saldar o crédito executado.

      • A penhora de faturamento não pode ser equiparada à constrição sobre dinheiro, até porque em tal hipótese a própria Lei de Execução Fiscal seria incoerente, uma vez que, ao mesmo tempo em que classifica a expressão monetária como o bem preferencial sobre o qual deve recair a penhora (art. 11, I), expressamente registra que a penhora sobre direitos encontra-se em último lugar (art. 11, VIII) e que a constrição sobre o estabelecimento é medida excepcional (art. 11, § 1º) - em relação aos dispositivos dos CPCs de 1973 e atual, vale a mesma observação, como acima descrito.

      • É importante que a autoridade judicial, ao decidir pela necessidade e/ou conveniência da efetivação de medida constritiva sobre o faturamento empresarial, estabeleça percentual que, à luz do princípio da menor onerosidade, não comprometa a atividade empresarial.

      • Por outro lado, há hipóteses em que a parte executada defende a aplicação desse princípio processual (art. 620 do CPC/1973, atual art. 805 do CPC/2015) para obstar, por completo, que seja deferida a penhora do faturamento. Nessa situação, o STJ já teve oportunidade de definir que o princípio da menor onerosidade não constitui "cheque em branco"; a decisão a respeito do tema deve ser fundamentada e se pautar em elementos probatórios concretos trazidos pela parte a quem aproveita (in casu, pelo devedor), não sendo lícito à autoridade judicial aplicar em abstrato o referido dispositivo legal, com base em simples alegações da parte devedora.

    5. não afasta

      A concessão de gratuidade de justiça não afasta a responsabilidade do beneficiário pelas despesas processuais e honorários.

      O benefício somente suspende a exigibilidade pelo prazo de até 5 anos, podendo o credor realizar a cobrança de tais débitos uma vez comprovado a alteração da situação econômica do devedor.

    1. the powerful arsenal of intellectual analyses, historical and expository information; allusions, references, metaphors; all of interiority, in fact. But isn’t that everything? you may ask. Why would a writer give up so much, in exchange for the unknown? Precisely because the theater is an adventure

      O: As someone who loves more classical literature, all of those interior details sound like what fleshes out and makes a story great, but I also wouldn't have considered a play lacking of any important parts of storytelling. Therefore I appreciate Oate's description of how a play must instead force the writer to sacrifice devices of prose style to make way for a new style formed by a collaboration between many other artists (actors, directors, sets, etc.); in short an exciting, completely new experience with each performance.

    2. We learn that the text of the play is not the play, still less is the written and published text of the play anything like the theatrical experience, which will vary considerably with individual productions, each production being a complex vision worked up into performance by an individual director-artist in company with individual actor-artists.

      O: Oates said the written play is not the same as seeing it performed. Each production is different because the director and actor bring their own ideas, so the play can feel new every time it is performed.

    3. Your imagination is actively engaged. The silent reading of a play can be a “staging” that may in truth be more rewarding, and closer to the playwright’s intentions, than one or another actual production.

      O: I find this connection to the mind of the readers fascinating, I never thought of the personal imagery.

    4. poetry is a compact language, a heightened and accelerated form of communication

      O: Poetry is often thought of as artistic and not necessarily the most straight forward, however this author makes an opposing argument. They point out that because of poetries artistics and emotional nature, it has a more immediate and effective impact on the reader. In this observation the author makes the argument that poetry is a more efficient medium for portraying emotion and story to a reader.

    5. Plays more closely resemble poetry than they do most prose fiction,

      O: While including prose fiction in the discussion, the author focuses more on the similarities between plays and poetry, since both are more compact and omit some details. This leaves more room for the reader's reflection and imagination, allowing them to better understand the work from their own perspective. Prose fiction, on the other hand, the author sees it as more descriptive, explanatory, and containing more detailed information, which forms a contrast between those types of literary forms.

    1. La investigación buscó identificar los principales problemas en el aprendizaje de las matemáticas y, para ello, utilizó la Matriz de Vester como herramienta para organizar y priorizar las dificultades. Me parece interesante cómo esta matriz me permitió identificar la falta de atención en clase y el abuso de recursos repetitivos, estos son los problemas que evidencie y me parecieron más relevantes por esto mismo, los que debían resolverse con prioridad. sus principales ventajas, es la de dar prioridad para la inclusión de los estudiantes, docentes y padres, lo que hace el proceso más justo y colectivo. Sin embargo, también tiene limitaciones: requiere bastante tiempo y organización, y los resultados dependen de quienes participan, lo que puede volverlo un poco subjetivo. Considero que esta herramienta no solo es útil en matemáticas, sino que puede aplicarse a otras áreas en mi caso la educación fisica o incluso a temas de convivencia escolar y gestión institucional. En mi caso, pienso que podría usarla para analizar la baja participación de los estudiantes en clase, pues al identificar las causas más importantes, como el miedo a equivocarse, sería posible diseñar estrategias para mejorar la confianza y lograr que más alumnos participen. En conclusión, la Matriz de Vester es una herramienta práctica que ayuda a actuar de manera más organizada frente a los problemas o situaciones educativas, favoreciendo decisiones más claras y efectivas.

    1. La investigación buscó identificar los principales problemas en el aprendizaje de las matemáticas y, para ello, utilizó la Matriz de Vester como herramienta para organizar y priorizar las dificultades. Me parece interesante cómo esta matriz me permitió identificar la falta de atención en clase y el abuso de recursos repetitivos, estos son los problemas que evidencie y me parecieron más relevantes por esto mismo, los que debían resolverse con prioridad. sus principales ventajas, es la de dar prioridad para la inclusión de los estudiantes, docentes y padres, lo que hace el proceso más justo y colectivo. Sin embargo, también tiene limitaciones: requiere bastante tiempo y organización, y los resultados dependen de quienes participan, lo que puede volverlo un poco subjetivo. Considero que esta herramienta no solo es útil en matemáticas, sino que puede aplicarse a otras áreas en mi caso la educación fisica o incluso a temas de convivencia escolar y gestión institucional. En mi caso, pienso que podría usarla para analizar la baja participación de los estudiantes en clase, pues al identificar las causas más importantes, como el miedo a equivocarse, sería posible diseñar estrategias para mejorar la confianza y lograr que más alumnos participen. En conclusión, la Matriz de Vester es una herramienta práctica que ayuda a actuar de manera más organizada frente a los problemas o situaciones educativas, favoreciendo decisiones más claras y efectivas.

    1. La investigación buscó identificar los principales problemas en el aprendizaje de las matemáticas y, para ello, utilizó la Matriz de Vester como herramienta para organizar y priorizar las dificultades. Me parece interesante cómo esta matriz me permitió identificar la falta de atención en clase y el abuso de recursos repetitivos, estos son los problemas que evidencie y me parecieron más relevantes por esto mismo, los que debían resolverse con prioridad. sus principales ventajas, es la de dar prioridad para la inclusión de los estudiantes, docentes y padres, lo que hace el proceso más justo y colectivo. Sin embargo, también tiene limitaciones: requiere bastante tiempo y organización, y los resultados dependen de quienes participan, lo que puede volverlo un poco subjetivo. Considero que esta herramienta no solo es útil en matemáticas, sino que puede aplicarse a otras áreas en mi caso la educación fisica o incluso a temas de convivencia escolar y gestión institucional. En mi caso, pienso que podría usarla para analizar la baja participación de los estudiantes en clase, pues al identificar las causas más importantes, como el miedo a equivocarse, sería posible diseñar estrategias para mejorar la confianza y lograr que más alumnos participen. En conclusión, la Matriz de Vester es una herramienta práctica que ayuda a actuar de manera más organizada frente a los problemas o situaciones educativas, favoreciendo decisiones más claras y efectivas.

    1. ӧ#4 ӧ4>ӧk ӧ)%ӧ(*ӧ ӧ.ō

      It is because of the notion of responsibility. Adults have more of such, so they lose the value of play compared to children

    Tags

    Annotators

    1. "Sentimientos de impotencia, inmovilidad y congelación. Si la hiperactivación es el acelerador del sistema nervioso, una sensación de impotencia abrumadora es su freno. La impotencia que se experimenta en esos momentos no es la sensación ordinaria de impotencia que puede afectar a cualquiera de vez en cuando. Es la sensación de estar colapsado, inmovilizado y completamente indefenso. No es una percepción, creencia o un truco de la imaginación. Es real". ― Peter A. Levine, Sanando el trauma: un programa pionero para restaurar la sabiduría de su cuerpo

      ,

    1. La naturaleza puede enseñar a la humanidad una o dos cosas sobre la curación del trauma y los trastornos por estrés. Es cierto que la investigación científica reciente ha sido fundamental para ayudar a eliminar parte del estigma asociado al trauma, mientras que los nuevos estudios y tratamientos inspiran esperanza para el alivio de los pacientes que sufren un trauma. Sin embargo, la psiquiatría no ha captado la esencia natural del trauma, ni ha cubierto si se puede curar y cómo hacerlo.

      .

  3. www.planalto.gov.br www.planalto.gov.br
    1. todas as Unidades

      Ou seja, é preciso unanimidade para a aprovação de benefícios fiscais em reuniões.

      No entanto, não será eficaz enquanto não for retificado por todos os Poderes Executivos expressa ou tacitamente. Acaso não haja a ratificação de todas as Unidades da Federação, será tido por rejeitado o convênio.

  4. www.planalto.gov.br www.planalto.gov.br
    1. Author response:

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

      Reviewer #1 (Public review): 

      Summary: 

      McDougal et al. aimed to characterize the antiviral activity of mammalian IFIT1 orthologs. They first performed three different evolutionary selection analyses within each major mammalian clade and identified some overlapping positive selection sites in IFIT1. They found that one site that is positively selected in primates is in the RNA-binding exit tunnel of IFIT1 and is tolerant of mutations to amino acids with similar biochemical properties. They then tested 9 diverse mammalian IFIT1 proteins against VEEV, VSV, PIV3, and SINV and found that each ortholog has distinct antiviral activities. Lastly, they compared human and chimpanzee IFIT1 and found that the determinant of their differential anti-VEEV activity may be partly attributed to their ability to bind Cap0 RNA. 

      Strengths: 

      The study is one of the first to test the antiviral activity of IFIT1 from diverse mammalian clades against VEEV, VSV, PIV3, and SINV. Cloning and expressing these 39 IFIT1 orthologs in addition to single and combinatorial mutants is not a trivial task. The positive connection between anti-VEEV activity and Cap0 RNA binding is interesting, suggesting that differences in RNA binding may explain differences in antiviral activity. 

      Weaknesses: 

      The evolutionary selection analyses yielded interesting results, but were not used to inform follow-up studies except for a positively selected site identified in primates. Since positive selection is one of the two major angles the authors proposed to investigate mammalian IFIT1 orthologs with, they should integrate the positive selection results with the rest of the paper more seamlessly, such as discussing the positive selection results and their implications, rather than just pointing out that positively selected sites were identified. The paper should elaborate on how the positive selection analyses PAML, FUBAR, and MEME complement one another to explain why the tests gave them different results. Interestingly, MEME which usually provides more sites did not identify site 193 in primates that was identified by both PAML and FUBAR. The authors should also provide the rationale for choosing to focus on the 3 sites identified in primates only. One of those sites, 193, was also found to be positively selected in bats, although the authors did not discuss or integrate that finding into the study. In Figure 1A, they also showed a dN/dS < 1 from PAML, which is confusing and would suggest negative selection instead of positive selection. Importantly, since the authors focused on the rapidly evolving site 193 in primates, they should test the IFIT1 orthologs against viruses that are known to infect primates to directly investigate the impact of the evolutionary arms race at this site on IFIT1 function. 

      We thank the reviewer for their assessment and for acknowledging the breadth of our dataset regarding diverse IFIT1s, number of viruses tested, and the functional data that may correlate biochemical properties of IFIT1 orthologous proteins with antiviral function. We have expanded the introduction and results sections to better explain and distinguish between PAML, FUBAR, and MEME analyses. Furthermore, we have expanded the discussion to incorporate the observation that site 193 is rapidly evolving in bats, as well as the observation that nearby sites to the TPR4 loop were identified as rapidly evolving in all clades of mammals tested. We also do observe an overall gene dN/dS of <1, however this is simply the average across all codons of the entire gene and does not rule out positive selection at specific sites. This is observed for other restriction factors, as many domains are undergoing purifying selection to retain core functions (e.g enzymatic function, structural integrity) while other domains (e.g. interfaces with viral antagonists or viral proteins) show strong positive selection. Specific examples include the restriction factors BST-2/Tetherin (PMID: 19461879) and MxA (PMID: 23084925). Furthermore, we agree that testing more IFIT1-sensitive viruses that naturally infect primates with our IFIT1 193 mutagenesis library would shed light on the influence of host-virus arms races at this site. However, VEEV naturally does also infect humans as well as at least one other species of primate (PMID: 39983680).

      Below we individually address the reviewers' claims of inaccurate data interpretation.

      Some of the data interpretation is not accurate. For example: 

      (1) Lines 232-234: "...western blot analysis revealed that the expression of IFIT1 orthologs was relatively uniform, except for the higher expression of orca IFIT1 and notably lower expression of pangolin IFIT1 (Figure 4B)." In fact, most of the orthologs are not expressed in a "relatively uniform" manner e.g. big brown bat vs. shrew are quite different. 

      We have now included quantification of the western blots to allow the reader to compare infection results with the infection data (Updated Figure 4B and 4G). We have also removed the phrase “relatively uniform” from the text and have instead included text describing the quantified expression differences.

      (2) Line 245: "...mammalian IFIT1 species-specific differences in viral suppression are largely independent of expression differences." While it is true that there is no correlation between protein expression and antiviral activity in each species, the authors cannot definitively conclude that the species-specific differences are independent of expression differences. Since the orthologs are clearly not expressed in the same amounts, it is impossible to fully assess their true antiviral activity. At the very least, the authors should acknowledge that the protein expression can affect antiviral activity. They should also consider quantifying the IFIT1 protein bands and normalizing each to GAPDH for readers to better compare protein expression and antiviral activity. The same issue is in Line 267. 

      We have now included quantification and normalization of the western blots to allow the reader to compare infection results with the infection data (Updated Figure 4B and 4G). Furthermore, we acknowledge in the text that expression differences may affect antiviral potency in infection experiments.

      (3) Line 263: "SINV... was modestly suppressed by pangolin, sheep, and chinchilla IFIT1 (Figure 4E)..." The term "modestly suppressed" does not seem fitting if there is 60-70% infection in cells expressing pangolin and chinchilla IFIT1. 

      We have modified the text to say “significantly suppressed” rather than “modestly suppressed.”

      (4) The study can be significantly improved if the authors can find a thread to connect each piece of data together, so the readers can form a cohesive story about mammalian IFIT1. 

      We appreciate the reviewer’s suggestion and have tried to make the story including more cohesive through commentary on positive selection and by using the computational analysis to first inform potential evolutionary consequences of IFIT1 functionality first by an intraspecies (human) approach, and then later an interspecies approach with diverse mammals that have great sequence diversity. Furthermore, we point out that almost all IFIT1s tested in the ortholog screen were also included in our computational analysis allowing for the potential to connect functional observations with those seen in the evolutionary analyses.

      Reviewer #2 (Public review): 

      McDougal et al. describe the surprising finding that IFIT1 proteins from different mammalian species inhibit the replication of different viruses, indicating that the evolution of IFIT1 across mammals has resulted in host speciesspecific antiviral specificity. Before this work, research into the antiviral activity and specificity of IFIT1 had mostly focused on the human ortholog, which was described to inhibit viruses including vesicular stomatitis virus (VSV) and Venezuelan equine encephalitis virus (VEEV) but not other viruses including Sindbis virus (SINV) and parainfluenza virus type 3 (PIV3). In the current work, the authors first perform evolutionary analyses on IFIT1 genes across a wide range of mammalian species and reveal that IFIT1 genes have evolved under positive selection in primates, bats, carnivores, and ungulates. Based on these data, they hypothesize that IFIT1 proteins from these diverse mammalian groups may show distinct antiviral specificities against a panel of viruses. By generating human cells that express IFIT1 proteins from different mammalian species, the authors show a wide range of antiviral activities of mammalian IFIT1s. Most strikingly, they find several IFIT1 proteins that have completely different antiviral specificities relative to human IFIT1, including IFIT1s that fail to inhibit VSV or VEEV, but strongly inhibit PIV3 or SINV. These results indicate that there is potential for IFIT1 to inhibit a much wider range of viruses than human IFIT1 inhibits. Electrophoretic mobility shift assays (EMSAs) suggest that some of these changes in antiviral specificity can be ascribed to changes in the direct binding of viral RNAs. Interestingly, they also find that chimpanzee IFIT1, which is >98% identical to human IFIT1, fails to inhibit any tested virus. Replacing three residues from chimpanzee IFIT1 with those from human IFIT1, one of which has evolved under positive selection in primates, restores activity to chimpanzee IFIT1. Together, these data reveal a vast diversity of IFIT1 antiviral specificity encoded by mammals, consistent with an IFIT1-virus evolutionary "arms race". 

      Overall, this is a very interesting and well-written manuscript that combines evolutionary and functional approaches to provide new insight into IFIT1 antiviral activity and species-specific antiviral immunity. The conclusion that IFIT1 genes in several mammalian lineages are evolving under positive selection is supported by the data, although there are some important analyses that need to be done to remove any confounding effects from gene recombination that has previously been described between IFIT1 and its paralog IFIT1B. The virology results, which convincingly show that IFIT1s from different species have distinct antiviral specificity, are the most surprising and exciting part of the paper. As such, this paper will be interesting for researchers studying mechanisms of innate antiviral immunity, as well as those interested in species-specific antiviral immunity. Moreover, it may prompt others to test a wide range of orthologs of antiviral factors beyond those from humans or mice, which could further the concept of host-specific innate antiviral specificity. Additional areas for improvement, which are mostly to clarify the presentation of data and conclusions, are described below. 

      Strengths: 

      (1) This paper is a very strong demonstration of the concept that orthologous innate immune proteins can evolve distinct antiviral specificities. Specifically, the authors show that IFIT1 proteins from different mammalian species are able to inhibit the replication of distinct groups of viruses, which is most clearly illustrated in Figure 4G. This is an unexpected finding, as the mechanism by which IFIT1 inhibits viral replication was assumed to be similar across orthologs. While the molecular basis for these differences remains unresolved, this is a clear indication that IFIT1 evolution functionally impacts host-specific antiviral immunity and that IFIT1 has the potential to inhibit a much wider range of viruses than previously described. 

      (2) By revealing these differences in antiviral specificity across IFIT1 orthologs, the authors highlight the importance of sampling antiviral proteins from different mammalian species to understand what functions are conserved and what functions are lineage- or species-specific. These results might therefore prompt similar investigations with other antiviral proteins, which could reveal a previously undiscovered diversity of specificities for other antiviral immunity proteins. 

      (3) The authors also surprisingly reveal that chimpanzee IFIT1 shows no antiviral activity against any tested virus despite only differing from human IFIT1 by eight amino acids. By mapping this loss of function to three residues on one helix of the protein, the authors shed new light on a region of the protein with no previously known function. 

      (4) Combined with evolutionary analyses that indicate that IFIT1 genes are evolving under positive selection in several mammalian groups, these functional data indicate that IFIT1 is engaged in an evolutionary "arms race" with viruses, which results in distinct antiviral specificities of IFIT1 proteins from different species. 

      Weaknesses: 

      (1) The evolutionary analyses the authors perform appear to indicate that IFIT1 genes in several mammalian groups have evolved under positive selection. However, IFIT1 has previously been shown to have undergone recurrent instances of recombination with the paralogous IFIT1B, which can confound positive selection analyses such as the ones the authors perform. The authors should analyze their alignments for evidence of recombination using a tool such as GARD (in the same HyPhy package along with MEME and FUBAR). Detection of recombination in these alignments would invalidate their positive selection inferences, in which case the authors need to either analyze individual non-recombining domains or limit the number of species to those that are not undergoing recombination. While it is likely that these analyses will still reveal a signature of positive selection, this step is necessary to ensure that the signatures of selection and sites of positive selection are accurate. 

      (2) The choice of IFIT1 homologs chosen for study needs to be described in more detail. Many mammalian species encode IFIT1 and IFIT1B proteins, which have been shown to have different antiviral specificity, and the evolutionary relationship between IFIT1 and IFIT1B paralogs is complicated by recombination. As such, the assertion that the proteins studied in this manuscript are IFIT1 orthologs requires additional support than the percent identity plot shown in Figure 3B. 

      (3) Some of the results and discussion text could be more focused on the model of evolution-driven changes in IFIT1 specificity. In particular, the chimpanzee data are interesting, but it would appear that this protein has lost all antiviral function, rather than changing its antiviral specificity like some other examples in this paper. As such, the connection between the functional mapping of individual residues with the positive selection analysis is somewhat confusing. It would be more clear to discuss this as a natural loss of function of this IFIT1, which has occurred elsewhere repeatedly across the mammalian tree. 

      (4) In other places in the manuscript, the strength of the differences in antiviral specificity could be highlighted to a greater degree. Specifically, the text describes a number of interesting examples of differences in inhibition of VSV versus VEEV from Figure 3C and 3D, but it is difficult for a reader to assess this as most of the dots are unlabeled and the primary data are not uploaded. A few potential suggestions would be to have a table of each ortholog with % infection by VSV and % infection by VEEV. Another possibility would be to plot these data as an XY scatter plot. This would highlight any species that deviate from the expected linear relationship between the inhibition of these two viruses, which would provide a larger panel of interesting IFIT1 antiviral specificities than the smaller number of species shown in Figure 4. 

      We thank the reviewer for their fair assessment of our manuscript. As the reviewer requested, we performed GARD analysis on our alignments used for PAML, FUBAR, and MEME (New Supp Fig 1). By GARD, we found 1 or 2 predicted breakpoints in each clade. However, much of the sequence was after or between the predicted breakpoints. Therefore, we were able to reanalyze for sites undergoing positive selection in the large region of the sequence that do not span the breakpoints. We were able to validate almost all sites originally identified as undergoing positive selection still exhibit signatures of positive selection taking these breakpoints into account: primates (11/12), bats (14/16), ungulates (30/37), and carnivores (2/4). To further validate our positive selection analysis, we used Recombination Detection Program 4 (RDP4) to remove inferred recombinant sequences from the primate IFIT1 alignment and performed PAML, FUBAR, and MEME. Once again, the sites in our original anlaysis were largely validated by this method. Importantly, sites 170, 193, and 366 in primates, which are discussed in our manuscript, were found to be undergoing positive selection in 2 of the 3 analyses using alignments after the indicated breakpoint in GARD and after removal of recombinant sequences by RDP4. We have updated the text to acknowledge IFIT1/IFIT1B recombination more clearly and include the GARD analysis as well as PAML, FUBAR, and MEME reanalysis taking into account predicted breakpoints by GARD and RDP4. Furthermore, to increase evidence that the sequences used in this study for both computational and functional analysis are IFIT1 orthologs rather than IFIT1B, we have included a maximum likelihood tree after aligning coding sequences on the C-terminal end (corresponding to bases 907-1437 of IFIT1). In Daughtery et al. 2016 (PMID: 27240734) this strategy was used to distinguish between IFIT1 and IFITB. All sequences used in our study grouped with IFIT1 sequences (including many confirmed IFIT1 sequences used in Daughterty et al.) rather than IFIT1B sequences or IFIT3. This new data, including the GARD, RDP4, and maximum likelihood tree is included as a new Supplementary Figure 1.

      We also agree with the reviewer that it is possible that chimpanzee IFIT1 has lost antiviral function due to the residues 364 and 366 that differ from human IFIT1. We have updated the discussion sections to include the possibility that chimpanzee IFIT1 is an example of a natural loss of function that has occurred in other species over evolution as well as the potential consequences of this occurrence. Regarding highlighting the strength of differences in antiviral activity between IFIT1 orthologs, we have included several updates to strengthen the ability of the reader to assess these differences. First, we have included a supplementary table that includes the infection data for each ortholog from the VEEV and VSV screen to allow for readers to evaluate ranked antiviral activity of the species that suppress these viruses. In addition, the silhouettes next to the dot plots indicate the top ranked hits in order of viral inhibition (with the top being the most inhibitory) giving the reader a visual representation in the figure of top antiviral orthologs during our screen. We have also updated the figure legend to inform the reader of this information.

      Reviewer #3 (Public Review):  

      Summary: 

      This manuscript by McDougal et al, demonstrates species-specific activities of diverse IFIT1 orthologs and seeks to utilize evolutionary analysis to identify key amino acids under positive selection that contribute to the antiviral activity of this host factor. While the authors identify amino acid residues as important for the antiviral activity of some orthologs and propose a possible mechanism by which these residues may function, the significance or applicability of these findings to other orthologs is unclear. However, the subject matter is of interest to the field, and these findings could be significantly strengthened with additional data.

      Strengths:

      Assessment of multiple IFIT1 orthologs shows the wide variety of antiviral activity of IFIT1, and identification of residues outside of the known RNA binding pocket in the protein suggests additional novel mechanisms that may regulate IFIT1 activity.

      Weaknesses:

      Consideration of alternative hypotheses that might explain the variable and seemingly inconsistent antiviral activity of IFIT1 orthologs was not really considered. For example, studies show that IFIT1 activity may be regulated by interaction with other IFIT proteins but was not assessed in this study.

      Given that there appears to be very little overlap observed in orthologs that inhibited the viruses tested, it's possible that other amino acids may be key drivers of antiviral activity in these other orthologs. Thus, it's difficult to conclude whether the findings that residues 362/4/6 are important for IFIT1 activity can be broadly applied to other orthologs, or whether these are unique to human and chimpanzee IFIT1. Similarly, while the hypothesis that these residues impact IFIT1 activity in an allosteric manner is an attractive one, there is no data to support this.  

      We thank the reviewer for their fair assessment of our manuscript. To address the weaknesses that the reviewer has pointed out we have expanded the discussion to more directly address alternate hypotheses, such as the possibility of IFIT1 activity being regulated by interaction with other IFIT proteins. Furthermore, we expanded the discussion to include an alternate hypothesis for the role of residues 364 and 366 in primate IFIT1 besides allosteric regulation. In addition, we did not intend to claim or imply that residues 364/6 are the key drivers of antiviral activity for all IFITs tested. However, we speculate that within primates these residues may play a key role as these residues differ between chimpanzee IFIT1 (which lacks significant antiviral activity towards the viruses tested in this study) and human IFIT1 (which possesses significant antiviral activity). In addition, these residues seem to be generally conserved in primate species, apart from chimpanzee IFIT1. We have included changes to the text to more clearly indicate that we highlight the importance of these residues specifically for primate IFIT1, but not necessarily for all IFIT1 proteins in all clades.

      Reviewer #1 (Recommendations for the authors): 

      (1) The readers would benefit from a more detailed background on the concept and estimation of positive selection for the readers, including the M7/8 models in PAML. 

      We have included more information in the text to provide a better background for the concepts of positive selection and how PAML tests for this using M7 and M8 models.

      (2) Presentation of data 

      a) Figure 3C and 3D: is there a better way to present the infection data so the readers can tell the ranked antiviral activity of the species that suppress VEEV? 

      We have included a supplementary table that includes the infection data for each ortholog from the VEEV and VSV screen to allow for readers to evaluate ranked antiviral activity of the species that suppress these viruses. In addition, the silhouettes next to the dot plots indicate the top ranked hits in order of viral inhibition (with the top being the most inhibitory). We have updated the figure legend to inform the reader of this information as well.

      b) Figure 4C and 4D: consider putting the western blot in Supplementary Figure 1 underneath the infection data or with the heatmap so readers can compare it with the antiviral activity. 

      We have also included quantification of the western blots performed to evaluate IFIT1 expression during the experiments shown in Figure 4C and 4D in an updated Figure 4B. We have also included normalized expression values with the heatmap shown in an updated Figure 4G so the reader can evaluate potential impact of protein expression on antiviral activity for all infection experiments shown in figure 4.

      (3) Line 269-270: as a rationale for narrowing the species to human, black flying fox, and chimp IFIT1, human and black flying fox were chosen because they strongly inhibit VEEV, but pangolin wasn't included even though it had the strongest anti-VEEV activity? 

      The rationale for narrowing the species to human, black flying fox, and chimpanzee IFIT1 was related to the availability of biological tools, high quality genome/transcriptome sequencing databases, and other factors. Specifically human and chimp IFIT1 are closely related but have variable antiviral activities, making their comparison highly relevant. Bats are well established as reservoirs for diverse viruses, whereas the reservoir status of many other mammals is less well defined. Furthermore, purifying large amounts of high quality IFIT1 protein after bacterial expression was another limitation to functional studies. We have added this information into the manuscript text.

      (4) Figure 5A: to strengthen the claim that "species-specific antiviral activities of IFIT1s can be partly explained by RNA binding potential", it would be good to include one more positive and one more negative control. In other words, test the cap0 RNA binding activity of an IFIT1 ortholog that strongly inhibits VEEV and an ortholog that does not. It would also be good to discuss why chimp IFIT1 still shows dose-dependent RNA binding yet it is one of the weakest at inhibiting VEEV. 

      We appreciate the reviewer's suggestion to include more controls and expand the dataset. While we understand the potential value of expanding the dataset, we believe that human IFIT1 serves as a robust positive control and human IFIT1 R187 (RNA-binding deficient) serves as an established negative control. Future experiments with other purified IFITs from other species will indeed strengthen evidence linking IFIT1 species-specific activity and RNA-binding.

      Regarding chimpanzee IFIT1, we acknowledge there appears to be some dose-dependent Cap0 RNA-binding. However, the binding affinity is much weaker than that of human or black flying fox IFIT1. We speculate that during viral infection reduced binding affinity could impair the ability of chimpanzee IFIT1 to efficiently sequester viral RNA and inhibit viral translation. This reduction in binding affinity may, therefore, allow the cell to be overwhelmed by the exponential increase in viral RNA during replication resulting in an ineffective antiviral IFIT1. In the literature, a similar phenomenon is observed by Hyde et. al (PMID: 24482115). In this study, the authors test mouse Ifit1 Cap0 RNA binding by EMSA of the 5’ UTR sequence of VEEV RNA containing an A or G at nucleotide position 3. EMSA shows binding of both the A3 and G3 Cap0 VEEV RNA sequences, however stronger Ifit1 binding is observed for A3 Cap0 RNA sequence. The consequences of the reduced Ifit1 binding of the G3 Cap0 VEEV RNA are observed in vitro by a substantial increase in viral titers produced from cells as well as an increase in protein produced in a luciferase-based translation assay. The authors also show in vivo relevance of this reduction of Ifit1 binding as WT B6 mice infected with VEEV containing the A3 UTR exhibited 100% survival, while WT B6 mice infected with VEEV containing the G3 UTR survived at a rate of only ~25%. Therefore, the literature supports that a decrease in Cap0 RNA binding by an IFIT protein (while still exhibiting Cap0 RNA binding) observed by EMSA can result in considerable alterations of viral infection both in vitro and in vivo.

      Minor: 

      (1) Line 82: "including 5' triphosphate (5'-ppp-RNA), or viral RNAs..." having a comma here will make the sentence clearer. 

      We have improved the clarity of this sentence. It now reads, “IFIT1 binds uncapped 5′triphosphate RNA (5′-ppp-RNA) and capped but unmethylated RNA (Cap0, an m<sup>7</sup>G cap lacking 2′-O methylation).”

      (2) Line 100: "...similar mechanisms have been at least partially evolutionarily conserved in IFIT proteins to restrict viral infection by IFIT proteins". 

      We have updated the text to improve clarity by revising the sentence to “VEEV TC-83 is sensitive to human IFIT1 and mouse Ifit1B, indicating at least partial conservation of antiviral function by IFIT proteins."

      (3) Line 109: "signatures of rapid evolution or positive selection" would put positive selection second because that is the more technical term that can benefit from the more layperson term (rapid evolution). 

      We have updated this sentence incorporating this suggestion. “Positive selection, or rapid evolution, is denoted by a high ratio of nonsynonymous to synonymous substitutions (dN/dS >1).”

      (4) Lines 116-117: "However, this was only assessed in a few species" would benefit from a citation. 

      We have inserted the citation.

      (5) Line 127 heading: "IFIT1 is rapidly evolving in mammals" would be more accurate to say "in major clades of mammals". 

      We have updated the text to include this suggestion.

      (6) Line 165: "IFIT1 L193 mutants". 

      We have updated the text to rephrase this for clarity.

      (7) Line 170: two strains of VEEV were mentioned in the Intro, so it would be good to specify which strain of VEEV was used?

      We have updated the text to clarify the VEEV strain. In this study, all experiments were performed using the VEEV TC-83 strain.

      (8) Line 174: "Indeed, all mutants at position 193, whether hydrophobic or positively charged, inhibited VEEV similarly to the WT..." It should read "all hydrophobic and positively charged mutants inhibited VEEV similarly to the WT...". 

      We corrected as suggested. 

      (9) Line 204: what are "control cells"? Cells that are mock-infected, or cells without IFIT1? 

      We have updated the text to improve clarity. What we refer to as control cells, were cells expressing an empty vector control rather than an IFIT1.

      (10) Need to clarify n=2 and n=3 replicates throughout the manuscript. Does that refer to three independent experiments? Or an experiment with triplicate wells/samples? 

      We have updated the text to say “independent experiments” instead of “biological replicates” to prevent any confusion.  All n=2 or n=3 replicates denote independent experiments.

      (11) Line 254: "dominant antiviral effector against the related human parainfluenza virus type 5..." 

      We have updated the text to improve clarity.

      (12) Line 271: "The black flying fox (Pteropus alecto), is a model megabat species..." scientific name was italicized here but not elsewhere. Remove comma.

      We have updated the text accordingly.

      (13) Line 293: "...chimpanzee IFIT1 lacked these properties" but chimp IFIT1 can bind cap0 RNA, just at a lower level. 

      We have updated the text to acknowledge that chimpanzee IFIT1 can bind cap0 RNA, albeit at a lower level than human IFIT1.

      (14) Figure 6B: please fix the x-axis labels. They're very cramped. 

      We have updated the x-axis labels for figure 6B and figure 6D to improve clarity.

      (15) Line 609: "...trimmed and aligned"? 

      Our phrasing is to indicate that coding sequences were aligned, and gaps were removed to reduce the chance of false positive signal by underrepresented codons such as gaps or short insertions. We have removed “trimmed” from the text and changed the text to say “aligned sequences” to increase clarity.

      Reviewer #2 (Recommendations for the authors): 

      (1) Numbers less than 10 should be spelled out throughout the manuscript (e.g. line 138). 

      We have updated the text to reflect the request.

      (2) Line 165: "expression of IFIT1 193 mutants" should be rephrased. 

      We have updated the text to rephrase this sentence for clarity.

      (3) A supplemental table or file should be included that contains the accession number and species names of sequences used for evolutionary analyses and for functional testing. In addition, the alignments that were used for positive selection can be included.  

      We have included a supplemental file containing accession numbers, species names for evolutionary analysis and functional studies. In addition, this table includes the infection data for each IFIT1 homolog for the screen performed in figure 3.

      (4) The discussion of potential functions of the C-terminus of IFIT1 should include possible interactions with other proteins. In particular, the C-terminus of IFIT1 has been shown to interact with IFIT3 in a way that modulates its activity (PMID: 29525521). Although residues 362-366 were not shown in that paper to interact with a fragment of IFIT3, it is possible that these residues may be important for interaction with full-length IFIT3 or some other IFIT1 binding partner. 

      We thank the reviewer for their suggestion. We have expanded the discussion to explore the possibility that residues 364 and 366 of IFIT1 may be involved in IFIT1-IFIT3 interactions and consequently Cap0 RNA-binding and antiviral activity.

      (5) The quantification of the EMSAs should be described in more detail. In particular, from looking at the images shown in Figure 5A, it would appear that human and chimpanzee IFIT1 show similar degrees of probe shift, while the human R187H panel shows no shifting at all. However, the quantification shows chimpanzee IFIT1 as being statistically indistinguishable from human R187H. Additional information on how bands were quantified and whether they were normalized to unshifted RNA would be helpful in attempting to resolve this visual discordance. 

      EMSAs were quantified by determining Adj. Vol. Intensity in ImageLab (BioRad), which subtracts background signal, after imaging at the same exposure and SYBR Gold staining time. To determine Adj. Vol. Intensity, we drew a box (same size for each gel and lane for each replicate) for each lane above the free probe. These values were not normalized to unshifted RNA, however equal RNA was loaded. While the ANOVA shows no significant difference, between human R187H and chimpanzee IFIT1 band shift intensity, this is potentially due to the between group variance in the ANOVA. The increase in the AUC value for chimpanzee IFIT1 is 36.4% higher than R187H.

      The AUC of Adj. Vol. Intensity of human IFIT1 band shift is roughly 2-fold more than that of chimpanzee IFIT1. We believe this matches with the visual representation as well, as human IFIT1 has a darker “upper” band in the shift, as well as a clear dark “lower” band that is not well defined in the chimpanzee shift. Furthermore, the upper band of the chimpanzee IFIT1 shift appears to be as intense in the 400nM as the upper band in the 240nM human IFIT1 lane, without taking into account the lower band seen for human IFIT1 as well. We included this quantification as kD was unable to be calculated due to no clear probe disappearance and we do not intend for this quantification to act as a substitute for binding affinity calculations, rather to aid the reader in data interpretation.

      Reviewer #3 (Recommendations for the authors): 

      (1) IFIT1 has been demonstrated to function in conjunction with other IFIT proteins, do you think the absence of antiviral activity is due to isolated expression of IFIT1 without these cofactors, and therefore might explain why there was little overlap observed in orthologs that inhibited the viruses tested (Figure 3, lines 209-210). 

      We do not believe that isolated expression of IFIT1 without cofactors (such as orthologous IFIT proteins) would fully explain the disparities in antiviral activity as many IFIT1s that expressed inhibited either VSV or VEEV in our screen. However, we acknowledge that the expression of IFIT1 alone does create a limitation in our study as IFIT1 antiviral activity and RNA-binding can be modulated by interactions with other IFIT proteins. Therefore, we do believe that it is possible that co-expression of IFIT1 with other IFITs from a given species might potentially enhance antiviral activity. Future studies may shed light on this.

      (2) Figure 5 - Calculating the Kd for each protein would be more informative. How does the binding affinity of these IFIT1 proteins compare to that which has previously been reported? 

      We are unable to accurately determine kD as there is not substantial diminished signal of the free probe. Therefore, we are only able to compare IFIT1 protein binding between species without accurate mathematical calculation of binding affinity. Our result does appear similar to that of mouse Ifit1 binding to VEEV RNA (PMID: 24482115), in which the authors also do not calculate a kD for their RNA EMSA.

      (3) Mutants 364 and 366 may not have direct contact with RNA, but RNA EMSA data presented suggest that the binding affinity may be different (though this is hard to conclude without Kd data). Additional biochemical data with these mutants might provide more insight here. 

      We agree that further studies using 364 and 366 double mutant human and chimpanzee protein in EMSAs would provide additional biochemical data and provide insight into the role of these residues in direct RNA binding. We acknowledge this is a limitation of our study as we provide only genetic data demonstrating the importance of these residues.

      (4) Given that there appears to be very little overlap observed in orthologs that inhibited the viruses tested, it's possible that other amino acids may be key drivers of antiviral activity in these other orthologs. Thus, it's difficult to conclude whether the findings that residues 362/4/6 are important for IFIT1 activity can be broadly applied to other orthologs. A more systematic assessment of the role of these mutations across multiple diverse orthologs would provide more insight here. Do other antiviral proteins show this trend (ie exhibit little overlap in orthologs that inhibit these viruses). What do you think might be driving this? 

      We agree that other residues outside of 364 and 366 may be key drivers of antiviral activity across the IFTI1 orthologs tested. We do not hypothesize that this will broadly apply across IFIT1 from diverse clades of mammals as overall amino acid identity can differ by over 30%. However, based on the chimpanzee and human IFIT1 data, as well as sequence alignment within primates specifically, we believe these residues may be key for primate (but not necessarily other clades of mammals) IFIT1 antiviral activity.

      Regarding if other antiviral proteins show little overlap in orthologs that inhibit a given virus, to our knowledge such a functional study with this large and divergent dataset of orthologs has not been performed. However, there are many examples of restriction factors exhibiting speciesspecific antiviral activity when ortholog screens have been performed. For example, HIV was reported to be suppressed by MX2 orthologs from human, rhesus macaque, and African green monkey, but not sheep or dog MX2 (PMID: 24760893). In addition, foamy virus was inhibited by the human and rhesus macaque orthologs of PHF11, but not the mouse and feline orthologs (PMID: 32678836). Furthermore, studies from our lab have shown variability in RTP4 ortholog antiviral activity inhibition towards viruses much as hepatitis C virus (HCV), West Nile virus (WNV), and Zika virus (ZIKV) (PMID: 33113352).

    1. Critica 1: "Onwuegbuzie y Leech (2005) añaden que actualmente los estudios que utilizan un solo método de investigación se convierten en una amenaza para el adelanto de las Ciencias Sociales".

      Se menciona que si un estudio es llevado a cabo bajo una sola metodología de investigación se considera una amenaza para las ciencias sociales. Pero yo creo que si un estudio se lleva a cabo bajo una sola metodología es porque esa metodología es la indicada para ejecutarse y cumplir los objetivos que se requieren.

      Critica 2: "Brown (2014) afirma que tomar decisiones correctas con respecto al tipo de instrumentos que se van a utilizar en una investigación puede requerir de mucho tiempo y consideración. Por otro lado, a más de decidir qué instrumentos darán a conocer información confiable, es necesario buscar la forma de reunir datos que finalmente sean fáciles y eficientes al momento de recopilarlos y analizarlos. "

      Critica 3: "Seidman (1998) recomienda a los investigadores que administren una secuencia de tres entrevistas con los mismos participantes para obtener suficiente información. La primera entrevista se aplica con el propósito de romper el hielo y crear un ambiente de empatía, a la vez que se realiza un rápido barrido de las áreas que serán investigadas posteriormente".

      Al momento de realizar 2 o más entrevistas a los mismos participantes, se puede llegar a dar el caso de que los participantes se empiecen a presentar fatiga por el tiempo que lleva respondiendo preguntas, es por esto que esta opción a mí consideración puede que no sea muy eficiente dependiendo el tipo de persona.

      Critica 4: " Luego del respectivo análisis de las transcripciones de las dos primeras entrevistas, el propósito de la tercera, según Seidman (1998), es cubrir ciertos temas que no fueron tratados en las sesiones previas"

      El realizar una tercera entrevista para abaracra temas que no fueron tocados creo que no es la mejor opción, ya que el tema que no fue abordado se pudiera abordar en las 2 entrevistas anteriores para evitar una sobrecarga en el entrevistado.

      Critica 5: "Mackey y Gass (2005) recomiendan a los investigadores novatos o principiantes que antes de conducir un análisis estadístico de datos, se familiaricen con la estadística de su estudio a través de cursos, textos o consultorías con expertos en el área".

      En esta parte el autor tiene razón en que los investigadores novatos que trabajan con datos estadisiticos previamente indaguen en la estadistica para una mejor interpretación de datos.

    2. 5

      Lo que el autor menciona sobre los cambios e innovación para realizar la recolección de datos cuantitativos hacen que las computadoras o software especializados nos permita realizarlo de una manera más eficiente

    3. reseña 5 el texto está bien para motivar, pero en algunos momentos se repite mucho con lo mismo de la importancia de la validez y la confiabilidad. Está padre que lo digan, pero hubiera estado mejor que mostraran un caso concreto o una mini guía paso a paso. Como que a ratos parece más un repaso de conceptos que un apoyo práctico para principiantes.

    4. Tenemos que conocer que tipos de instrumentos o herramientas (cuestionarios, entrevistas, segmentación, etc.) vamos a utilizar ya estos nos van a dar información más certera acerca del problema o tema que estemos tratando, gracias a estos instrumentos tendremos datos cualitativos o cuantitativos.

    5. Concuerdo que siempre tenemos que tener a una cierta población o hacer un segmento de mercado para que nuestra investigación sea más eficaz y más detallada

    6. Critica 4.

      la exploración de datos en el análisis cualitativo involucra una lectura completa de toda la información con el propósito de desarrollar un entendimiento general de la base de datos. Aunque los datos cualitativos son un poco complejos por las ideas principales o iniciales que se deben plantear

    7. Seidman, considera contar con tres entrevistas, siento que muchos autores no consideraron mucho el sesgo, teniendo en cuanta el amplio espectro de búsqueda de respuestas. El hecho de que la mayoría tiene una forma de entrevista o recolección de datos muy compleja me hace pensar más que sera una prueba y error y no como tal un descubrimiento, buscar efectividad es bueno, pero hacer pensar que tu modelo es el correcto, es el peor error humano posible.

    8. Critica 3.

      Aunque en el artículo hagan varias recomendaciones se centran en muchos datos, aunque su propósito es determinar la orientación de estos datos, saber si es apropiado para el tipo de análisis de que este buscando que hago observación de que no hay mucho énfasis en esos tipos o como identificarlos

    9. Creswell y Plano Clark destacan la rigurosidad necesaria en la recolección de datos dentro de la metodología mixta, se corre el riesgo de que el proceso se vuelva demasiado complejo y demandante para el investigador. Coordinar de manera exhaustiva los procedimientos cuantitativos y cualitativos no solo exige más tiempo y recursos, sino que también puede generar dificultades en la integración final de los datos, lo cual pone en cuestión la viabilidad práctica de este enfoque cuando no se cuenta con una planeación clara o con las competencias suficientes.

    10. Concuerdo que ambos métodos tienen que ir de la mano ya que buscan el conocimiento y la comprensión de un tema en especifico o buscarle una solución a un problema. Al analizar los datos es contribuir al conocimiento

    11. Los autores destacan las ventajas de cuestionarios y entrevistas como instrumentos de recolección de datos, pero no profundizan en sus limitaciones. La elección de instrumentos no debería centrarse solo en la accesibilidad o versatilidad, sino también en la calidad y pertinencia de los resultados que se esperan obtener.

    12. Aunque resaltan la validez de trabajar con muestras pequeñas en la investigación cualitativa, pueden tener limitaciones. Un número reducido de participantes puede introducir sesgos o dejar fuera perspectivas relevantes del fenómeno estudiado. Además, la falta de criterios claros y uniformes sobre el tamaño muestral en estudios cualitativos puede afectar y la comparabilidad entre investigaciones.

    13. POST 3, PIENSO DIFERENTE A LORD RUTHERFORD YA QUE SI BIEN LOS DATOS CUANTITATIVOS SON HASTA CIERTA PARTE EXACTOS, NO QUIERE DECIR QUE LOS OTROS TIPOS DE INVESTIGACIÓN NO LO SEAN O QUE SEAN MENOS IMPORTANTES, YA QUE SIRVEN PARA DIFERENTES TIPOS DE INVESTIGACIONES Y TODAS SON BUENAS DEPENDEINDO DONDE LAS DESENVUELVAS

    14. POST 2. AÚN EN EL METODO CIENTIFICO PUEDE HABER SESGO PORQUE LOS INVESTIGADORES OSMOS SERES HUMANOS, TENEMOS CREENCIAS, PREFERENCIAS Y SOMO SECEPTIBLES, ASI QUE NO ES 100% CONFIABLE O VERIDICO

    15. Los autores presentan la triangulación casi como una garantía de validez, cuando en la práctica no siempre es así. La convergencia de resultados puede fortalecer una investigación, pero también puede ocultar tensiones, contradicciones o aspectos únicos que cada método revela por separado. En este sentido, el énfasis excesivo en la validación podría limitar el potencial de la triangulación como herramienta para enriquecer la comprensión del fenómeno más allá de la mera confirmación de hipótesis.

    16. Post #2 Estoy totalmente de acuerdo con el pensamiento de Bernal (2013) que una investigación cualitativa no tiene que ser generalizada, bien dicen cada cabeza es un mundo y cada quien puede verlo desde un panorama o punto de vista diferente e incluso otros investigadores o lectores encuentren similitudes y apliquen lo aprendido en contextos parecidos.

    17. Considero que es relevante que se aclare un hecho importante que la mayor parte de los investigadores se vuelven profesores, pero eso no significa que sean buenos enseñando, pienso que ese campo es poco explorado, pero que debería saberse si es mejor o peor.

    1. Author response:

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

      Reviewer #1 (Public review):

      Summary:

      Parise presents another instantiation of the Multisensory Correlation Detector model that can now accept stimulus-level inputs. This is a valuable development as it removes researcher involvement in the characterization/labeling of features and allows analysis of complex stimuli with a high degree of nuance that was previously unconsidered (i.e., spatial/spectral distributions across time). The author demonstrates the power of the model by fitting data from dozens of previous experiments, including multiple species, tasks, behavioral modalities, and pharmacological interventions.

      Thanks for the kind words!

      Strengths:

      One of the model's biggest strengths, in my opinion, is its ability to extract complex spatiotemporal co-relationships from multisensory stimuli. These relationships have typically been manually computed or assigned based on stimulus condition and often distilled to a single dimension or even a single number (e.g., "-50 ms asynchrony"). Thus, many models of multisensory integration depend heavily on human preprocessing of stimuli, and these models miss out on complex dynamics of stimuli; the lead modality distribution apparent in Figures 3b and c is provocative. I can imagine the model revealing interesting characteristics of the facial distribution of correlation during continuous audiovisual speech that have up to this point been largely described as "present" and almost solely focused on the lip area.

      Another aspect that makes the MCD stand out among other models is the biological inspiration and generalizability across domains. The model was developed to describe a separate process - motion perception - and in a much simpler organism - Drosophila. It could then describe a very basic neural computation that has been conserved across phylogeny (which is further demonstrated in the ability to predict rat, primate, and human data) and brain area. This aspect makes the model likely able to account for much more than what has already been demonstrated with only a few tweaks akin to the modifications described in this and previous articles from Parise.

      What allows this potential is that, as Parise and colleagues have demonstrated in those papers since our (re)introduction of the model in 2016, the MCD model is modular - both in its ability to interface with different inputs/outputs and its ability to chain MCD units in a way that can analyze spatial, spectral, or any other arbitrary dimension of a stimulus. This fact leaves wide open the possibilities for types of data, stimuli, and tasks a simplistic, neutrally inspired model can account for.

      And so it's unsurprising (but impressive!) that Parise has demonstrated the model's ability here to account for such a wide range of empirical data from numerous tasks (synchrony/temporal order judgement, localization, detection, etc.) and behavior types (manual/saccade responses, gaze, etc.) using only the stimulus and a few free parameters. This ability is another of the model's main strengths that I think deserves some emphasis: it represents a kind of validation of those experiments, especially in the context of cross-experiment predictions (but see some criticism of that below).

      Finally, what is perhaps most impressive to me is that the MCD (and the accompanying decision model) does all this with very few (sometimes zero) free parameters. This highlights the utility of the model and the plausibility of its underlying architecture, but also helps to prevent extreme overfitting if fit correctly (but see a related concern below).

      We sincerely thank the reviewer for their thoughtful and generous comments. We are especially pleased that the core strengths of the model—its stimulus-computable architecture, biological grounding, modularity, and cross-domain applicability—were clearly recognized. As the reviewer rightly notes, removing researcher-defined abstractions and working directly from naturalistic stimuli opens the door to uncovering previously overlooked dynamics in complex multisensory signals, such as the spatial and temporal richness of audiovisual speech.

      We also appreciate the recognition of the model’s origins in a simple organism and its generalization across species and behaviors. This phylogenetic continuity reinforces our view that the MCD captures a fundamental computation with wide-ranging implications. Finally, we are grateful for the reviewer’s emphasis on the model’s predictive power across tasks and datasets with few or no free parameters—a property we see as key to both its parsimony and explanatory utility.

      We have highlighted these points more explicitly in the revised manuscript, and we thank the reviewer for their generous and insightful endorsement of the work.

      Weaknesses:

      There is an insufficient level of detail in the methods about model fitting. As a result, it's unclear what data the models were fitted and validated on. Were models fit individually or on average group data? Each condition separately? Is the model predictive of unseen data? Was the model cross-validated? Relatedly, the manuscript mentions a randomization test, but the shuffled data produces model responses that are still highly correlated to behavior despite shuffling. Could it be that any stimulus that varies in AV onset asynchrony can produce a psychometric curve that matches any other task with asynchrony judgements baked into the task? Does this mean all SJ or TOJ tasks produce correlated psychometric curves? Or more generally, is Pearson's correlation insensitive to subtle changes here, considering psychometric curves are typically sigmoidal? Curves can be non-overlapping and still highly correlated if one is, for example, scaled differently. Would an error term such as mean-squared or root mean-squared error be more sensitive to subtle changes in psychometric curves? Alternatively, perhaps if the models aren't cross-validated, the high correlation values are due to overfitting?

      The reviewer is right: the current version of the manuscript only provides limited information about parameter fitting. In the revised version of the manuscript, we included a parameter estimation and generalizability section that includes all information requested by the reviewer.

      To test whether using the MSE instead of Pearson correlation led to a similar estimated set of parameter values, we repeated the fitting using the MSE. The parameter estimated with this method (TauV, TauA, TauBim) closely followed those estimated using Pearson correlation (TauV, TauA, TauBim). Given the similarity of these results, we have chosen not to include further figures, however this analysis is now included in the new section (pages 23-24).

      Regarding the permutation test, it is expected that different stimuli produce analogous psychometric functions: after all, all studies relied on stimuli containing identical manipulation of lags. As a result, MCD population responses tend to be similar across experiments. Therefore, it is not a surprise that the permuted distribution of MCD-data correlation in Supplementary Figure 1K has a mean as high as 0.97. However, what is important is to demonstrate that the non-permuted dataset has an even higher goodness of fit. Supplementary Figure 1K demonstrates that none of the permuted stimuli could outperform the non-permuted dataset; the mean of the non-permuted distribution is 4.7 (standard deviations) above the mean of the already high  permuted distribution.

      We believe the new section, along with the present response, fully addresses the legitimate concerns of the reviewer.

      While the model boasts incredible versatility across tasks and stimulus configurations, fitting behavioral data well doesn't mean we've captured the underlying neural processes, and thus, we need to be careful when interpreting results. For example, the model produces temporal parameters fitting rat behavior that are 4x faster than when fitting human data. This difference in slope and a difference at the tails were interpreted as differences in perceptual sensitivity related to general processing speeds of the rat, presumably related to brain/body size differences. While rats no doubt have these differences in neural processing speed/integration windows, it seems reasonable that a lot of the differences in human and rat psychometric functions could be explained by the (over)training and motivation of rats to perform on every trial for a reward - increasing attention/sensitivity (slope) - and a tendency to make mistakes (compression evident at the tails). Was there an attempt to fit these data with a lapse parameter built into the decisional model as was done in Equation 21? Likewise, the fitted parameters for the pharmacological manipulations during the SJ task indicated differences in the decisional (but not the perceptual) process and the article makes the claim that "all pharmacologically-induced changes in audiovisual time perception" can be attributed to decisional processes "with no need to postulate changes in low-level temporal processing." However, those papers discuss actual sensory effects of pharmacological manipulation, with one specifically reporting changes to response timing. Moreover, and again contrary to the conclusions drawn from model fits to those data, both papers also report a change in psychometric slope/JND in the TOJ task after pharmacological manipulation, which would presumably be reflected in changes to the perceptual (but not the decisional) parameters.

      Fitting or predicting behaviour does not in itself demonstrate that a model captures the underlying neural computations—though it may offer valuable constraints and insights. In line with this, we were careful not to extrapolate the implications of our simulations to specific neural mechanisms.

      Temporal sensitivity is, by definition, a behavioural metric, and—as the reviewer correctly notes—its estimation may reflect a range of contributing factors beyond low-level sensory processing, including attention, motivation, and lapse rates (i.e., stimulus-independent errors). In Equation 21, we introduced a lapse parameter specifically to account for such effects in the context of monkey eye-tracking data. For the rat datasets, however, the inclusion of a lapse term was not required to achieve a close fit to the psychometric data (ρ = 0.981). While it is likely that adding a lapse component would yield a marginally better fit, the absence of single-trial data prevents us from applying model comparison criteria such as AIC or BIC to justify the additional parameter. In light of this, and to avoid unnecessary model complexity, we opted not to include a lapse term in the rat simulations.

      With respect to the pharmacological manipulation data, we acknowledge the reviewer’s point that observed changes in slope and bias could plausibly arise from alterations at either the sensory or decisional level—or both. In our model, low-level sensory processing is instantiated by the MCD architecture, which outputs the MCDcorr and MCDlag signals that are then scaled and integrated during decision-making. Importantly, this scaling operation influences the slope of the resulting psychometric functions, such that changes in slope can arise even in the absence of any change to the MCD’s temporal filters. In our simulations, the temporal constants of the MCD units were fixed to the values estimated from the non-pharmacological condition (see parameter estimation section above), and only the decision-related parameters were allowed to vary. From this modelling perspective, the behavioural effects observed in the pharmacological datasets can be explained entirely by changes at the decisional level. However, we do not claim that such an explanation excludes the possibility of genuine sensory-level changes. Rather, we assert that our model can account for the observed data without requiring modifications to early temporal tuning.

      To rigorously distinguish sensory from decisional effects, future experiments will need to employ stimuli with richer temporal structure—e.g., temporally modulated sequences of clicks and flashes that vary in frequency, phase, rhythm, or regularity (see Fujisaki & Nishida, 2007; Denison et al., 2012; Parise & Ernst, 2016, 2025; Locke & Landy, 2017; Nidiffer et al., 2018). Such stimuli engage the MCD in a more stimulus-dependent manner, enabling a clearer separation between early sensory encoding and later decision-making processes. Unfortunately, the current rat datasets—based exclusively on single click-flash pairings—lack the complexity needed for such disambiguation. As a result, while our simulations suggest that the observed pharmacologically induced effects can be attributed to changes in decision-level parameters, they do not rule out concurrent sensory-level changes.

      In summary, our results indicate that changes in the temporal tuning of MCD units are not necessary to reproduce the observed pharmacological effects on audiovisual timing behaviour. However, we do not assert that such changes are absent or unnecessary in principle. Disentangling sensory and decisional contributions will ultimately require richer datasets and experimental paradigms designed specifically for this purpose. We have now modified the results section (page 6) and the discussion (page 11) to clarify these points.

      The case for the utility of a stimulus-computable model is convincing (as I mentioned above), but its framing as mission-critical for understanding multisensory perception is overstated, I think. The line for what is "stimulus computable" is arbitrary and doesn't seem to be followed in the paper. A strict definition might realistically require inputs to be, e.g., the patterns of light and sound waves available to our eyes and ears, while an even more strict definition might (unrealistically) require those stimuli to be physically present and transduced by the model. A reasonable looser definition might allow an "abstract and low-dimensional representation of the stimulus, such as the stimulus envelope (which was used in the paper), to be an input. Ultimately, some preprocessing of a stimulus does not necessarily confound interpretations about (multi)sensory perception. And on the flip side, the stimulus-computable aspect doesn't necessarily give the model supreme insight into perception. For example, the MCD model was "confused" by the stimuli used in our 2018 paper (Nidiffer et al., 2018; Parise & Ernst, 2025). In each of our stimuli (including catch trials), the onset and offset drove strong AV temporal correlations across all stimulus conditions (including catch trials), but were irrelevant to participants performing an amplitude modulation detection task. The to-be-detected amplitude modulations, set at individual thresholds, were not a salient aspect of the physical stimulus, and thus only marginally affected stimulus correlations. The model was of course, able to fit our data by "ignoring" the on/offsets (i.e., requiring human intervention), again highlighting that the model is tapping into a very basic and ubiquitous computational principle of (multi)sensory perception. But it does reveal a limitation of such a stimulus-computable model: that it is (so far) strictly bottom-up.

      We appreciate the reviewer’s thoughtful engagement with the concept of stimulus computability. We agree that the term requires careful definition and should not be taken as a guarantee of perceptual insight or neural plausibility. In our work, we define a model as “stimulus-computable” if all its inputs are derived directly from the stimulus, rather than from experimenter-defined summary descriptors such as temporal lag, spatial disparity, or cue reliability. In the context of multisensory integration, this implies that a model must account not only for how cues are combined, but also for how those cues are extracted from raw inputs—such as audio waveforms and visual contrast sequences.

      This distinction is central to our modelling philosophy. While ideal observer models often specify how information should be combined once identified, they typically do not address the upstream question of how this information is extracted from sensory input. In that sense, models that are not stimulus-computable leave out a key part of the perceptual pipeline. We do not present stimulus computability as a marker of theoretical superiority, but rather as a modelling constraint that is necessary if one’s aim is to explain how structured sensory input gives rise to perception. This is a view that is also explicitly acknowledged and supported by Reviewer 2.

      Framed in Marr’s (1982) terms, non–stimulus-computable models tend to operate at the computational level, defining what the system is doing (e.g., computing a maximum likelihood estimate), whereas stimulus-computable models aim to function at the algorithmic level, specifying how the relevant representations and operations might be implemented. When appropriately constrained by biological plausibility, such models may also inform hypotheses at the implementational level, pointing to potential neural substrates that could instantiate the computation.

      Regarding the reviewer’s example illustrating a limitation of the MCD model, we respectfully note that the account appears to be based on a misreading of our prior work. In Parise & Ernst (2025), where we simulated the stimuli from Nidiffer et al. (2018), the MCD model reproduced participants’ behavioural data without any human intervention or adjustment. The model was applied in a fully bottom-up, stimulus-driven manner, and its output aligned with observer responses as-is. We suspect the confusion may stem from analyses shown in Figure 6 - Supplement Figure 5 of Parise & Ernst (2025), where we investigated the lack of a frequency-doubling effect in the Nidiffer et al. data. However, those analyses were based solely on the Pearson correlation between auditory and visual stimulus envelopes and did not involve the MCD model. No manual exclusion of onset/offset events was applied, nor was the MCD used in those particular figures. We also note that Parise & Ernst (2025) is a separate, already published study and is not the manuscript currently under review. 

      In summary, while we fully agree that stimulus computability does not resolve all the complexities of multisensory perception (see comments below about speech), we maintain that it provides a valuable modelling constraint—one that enables robust, generalisable predictions when appropriately scoped. 

      The manuscript rightly chooses to focus a lot of the work on speech, fitting the MCD model to predict behavioral responses to speech. The range of findings from AV speech experiments that the MCD can account for is very convincing. Given the provided context that speech is "often claimed to be processed via dedicated mechanisms in the brain," a statement claiming a "first end-to-end account of multisensory perception," and findings that the MCD model can account for speech behaviors, it seems the reader is meant to infer that energetic correlation detection is a complete account of speech perception. I think this conclusion misses some facets of AV speech perception, such as integration of higher-order, non-redundant/correlated speech features (Campbell, 2008) and also the existence of top-down and predictive processing that aren't (yet!) explained by MCD. For example, one important benefit of AV speech is interactions on linguistic processes - how complementary sensitivity to articulatory features in the auditory and visual systems (Summerfield, 1987) allow constraint of linguistic processes (Peelle & Sommers, 2015; Tye-Murray et al., 2007).

      We thank the reviewer for their thoughtful comments, and especially for the kind words describing the range of findings from our AV speech simulations as “very convincing.”

      We would like to clarify that it is not our view that speech perception can be reduced to energetic correlation detection. While the MCD model captures low- to mid-level temporal dependencies between auditory and visual signals, we fully agree that a complete account of audiovisual speech perception must also include higher-order processes—including linguistic mechanisms and top-down predictions. These are critical components of AV speech comprehension, and lie beyond the scope of the current model.

      Our use of the term “end-to-end” is intended in a narrow operational sense: the model transforms raw audiovisual input (i.e., audio waveforms and video frames) directly into behavioural output (i.e., button press responses), without reliance on abstracted stimulus parameters such as lag, disparity or reliability. It is in this specific technical sense that the MCD offers an end-to-end model. We have revised the manuscript to clarify this usage to avoid any misunderstanding.

      In light of the reviewer’s valuable point, we have now edited the Discussion to acknowledge the importance of linguistic processes (page 13) and to clarify what we mean by end-to-end account (page 11). We agree that future work will need to explore how stimulus-computable models such as the MCD can be integrated with broader frameworks of linguistic and predictive processing (e.g., Summerfield, 1987; Campbell, 2008; Peelle & Sommers, 2015; Tye-Murray et al., 2007).

      References

      Campbell, R. (2008). The processing of audio-visual speech: empirical and neural bases. Philosophical Transactions of the Royal Society B: Biological Sciences, 363(1493), 1001-1010. https://doi.org/10.1098/rstb.2007.2155

      Nidiffer, A. R., Diederich, A., Ramachandran, R., & Wallace, M. T. (2018). Multisensory perception reflects individual differences in processing temporal correlations. Scientific Reports 2018 8:1, 8(1), 1-15. https://doi.org/10.1038/s41598-018-32673-y

      Parise, C. V, & Ernst, M. O. (2025). Multisensory integration operates on correlated input from unimodal transient channels. ELife, 12. https://doi.org/10.7554/ELIFE.90841

      Peelle, J. E., & Sommers, M. S. (2015). Prediction and constraint in audiovisual speech perception. Cortex, 68, 169-181. https://doi.org/10.1016/j.cortex.2015.03.006

      Summerfield, Q. (1987). Some preliminaries to a comprehensive account of audio-visual speech perception. In B. Dodd & R. Campbell (Eds.), Hearing by Eye: The Psychology of Lip-Reading (pp. 3-51). Lawrence Erlbaum Associates.

      Tye-Murray, N., Sommers, M., & Spehar, B. (2007). Auditory and Visual Lexical Neighborhoods in Audiovisual Speech Perception: Trends in Amplification, 11(4), 233-241. https://doi.org/10.1177/1084713807307409

      Reviewer #2 (Public review):

      Summary:

      Building on previous models of multisensory integration (including their earlier correlation-detection framework used for non-spatial signals), the author introduces a population-level Multisensory Correlation Detector (MCD) that processes raw auditory and visual data. Crucially, it does not rely on abstracted parameters, as is common in normative Bayesian models," but rather works directly on the stimulus itself (i.e., individual pixels and audio samples). By systematically testing the model against a range of experiments spanning human, monkey, and rat data, the authors show that their MCD population approach robustly predicts perception and behavior across species with a relatively small (0-4) number of free parameters.

      Strengths:

      (1) Unlike prior Bayesian models that used simplified or parameterized inputs, the model here is explicitly computable from full natural stimuli. This resolves a key gap in understanding how the brain might extract "time offsets" or "disparities" from continuously changing audio-visual streams.

      (2) The same population MCD architecture captures a remarkable range of multisensory phenomena, from classical illusions (McGurk, ventriloquism) and synchrony judgments, to attentional/gaze behavior driven by audio-visual salience. This generality strongly supports the idea that a single low-level computation (correlation detection) can underlie many distinct multisensory effects.

      (3) By tuning model parameters to different temporal rhythms (e.g., faster in rodents, slower in humans), the MCD explains cross-species perceptual data without reconfiguring the underlying architecture.

      We thank the reviewer for their positive evaluation of the manuscript, and particularly for highlighting the significance of the model's stimulus-computable architecture and its broad applicability across species and paradigms. Please find our responses to the individual points below.

      Weaknesses:

      (1) The authors show how a correlation-based model can account for the various multisensory integration effects observed in previous studies. However, a comparison of how the two accounts differ would shed light on the correlation model being an implementation of the Bayesian computations (different levels in Marr's hierarchy) or making testable predictions that can distinguish between the two frameworks. For example, how uncertainty in the cue combined estimate is also the harmonic mean of the unimodal uncertainties is a prediction from the Bayesian model. So, how the MCD framework predicts this reduced uncertainty could be one potential difference (or similarity) to the Bayesian model.

      We fully agree with the reviewer that a comparison between the correlation-based MCD model and Bayesian accounts is valuable—particularly for clarifying how the two frameworks differ conceptually and where they may converge.

      As noted in the revised manuscript, the key distinction lies in the level of analysis described by Marr (1982). Bayesian models operate at the computational level, describing what the system is aiming to compute (e.g., optimal cue integration). In contrast, the MCD functions at the algorithmic level, offering a biologically plausible mechanism for how such integration might emerge from stimulus-driven representations.

      In this context, the MCD provides a concrete, stimulus-grounded account of how perceptual estimates might be constructed—potentially implementing computations with Bayesian-like characteristics (e.g., reduced uncertainty, cue weighting). Thus, the two models are not mutually exclusive but can be seen as complementary: the MCD may offer an algorithmic instantiation of computations that, at the abstract level, resemble Bayesian inference.

      We have now updated the manuscript to explicitly highlight this relationship (pages 2 and 11). In the revised manuscript, we also included a new figure (Figure 5) and movie (Supplementary Movie 3), to show how the present approach extends previous Bayesian models for the case of cue integration (i.e., the ventriloquist effect).

      (2) The authors show a good match for cue combination involving 2 cues. While Bayesian accounts provide a direction for extension to more cues (also seen empirically, for eg, in Hecht et al. 2008), discussion on how the MCD model extends to more cues would benefit the readers.

      We thank the reviewer for this insightful comment: extending the MCD model to include more than two sensory modalities is a natural and valuable next step. Indeed, one of the strengths of the MCD framework lies in its modularity. Let us consider the MCDcorr​ output (Equation 6), which is computed as the pointwise product of transient inputs across modalities. Extending this to include a third modality, such as touch, is straightforward: MCD units would simply multiply the transient channels from all three modalities, effectively acting as trimodal coincidence detectors that respond when all inputs are aligned in time and space.

      By contrast, extending MCDlag is less intuitive, due to its reliance on opponency between two subunits (via subtraction). A plausible solution is to compute MCDlag in a pairwise fashion (e.g., AV, VT, AT), capturing relative timing across modality pairs.

      Importantly, the bulk of the spatial integration in our framework is carried by MCDcorr, which generalises naturally to more than two modalities. We have now formalised this extension and included a graphical representation in a supplementary section of the revised manuscript.

      Likely Impact and Usefulness:

      The work offers a compelling unification of multiple multisensory tasks- temporal order judgments, illusions, Bayesian causal inference, and overt visual attention - under a single, fully stimulus-driven framework. Its success with natural stimuli should interest computational neuroscientists, systems neuroscientists, and machine learning scientists. This paper thus makes an important contribution to the field by moving beyond minimalistic lab stimuli, illustrating how raw audio and video can be integrated using elementary correlation analyses.

      Reviewer #1 (Recommendations for the authors):

      Recommendations:

      My biggest concern is a lack of specificity about model fitting, which is assuaged by the inclusion of sufficient detail to replicate the analysis completely or the inclusion of the analysis code. The code availability indicates a script for the population model will be included, but it is unclear if this code will provide the fitting details for the whole of the analysis.

      We thank the reviewer for raising this important point. A new methodological section has been added to the manuscript, detailing the model fitting procedures used throughout the study. In addition, the accompanying code repository now includes MATLAB scripts that allow full replication of the spatiotemporal MCD simulations.

      Perhaps it could be enlightening to re-evaluate the model with a measure of error rather than correlation? And I think many researchers would be interested in the model's performance on unseen data.

      The model has now been re-evaluated using mean squared error (MSE), and the results remain consistent with those obtained using Pearson correlation. Additionally, we have clarified which parts of the study involve testing the model on unseen data (i.e., data not used to fit the temporal constants of the units). These analyses are now included and discussed in the revised fitting section of the manuscript (pages 23-24).

      Otherwise, my concerns involve the interpretation of findings, and thus could be satisfied with minor rewording or tempering conclusions.

      The manuscript has been revised to address these interpretative concerns, with several conclusions reworded or tempered accordingly. All changes are marked in blue in the revised version.

      Miscellanea:

      Should b0 in equation 10 be bcrit to match the below text?

      Thank you for catching this inconsistency. We have corrected Equation 10 (and also Equation 21) to use the more transparent notation bcrit instead of b0, in line with the accompanying text.

      Equation 23, should time be averaged separately? For example, if multiple people are speaking, the average correlation for those frames will be higher than the average correlation across all times.

      We thank the reviewer for raising this thoughtful and important point. In response, we have clarified the notation of Equation 23 in the revised manuscript (page 20). Specifically, we now denote the averaging operations explicitly as spatial means and standard deviations across all pixel locations within each frame.

      This equation computes the z-score of the MCD correlation value at the current gaze location, normalized relative to the spatial distribution of correlation values in the same frame. That is, all operations are performed at the frame level, not across time. This ensures that temporally distinct events are treated independently and that the final measure reflects relative salience within each moment, not a global average over the stimulus. In other words, the spatial distribution of MCD activity is re-centered and rescaled at each frame, exactly to avoid the type of inflation or confounding the reviewer rightly cautioned against.

      Reviewer #2 (Recommendations for the authors):

      The authors have done a great job of providing a stimulus computable model of cue combination. I had just a few suggestions to strengthen the theoretical part of the paper:

      (1) While the authors have shown a good match between MCD and cue combination, some theoretical justification or equivalence analysis would benefit readers on how the two relate to each other. Something like Zhang et al. 2019 (which is for motion cue combination) would add to the paper.

      We agree that it is important to clarify the theoretical relationship between the Multisensory Correlation Detector (MCD) and normative models of cue integration, such as Bayesian combination. In the revised manuscript, we have now modified the introduction and added a paragraph in the Discussion addressing this link more explicitly. In brief, we see the MCD as an algorithmic-level implementation (in Marr’s terms) that may approximate or instantiate aspects of Bayesian inference.

      (2) Simulating cue combination for tasks that require integration of more than two cues (visual, auditory, haptic cues) would more strongly relate the correlation model to Bayesian cue combination. If that is a lot of work, at least discussing this would benefit the paper

      This point has now been addressed, and a new paragraph discussing the extension of the MCD model to tasks involving more than two sensory modalities has been added to the Discussion section.

  5. physerver.hamilton.edu physerver.hamilton.edu
    1. found in every case t h e originalcharge o n t h e d r o p a n exact m u l t i p l e o f t h esmallest charge w h i c h w e f o u n d t h a t t h e d r o pc a u g h t f r o m t h e air.

      Kind of gives me chills reading about quantization of charge from the people who first discovered it...

    1. eLife Assessment

      This is an important study that takes a key step towards understanding developmental disorders linked to mutations in the O-GlcNAc transferase enzyme by generating a mouse model harboring the C921Y mutation. The study thoroughly examines behavioral and anatomical differences in these mice and finds behavioral hyperactivity and learning/memory deficits, as well as phenotypic differences in skull and brain formation. However, the experimental evidence is incomplete owing to discrepancy in OGT protein/RNA levels in the C921Y mutant mice in this paper and the previous paper ("Neurodevelopmental defects in a mouse model of O-GlcNAc transferase intellectual disability "). This line of research will benefit from investigation of the differences in associated glycoproteins and mechanistic insights. This study will be of interest to those studying neurodevelopment, learning and behavior, or associated brain mechanisms.

    2. Reviewer #1 (Public review):

      This study established a C921Y OGT-ID mouse model, systematically demonstrating in mammals the pathological link between O-GlcNAc metabolic imbalance and neurodevelopmental disorders (cortical malformation, microcephaly) as well as behavioral abnormalities (hyperactivity, impulsivity, learning/memory deficits). However, critical flaws in the current findings require resolution to ensure scientific rigor.

      The most concerning finding appears in Figure S12. While Supplementary Figure S12 demonstrates decreased OGA expression without significant OGT level changes in C921Y mutants via Western blot/qPCR, previous reports (Florence Authier, et al., Dis Model Mech. 2023) described OGT downregulation in Western blot and an increase in qPCR in the same models. The opposite OGT expression outcomes in supposedly identical mouse models directly challenge the model's reliability. This discrepancy raises serious concerns about either the experimental execution or the interpretation of results. The authors must revalidate the data with rigorous controls or provide a molecular biology-based explanation.

      A few additional comments to the author may be helpful to improve the study.

      Major

      (1) While this study systematically validated multi-dimensional phenotypes (including neuroanatomical abnormalities and behavioral deficits) in OGT C921Y mutant mice, there is a lack of relevant mechanisms and intervention experiments. For example, the absence of targeted intervention studies on key signaling pathways prevents verification of whether proteomics-identified molecular changes directly drive phenotypic manifestations.

      (2) Although MRI detected nodular dysplasia and heterotopia in the cingulate cortex, the cellular basis remains undefined. Spatiotemporal immunofluorescence analysis using neuronal (NeuN), astrocytic (GFAP), and synaptic (Synaptophysin) markers is recommended to identify affected cell populations (e.g., radial glial migration defects or intermediate progenitor differentiation abnormalities).

      (3) While proteomics revealed dysregulation in pathways including Wnt/β-catenin and mTOR signaling, two critical issues remain unresolved: a) O-GlcNAc glycoproteomic alterations remain unexamined; b) The causal relationship between pathway changes and O-GlcNAc imbalance lacks validation. It is recommended to use co-immunoprecipitation or glycosylation sequencing to confirm whether the relevant proteins undergo O-GlcNAc modification changes, identify specific modification sites, and verify their interactions with OGT.

      (4) Given that OGT-ID neuropathology likely originates embryonically, we recommend serial analyses from E14.5 to P7 to examine cellular dynamics during critical corticogenesis phases.

      (5) The interpretation of Figure 8A constitutes overinterpretation. Current data fail to conclusively demonstrate impairment of OGT's protein interaction network and lack direct evidence supporting the proposed mechanisms of HCF1 misprocessing or OGA loss.

    3. Reviewer #2 (Public review):

      Summary:

      The authors are trying to understand why certain mutants of O-GlcNAc transferase (OGT) appear to cause developmental disorders in humans. As an important step towards that goal, the authors generated a mouse model with one of these mutations that disrupts OGT activity. They then go on to test these mice for behavioral differences, finding that the mutant mice exhibit some signs of hyperactivity and differences in learning and memory. They then examine alterations to the structure of the brain and skull, and again find changes in the mutant mice that have been associated with developmental disorders. Finally, they identify proteins that are up- or down-regulated between the two mice as potential mechanisms to explain the observations.

      Strengths:

      The major strength of this manuscript is the creation of this mouse model, as a key step in beginning to understand how OGT mutants cause developmental disorders. This line will prove important for not only the authors but other investigators as well, enabling the testing of various hypotheses and potentially treatments. The experiments are also rigorously performed, and the conclusions are well supported by the data.

      Weaknesses:

      The only weakness identified is a lack of mechanistic insight. However, this certainly may come in the future through more targeted experimentation using this mouse model.

    4. Author response:

      Reviewer #1 (Public review):

      This study established a C921Y OGT-ID mouse model, systematically demonstrating in mammals the pathological link between O-GlcNAc metabolic imbalance and neurodevelopmental disorders (cortical malformation, microcephaly) as well as behavioral abnormalities (hyperactivity, impulsivity, learning/memory deficits). However, critical flaws in the current findings require resolution to ensure scientific rigor.

      The most concerning finding appears in Figure S12. While Supplementary Figure S12 demonstrates decreased OGA expression without significant OGT level changes in C921Y mutants via Western blot/qPCR, previous reports (Florence Authier, et al., Dis Model Mech. 2023) described OGT downregulation in Western blot and an increase in qPCR in the same models. The opposite OGT expression outcomes in supposedly identical mouse models directly challenge the model's reliability. This discrepancy raises serious concerns about either the experimental execution or the interpretation of results. The authors must revalidate the data with rigorous controls or provide a molecular biology-based explanation.

      The referee’s assessment is based on a misunderstanding – these are certainly not the same experiment repeated twice with different answers. In the previous report of the OGT-C921Y mutant mice (Florence Authier, et al., Dis Model Mech. 2023), OGT and OGA mRNA/protein expression have been assessed in total brain protein extract from 3 months old male mice. In that study we observed a significant reduction in OGT protein levels while OGT mRNA levels were significantly increased in the mutant compared to WT controls. However, in our the current study (Figure S12), OGA and OGT mRNA/protein expression have been a) restricted to the pre-frontal cortex and b) are from 4 months old male mice, which does not allow a direct comparison of the two studies. In the pre-frontal cortex, OGT protein levels are not changed while OGT mRNA levels are increased (similarly to the total brain data), albeit not significantly. The different outcomes of OGT protein levels in both total brain and prefrontal cortex could suggest regional differences in OGT protein levels/stability as OGT mRNA levels are increased in both cases. Three other brain regions (hippocampus, striatum and cerebellum) have now also been assessed for OGT mRNA/protein expression, supporting such regional differences in OGT protein levels and these data will be included in the new version of the manuscript.

      A few additional comments to the author may be helpful to improve the study.

      Major

      (1) While this study systematically validated multi-dimensional phenotypes (including neuroanatomical abnormalities and behavioral deficits) in OGT C921Y mutant mice, there is a lack of relevant mechanisms and intervention experiments. For example, the absence of targeted intervention studies on key signaling pathways prevents verification of whether proteomics-identified molecular changes directly drive phenotypic manifestations.

      We agree with the referee that these experiments would further strenghten the work. They would, however, result in a 1-5 year delay in sharing this work with the scientific and patient communities. We will continue to work along these lines and report separately in the future.

      (2) Although MRI detected nodular dysplasia and heterotopia in the cingulate cortex, the cellular basis remains undefined. Spatiotemporal immunofluorescence analysis using neuronal (NeuN), astrocytic (GFAP), and synaptic (Synaptophysin) markers is recommended to identify affected cell populations (e.g., radial glial migration defects or intermediate progenitor differentiation abnormalities).

      We are currently performing these experiments so that they can be included in the version of record of this manuscript.

      (3) While proteomics revealed dysregulation in pathways including Wnt/β-catenin and mTOR signaling, two critical issues remain unresolved: a) O-GlcNAc glycoproteomic alterations remain unexamined; b) The causal relationship between pathway changes and O-GlcNAc imbalance lacks validation. It is recommended to use co-immunoprecipitation or glycosylation sequencing to confirm whether the relevant proteins undergo O-GlcNAc modification changes, identify specific modification sites, and verify their interactions with OGT.

      We agree with the referee that these experiments would further strenghten the work and will perform further experiments to explore whether these pathways are functionally affected. However, it is important to note that the inference that these proteins must themselves be O-GlcNAc modified is incorrect – indeed, O-GlcNAcylation of unknown protein kinase X, E3 ligase/DUB, Y or transcription factor Z could indirectly affect these pathways/proteins.

      (4) Given that OGT-ID neuropathology likely originates embryonically, we recommend serial analyses from E14.5 to P7 to examine cellular dynamics during critical corticogenesis phases.

      We agree with the referee that these experiments would further strenghten the work. They would, however, result in a significant delay in sharing this work with the scientific and patient communities. We will continue to work along these lines and report separately in the future.

      (5) The interpretation of Figure 8A constitutes overinterpretation. Current data fail to conclusively demonstrate impairment of OGT's protein interaction network and lack direct evidence supporting the proposed mechanisms of HCF1 misprocessing or OGA loss.

      For clarity, we will remove panel A from Figure 8 in the version of record – this panel was only ever meant to represent a priori hypotheses for OGT-CDG mechanisms, none of which have been either excluded or confirmed.

      Reviewer #2 (Public review):

      Summary:

      The authors are trying to understand why certain mutants of O-GlcNAc transferase (OGT) appear to cause developmental disorders in humans. As an important step towards that goal, the authors generated a mouse model with one of these mutations that disrupts OGT activity. They then go on to test these mice for behavioral differences, finding that the mutant mice exhibit some signs of hyperactivity and differences in learning and memory. They then examine alterations to the structure of the brain and skull, and again find changes in the mutant mice that have been associated with developmental disorders. Finally, they identify proteins that are up- or down-regulated between the two mice as potential mechanisms to explain the observations.

      Strengths:

      The major strength of this manuscript is the creation of this mouse model, as a key step in beginning to understand how OGT mutants cause developmental disorders. This line will prove important for not only the authors but other investigators as well, enabling the testing of various hypotheses and potentially treatments. The experiments are also rigorously performed, and the conclusions are well supported by the data.

      Weaknesses:

      The only weakness identified is a lack of mechanistic insight. However, this certainly may come in the future through more targeted experimentation using this mouse model.

      We agree with the referee that these experiments would further strenghten the work. They would, however, result in a 1-5 year delay in sharing this work with the scientific and patient communities. We will continue to work along these lines and report separately in the future.

    1. Author response:

      The following is the authors’ response to the previous reviews

      Reviewer #1 (Public Review):

      Summary:

      Previous studies have shown that treatment with 17α-estradiol (a stereoisomer of the 17β-estradiol) extends lifespan in male mice but not in females. The current study by Li et al, aimed to identify cell-specific clusters and populations in the hypothalamus of aged male rats treated with 17α-estradiol (treated for 6 months). This study identifies genes and pathways affected by 17α-estradiol in the aged hypothalamus.

      Strengths:

      Using single-nucleus transcriptomic sequencing (snRNA-seq) on the hypothalamus from aged male rats treated with 17α-estradiol they show that 17α-estradiol significantly attenuated age-related increases in cellular metabolism, stress, and decreased synaptic activity in neurons.

      Thanks.

      Moreover, sc-analysis identified GnRH as one of the key mediators of 17α-estradiol's effects on energy homeostasis. Furthermore, they show that CRH neurons exhibited a senescent phenotype, suggesting a potential side effect of the 17α-estradiol. These conclusions are supported by supervised clustering by neuropeptides, hormones, and their receptors.

      Thanks.

      Weaknesses:

      However, the study has several limitations that reduce the strength of the key claims in the manuscript. In particular:

      (1) The study focused only on males and did not include comparisons with females. However, previous studies have shown that 17α-estradiol extends lifespan in a sex-specific manner in mice, affecting males but not females. Without the comparison with the female data, it's difficult to assess its relevance to the lifespan.

      This study was originally designed based on previous findings indicating that lifespan extension is only effective in males, leading to the exclusion of females from the analysis. The primary focus of our research was on the transcriptional changes and serum endocrine alterations induced by 17α-estradiol in aged males compared to untreated aged males. We believe that even in the absence of female subjects, the significant effects of 17α-estradiol on metabolism in the hypothalamus, synapses, and endocrine system remain evident, particularly regarding the expression levels of GnRH and testosterone. Notably, lower overall metabolism, increased synaptic activity, and elevated levels of GnRH and testosterone are strong indicators of health and well-being in males, supporting the validity of our primary conclusions. However, including female controls would enhance the depth of our findings. If female controls were incorporated, we propose redesigning the sample groups to include aged male control, aged female control, aged female treated, aged male treated, as well as young male control, young male treated, young female control, and young female treated. We regret that we cannot provide this data in the short term. Nevertheless, we believe this reviewer’s creative idea presents a valuable avenue for future research on this topic. In this study, we emphasize the role of 17α-estradiol in overall metabolism, synaptic function, GnRH, and testosterone in aged males and underscore the importance of supervised clustering of neuropeptide-secreting neurons in the hypothalamus.

      (2) It is not known whether 17α-estradiol leads to lifespan extension in male rats similar to male mice. Therefore, it is not possible to conclude that the observed effects in the hypothalamus, are linked to the lifespan extension.

      Thanks for the reminding. 17α-estradiol was reported to extend lifespan in male rats similar to male mice (PMID: 33289482). We have added the valuable reference to introduction in the new version.  

      (3) The effect of 17α-estradiol on non-neuronal cells such as microglia and astrocytes is not well-described (Figure 1). Previous studies demonstrated that 17α-estradiol reduces microgliosis and astrogliosis in the hypothalamus of aged male mice. Current data suggest that the proportion of oligo, and microglia were increased by the drug treatment, while the proportions of astrocytes were decreased. These data might suggest possible species differences, differences in the treatment regimen, or differences in drug efficiency. This has to be discussed.

      We have reviewed reports describing changes in cell numbers following 17α-estradiol treatment in the brain, using the keywords "17α-estradiol," "17alpha-estradiol," and "microglia" or "astrocyte." Only a limited amount of data was obtained. We found one article indicating that 17α-estradiol treatment in Tg (AβPP(swe)/PS1(ΔE9)) model mice resulted in a decreased microglial cell number compared to the placebo (AβPP(swe)/PS1(ΔE9) mice), but this change was not significant when compared to the non-transgenic control (PMID: 21157032). The transgenic AβPP(swe)/PS1(ΔE9) mouse model may differ from our wild-type aging rat model in this context.

      Moreover, the calculation of cell numbers was based on visual observation under a microscope across several brain tissue slices. This traditional method often yields controversial results. For example, oligodendrocytes in the corpus callosum, fornix, and spinal cord have been reported to be 20-40% more numerous in males than in females based on microscopic observations (PMID: 16452667). In contrast, another study found no significant difference in the number of oligodendrocytes between sexes when using immunohistochemistry staining (PMID: 18709647). Such discrepancies arising from traditional observational methods are inevitable.

      We believe the data presented in this article are reliable because the cell number and cell ratio data were derived from high-throughput cell counting of the entire hypothalamus using single-cell suspension and droplet wrapping (10x Genomics).

      (4) A more detailed analysis of glial cell types within the hypothalamus in response to drugs should be provided.

      We provided more enrichment analysis data of differentially expressed genes between Y, O, and O.T in microglia and astrocytes in Figure 2—figure supplement 3. In this supplemental data, we found unlike that in neurons, Micro displayed lower levels of synapse-related cellular processes in O.T. compared to O.

      (5) The conclusion that CRH neurons are going into senescence is not clearly supported by the data. A more detailed analysis of the hypothalamus such as histological examination to assess cellular senescence markers in CRH neurons, is needed to support this claim.

      We also noted the inappropriate claim and have changed "senescent phenotype" to "stressed phenotype" and "abnormal phenotype" in both the abstract and results sections. The stressed phenotype could be induced by heightened functional activity in the cells, potentially indicating higher cellular activity. The GnRH and CRH neurons discussed in this paper may represent such a case, as illustrated by the observed high serum GnRH, testosterone, and cortisol levels. This revision suggestion is highly valuable and constructive for our understanding of the unique physiological characteristics revealed by these data.

      Reviewer #2 (Public Review):

      Summary:

      Li et al. investigated the potential anti-ageing role of 17α-Estradiol on the hypothalamus of aged rats. To achieve this, they employed a very sophisticated method for single-cell genomic analysis that allowed them to analyze effects on various groups of neurons and non-neuronal cells. They were able to sub-categorize neurons according to their capacity to produce specific neurotransmitters, receptors, or hormones. They found that 17α-Estradiol treatment led to an improvement in several factors related to metabolism and synaptic transmission by bringing the expression levels of many of the genes of these pathways closer or to the same levels as those of young rats, reversing the ageing effect. Interestingly, among all neuronal groups, the proportion of Oxytocin-expressing neurons seems to be the one most significantly changing after treatment with 17α-Estradiol, suggesting an important role of these neurons in mediating its anti-ageing effects. This was also supported by an increase in circulating levels of oxytocin. It was also found that gene expression of corticotropin-releasing hormone neurons was significantly impacted by 17α-Estradiol even though it was not different between aged and young rats, suggesting that these neurons could be responsible for side effects related to this treatment. This article revealed some potential targets that should be further investigated in future studies regarding the role of 17α-Estradiol treatment in aged males.

      Strengths:

      (1) Single-nucleus mRNA sequencing is a very powerful method for gene expression analysis and clustering. The supervised clustering of neurons was very helpful in revealing otherwise invisible differences between neuronal groups and helped identify specific neuronal populations as targets.

      Thanks.

      (2) There is a variety of functions used that allow the differential analysis of a very complex type of data. This led to a better comparison between the different groups on many levels.

      Thanks.

      (3) There were some physiological parameters measured such as circulating hormone levels that helped the interpretation of the effects of the changes in hypothalamic gene expression

      Thanks.

      Weaknesses

      (1) One main control group is missing from the study, the young males treated with 17α-Estradiol.

      Given that the treatment period lasts six months, which extends beyond the young male rats' age range, we aimed to investigate the perturbation of 17α-Estradiol on the normal aging process. Including data from young males could potentially obscure the treatment's effects in aged males due to age effects, though similar effects between young and aged animals may exist. Long-term treatment of hormone may exert more developmental effects on the young than the old. Consequently, we decided to exclude this group from our initial sample design. We apologize for this omission.

      (2) Even though the technical approach is a sophisticated one, analyzing the whole rat hypothalamus instead of specific nuclei or subregions makes the study weaker.

      The precise targets of 17α-Estradiol within the hypothalamus remain unresolved. Selecting a specific nucleus for study is challenging. The supervised clustering method described in this manuscript allows us to identify the more sensitive neuron subtypes influenced by 17α-Estradiol and aging across the entire hypothalamus, without the need to isolate specific nuclei in a disturbed hypothalamic environment.

      (3) Although the authors claim to have several findings, the data fail to support these claims. You may mean the claim as the senescent phenotype in Crh neuron induced by 17a-estradiol.

      Thanks. We have changed the "senescent phenotype" to "stressed phenotype" in the abstract and results to avoid such claim. The stressed phenotype may be induced by heightened functional activity in the cells, potentially indicating higher cellular activity.

      (4) The study is about improving ageing but no physiological data from the study demonstrated such a claim with the exception of the testes histology which was not properly analyzed and was not even significantly different between the groups.

      The primary objective of this study is to elucidate the effects of 17α-Estradiol on the endocrine system in the aging hypothalamus; exploring anti-aging effects is not the main focus. From the characteristics of the aging hypothalamus, we know that down-regulated GnRH and testosterone levels, along with elevated mTOR signaling, are indicators of aging in these organs from previous publications (PMID: 37886966, PMID: 37048056, PMID: 22884327). The contrasting signaling networks related to metabolism and synaptic processes significantly differentiate young and aging hypothalami, and 17α-Estradiol helps rebalance these networks, suggesting its potential anti-aging effects.

      (5) Overall, the study remains descriptive with no physiological data to demonstrate that any of the effects on hypothalamic gene expression are related to metabolic, synaptic, or other functions.

      The study focuses on investigating cellular responses and endocrine changes in the aging hypothalamus induced by 17α-estradiol, utilizing single-nucleus RNA sequencing (snRNA-seq) and a novel data mining methodology to analyze various neuron subtypes. It is important to note that this study does not mainly aim to explore the anti-aging effects. Consequently, we have revised the claim in the abstract from “the effects of 17α-estradiol in anti-aging in neurons” to “the effects of 17α-estradiol on aging neurons.” We observed that the lower overall metabolism and increased expression levels of cellular processes in the synapses align with findings previously reported regarding 17α-estradiol. To address the lack of physiological data and the challenges in measuring multiple endocrine factors due to their volatile nature, we employed several bidirectional Mendelian analyses of various genome-wide association study (GWAS) data related to these serum endocrine factors to identify their mutual causal effects.

      Reviewing Editor Comment:

      Based on the Public Reviews and Recommendations for Authors, the Reviewers strongly recommend that revisions include an experimental demonstration of the physiological effects of the treatment on ageing in rats as well as the CRH-senescence link. Additional analysis of the glia would greatly strengthen the study, as would inclusion of females and young male controls. The important point was also raised that the work linking 17a-estradiol was performed in mice, and the link with lifespan in rats is not known. Discussion of this point is recommended.

      We thank the reviewers for their constructive feedback. Regarding the recommendations in the Public Reviews and Recommendations for Authors:

      a)  Physiological effects & CRH-senescence link:

      We acknowledge that 17α-estradiol has been reported to extend lifespan in male rats, consistent with findings in male mice (PMID: 33289482). This point has now been noted in the Introduction. We regret that further experimental validation of the treatment's physiological effects on aging in rats was beyond the scope of this study.

      b) Phenotype terminology:

      In response to concerns about the "senescent" characterization of CRH neurons, we have revised this terminology to "stressed phenotype" throughout the abstract and results. While we were unable to conduct additional experiments to confirm senescence markers, this revised description better reflects the heightened cellular activity observed (as evidenced by elevated serum GnRH and testosterone levels), without implying confirmed senescence.

      c) Glial cell analysis:

      To address questions about glial cell function during treatment, we have added new enrichment analysis data of differentially expressed genes in microglia and astrocytes from young (Y), old (O), and old treated (O.T) groups in Figure 2—figure supplement 3. This analysis reveals that microglia exhibit contrasting synaptic-related cellular processes compared to total neurons.

      d) Female and young controls:

      We sincerely apologize for the absence of female subjects and young male controls in the current study. The reviewers' suggestion to examine the male-specific effects of 17α-estradiol using female controls represents an excellent direction for future research, which we plan to pursue in upcoming studies.

      Reviewer #2 (Recommendations For The Authors):

      General comments:

      (1) The manuscript is very hard to read. Proofreading and editing by software or a professional seems necessary. The words "enhanced", "extensive" etc. are not always used in the right way.

      Thanks for the suggestion. We have revised the proofreading and editing. The words "enhanced" and "extensive" were also revised in most sentences.

      (2) The numbers of animals and samples are not well explained. Is it 9 rats overall or per group? If there are 8 testes samples per group, should we assume that there were 4 rats per group? The pooling of the hypothalamic how was it done? Were all the hypothalamic from each group pooled together? A small table with the animals per group and the samples would help.

      We appreciate your reminder regarding the initial mistake in our manuscript preparation. In the preliminary submission, we reported 9 rats based solely on sequencing data and data mining. The revised version (v1) now includes additional experimental data, with an effective total of 12 animals (4 per group). Unfortunately, we overlooked updating this information in the v1 submission. We have since added detailed information in the Materials and Methods sections: Animals, Treatment and Tissues, and snRNA-seq Data Processing, Batch Effect Correction, and Cell Subset Annotation.

      (3) The Clustering is wrong. There are genes in there that do not fall into any of the 3 categories: Neurotransmitters, Receptors, Hormones.

      We acknowledge the error in gene clustering and have implemented the following corrections:

      (a) The description has been updated to state: 'Vast majority of these subtypes were clustered by neuropeptides, hormones, and their receptors among all neurons.'

      (b) Genes not belonging to these three categories have been substantially removed.

      (c) The neuropeptide category (now including several growth hormones) has been expanded to 104 genes, while their corresponding receptors (including several sex hormone receptors) now comprise 105 genes.

      (4) The coloring of groups in the graphs is inconsistent. It must be more homogeneous to make it easier to identify.

      We have changed the colors of groups in Fig. 1D to make the color of cell clusters consistent in Fig. 1A-D.

      (5) The groups c1-c4 are not well explained. How did the authors come up with these?

      We have added more descriptions of c1-c4 in materials and methods in the new version.

      (6) In most cases it's not clear if the authors are talking about cell numbers that express a certain mRNA, the level of expression of a certain mRNA, or both. They need to do a better job using more precise descriptions instead of using general terms such as "signatures", "expression profiles", "affected neurons" etc. It is very hard to understand if the number of neurons is compared between the groups or the gene expression.

      We have changed the "signatures" to "gene signatures" to make it more accurate in meaning. The "affected neurons" were also changed to "sensitive neurons". But sorry that we were not able to find better alternatives to the "expression profiles".

      (7) Sometimes there are claims made without justification or a reference. For example, the claim about the senescence of CRH neurons due to the upregulation of mitochondrial genes and downregulation of adherence junction genes (lines 326-328) should be supported by a reference or own findings.

      The "senescence" here is not appropriate. We have changed it to "stressed phenotype" or "aberrant changes" in abstract and results.

      (8) Young males treated with Estradiol as a control group is necessary and it is missing.

      Your suggestion is appreciated; however, the treatment duration for aged mice (O.T) was set at 6 months, while the young mice were only 4 months old. This disparity makes it challenging to align treatment timelines for the young animals. The primary aim of this study is to investigate the perturbation of 17α-estradiol on the aging process, and any distinct effects due to age effect observed in young males might complicate our understanding of its role in aged males, though similar endocrine effects may exist in the young animals. Long-term treatment of hormone may exert more developmental effects on the young than the old. Therefore, we made the decision to exclude the young samples in our initial study design. We apologize for any confusion this may have caused.

      Specific Comments:

      Line 28: "elevated stresses and decreased synaptic activity": Please make this clearer. Can't claim changes in synaptic activity by gene expression.

      We have changed it to "the expression level of pathways involved in synapse"

      Line 32: "increased Oxytocin": serum Oxytocin.

      We have added the “serum”.

      Line 52 - 54: Any studies from rats?

      Thanks. In rats there is also reported that 17α-estradiol has similar metabolic roles as that in mice (PMID: 33289482) and we have added it to the refences. It’s very useful for this manuscript.

      Line 62 - 65: It wasn't investigated thoroughly in this paper so why was it suggested in the introduction?

      We have deleted this sentence as being suggested.

      Line 70: "synaptic activity" Same as line 28.

      We have changed it to "pathways involved in synaptic activity".

      Line 79: Why were aged rats caged alone and young by two? Could that introduce hypothalamic gene expression effects?

      The young males were bred together in peace. But the aged males will fight and should be kept alone.

      Lines 78, 99, 109-110: It is not clear how many animals per group were used and how many samples per group were used separately and/or grouped. Please be more specific.

      We have added these information to Materials and methods/Animals, treatment and tissues and Materials and methods/snRNA-seq data processing, batch effect correction, and cell subset annotation.

      Line 205: "in O" please add "versus young.".

      We have changed accordingly.

      Line 207: replace "were" with "was"

      We have alternatively changed the "proportion" to "proportions".

      Line 208: replace "that" with "compared to" and after "in O.T." add "compared to?"

      We have changed accordingly.

      Line 223: "O.T." compared to what? Figure?

      We have changed it accordingly.

      Line 227: Figure?

      We have added (Figure 1E) accordingly.

      Line 229: "synaptic activity" Same as line 28.

      We have revised it.

      Line 235: "synaptic activity" and "neuropeptide secretion" Same as line 28.

      We have revised it.

      Line 256:" interfered" please revise.

      We changed to "exerted".

      Line 263: "on the contrary" please revise.

      We have changed "on the contrary" to "opposite".

      Line 270: "conversed" did you mean "conserved"?

      We have changed "conversed" to "inversed".

      Line 296-298: Please explain. Why would these be side effects?

      It’s hard to explain, therefore, we deleted the words "side effects".

      Line 308: "synaptic activity" Same as line 28.

      We have changed it to "expression levels of synapse-related cellular processes".

      Line 314: "and sex hormone secretion and signaling"Isn't this expected?

      Yes, it is expected. We have added it to the sentence "and, as expected, sex hormone secretion and signaling".

      Line 325-328: Why is this senescence? Reference?

      We have added “potent” to it.

      Line 360-361: This doesn't show elevated synaptic activity.

      "elevated synaptic activity" was changed to "The elevated expression of synapse-related pathways"

      Line 363-364: "Unfortunately" is not a scientific expression and show bias.

      We have changed it to "Notably".

      Line 376: Similar as above.

      Yes, we have change it to "in contrast".

      Lines 382-385: This is speculation. Please move to discussion.

      Sorry for that. We think the causal effects derived from MR result is evidence. As such, we have not changed it.

      Line 389: Please revise "hormone expressing".

      We have changed it accordingly.

      Line 401: Isn't this effect expected due to feedback inhibition of the biochemical pathway? Please comment.

      The binding capability of 17alpha-estradiol to estrogen receptors and its role in transcriptional activation remain core questions surrounded by controversy. Earlier studies suggest that 17alpha-estradiol exhibits at least 200 times less activity than 17beta-estradiol (PMID: 2249627, PMID: 16024755). However, recent data indicate that 17alpha-estradiol shows comparable genomic binding and transcriptional activation through estrogen receptor α (Esr1) to that of 17beta-estradiol (PMID: 33289482). Additionally, there is evidence that 17alpha-estradiol has anti-estrogenic effects in rats (PMID: 16042770). These findings imply possible feedback inhibition via estrogen receptors. Furthermore, 17alpha-estradiol likely differs from 17beta-estradiol due to its unique metabolic consequences and its potential to slow aging in males, an effect not attributed to 17beta-estradiol. For instance, neurons are also targets of 17alpha-estradiol, with Esr1 not being the sole target (PMID: 38776045). Intriguingly, neurons expressing Ar and Esr1 ranked among the top 20 most perturbed receptor subtypes during aging (O vs Y), but were no longer ranked in this group following treatment (O.T vs Y and O.T vs O comparisons). This indicates that 17α-estradiol administration attenuated age-associated perturbation in these neuronal subtypes, which may be a consequence of potential feedback (Figure 3D). Nevertheless, the precise effective targets of 17alpha-estradiol are still unresolved.

      Line 409: This conclusion cannot be made because the effect is not statistically significant. Can say "trend" etc.

      Thanks for the recommendation. We have added "potential" in front of the conclusion.

      Line 426: "suggesting" please revise.

      sorry, it’s a verb.

      Lines 426-428: This is speculation. Please move to discussion.

      The elevated GnRH levels in O.T., observed through EIA analysis, suggest a deduction regarding the direct causal effects of 17alpha-estradiol on various endocrine factors related to feeding, energy homeostasis, reproduction, osmotic regulation, stress response, and neuronal plasticity through MR analysis. Thus, we have not amended our position. We apologize for any confusion.

      Lines 431-432: improved compared to what?

      The statement have been revised as " The most striking role of 17α-estradiol treatment revealed in this study showed that HPG axis was substantially improved in the levels of serum Gnrh and testosterone".

      Line 435: " Estrogen Receptor Antagonists". Please revise.

      Thanks for the recommendation. We have changed it to "estrogen receptor antagonists".

      Line 438" "Secrete". Please revise

      Sorry, it is "secret".

      Lines 439-449: None of this has been demonstrated. Please remove these conclusions.

      We appreciate the reviewer's scrutiny regarding lines 439-449. While these statements should not be interpreted as definitive conclusions from our current data, we propose they serve as clinically relevant discussion points worthy of exploration. Our findings demonstrate 17α-estradiol's role in modulating testosterone levels in aged males. This mechanistic insight warrants consideration of its therapeutic potential for age-related hypogonadism - a hypothesis we believe merits discussion given the compound's specific endocrine effects.

      Lines 450-457: No females were included in this study. Why? Also, why is this discussed? It is relevant but doesn't belong in this manuscript since it was not studied here.

      Testosterone levels are crucial for male health, while estradiol levels are essential for the health and fertility of females. Previous studies have demonstrated that 17α-estradiol does not contribute to lifespan extension in females. Given the effects of 17α-estradiol on males—specifically, its role in promoting testosterone and reducing estradiol levels—we believe it is important to discuss the potential sex-biased effects of 17α-estradiol, as this could inform future investigations. We have refined this section to clarify that these points represent mechanistic hypotheses derived from our male data and existing literature, not conclusions about unstudied female physiology. This framing maintains the discussion's scientific value while respecting the study's scope.

      Lines 458-459: This was not demonstrated in this article. Please remove.

      We have restricted the claim to "expression level of energy metabolism in hypothalamic neurons".

      Line 464: "Promoted lifespan extension" Not demonstrated. Please remove.

      At the end of the sentence it was revised as "which may be a contributing factor in promoting lifespan extension".

      Line 466: "Showed" No.

      The whole sentence was deleted in the new version.

      Line 483: "the sex-based effects". Not studied here.

      Since the changes in testosterone levels are significant in this dataset and this hormone has a sex-biased nature, we find it worthwhile to suggest this as a topic for future investigation. We have added "which needs further verification in the future" at the end of this sentence.

    1. Author response:

      The following is the authors’ response to the original reviews

      Reviewer #1 (Public review):

      This is a well-designed and very interesting study examining the impact of imprecise feedback on outcomes in decision-making. I think this is an important addition to the literature, and the results here, which provide a computational account of several decision-making biases, are insightful and interesting.

      We thank the reviewer for highlighting the strengths of this work.

      I do not believe I have substantive concerns related to the actual results presented; my concerns are more related to the framing of some of the work. My main concern is regarding the assertion that the results prove that non-normative and non-Bayesian learning is taking place. I agree with the authors that their results demonstrate that people will make decisions in ways that demonstrate deviations from what would be optimal for maximizing reward in their task under a strict application of Bayes' rule. I also agree that they have built reinforcement learning models that do a good job of accounting for the observed behavior. However, the Bayesian models included are rather simple, per the author's descriptions, applications of Bayes' rule with either fixed or learned credibility for the feedback agents. In contrast, several versions of the RL models are used, each modified to account for different possible biases. However, more complex Bayes-based models exist, notably active inference, but even the hierarchical Gaussian filter. These formalisms are able to accommodate more complex behavior, such as affect and habits, which might make them more competitive with RL models. I think it is entirely fair to say that these results demonstrate deviations from an idealized and strict Bayesian context; however, the equivalence here of Bayesian and normative is, I think, misleading or at least requires better justification/explanation. This is because a great deal of work has been done to show that Bayes optimal models can generate behavior or other outcomes that are clearly not optimal to an observer within a given context (consider hallucinations for example), but which make sense in the context of how the model is constructed as well as the priors and desired states the model is given.

      As such, I would recommend that the language be adjusted to carefully define what is meant by normative and Bayesian and to recognize that work that is clearly Bayesian could potentially still be competitive with RL models if implemented to model this task. An even better approach would be to directly use one of these more complex modelling approaches, such as active inference, as the comparator to the RL models, though I would understand if the authors would want this to be a subject for future work.

      We thank the reviewer for raising this crucial and insightful point regarding the framing of our results and the definitions of 'normative' and 'Bayesian' learning. Our primary aim in this work was to characterize specific behavioral signatures that demonstrate deviations from predictions generated by a strict, idealized Bayesian framework when learning from disinformation (which we term “biases”). We deliberately employed relatively simple Bayesian models as benchmarks to highlight these specific biases. We fully agree that more sophisticated Bayes-based models (as mentioned by the reviewer, or others) could potentially offer alternative mechanistic explanations for participant behavior. However, we currently do not have a strong notion about which Bayesian models can encompass our findings, and hence, we leave this important question for future work.

      To enhance clarity within the current manuscript we now avoided the use of the term “normative” to refer to our Bayesian models, using the term “ideal” instead. We also define more clearly what exactly we mean by that notion when the idea model is described:

      “This model is based on an idealized assumptions that during the feedback stage of each trial, the value of the chosen bandit is updated (based on feedback valence and credibility) according to Bayes rule reflecting perfect adherence to the instructed task structure (i.e., how true outcomes and feedback are generated).”

      Moreover, we have added a few sentences in the discussion commenting on how more complex Bayesian models might account for our empirical findings:

      “However, as hypothesized, when facing potential disinformation, we also find that individuals exhibit several important biases i.e., deviations from strictly idealized Bayesian strategies. Future studies should explore if and under what assumptions, about the task’s generative structure and/or learner’s priors and objectives, more complex Bayesian models (e.g., active inference (58)) might account for our empirical findings.”

      Abstract:

      The abstract is lacking in some detail about the experiments done, but this may be a limitation of the required word count. If word count is not an issue, I would recommend adding details of the experiments done and the results.

      We thank the reviewer for their valuable suggestion. We have now included more details about the experiment in the abstract:

      “In two experiments, participants completed a two-armed bandit task, where they repeatedly chose between two lotteries and received outcome-feedback from sources of varying credibility, who occasionally disseminated disinformation by lying about true choice outcome (e.g., reporting non reward when a reward was truly earned or vice versa).”

      One comment is that there is an appeal to normative learning patterns, but this suggests that learning patterns have a fixed optimal nature, which may not be true in cases where the purpose of the learning (e.g. to confirm the feeling of safety of being in an in-group) may not be about learning accurately to maximize reward. This can be accommodated in a Bayesian framework by modelling priors and desired outcomes. As such, the central premise that biased learning is inherently non-normative or non-Bayesian, I think, would require more justification. This is true in the introduction as well.

      Introduction:

      As noted above, the conceptualization of Bayesian learning being equivalent to normative learning, I think requires further justification. Bayesian belief updating can be biased and non-optimal from an observer perspective, while being optimal within the agent doing the updating if the priors/desired outcomes are set up to advantage these "non-optimal" modes of decision making.

      We appreciate the reviewer's thoughtful comment regarding the conceptualization of "normative" and "Bayesian" learning. We fully agree that the definition of "normative" is nuanced and can indeed depend on whether one considers reward-maximization or the underlying principles of belief updating. As explained above we now restrict our presentation to deviations from “ideal Bayes” learning patterns and we acknowledge the reviewer’s concern in a caveat in our discussion.

      Results:

      I wonder why the agent was presented before the choice, since the agent is only relevant to the feedback after the choice is made. I wonder if that might have induced any false association between the agent identity and the choice itself. This is by no means a critical point, but it would be interesting to get the authors' thoughts.

      We thank the reviewer for raising this interesting point regarding the presentation of the agent before the choice. Our decision to present the agent at this stage was intentional, as our original experimental design aimed to explore the possible effects of "expected source credibility" on participants' choices (e.g., whether knowledge of feedback credibility will affect choice speed and accuracy). However, we found nothing that would be interesting to report.

      The finding that positive feedback increases learning is one that has been shown before and depends on valence, as the authors note. They expanded their reinforcement learning model to include valence, but they did not modify the Bayesian model in a similar manner. This lack of a valence or recency effect might also explain the failure of the Bayesian models in the preceding section, where the contrast effect is discussed. It is not unreasonable to imagine that if humans do employ Bayesian reasoning that this reasoning system has had parameters tuned based on the real world, where recency of information does matter; affect has also been shown to be incorporable into Bayesian information processing (see the work by Hesp on affective charge and the large body of work by Ryan Smith). It may be that the Bayesian models chosen here require further complexity to capture the situation, just like some of the biases required updates to the RL models. This complexity, rather than being arbitrary, may be well justified by decision-making in the real world.

      Thanks for these additional important ideas which speak more to the notion that more complex Bayesian frameworks may account for biases we report.

      The methods mention several symptom scales- it would be interesting to have the results of these and any interesting correlations noted. It is possible that some of the individual variability here could be related to these symptoms, which could introduce precision parameter changes in a Bayesian context and things like reward sensitivity changes in an RL context.

      We included these questionnaires for exploratory purposes, with the aim of generating informed hypotheses for future research into individual differences in learning. Given the preliminary nature of these analyses, we believe further research is required about this important topic.

      Discussion:

      (For discussion, not a specific comment on this paper): One wonders also about participants' beliefs about the experiment or the intent of the experimenters. I have often had participants tell me they were trying to "figure out" a task or find patterns even when this was not part of the experiment. This is not specific to this paper, but it may be relevant in the future to try and model participant beliefs about the experiment especially in the context of disinformation, when they might be primed to try and "figure things out".

      We thank the reviewer for this important recommendation. We agree and this point is included in our caveat (cited above) that future research should address what assumptions about the generative task structure can allow Bayesian models to account for our empirical patterns.

      As a general comment, in the active inference literature, there has been discussion of state-dependent actions, or "habits", which are learned in order to help agents more rapidly make decisions, based on previous learning. It is also possible that what is being observed is that these habits are at play, and that they represent the cognitive biases. This is likely especially true given, as the authors note, the high cognitive load of the task. It is true that this would mean that full-force Bayesian inference is not being used in each trial, or in each experience an agent might have in the world, but this is likely adaptive on the longer timescale of things, considering resource requirements. I think in this case you could argue that we have a departure from "normative" learning, but that is not necessarily a departure from any possible Bayesian framework, since these biases could potentially be modified by the agent or eschewed in favor of more expensive full-on Bayesian learning when warranted.<br /> Indeed, in their discussion on the strategy of amplifying credible news sources to drown out low-credibility sources, the authors hint at the possibility of longer-term strategies that may produce optimal outcomes in some contexts, but which were not necessarily appropriate to this task. As such, the performance on this task- and the consideration of true departure from Bayesian processing- should be considered in this wider context.

      Another thing to consider is that Bayesian inference is occurring, but that priors present going in produce the biases, or these biases arise from another source, for example, factoring in epistemic value over rewards when the actual reward is not large. This again would be covered under an active inference approach, depending on how the priors are tuned. Indeed, given the benefit of social cohesion in an evolutionary perspective, some of these "biases" may be the result of adaptation. For example, it might be better to amplify people's good qualities and minimize their bad qualities in order to make it easier to interact with them; this entails a cost (in this case, not adequately learning from feedback and potentially losing out sometimes), but may fulfill a greater imperative (improved cooperation on things that matter). Given the right priors/desired states, this could still be a Bayes-optimal inference at a social level and, as such, may be ingrained as a habit that requires effort to break at the individual level during a task such as this.

      We thank the reviewer for these insightful suggestions speaking further to the point about more complex Bayesian models.

      The authors note that this task does not relate to "emotional engagement" or "deep, identity-related issues". While I agree that this is likely mostly true, it is also possible that just being told one is being lied to might elicit an emotional response that could bias responses, even if this is a weak response.

      We agree with the reviewer that a task involving performance-based bonuses, and particularly one where participants are explicitly told they are being lied to, might elicit weak emotional response. However, our primary point is that the degree of these responses is expected to be substantially weaker than those typically observed in the broader disinformation literature, which frequently deals with highly salient political, social, or identity-related topics that inherently carry strong emotional and personal ties for participants, leading to much more pronounced affective engagement and potential biases. Our task deliberately avoids such issues thus minimizing the potential for significant emotion-driven biases. We have toned down the discussion accordingly:

      “This occurs even when the decision at hand entails minimal emotional engagement or pertinence to deep, identity-related, issues.”

      Reviewer #2 (Public review):

      This valuable paper studies the problem of learning from feedback given by sources of varying credibility. The solid combination of experiment and computational modeling helps to pin down properties of learning, although some ambiguity remains in the interpretation of results.

      Summary:

      This paper studies the problem of learning from feedback given by sources of varying credibility. Two banditstyle experiments are conducted in which feedback is provided with uncertainty, but from known sources. Bayesian benchmarks are provided to assess normative facets of learning, and alternative credit assignment models are fit for comparison. Some aspects of normativity appear, in addition to deviations such as asymmetric updating from positive and negative outcomes.

      Strengths:

      The paper tackles an important topic, with a relatively clean cognitive perspective. The construction of the experiment enables the use of computational modeling. This helps to pinpoint quantitatively the properties of learning and formally evaluate their impact and importance. The analyses are generally sensible, and parameter recovery analyses help to provide some confidence in the model estimation and comparison.

      We thank the reviewer for highlighting the strengths of this work.

      Weaknesses:

      (1) The approach in the paper overlaps somewhat with various papers, such as Diaconescu et al. (2014) and Schulz et al. (forthcoming), which also consider the Bayesian problem of learning and applying source credibility, in terms of theory and experiment. The authors should discuss how these papers are complementary, to better provide an integrative picture for readers.

      Diaconescu, A. O., Mathys, C., Weber, L. A., Daunizeau, J., Kasper, L., Lomakina, E. I., ... & Stephan, K. E. (2014). Inferring the intentions of others by hierarchical Bayesian learning. PLoS computational biology, 10(9), e1003810.

      Schulz, L., Schulz, E., Bhui, R., & Dayan, P. Mechanisms of Mistrust: A Bayesian Account of Misinformation Learning. https://doi.org/10.31234/osf.io/8egxh

      We thank the reviewers for pointing us to this relevant work. We have updated the introduction, mentioning these precedents in the literature and highlighting our specific contributions:

      “To address these questions, we adopt a novel approach within the disinformation literature by exploiting a Reinforcement Learning (RL) experimental framework (36). While RL has guided disinformation research in recent years (37–41), our approach is novel in using one of its most popular tasks: the “bandit task”.”

      We also explain in the discussion how these papers relate to the current study:

      “Unlike previous studies wherein participants had to infer source credibility from experience (30,37,72), we took an explicit-instruction approach, allowing us to precisely assess source-credibility impact on learning, without confounding it with errors in learning about the sources themselves. More broadly, our work connects with prior research on observational learning, which examined how individuals learn from the actions or advice of social partners (72–75). This body of work has demonstrated that individuals integrate learning from their private experiences with learning based on others’ actions or advice—whether by inferring the value others attribute to different options or by mimicking their behavior (57,76). However, our task differs significantly from traditional observational learning. Firstly, our feedback agents interpret outcomes rather than demonstrating or recommending actions (30,37,72).”

      (2) It isn't completely clear what the "cross-fitting" procedure accomplishes. Can this be discussed further?

      We thank the reviewer for requesting further clarification on the cross-fitting procedure. Our study utilizes two distinct model families: Bayesian models and CA models. The credit assignment parameters from the CA models can be treated as “data/behavioural features” corresponding to how choice feedback affects choice-propensities. The cross fitting-approach allows us in effect to examine whether these propensity features are predicted from our Bayesian models. To the extent they are not, we can conclude empirical behavior is “biased”.

      Thus, in our cross-fitting procedure we compare the CA model parameters extracted from participant data (empirical features) with those that would be expected if our Bayesian agents performed the task. Specifically, we first fit participant behavior with our Bayesian models, then simulate this model using the best-fitted parameters and fit those simulations with our CA models. This generates a set of CA parameters that would be predicted if participants behavior is reduced to a Bayesian account. By comparing these predicted Bayesian CA parameters with the actual CA parameters obtained from human participants, the cross-fitting procedure allows us to quantitatively demonstrate that the observed participant parameters are indeed statistically significant deviations from normative Bayesian processing. This provides a robust validation that the biases we identify are not artifacts of the CA model's structure but true departures from normative learning.

      We also note that Reviewer 3 suggested an intuitive way to think about the CA parameters—as analogous to logistic regression coefficients in a “sophisticated regression” of choice on (recencyweighted) choice-feedback. We find this suggestion potentially helpful for readers. Under this interpretation, the purpose of the cross-fitting method can be seen simply as estimating the regression coefficients that would be predicted by our Bayesian agents, and comparing those to the empirical coefficients.

      In our manuscript we now explain this issues more clearly by explaining how our model is analogous to a logistic regression:

      “The probability to choose a bandit (say A over B) in this family of models is a logistic function of the contrast choice-propensities between these two bandits. One interpretation of this model is as a “sophisticated” logistic regression, where the CA parameters take the role of “regression coefficients” corresponding to the change in log odds of repeating the just-taken action in future trials based on the feedback (+/- CA for positive or negative feedback, respectively; the model also includes gradual perseveration which allows for constant log-odd changes that are not affected by choice feedback) . The forgetting rate captures the extent to which the effect of each trial on future choices diminishes with time. The Q-values are thus exponentially decaying sums of logistic choice propensities based on the types of feedback a bandit received.”

      We also explain our cross-fitting procedure in more detail:

      “To further characterise deviations between behaviour and our Bayesian learning models, we used a “crossfitting” method. Treating CA parameters as data-features of interest (i.e., feedback dependent changes in choice propensity), our goal was to examine if and how empirical features differ from features extracted from simulations of our Bayesian learning models. Towards that goal, we simulated synthetic data based on Bayesian agents (using participants’ best fitting parameters), but fitted these data using the CA-models, obtaining what we term “Bayesian-CA parameters” (Fig. 2d; Methods). A comparison of these BayesianCA parameters, with empirical-CA parameters obtained by fitting CA models to empirical data, allowed us to uncover patterns consistent with, or deviating from, ideal-Bayesian value-based inference. Under the sophisticated logistic-regression interpretation of the CA-model family the cross-fitting method comprises a comparison between empirical regression coefficients (i.e., empirical CA parameters) and regression coefficient based on simulations of Bayesian models (Bayesian CA parameters).”

      (3) The Credibility-CA model seems to fit the same as the free-credibility Bayesian model in the first experiment and barely better in the second experiment. Why not use a more standard model comparison metric like the Bayesian Information Criterion (BIC)? Even if there are advantages to the bootstrap method (which should be described if so), the BIC would help for comparability between papers.

      We thank the reviewer for this important comment regarding our model comparison approach. We acknowledge that classical information criteria like AIC and BIC are widely used in RL studies. However, we argue our method for model-comparison is superior.

      We conducted a model recovery analysis demonstrating a significant limitation of using AIC or BIC for model-comparison in our data. Both these methods are strongly biased in favor of the Bayesian models. Our PBCM method, on the other hand, is both unbiased and more accurate. We believe this is because “off the shelf” methods like AIC and BIC rely on strong assumptions (such as asymptotic sample size and trial-independence) that are not necessarily met in our tasks (Data is finite; Trials in RL tasks depend on previous trials). PBCM avoids such assumptions to obtain comparison criteria specifically tailored to the structure and size of our empirical data. We have now mentioned this fact in the results section of the main text:

      “We considered using AIC and BIC, which apply “off-the shelf” penalties for model-complexity. However, these methods do not adapt to features like finite sample size (relying instead on asymptotic assumption) or temporal dependence (as is common in reinforcement learning experiments). In contrast, the parametric bootstrap cross-fitting method replaces these fixed penalties with empirical, data-driven criteria for modelselection. Indeed, model-recovery simulations confirmed that whereas AIC and BIC were heavily biased in favour of the Bayesian models, the bootstrap method provided excellent model-recovery (See Fig. S20).”

      We have also included such model recovery in the SI document:

      (4) As suggested in the discussion, the updating based on random feedback could be due to the interleaving of trials. If one is used to learning from the source on most trials, the occasional random trial may be hard to resist updating from. The exact interleaving structure should also be clarified (I assume different sources were shown for each bandit pair). This would also relate to work on RL and working memory: Collins, A. G., & Frank, M. J. (2012). How much of reinforcement learning is working memory, not reinforcement learning? A behavioral, computational, and neurogenetic analysis. European Journal of Neuroscience, 35(7), 10241035.

      We thank the reviewer for this point. The specific interleaved structure of the agents is described in the main text:

      “Each agent provided feedback for 5 trials for each bandit pair (with the agent order interleaved within the bandit pair).”

      As well as in the methods section:

      “Feedback agents were randomly interleaved across trials subject to the constraint that each agent appeared on 5-trials for each bandit pair.”

      We also thank the reviewer for mentioning the relevant work on working memory. We have now added it to our discussion point:

      “In our main study, we show that participants revised their beliefs based on entirely non-credible feedback, whereas an ideal Bayesian strategy dictates such feedback should be ignored. This finding resonates with the “continued-influence effect” whereby misleading information continues to influence an individual's beliefs even after it has been retracted (59,60). One possible explanation is that some participants failed to infer that feedback from the 1-star agent was statistically void of information content, essentially random (e.g., the group-level credibility of this agent was estimated by our free-credibility Bayesian model as higher than 50%). Participants were instructed that this feedback would be “a lie” 50% of the time but were not explicitly told that this meant it was random and should therefore be disregarded. Notably, however, there was no corresponding evidence random feedback affected behaviour in our discovery study. It is possible that an individual’s ability to filter out random information might have been limited due to a high cognitive load induced by our main study task, which required participants to track the values of three bandit pairs and juggle between three interleaved feedback agents (whereas in our discovery study each experimental block featured a single bandit pair). Future studies should explore more systematically how the ability to filter random feedback depends on cognitive load (61).”

      (5) Why does the choice-repetition regression include "only trials for which the last same-pair trial featured the 3-star agent and in which the context trial featured a different bandit pair"? This could be stated more plainly.

      We thank the reviewer for this question. When we previously submitted our manuscript, we thought that finding enhanced credit-assignment for fully credible feedback following potential disinformation from a different context would constitute a striking demonstration of our “contrast effect”. However, upon reexamining this finding we found out we had a coding error (affecting how trials were filtered). We have now rerun and corrected this analysis. We have assessed the contrast effect for both "same-context" trials (where the contextual trial featured the same bandit pair as the learning trial) and "different-context" trials (where the contextual trial featured a different bandit pair). Our re-analysis reveals a selective significant contrast effect in the samecontext condition, but no significant effect in the different-context condition. We have updated the main text to reflect these corrected findings and provide a clearer explanation of the analysis:

      “A comparison of empirical and Bayesian credit-assignment parameters revealed a further deviation from ideal Bayesian learning: participants showed an exaggerated credit-assignment for the 3-star agent compared with Bayesian models [Wilcoxon signed-rank test, instructed-credibility Bayesian model (median difference=0.74, z=11.14); free-credibility Bayesian model (median difference=0.62, z=10.71), all p’s<0.001] (Fig. 3a). One explanation for enhanced learning for the 3-star agents is a contrast effect, whereby credible information looms larger against a backdrop of non-credible information. To test this hypothesis, we examined whether the impact of feedback from the 3-star agent is modulated by the credibility of the agent in the trial immediately preceding it. More specifically, we reasoned that the impact of a 3-star agent would be amplified by a “low credibility context” (i.e., when it is preceded by a low credibility trial). In a binomial mixed effects model, we regressed choice-repetition on feedback valence from the last trial featuring the same bandit pair (i.e., the learning trial) and the feedback agent on the trial immediately preceding that last trial (i.e., the contextual credibility; see Methods for model-specification). This analysis included only learning trials featuring the 3-star agent, and context trials featuring the same bandit pair as the learning trial (Fig. 4a). We found that feedback valence interacted with contextual credibility (F(2,2086)=11.47, p<0.001) such that the feedback-effect (from the 3-star agent) decreased as a function of the preceding context-credibility (3-star context vs. 2-star context: b= -0.29, F(1,2086)=4.06, p=0.044; 2star context vs. 1-star context: b=-0.41, t(2086)=-2.94, p=0.003; and 3-star context vs. 1-star context: b=0.69, t(2086)=-4.74, p<0.001) (Fig. 4b). This contrast effect was not predicted by simulations of our main models of interest (Fig. 4c). No effect was found when focussing on contextual trials featuring a bandit pair different than the one in the learning trial (see SI 3.5). Thus, these results support an interpretation that credible feedback exerts a greater impact on participants’ learning when it follows non-credible feedback, in the same learning context.”

      We have modified the discussion accordingly as well:

      “A striking finding in our study was that for a fully credible feedback agent, credit assignment was exaggerated (i.e., higher than predicted by our Bayesian models). Furthermore, the effect of fully credible feedback on choice was further boosted when it was preceded by a low-credibility context related to current learning. We interpret this in terms of a “contrast effect”, whereby veridical information looms larger against a backdrop of disinformation (21). One upshot is that exaggerated learning might entail a risk of jumping to premature conclusions based on limited credible evidence (e.g., a strong conclusion that a vaccine is produces significant side-effect risks based on weak credible information, following non-credible information about the same vaccine). An intriguing possibility, that could be tested in future studies, is that participants strategically amplify the extent of learning from credible feedback to dilute the impact of learning from noncredible feedback. For example, a person scrolling through a social media feed, encountering copious amounts of disinformation, might amplify the weight they assign to credible feedback in order to dilute effects of ‘fake news’. Ironically, these results also suggest that public campaigns might be more effective when embedding their messages in low-credibility contexts , which may boost their impact.”

      And we have included some additional analyses in the SI document:

      “3.5 Contrast effects for contexts featuring a different bandit

      Given that we observed a contrast effect when both the learning and the immediately preceding "context trial” involved the same pair of bandits, we next investigated whether this effect persisted when the context trial featured a different bandit pair – a situation where the context would be irrelevant to the current learning. Again, we used in a binomial mixed effects model, regressing choice-repetition on feedback valence in the learning trial and the feedback agent in the context trial. This analysis included only learning trials featuring the 3-star agent, and context trials featuring a different bandit pair than the learning trial (Fig. S22a). We found no significant evidence of an interaction between feedback valence and contextual credibility (F(2,2364)=0.21, p=0.81) (Fig. S22b). This null result was consistent with the range of outcomes predicted by our main computational models (Fig. S22c).

      We aimed to formally compare the influence of two types of contextual trials: those featuring the same bandit pair as the learning trial versus those featuring a different pair. To achieve this, we extended our mixedeffects model by incorporating a new predictor variable, "CONTEXT_TYPE" which coded whether the contextual trial involved the same bandit pair (coded as -0.5) or a different bandit pair (+0.5) compared to the learning trial. The Wilkinson notation for this expanded mixed-effects model is:

      𝑅𝐸𝑃𝐸𝐴𝑇 ~ 𝐶𝑂𝑁𝑇𝐸𝑋𝑇_𝑇𝑌𝑃𝐸 ∗ 𝐹𝐸𝐸𝐷𝐵𝐴𝐶𝐾 ∗ (𝐶𝑂𝑁𝑇𝐸𝑋𝑇<sub>2-star</sub> + 𝐶𝑂𝑁𝑇𝐸𝑋𝑇<sub>3-star</sub>) + 𝐵𝐸𝑇𝑇𝐸𝑅 + (1|𝑝𝑎𝑟𝑡𝑖𝑐𝑖𝑝𝑎𝑛𝑡)

      This expanded model revealed a significant three-way interaction between feedback valence, contextual credibility, and context type (F(2,4451) = 7.71, p<0.001). Interpreting this interaction, we found a 2-way interaction between context-source and feedback valence when the context was the same (F(2,4451) = 12.03, p<0.001), but not when context was different (F(2,4451) = 0.23, p = 0.79). Further interpreting the double feedback-valence * context-source interaction (for the same context) we obtained the same conclusions as reported in the main text.”

      (6) Why apply the "Truth-CA" model and not the Bayesian variant that it was motivated by?

      Thanks for this very useful suggestion. We are unsure if we fully understand the question. The Truth-CA model was not motivated by a new Bayesian model. Our Bayesian models were simply used to make the point that participants may partially discriminate between truthful and untruthful feedback (for a given source). This led to the idea that perhaps more credit is assigned for truth (than lie) trials, which is what we found using our Truth-CA model. Note we show that our Bayesian models cannot account for this modulation.

      We have now improved our "Truth-CA" model. Previously, our Truth-CA model considered whether feedback on each trial was true or not based on realized latent true outcomes. However, it is possible that the very same feedback would have had an opposite truth-status if the latent true outcome was different (recall true outcomes are stochastic). This injects noise into the trial classification in our previous model. To avoid this, in our new model feedback is modulated by the probability the reported feedback is true (marginalized over stochasticity of true outcome).

      We have described this new model in the methods section:

      “Additionally, we formulated a “Truth-CA” model, which worked as our Credibility-CA model, but incorporated a free truth-bonus parameter (TB). This parameter modulates the extent of credit assignment for each agent based on the posterior probability of feedback being true (given the credibility of the feedback agent, and the true reward probability of the chosen bandit). The chosen bandit was updated as follows:

      𝑄 ← (1 – 𝑓<sub>Q</sub>) ∗ 𝑄 + [𝐶𝐴(𝑎𝑔𝑒𝑛𝑡) + 𝑇𝐵 ∗ (𝑃(𝑡𝑟𝑢𝑡ℎ) − 0.5)] ∗ 𝐹

      where P(truth) is the posterior probability of the feedback being true in the current trial (for exact calculation of P(truth) see “Methods: Bayesian estimation of posterior belief that feedback is true”).”

      All relevant results have been updated accordingly in the main text:

      “To formally address whether feedback truthfulness modulates credit assignment, we fitted a new variant of the CA model (the “Truth-CA” model) to the data. This variant works as our Credibility-CA model but incorporated a truth-bonus parameter (TB) which increases the degree of credit assignment for feedback as a function of the experimenter-determined likelihood the feedback is true (which is read from the curves in Fig 6a when x is taken to be the true probability the bandit is rewarding). Specifically, after receiving feedback, the Q-value of the chosen option is updated according to the following rule: 𝑄 ← (1 – 𝑓<sub>Q</sub>) ∗ 𝑄 + [𝐶𝐴(𝑎𝑔𝑒𝑛𝑡) + 𝑇𝐵 ∗ (𝑃(𝑡𝑟𝑢𝑡ℎ) − 0.5)] ∗ 𝐹 where 𝑇𝐵 is the free parameter representing the truth bonus, and 𝑃(𝑡𝑟𝑢𝑡ℎ) is the probability the received feedback being true (from the experimenter’s perspective). We acknowledge that this model falls short of providing a mechanistically plausible description of the credit assignment process, because participants have no access to the experimenter’s truthfulness likelihoods (as the true bandit reward probabilities are unknown to them). Nonetheless, we use this ‘oracle model’ as a measurement tool to glean rough estimates for the extent to which credit assignment Is boosted as a function of its truthfulness likelihood. Fitting this Truth-CA model to participants' behaviour revealed a significant positive truth-bonus (mean=0.21, t(203)=3.12, p=0.002), suggesting that participants indeed assign greater weight to feedback that is likely to be true (Fig. 6c; see SI 3.3.1 for detailed ML parameter results). Notably, simulations using our other models (Methods) consistently predicted smaller truth biases (compared to the empirical bias) (Fig. 6d). Moreover, truth bias was still detected even in a more flexible model that allowed for both a positivity bias and truth-bias (see SI 3.7). The upshot is that participants are biased to assign higher credit based on feedback that is more likely to be true in a manner that is inconsistent with out Bayesian models and above and beyond the previously identified positivity biases.“

      Finally, the Supplementary Information for the discovery study has also been revised to feature this analysis:

      “We next assessed whether participants infer whether the feedback they received on each trial was true or false and adjust their credit assignment based on this inference. We again used the “Truth-CA” model to obtain estimates for the truth bonus (TB), the increase in credit assignment as a function of the posterior probability of feedback being true. As in our main study, the fitted truth bias parameter was significantly positive, indicating that participants assign greater weight to feedback they believe is likely to be true (Fig, S4a; see SI 3.3.1 for detailed ML parameter results). Strikingly, model-simulations (Methods) predicted a lower truth bonus than the one observed in participants (Fig. S4b).”

      (7) "Overall, the results from this study support the exact same conclusions (See SI section 1.2) but with one difference. In the discovery study, we found no evidence for learning based on 50%-credibility feedback when examining either the feedback effect on choice repetition or CA in the credibility-CA model (SI 1.2.3)" - this seems like a very salient difference, when the paper reports the feedback effect as a primary finding of interest, though I understand there remains a valence-based difference.

      We agree with the reviewer and thank them for this suggestion. We now state explicitly throughout the manuscript that this finding was obtained only in one of our two studies. In the section “Discovery study” of the results we state explicitly this finding was not found in the discovery study:

      “However, we found no evidence for learning based on 50%-credibility feedback when examining either the feedback effect on choice repetition or CA in the credibility-CA model (SI 1.2.3).”

      We also note that related to another concern from R3 (that perseveration may masquerade as positivity bias) we conducted additional analyses (detailed in SI 3.6.2). These analyses revealed that the observed positivity bias for the 1-star agent in the discovery study falls within the range predicted by simple choice-perseveration. Consequently, we have removed the suggestion that participants still learn from the random agent in the discovery study. Furthermore, we have modified the discussion section to include a possible explanation for this discrepancy between the two studies:

      “Notably, however, there was no corresponding evidence random feedback affected behaviour in our discovery study. It is possible that an individual’s ability to filter out random information might have been limited due to a high cognitive load induced by our main study task, which required participants to track the values of three bandit pairs and juggle between three interleaved feedback agents (whereas in our discovery study each experimental block featured a single bandit pair). Future studies should explore more systematically how the ability to filter random feedback depends on cognitive load (61).”

      (8) "Participants were instructed that this feedback would be "a lie 50% of the time but were not explicitly told that this meant it was random and should therefore be disregarded." - I agree that this is a possible explanation for updating from the random source. It is a meaningful caveat.

      Thank you for this thought. While this can be seen as a caveat—since we don’t know what would have happened with explicit instructions—we also believe it is interesting from another perspective. In many real-life situations, individuals may have all the necessary information to infer that the feedback they receive is uninformative, yet still fail to do so, especially when they are not explicitly told to ignore it.

      In future work, we plan to examine how behaviour changes when participants are given more explicit instructions—for example, that the 50%-credibility agent provides purely random feedback.

      (9) "Future studies should investigate conditions that enhance an ability to discard disinformation, such as providing explicit instructions to ignore misleading feedback, manipulations that increase the time available for evaluating information, or interventions that strengthen source memory." - there is work on some of this in the misinformation literature that should be cited, such as the "continued influence effect". For example: Johnson, H. M., & Seifert, C. M. (1994). Sources of the continued influence effect: When misinformation in memory affects later inferences. Journal of experimental psychology: Learning, memory, and cognition, 20(6), 1420.

      We thank the reviewer for pointing us towards the relevant literature. We have now included citations about the “continued influence effect” of misinformation in the discussion:

      “In our main study, we show that participants revised their beliefs based on entirely non-credible feedback, whereas an ideal Bayesian strategy dictates such feedback should be ignored. This finding resonates with the “continued-influence effect” whereby misleading information continues to influence an individual's beliefs even after it has been retracted (59,60).”

      (10) Are the authors arguing that choice-confirmation bias may be at play? Work on choice-confirmation bias generally includes counterfactual feedback, which is not present here.

      We agree with the reviewer that a definitive test for choice-confirmation bias typically requires counterfactual feedback, which is not present in our current task. In our discussion, we indeed suggest that the positivity bias we observe may stem from a form of choice-confirmation, drawing on the extensive literature on this bias in reinforcement learning (Lefebvre et al., 2017; Palminteri et al., 2017; Palminteri & Lebreton, 2022). However, we fully acknowledge that this link is a hypothesis and that explicitly testing for choice-confirmation bias would necessitate a future study specifically incorporating counterfactual feedback. We have included a clarification of this point in the discussion:

      “Previous reinforcement learning studies, report greater credit-assignment based on positive compared to negative feedback, albeit only in the context of veridical feedback (43,44,62). Here, supporting our a-priori hypothesis we show that this positivity bias is amplified for information of low and intermediate credibility (in absolute terms in the discovery study, and relative to the overall extent of CA in both studies) . Of note, previous literature has interpreted enhanced learning for positive outcomes in reinforcement learning as indicative of a confirmation bias (42,44). For example, positive feedback may confirm, to a greater extent than negative feedback one’s choice as superior (e.g., “I chose the better of the two options”). Leveraging the framework of motivated cognition (35), we posited that feedback of uncertain veracity (e.g., low credibility) amplifies this bias by incentivising individuals to self-servingly accept positive feedback as true (because it confers positive, desirable outcomes), and explain away undesirable, choice-disconfirming, negative feedback as false. This could imply an amplified confirmation bias on social media, where content from sources of uncertain credibility, such as unknown or unverified users, is more easily interpreted in a self-serving manner, disproportionately reinforcing existing beliefs (63). In turn, this could contribute to an exacerbation of the negative social outcomes previously linked to confirmation bias such as polarization (64,65), the formation of ‘echo chambers’ (19), and the persistence of misbelief regarding contemporary issues of importance such as vaccination (66,67) and climate change (68–71). We note however, that further studies are required to determine whether positivity bias in our task is indeed a form of confirmation bias.”

      Reviewer #3 (Public review):

      Summary

      This paper investigates how disinformation affects reward learning processes in the context of a two-armed bandit task, where feedback is provided by agents with varying reliability (with lying probability explicitly instructed). They find that people learn more from credible sources, but also deviate systematically from optimal Bayesian learning: They learned from uninformative random feedback, learned more from positive feedback, and updated too quickly from fully credible feedback (especially following low-credibility feedback). Overall, this study highlights how misinformation could distort basic reward learning processes, without appeal to higher-order social constructs like identity.

      Strengths

      (1) The experimental design is simple and well-controlled; in particular, it isolates basic learning processes by abstracting away from social context.

      (2) Modeling and statistics meet or exceed the standards of rigor.

      (3) Limitations are acknowledged where appropriate, especially those regarding external validity.

      (4) The comparison model, Bayes with biased credibility estimates, is strong; deviations are much more compelling than e.g., a purely optimal model.

      (5) The conclusions are interesting, in particular the finding that positivity bias is stronger when learning from less reliable feedback (although I am somewhat uncertain about the validity of this conclusion)

      We deeply thank the reviewer for highlighting the strengths of this work.

      Weaknesses

      (1) Absolute or relative positivity bias?

      In my view, the biggest weakness in the paper is that the conclusion of greater positivity bias for lower credible feedback (Figure 5) hinges on the specific way in which positivity bias is defined. Specifically, we only see the effect when normalizing the difference in sensitivity to positive vs. negative feedback by the sum. I appreciate that the authors present both and add the caveat whenever they mention the conclusion (with the crucial exception of the abstract). However, what we really need here is an argument that the relative definition is the right way to define asymmetry....

      Unfortunately, my intuition is that the absolute difference is a better measure. I understand that the relative version is common in the RL literature; however previous studies have used standard TD models, whereas the current model updates based on the raw reward. The role of the CA parameter is thus importantly different from a traditional learning rate - in particular, it's more like a logistic regression coefficient (as described below) because it scales the feedback but not the decay. Under this interpretation, a difference in positivity bias across credibility conditions corresponds to a three-way interaction between the exponentially weighted sum of previous feedback of a given type (e.g., positive from the 75% credible agent), feedback positivity, and condition (dummy coded). This interaction corresponds to the nonnormalized, absolute difference.

      Importantly, I'm not terribly confident in this argument, but it does suggest that we need a compelling argument for the relative definition.

      We thank the reviewer for raising this important point about the definition of positivity bias, and for their thoughtful discussion on the absolute versus relative measures. We believe that the relative valence bias offers a distinct and valuable perspective on positivity bias. Conceptually, this measure describes positivity bias in a manner akin to a “percentage difference” relative to the overall level of learning which allows us to control for the overall decreases in the overall amount of credit assignment as feedback becomes less credible. We are unsure if one measure is better or more correct than the other and we believe that reporting both measures enriches the understanding of positivity bias and allows for a more comprehensive characterization of this phenomenon (as long as these measures are interpreted carefully). We have stated the significance of the relative measure in the results section:

      “Following previous research, we quantified positivity bias in 2 ways: 1) as the absolute difference between credit-assignment based on positive or negative feedback, and 2) as the same difference but relative to the overall extent of learning. We note that the second, relative, definition, is more akin to “percentage change” measurements providing a control for the overall lower levels of credit-assignment for less credible agent.”

      We also wish to point out that in our discovery study we had some evidence for amplification of positivity bias in absolute sense.

      (2) Positivity bias or perseveration?

      A key challenge in interpreting many of the results is dissociating perseveration from other learning biases. In particular, a positivity bias (Figure 5) and perseveration will both predict a stronger correlation between positive feedback and future choice. Crucially, the authors do include a perseveration term, so one would hope that perseveration effects have been controlled for and that the CA parameters reflect true positivity biases. However, with finite data, we cannot be sure that the variance will be correctly allocated to each parameter (c.f. collinearity in regressions). The fact that CA- is fit to be negative for many participants (a pattern shown more strongly in the discovery study) is suggestive that this might be happening. A priori, the idea that you would ever increase your value estimate after negative feedback is highly implausible, which suggests that the parameter might be capturing variance besides that it is intended to capture.

      The best way to resolve this uncertainty would involve running a new study in which feedback was sometimes provided in the absence of a choice - this would isolate positivity bias. Short of that, perhaps one could fit a version of the Bayesian model that also includes perseveration. If the authors can show that this model cannot capture the pattern in Figure 5, that would be fairly convincing.

      We thank the reviewer for this very insightful and crucial point regarding the potential confound between positivity bias and perseveration. We entirely agree that distinguishing these effects can be challenging. To rigorously address this concern and ascertain that our observed positivity bias, particularly its inflation for low-credibility feedback, is not merely an artifact of perseveration, we conducted additional analyses as suggested.

      First, following the reviewer’s suggestion we simulated our Bayesian models, including a perseveration term, for both our main and discovery studies. Crucially, none of these simulations predicted the specific pattern of inflated positivity bias for low-credibility feedback that we identified in participants.

      Additionally, taking a “devil’s advocate” approach, we tested whether our credibility-CA model (which includes perseveration but not a feedback valence bias) can predict our positivity bias findings. Thus, we simulated 100 datasets using our Credibility-CA model (based on empirical best-fitting parameters). We then fitted each of these simulated datasets using our CredibilityValence CA model. By examining the distribution of results across these synthetic datasets fits and comparing them to the actual results from participants, we found that while perseveration could indeed lead (as the reviewer suspected) to an artifactual positivity bias, it could not predict the magnitude of the observed inflation of positivity bias for low-credibility feedback (whether measured in absolute or relative terms).

      Based on these comprehensive analyses, we are confident that our main results concerning the modulation of a valence bias as a function of source-credibility cannot be accounted by simple choice-perseveration. We have briefly explained these analyses in the main results section:

      “Previous research has suggested that positivity bias may spuriously arise from pure choice-perseveration (i.e., a tendency to repeat previous choices regardless of outcome) (49,50). While our models included a perseveration-component, this control may not be preferent. Therefore, in additional control analyses, we generated synthetic datasets using models including choice-perseveration but devoid of feedback-valence bias, and fitted them with our credibility-valence model (see SI 3.6.1). These analyses confirmed that perseveration can masquerade as an apparent positivity bias. Critically, however, these analyses also confirmed that perseveration cannot account for our main finding of increased positivity bias, relative to the overall extent of CA, for low-credibility feedback.”

      Additionally, we have added a detailed description of these additional analyses and their findings to the Supplementary Information document:

      “3.6 Positivity bias results cannot be explained by a pure perseveration

      3.6.1 Main study

      Previous research has suggested it may be challenging to dissociate between a feedback-valence positivity bias and perseveration (i.e., a tendency to repeat previous choices regardless of outcome). While our Credit Assignment (CA) models already include a perseveration mechanism to account for this, this control may not be perfect. We thus conducted several tests to examine if our positivity-bias related results could be accounted for by perseveration.

      First we examined whether our Bayesian-models, augmented by a perseveration mechanism (as in our CA model) can generate predictions similar to our empirical results. We repeated our cross-fitting procedure to these extended Bayesian models. To briefly recap, this involved fitting participant behavior with them, generating synthetic datasets based on the resulting maximum likelihood (ML) parameters, and then fitting these simulated datasets with our Credibility-Valence CA model (which is designed to detect positivity bias). This test revealed that adding perseveration to our Bayesian models did not predict a positivity bias in learning. In absolute terms there was a small negativity bias (instructed-credibility Bayesian: b=−0.19, F(1,1218)=17.78, p<0.001, Fig. S23a-b; free-credibility Bayesian: b=−0.17, F(1,1218)=13.74, p<0.001, Fig. S23d-e). In relative terms we detected no valence related bias (instructed-credibility Bayesian: b=−0.034, F(1,609)=0.45, p=0.50, Fig. S22c; free-credibility Bayesian: b=−0.04, F(1,609)=0.51, p=0.47, Fig. S23f). More critically, these simulations also did not predict a change in the level of positivity bias as a function of feedback credibility, neither at an absolute level (instructed-credibility Bayesian: F(2,1218)=0.024, p=0.98, Fig. S23b; free-credibility Bayesian: F(2,1218)=0.008, p=0.99, Fig. S23e), nor at a relative level (instructedcredibility Bayesian: F(2,609)=1.57, p=0.21, Fig. S23c; free-credibility Bayesian: F(2,609)=0.13, p=0.88, Fig. S23f). The upshot is that our positivity-bias findings cannot be accounted for by our Bayesian models even when these are augmented with perseveration.

      However, it is still possible that empirical CA parameters from our credibility-valence model (reported in main text Fig. 5) were distorted, absorbing variance from a perseveration. To address this, we took a “devil's advocate” approach testing the assumption that CA parameters are not truly affected by feedback valance and that there is only perseveration in our data. Towards that goal, we simulated data using our CredibilityCA model (which includes perseveration but does not contain a valence bias in its learning mechanism) and then fitted these synthetic datasets using our Credibility-Valence CA model to see if the observed positivity bias could be explained by perseveration alone. Specifically, we generated 101 “group-level” synthetic datasets (each including one simulation for each participant, based on their empirical ML parameters), and fitted each dataset with our Credibility-Valence CA model. We then analysed the resulting ML parameters in each dataset using the same mixed-effects models as described in the main text, examining the distribution of effects of interest across these simulated datasets. Comparing these simulation results to the data from participants revealed a nuanced picture. While the positivity bias observed in participants is within the range predicted by a pure perseveration account when measured in absolute terms (Fig. S24a), it is much higher than predicted by pure perseveration when measured relative to the overall level of learning (Fig. S24c). More importantly, the inflation in positivity bias for lower credibility feedback is substantially higher in participants than what would be predicted by a pure perseveration account, a finding that holds true for both absolute (Fig. S24b) and relative (Fig. S24d) measures.”

      “3.6.2 Discovery study

      We then replicated these analyses in our discovery study to confirm our findings. We again checked whether extended versions of the Bayesian models (including perseveration) predicted the positivity bias results observed. Our cross-fitting procedure showed that the instructed-credibility Bayesian model with perseveration did predict a positivity bias for all credibility levels in this discovery study, both when measured in absolute terms [50% credibility (b=1.74,t(824)=6.15), 70% credibility (b=2.00,F(1,824)=49.98), 85% credibility (b=1.81,F(1,824)=40.78), 100% credibility (b=2.42,F(1,824)=72.50), all p's<0.001], and in relative terms [50% credibility (b=0.25,t(412)=3.44), 70% credibility (b=0.31,F(1,412)=17.72), 85% credibility (b=0.34,F(1,412)=21.06), 100% credibility (b=0.42,F(1,412)=31.24), all p's<0.001]. However, importantly, these simulations did not predict a change in the level of positivity bias as a function of feedback credibility, neither at an absolute level (F(3,412)=1.43,p=0.24), nor at a relative level (F(3,412)=2.06,p=0.13) (Fig. S25a-c). In contrast, simulations of the free-credibility Bayesian model (with perseveration) predicted a slight negativity bias when measured in absolute terms (b=−0.35,F(1,824)=5.14,p=0.024), and no valence bias when measured relative to the overall degree of learning (b=0.05,F(1,412)=0.55,p=0.46). Crucially, this model also did not predict a change in the level of positivity bias as a function of feedback credibility, neither at an absolute level (F(3,824)=0.27,p=0.77), nor at a relative level (F(3,412)=0.76,p=0.47) (Fig. S25d-f).

      As in our main study, we next assessed whether our Credibility-CA model (which includes perseveration but no valence bias) predicted the positivity bias results observed in participants in the discovery study. This analysis revealed that the average positivity bias in participants is higher than predicted by a pure perseveration account, both when measured in absolute terms (Fig. S26a) and in relative terms (Fig. S26c). Specifically, only the aVBI for the 70% credibility agent was above what a perseveration account would predict, while the rVBI for all agents except the completely credible one exceeded that threshold. Furthermore, the inflation in positivity bias for lower credibility feedback (compared to the 100% credibility agent) is significantly higher in participants than would be predicted by a pure perseveration account, in both absolute (Fig. S26b) and relative (Fig. S26d) terms.

      Together, these results show that the general positivity bias observed in participants could be predicted by an instructed-credibility Bayesian model with perseveration, or by a CA model with perseveration. Moreover, we find that these two models can predict a positivity bias for the 50% credibility agent, raising a concern that our positivity bias findings for this source may be an artefact of not-fully controlled for perseveration. However, the credibility modulation of this positivity bias, where the bias is amplified for lower credibility feedback, is consistently not predicted by perseveration alone, regardless of whether perseveration is incorporated into a Bayesian or a CA model. This finding suggests that participants are genuinely modulating their learning based on feedback credibility, and that this modulation is not merely an artifact of choice perseveration.”

      (3) Veracity detection or positivity bias?

      The "True feedback elicits greater learning" effect (Figure 6) may be simply a re-description of the positivity bias shown in Figure 5. This figure shows that people have higher CA for trials where the feedback was in fact accurate. But assuming that people tend to choose more rewarding options, true-feedback cases will tend to also be positive-feedback cases. Accordingly, a positivity bias would yield this effect, even if people are not at all sensitive to trial-level feedback veracity. Of course, the reverse logic also applies, such that the "positivity bias" could actually reflect discounting of feedback that is less likely to be true. This idea has been proposed before as an explanation for confirmation bias (see Pilgrim et al, 2024 https://doi.org/10.1016/j.cognition.2023.105693and much previous work cited therein). The authors should discuss the ambiguity between the "positivity bias" and "true feedback" effects within the context of this literature....

      Before addressing these excellent comments, we first note that we have now improved our "TruthCA" model. Previously, our Truth-CA model considered whether feedback on each trial was true or not based on realized latent true outcomes. However, it is possible that the very same feedback would have had an opposite truth-status if the latent true outcome was different (recall true outcomes are stochastic). This injects noise into the trial classification in our former model. To avoid this, in our new model feedback is modulated by the probability the reported feedback is true (marginalized over stochasticity of true outcome). Please note in our responses below that we conducted extensive analysis to confirm that positivity bias doesn’t in fact predict the truthbias we detect using our truth biased model

      We have described this new model in the methods section:

      “Additionally, we formulated a “Truth-CA” model, which worked as our Credibility-CA model, but incorporated a free truth-bonus parameter (TB). This parameter modulates the extent of credit assignment for each agent based on the posterior probability of feedback being true (given the credibility of the feedback agent, and the true reward probability of the chosen bandit). The chosen bandit was updated as follows:

      𝑄 ← (1 – 𝑓<sub>Q</sub>) ∗ 𝑄 + [𝐶𝐴(𝑎𝑔𝑒𝑛𝑡) + 𝑇𝐵 ∗ (𝑃(𝑡𝑟𝑢𝑡ℎ) − 0.5)] ∗ 𝐹

      where P(truth) is the posterior probability of the feedback being true in the current trial (for exact calculation of P(truth) see “Methods: Bayesian estimation of posterior belief that feedback is true”).”

      All relevant results have been updated accordingly in the main text:

      To formally address whether feedback truthfulness modulates credit assignment, we fitted a new variant of the CA model (the “Truth-CA” model) to the data. This variant works as our Credibility-CA model, but incorporated a truth-bonus parameter (TB) which increases the degree of credit assignment for feedback as a function of the experimenter-determined likelihood the feedback is true (which is read from the curves in Fig 6a when x is taken to be the true probability the bandit is rewarding). Specifically, after receiving feedback, the Q-value of the chosen option is updated according to the following rule:

      𝑄 ← (1 – 𝑓<sub>Q</sub>) ∗ 𝑄 + [𝐶𝐴(𝑎𝑔𝑒𝑛𝑡) + 𝑇𝐵 ∗ (𝑃(𝑡𝑟𝑢𝑡ℎ) − 0.5)] ∗ 𝐹

      where 𝑇𝐵 is the free parameter representing the truth bonus, and 𝑃(𝑡𝑟𝑢𝑡ℎ) is the probability the received feedback being true (from the experimenter’s perspective). We acknowledge that this model falls short of providing a mechanistically plausible description of the credit assignment process, because participants have no access to the experimenter’s truthfulness likelihoods (as the true bandit reward probabilities are unknown to them). Nonetheless, we use this ‘oracle model’ as a measurement tool to glean rough estimates for the extent to which credit assignment Is boosted as a function of its truthfulness likelihood.

      Fitting this Truth-CA model to participants' behaviour revealed a significant positive truth-bonus (mean=0.21, t(203)=3.12, p=0.002), suggesting that participants indeed assign greater weight to feedback that is likely to be true (Fig. 6c; see SI 3.3.1 for detailed ML parameter results). Notably, simulations using our other models (Methods) consistently predicted smaller truth biases (compared to the empirical bias) (Fig. 6d). Moreover, truth bias was still detected even in a more flexible model that allowed for both a positivity bias and truth-bias (see SI 3.7). The upshot is that participants are biased to assign higher credit based on feedback that is more likely to be true in a manner that is inconsistent with out Bayesian models and above and beyond the previously identified positivity biases.”

      Finally, the Supplementary Information for the discovery study has also been revised to feature this analysis:

      “We next assessed whether participants infer whether the feedback they received on each trial was true or false and adjust their credit assignment based on this inference. We again used the “Truth-CA” model to obtain estimates for the truth bonus (TB), the increase in credit assignment as a function of the posterior probability of feedback being true. As in our main study, the fitted truth bias parameter was significantly positive, indicating that participants assign greater weight to feedback they believe is likely to be true (Fig, S4a; see SI 3.3.1 for detailed ML parameter results). Strikingly, model-simulations (Methods) predicted a lower truth bonus than the one observed in participants (Fig. S4b).”

      Additionally, we thank the reviewer for pointing us to the relevant work by Pilgrim et al. (2024). We agree that the relationship between "true feedback" and "positivity bias" effects is nuanced, and their potential overlap warrants careful consideration. Note our analyses suggest that this is not solely the case. Firstly, simulations of our Credibility-Valence CA model predict only a small "truth bonus" effect, which is notably smaller than what we observed in participants. Secondly, we formulated an extension of our "Truth-CA" model that includes a valence bias in credit assignment. If our truth bonus results were merely an artifact of positivity bias, this extended model should absorb that variance, producing a null truth bonus parameter. However, fitting this model to participant data still revealed a significant positive truth bonus, which again exceeds the range predicted by simulations of our Credibility CA model:

      “3.7 Truth inference is still detected when controlling for valence bias

      Given that participants frequently select bandits that are, on average, mostly rewarding, it is reasonable to assume that positive feedback is more likely to be objectively true than negative feedback. This raises a question if the "truth inference" effect we observed in participants might simply be an alternative description of a positivity bias in learning. To directly test this idea, we extended our Truth-CA model to explicitly account for a valence bias in credit assignment. This extended model features separate CA parameters for positive and negative feedback for each agent. When we fitted this new model to participant behavior, it still revealed a significant truth bonus in both the main study (Wilkoxon’s signrank test: median = 0.09, z(202)=2.12, p=0.034; Fig. S27a) and the discovery study (median = 3.52, z(102)=7.86, p<0.001; Fig. S27c). Moreover, in the main study, this truth bonus remained significantly higher than what was predicted by all the alternative models, with the exception of the instructed-credibility bayesian model (Fig. S27b). In the discovery study, the truth bonus was significantly higher than what was predicted by all the alternative models (Fig. S27d).”

      Together, these findings suggest that our truth inference results are not simply a re-description of a positivity bias.

      Conversely, we acknowledge the reviewer's point that our positivity bias results could potentially stem from a more general truth inference mechanism. We believe that this possibility should be addressed in a future study where participants rate their belief that received feedback is true (rather than a lie).We have extended our discussion to clarify this possibility and to include the suggested citation:

      “Our findings show that individuals increase their credit assignment for feedback in proportion to the perceived probability that the feedback is true, even after controlling for source credibility and feedback valence. Strikingly, this learning bias was not predicted by any of our Bayesian or credit-assignment (CA) models. Notably, our evidence for this bias is based on a “oracle model” that incorporates the probability of feedback truthfulness from the experimenter's perspective, rather than the participant’s. This raises an important open question: how do individuals form beliefs about feedback truthfulness, and how do these beliefs influence credit assignment? Future research should address this by eliciting trial-by-trial beliefs about feedback truthfulness. Doing so would also allow for testing the intriguing possibility that an exaggerated positivity bias for non-credible sources reflects, to some extent, a truth-based discounting of negative feedback—i.e., participants may judge such feedback as less likely to be true. However, it is important to note that the positivity bias observed for fully credible sources (here and in other literature) cannot be attributed to a truth bias—unless participants were, against instructions, distrustful of that source.”

      The authors get close to this in the discussion, but they characterize their results as differing from the predictions of rational models, the opposite of my intuition. They write:

      “Alternative "informational" (motivation-independent) accounts of positivity and confirmation bias predict a contrasting trend (i.e., reduced bias in low- and medium credibility conditions) because in these contexts it is more ambiguous whether feedback confirms one's choice or outcome expectations, as compared to a full-credibility condition.”

      I don't follow the reasoning here at all. It seems to me that the possibility for bias will increase with ambiguity (or perhaps will be maximal at intermediate levels). In the extreme case, when feedback is fully reliable, it is impossible to rationally discount it (illustrated in Figure 6A). The authors should clarify their argument or revise their conclusion here.

      We apologize for the lack of clarity in our previous explanation. We removed the sentence you cited (it was intended to make a different point which we now consider non-essential). Our current narration is consistent with the point you are making.

      (4) Disinformation or less information?

      Zooming out, from a computational/functional perspective, the reliability of feedback is very similar to reward stochasticity (the difference is that reward stochasticity decreases the importance/value of learning in addition to its difficulty). I imagine that many of the effects reported here would be reproduced in that setting. To my surprise, I couldn't quickly find a study asking that precise question, but if the authors know of such work, it would be very useful to draw comparisons. To put a finer point on it, this study does not isolate which (if any) of these effects are specific to disinformation, rather than simply less information. I don't think the authors need to rigorously address this in the current study, but it would be a helpful discussion point.

      We thank the reviewer for highlighting the parallel (and difference) between feedback reliability and reward stochasticity. However, we have not found any comparable results in the literature. We also note that our discussion includes a paragraph addressing the locus of our effects making the point that more studies are necessary to determine whether our findings are due to disinformation per se or sources being less informative. While this paragraph was included in the previous version it led us to infer our Discussion was too long and we therefore shortened it considerably:

      “An important question arises as to the psychological locus of the biases we uncovered. Because we were interested in how individuals process disinformation—deliberately false or misleading information intended to deceive or manipulate—we framed the feedback agents in our study as deceptive, who would occasionally “lie” about the true choice outcome. However, statistically (though not necessarily psychologically), these agents are equivalent to agents who mix truth-telling with random “guessing” or “noise” where inaccuracies may arise from factors such as occasionally lacking access to true outcomes, simple laziness, or mistakes, rather than an intent to deceive. This raises the question of whether the biases we observed are driven by the perception of potential disinformation as deceitful per se or simply as deviating from the truth. Future studies could address this question by directly comparing learning from statistically equivalent sources framed as either lying or noisy. Unlike previous studies wherein participants had to infer source credibility from experience (30,37,72), we took an explicit-instruction approach, allowing us to precisely assess source-credibility impact on learning, without confounding it with errors in learning about the sources themselves. More broadly, our work connects with prior research on observational learning, which examined how individuals learn from the actions or advice of social partners (72–75). This body of work has demonstrated that individuals integrate learning from their private experiences with learning based on others’ actions or advice—whether by inferring the value others attribute to different options or by mimicking their behavior (57,76). However, our task differs significantly from traditional observational learning. Firstly, our feedback agents interpret outcomes rather than demonstrating or recommending actions (30,37,72). Secondly, participants in our study lack private experiences unmediated by feedback sources. Finally, unlike most observational learning paradigms, we systematically address scenarios with deliberately misleading social partners. Future studies could bridge this by incorporating deceptive social partners into observational learning, offering a chance to develop unified models of how individuals integrate social information when credibility is paramount for decision-making.”

      (5) Over-reliance on analyzing model parameters

      Most of the results rely on interpreting model parameters, specifically, the "credit assignment" (CA) parameter. Exacerbating this, many key conclusions rest on a comparison of the CA parameters fit to human data vs. those fit to simulations from a Bayesian model. I've never seen anything like this, and the authors don't justify or even motivate this analysis choice. As a general rule, analyses of model parameters are less convincing than behavioral results because they inevitably depend on arbitrary modeling assumptions that cannot be fully supported. I imagine that most or even all of the results presented here would have behavioral analogues. The paper would benefit greatly from the inclusion of such results. It would also be helpful to provide a description of the model in the main text that makes it very clear what exactly the CA parameter is capturing (see next point).

      We thank the reviewer for this important suggestion which we address together with the following point.

      (6) RL or regression?

      I was initially very confused by the "RL" model because it doesn't update based on the TD error. Consequently, the "Q values" can go beyond the range of possible reward (SI Figure 5). These values are therefore not Q values, which are defined as expectations of future reward ("action values"). Instead, they reflect choice propensities, which are sometimes notated $h$ in the RL literature. This misuse of notation is unfortunately quite common in psychology, so I won't ask the authors to change the variable. However, they should clarify when introducing the model that the Q values are not action values in the technical sense. If there is precedent for this update rule, it should be cited.

      Although the change is subtle, it suggests a very different interpretation of the model.

      Specifically, I think the "RL model" is better understood as a sophisticated logistic regression, rather than a model of value learning. Ignoring the decay term, the CA term is simply the change in log odds of repeating the just-taken action in future trials (the change is negated for negative feedback). The PERS term is the same, but ignoring feedback. The decay captures that the effect of each trial on future choices diminishes with time. Importantly, however, we can re-parameterize the model such that the choice at each trial is a logistic regression where the independent variables are an exponentially decaying sum of feedback of each type (e.g., positive-cred50, positive-cred75, ... negative-cred100). The CA parameters are simply coefficients in this logistic regression.

      Critically, this is not meant to "deflate" the model. Instead, it clarifies that the CA parameter is actually not such an assumption-laden model estimate. It is really quite similar to a regression coefficient, something that is usually considered "model agnostic". It also recasts the non-standard "cross-fitting" approach as a very standard comparison of regression coefficients for model simulations vs. human data. Finally, using different CA parameters for true vs false feedback is no longer a strange and implausible model assumption; it's just another (perfectly valid) regression. This may be a personal thing, but after adopting this view, I found all the results much easier to understand.

      We thank the reviewer for their insightful and illuminating comments, particularly concerning the interpretation of our model parameters and the nature of our Credit assignment model. We believe your interpretation of the model is accurate and we now narrate it to readers in the hope that our modelling will become clearer and more intuitively. We also present to readers how these recasts our “cross-fitting” approach in the way you suggested (we return to this point below).

      Broadly, while we agree that modelling results depend on underlying assumptions, we believe that “model-agnostic” approaches also have important limitations—especially in reinforcement learning (RL), where choices are shaped by histories of past events, which such approaches often fail to fully account for. As students of RL, we are frequently struck by how careful modelling demonstrates that seemingly meaningful “model-agnostic” patterns can emerge as artefacts of unaccounted-for variables. We also note that the term “model-agnostic” is difficult to define—after all, even regression models rely on assumptions, and some computational models make richer or more transparent assumptions than others. Ideally, we aim to support our findings using converging methods wherever possible.

      We want to clarify that many of our reported findings indeed stem from straightforward behavioral analyses (e.g., simple regressions of choice-repetition), which do not rely on complex modeling assumptions. The two key results that primarily depend on the analysis of model parameters are our findings related to positivity bias and truth inference.

      Regarding the positivity bias, identifying truly model-agnostic behavioral signatures, distinct from effects like choice-perseveration, has historically been a significant challenge in the literature. Classical research on this bias rests on the interpretation of model parameters (Lefebvre et al., 2017; Palminteri et al., 2017), or at least on the use of models to assess what an “unbiased learner” baseline should look like (Palminteri & Lebreton, 2022). Some researchers have suggested possible regressions incorporating history effects to detect positivity bias from choicerepetition behavior, but these regressions (as our model) rely on subtle assumptions about forgetting and history effects (Toyama et al., 2019). Specifically, in our case, this issue is also demonstrated by analysis we conducted related to the previous point the reviewer made (about perseveration masquerading as positivity bias). We believe that dissociating clearly positivity bias from perseveration is an important challenge for the field going forward.

      For our truth inference results, obtaining purely behavioral signatures is similarly challenging due to the intricate interdependencies (the reviewer has identified in previous points) between agent credibility, feedback valence, feedback truthfulness, and choice accuracy within our task design.

      Finally, we agree with the reviewer that regression coefficients are often interpreted as a “modelagnostic” pattern. From this perspective even our findings regarding positivity and truth bias are not a case of over-reliance on complex model assumptions but are rather a way to expose deviations between empirical “sophisticated” regression coefficients and coefficients predicted from Bayesian models.

      We have now described the main learning rule of our model in the main text to ensure that the meaning of the CA parameters is clearer for readers:

      “Next, we formulated a family of non-Bayesian computational RL models. Importantly, these models can flexibly express non-Bayesian learning patterns and, as we show in following sections, can serve to identify learning biases deviating from an idealized Bayesian strategy. Here, an assumption is that during feedback, the choice propensity for the chosen bandit (which here is represented by a point estimate, “Q value“, rather than a distribution) either increases or decreases (for positive or negative feedback, respectively) according to a magnitude quantified by the free “Credit-Assignment (CA)” model parameters (47):

      𝑄(𝑐ℎ𝑜𝑠𝑒𝑛) ← (1 – 𝑓<sub>Q</sub>) ∗ 𝑄(𝑐ℎ𝑜𝑠𝑒𝑛) + 𝐶𝐴(𝑎𝑔𝑒𝑛𝑡, 𝑣𝑎𝑙𝑒𝑛𝑐𝑒) ∗ 𝐹

      where F is the feedback received from the agents (coded as 1 for reward feedback and -1 for non-reward feedback), while fQ (∈[0,1]) is the free parameter representing the forgetting rate of the Q-value (Fig. 2a, bottom panel; Fig. S5b; Methods). The probability to choose a bandit (say A over B) in this family of models is a logistic function of the contrast choice-propensities between these two bandits. One interpretation of this model is as a “sophisticated” logistic regression, where the CA parameters take the role of “regression coefficients” corresponding to the change in log odds of repeating the just-taken action in future trials based on the feedback (+/- CA for positive or negative feedback, respectively; the model also includes gradual perseveration which allows for constant log-odd changes that are not affected by choice feedback; see “Methods: RL models”) . The forgetting rate captures the extent to which the effect of each trial on future choices diminishes with time. The Q-values are thus exponentially decaying sums of logistic choice propensities based on the types of feedback a bandit received.”

      We also explain the implications of this perspective for our cross-fitting procedure:

      “To further characterise deviations between behaviour and our Bayesian learning models, we used a “crossfitting” method. Treating CA parameters as data-features of interest (i.e., feedback dependent changes in choice propensity), our goal was to examine if and how empirical features differ from features extracted from simulations of our Bayesian learning models. Towards that goal, we simulated synthetic data based on Bayesian agents (using participants’ best fitting parameters), but fitted these data using the CA-models, obtaining what we term “Bayesian-CA parameters” (Fig. 2d; Methods). A comparison of these BayesianCA parameters, with empirical-CA parameters obtained by fitting CA models to empirical data, allowed us to uncover patterns consistent with, or deviating from, ideal-Bayesian value-based inference. Under the sophisticated logistic-regression interpretation of the CA-model family the cross-fitting method comprises a comparison between empirical regression coefficients (i.e., empirical CA parameters) and regression coefficient based on simulations of Bayesian models (Bayesian CA parameters). Using this approach, we found that both the instructed-credibility and free-credibility Bayesian models predicted increased BayesianCA parameters as a function of agent credibility (Fig. 3c; see SI 3.1.1.2 Tables S8 and S9). However, an in-depth comparison between Bayesian and empirical CA parameters revealed discrepancies from ideal Bayesian learning, which we describe in the following sections.”

      Recommendations for the authors:

      Reviewer #3 (Recommendations for the authors):

      (1) Keep terms consistent, e.g., follow-up vs. main; hallmark vs. traditional.

      We have now changed the text to keep terms consistent.

      (2) CA model is like a learning rate; but it's based on the raw reward, not the TD error - this seems strange.

      We thank the reviewer for this comment. We understand that the use of a CA model instead of a TD error model may seem unusual at first glance. However, the CA model offers an important advantage: it more easily accommodates what we term "negative learning rates". This means that some participants may treat certain agents (especially the random one) as consistently deceitful, leading them to effectively increase/reduce choice tendencies following negative/positive feedback. A CA model handles this naturally by allowing negative CA parameters as a simple extension of positive ones. In contrast, adapting a TD error model to account for this is more complex. For instance, attempting to introduce a "negative learning rate" makes the RW model behave in a non-stable manner (e.g., Q values become <0 or >1). At the initial stages of our project, we explored different approaches to dealing with this issue and we found the CA model provides the best approach. For these reasons, we decided to proceed with our CA model.

      Additionally, we used the CA model in previous studies (e.g., Moran, Dayan & Dolan (2021)) where we included (in SI) a detailed discussion of the similarities and difference between creditassignment and Rescorla-Wagner models

      (3) Why was the follow-up study not pre-registered?

      We appreciate the reviewer's comment regarding preregistration, which we should have done. Unfortunately, this is now “water under the bridge” but going forward we hope to pre-register increasing parts of our work.

      (4) Other work looking at reward stochasticity?

      As noted in point 4 of the main weaknesses, previous work on reward stochasticity primarily focused on explaining the increase/decrease in learning and its mechanistic bases under varying stochasticity levels. In our study, we uniquely characterize several specific learning biases that are modulated by source credibility, a topic not extensively explored within the existing reward stochasticity framework, as far as we know.

      (5) Equation 1 is different from the one in the figure?

      The reviewer is completely correct. The figure provides a simplified visual representation, primarily focusing on the feedback-based update of the Q-value, and for simplicity, it omits the forgetting term present in the full Equation 1. To ensure complete clarity and prevent any misunderstanding, we have now incorporated a more detailed explanation of the model, including the complete Equation 1 and its components, directly within the main text. This comprehensive description will ensure that readers are fully aware of how the model operates.

      “Next, we formulated a family of non-Bayesian computational RL models. Importantly, these models can flexibly express non-Bayesian learning patterns and, as we show in following sections, can serve to identify learning biases deviating from an idealized Bayesian strategy. Here, an assumption is that during feedback, the choice propensity for the chosen bandit (which here is represented by a point estimate, “Q value“, rather than a distribution) either increases or decreases (for positive or negative feedback, respectively) according to a magnitude quantified by the free “Credit-Assignment (CA)” model parameters (47):

      𝑄(𝑐ℎ𝑜𝑠𝑒𝑛) ← (1 – 𝑓<sub>Q</sub>) ∗ 𝑄(𝑐ℎ𝑜𝑠𝑒𝑛) + 𝐶𝐴(𝑎𝑔𝑒𝑛𝑡, 𝑣𝑎𝑙𝑒𝑛𝑐𝑒) ∗ 𝐹

      where F is the feedback received from the agents (coded as 1 for reward feedback and -1 for non-reward feedback), while fQ (∈[0,1]) is the free parameter representing the forgetting rate of the Q-value (Fig. 2a, bottom panel; Fig. S5b; Methods).”

      (6) Please describe/plot the distribution of all fitted parameters in the supplement. I would include the mean and SD in the main text (methods) as well.

      Following the reviewer’s suggestions, we have included in the Supplementary Document tables displaying the mean and SD of fitted parameters from participants for our main models of interest. We have also plotted the distributions of such parameters. Both for the main study:

      (7) "A novel approach within the disinformation literature by exploiting a Reinforcement Learning (RL) experimental framework".

      The idea of applying RL to disinformation is not new. Please tone down novelty claims. It would be nice to cite/discuss some of this work as well.

      https://arxiv.org/abs/2106.05402?utm_source=chatgpt.com https://www.scirp.org/pdf/jbbs_2022110415273931.pdf https://papers.ssrn.com/sol3/papers.cfm?abstract_id=4173312

      We thank the reviewer for pointing us towards relevant literature. We have now toned down the sentence in the introduction and cited the references provided:

      “To address these questions, we adopt a novel approach within the disinformation literature by exploiting a Reinforcement Learning (RL) experimental framework (36). While RL has guided disinformation research in recent years (37–40), our approach is novel in using one of its most popular tasks: the “bandit task”.”

      (8) Figure 3a - The figures should be in the order that they're referenced (3 is referenced before 2).

      We generally try to stick to this important rule but, in this case, we believe that our ordering serves better the narrative and hope the reviewer will excuse this small violation.

      (9) "Additionally, we found a positive feedback-effect for the 3-star agent"

      What is the analysis here? To avoid confusion with the "positive feedback" effect, consider using "positive effect of feedback". The dash wasn't sufficient to avoid confusion in my case.

      We have now updated the terms in the text to avoid confusion.

      (10) The discovery study revealed even stronger results supporting a conclusion that the credibility-CA model was superior to both Bayesian models for most subjects

      This is very subjective, but I'll just mention that my "cherry-picking" flag was raised by this sentence. Are you only mentioning cases where the discovery study was consistent with the main study? Upon a closer read, I think the answer is most likely "no", but you might consider adopting a more systematic (perhaps even explicit) policy on when and how you reference the discovery study to avoid creating this impression in a more casual reader.

      We thank the reviewer for this valuable suggestion. To prevent any impression of "cherry-picking", we have removed specific references to the discovery study from the main body of the text. Instead, all discussions regarding the convergence and divergence of results between the two studies are now in the dedicated section focusing on the discovery study:

      “The discovery study (n=104) used a disinformation task structurally similar to that used in our main study, but with three notable differences: 1) it included 4 feedback agents, with credibilities of 50%, 70%, 85% and 100%, represented by 1, 2, 3, and 4 stars, respectively; 2) each experimental block consisted of a single bandit pair, presented over 16 trials (with 4 trials for each feedback agent); and 3) in certain blocks, unbeknownst to participants, the two bandits within a pair were equally rewarding (see SI section 1.1). Overall, this study's results supported similar conclusions as our main study (see SI section 1.2) with a few differences. We found convergent support for increased learning from more credible sources (SI 1.2.1), superior fit for the CA model over Bayesian models (SI 1.2.2) and increased learning from feedback inferred to be true (SI 1.2.6). Additionally, we found an inflation of positivity bias for low-credibility both when measured relative to the overall level of credit assignment (as in our main study), or in absolute terms (unlike in our main study) (Fig. S3; SI 1.2.5). Moreover, choice-perseveration could not predict an amplification of positivity bias for low-credibility sources (see SI 3.6.2). However, we found no evidence for learning based on 50%-credibility feedback when examining either the feedback effect on choice repetition or CA in the credibility-CA model (SI 1.2.3).”

      (11) An in-depth comparison between Bayesian and empirical CA parameters revealed discrepancies from normative Bayesian learning.

      Consider saying where this in-depth comparison can be found (based on my reading, I think you're referring to the next section?

      We have now modified the sentence for better clarity:

      “However, an in-depth comparison between Bayesian and empirical CA parameters revealed discrepancies from ideal Bayesian learning, which we describe in the following sections.”

      (12) "which essentially provides feedback" Perhaps you meant "random feedback"?

      We have modified the text as suggested by the reviewer.

      <(13) Essentially random

      Why "essentially"? Isn't it just literally random?

      We have modified the text as suggested by the reviewer.

      (14) Both Bayesian models predicted an attenuated credit-assignment for the 3-star agent

      Attenuated relative to what? I wouldn't use this word if you mean weaker than what we see in the human data. Instead, I would say people show an exaggerated credit-assignment, since Bayes is the normative baseline.

      We changed the text according to the reviewer’s suggestion:

      “A comparison of empirical and Bayesian credit-assignment parameters revealed a further deviation from ideal Bayesian learning: participants showed an exaggerated credit-assignment for the 3-star agent compared with Bayesian models.”

      (15) "there was no difference between 2-star and 3-star agent contexts (b=0.051, F(1,2419)=0.39, p=0.53)"

      You cannot confirm the null hypothesis! Instead, you can write "The difference between 2-star and 3-star agent contexts was not significant". Although even with this language, you should be careful that your conclusions don't rest on the lack of a difference (the next sentence is somewhat ambiguous on this point).

      Additionally, the reported b coefs do not match the figure, which if anything, suggests a larger drop from 0.75 (2-star) to 1 (3-star). Is this a mixed vs fixed effects thing? It would be helpful to provide an explanation here.

      We thank the reviewer for this question. When we previously submitted our manuscript, we thought that finding enhanced credit-assignment for fully credible feedback following potential disinformation from a DIFFERENT context would constitute a striking demonstration of our “contrast effect”. However, upon reexamining this finding we found out we had a coding error (affecting how trials were filtered). We have now rerun and corrected this analysis. We have assessed the contrast effect for both "same-context" trials (where the contextual trial featured the same bandit pair as the learning trial) and "different-context" trials (where the contextual trial featured a different bandit pair). Our re-analysis reveals a selective significant contrast effect in the same-context condition, but no significant effect in the different-context condition. We have updated the main text to reflect these corrected findings and provide a clearer explanation of the analysis:

      “A comparison of empirical and Bayesian credit-assignment parameters revealed a further deviation from ideal Bayesian learning: participants showed an exaggerated credit-assignment for the 3-star agent compared with Bayesian models [Wilcoxon signed-rank test, instructed-credibility Bayesian model (median difference=0.74, z=11.14); free-credibility Bayesian model (median difference=0.62, z=10.71), all p’s<0.001] (Fig. 3a). One explanation for enhanced learning for the 3-star agents is a contrast effect, whereby credible information looms larger against a backdrop of non-credible information. To test this hypothesis, we examined whether the impact of feedback from the 3-star agent is modulated by the credibility of the agent in the trial immediately preceding it. More specifically, we reasoned that the impact of a 3-star agent would be amplified by a “low credibility context” (i.e., when it is preceded by a low credibility trial). In a binomial mixed effects model, we regressed choice-repetition on feedback valence from the last trial featuring the same bandit pair (i.e., the learning trial) and the feedback agent on the trial immediately preceding that last trial (i.e., the contextual credibility; see Methods for model-specification). This analysis included only learning trials featuring the 3-star agent, and context trials featuring the same bandit pair as the learning trial (Fig. 4a). We found that feedback valence interacted with contextual credibility (F(2,2086)=11.47, p<0.001) such that the feedback-effect (from the 3-star agent) decreased as a function of the preceding context-credibility (3-star context vs. 2-star context: b= -0.29, F(1,2086)=4.06, p=0.044; 2star context vs. 1-star context: b=-0.41, t(2086)=-2.94, p=0.003; and 3-star context vs. 1-star context: b=0.69, t(2086)=-4.74, p<0.001) (Fig. 4b). This contrast effect was not predicted by simulations of our main models of interest (Fig. 4c). No effect was found when focussing on contextual trials featuring a bandit pair different than the one in the learning trial (see SI 3.5). Thus, these results support an interpretation that credible feedback exerts a greater impact on participants’ learning when it follows non-credible feedback, in the same learning context.”

      We have modified the discussion accordingly as well:

      “A striking finding in our study was that for a fully credible feedback agent, credit assignment was exaggerated (i.e., higher than predicted by our Bayesian models). Furthermore, the effect of fully credible feedback on choice was further boosted when it was preceded by a low-credibility context related to current learning. We interpret this in terms of a “contrast effect”, whereby veridical information looms larger against a backdrop of disinformation (21). One upshot is that exaggerated learning might entail a risk of jumping to premature conclusions based on limited credible evidence (e.g., a strong conclusion that a vaccine produces significant side-effect risks based on weak credible information, following non-credible information about the same vaccine). An intriguing possibility, that could be tested in future studies, is that participants strategically amplify the extent of learning from credible feedback to dilute the impact of learning from noncredible feedback. For example, a person scrolling through a social media feed, encountering copious amounts of disinformation, might amplify the weight they assign to credible feedback in order to dilute effects of ‘fake news’. Ironically, these results also suggest that public campaigns might be more effective when embedding their messages in low-credibility contexts, which may boost their impact.”

      And we have included some additional analyses in the SI document:

      “3.5 Contrast effects for contexts featuring a different bandit Given that we observed a contrast effect when both the learning and the immediately preceding "context trial” involved the same pair of bandits, we next investigated whether this effect persisted when the context trial featured a different bandit pair – a situation where the context would be irrelevant to the current learning. Again, we used in a binomial mixed effects model, regressing choice-repetition on feedback valence in the learning trial and the feedback agent in the context trial. This analysis included only learning trials featuring the 3-star agent, and context trials featuring a different bandit pair than the learning trial (Fig. S22a). We found no significant evidence of an interaction between feedback valence and contextual credibility (F(2,2364)=0.21, p=0.81) (Fig. S22b). This null result was consistent with the range of outcomes predicted by our main computational models (Fig. S22c).”

      We aimed to formally compare the influence of two types of contextual trials: those featuring the same bandit pair as the learning trial versus those featuring a different pair. To achieve this, we extended our mixedeffects model by incorporating a new predictor variable, "CONTEXT_TYPE" which coded whether the contextual trial involved the same bandit pair (coded as -0.5) or a different bandit pair (+0.5) compared to the learning trial. The Wilkinson notation for this expanded mixed-effects model is:

      𝑅𝐸𝑃𝐸𝐴𝑇 ~ 𝐶𝑂𝑁𝑇𝐸𝑋𝑇_𝑇𝑌𝑃𝐸 ∗ 𝐹𝐸𝐸𝐷𝐵𝐴𝐶𝐾 ∗ (𝐶 𝐶𝑂𝑁𝑇𝐸𝑋𝑇<sub>2-star</sub> + 𝐶𝑂𝑁𝑇𝐸𝑋𝑇<sub>3-star</sub>) + 𝐵𝐸𝑇𝑇𝐸𝑅 + (1|𝑝𝑎𝑟𝑡𝑖𝑐𝑖𝑝𝑎𝑛𝑡)

      This expanded model revealed a significant three-way interaction between feedback valence, contextual credibility, and context type (F(2,4451) = 7.71, p<0.001). Interpreting this interaction, we found a 2-way interaction between context-source and feedback valence when the context was the same (F(2,4451) = 12.03, p<0.001), but not when context was different (F(2,4451) = 0.23, p = 0.79). Further interpreting the double feedback-valence * context-source interaction (for the same context) we obtained the same conclusions as reported in the main text.”

      (16) "Strikingly, model-simulations (Methods) showed this pattern is not predicted by any of our other models"

      Why doesn't the Bayesian model predict this?

      Thanks for the comment. Overall, Bayesian models do predict a slight truth inference effect (see Figure 6d). However, these effects are not as strong as the ones observed in participants, suggesting that our results go beyond what would be predicted by a Bayesian model.

      Conceptually, it's important to note that the Bayesian model can infer (after controlling for source credibility and feedback valence) whether feedback is truthful based solely on prior beliefs about the chosen bandit. Using this inferred truth to amplify the weight of truthful feedback would effectively amount to “bootstrapping on one’s own beliefs.” This is most clearly illustrated with the 50% agent: if one believes that a chosen bandit yields rewards 70% of the time, then positive feedback is more likely to be truthful than negative feedback. However, a Bayesian observer would also recognize that, given the agent’s overall unreliability, such feedback should be ignored regardless.

      (17) "A striking finding in our study was that for a fully credible feedback agent, credit assignment was exaggerated (i.e., higher than predicted by a Bayesian strategy)".

      "Since we did not find any significant interactions between BETTER and the other regressors, we decided to omit it from the model formulation".

      Was this decision made after seeing the data? If so, please report the original analysis as well.

      We have included the BETTER regressor again, and we have re-run the analyses. We now report the results of such regression. We have also changed the methods section accordingly:

      “We used a different mixed-effects binomial regression model to test whether value learning from the 3-star agent was modulated by contextual credibility. We focused this analysis on instances where the previous trial with the same bandit pair featured the 3-star agent. We regressed the variable REPEAT, which indicated whether the current trial repeated the choice from the previous trial featuring the same bandit-pair (repeated choice=1, non-repeated choice=0). We included the following regressors: FEEDBACK coding the valence of feedback in the previous trial with the same bandit pair (positive=0.5, negative=-0.5), CONTEXT2-star indicating whether the trial immediately preceding the previous trial with the same bandit pair (context trial) featured the 2-star agent (feedback from 2-star agent=1, otherwise=0), and CONTEXT3star indicating whether the trial immediately preceding the previous trial with the same bandit pair featured the 3-star agent. We also included a regressor (BETTER) coding whether the bandit chosen in the learning trial was the better -mostly rewarding- or the worse -mostly unrewarding- bandit within the pair. We included in this analysis only current trials where the context trial featured a different bandit pair. The model in Wilkinson’s notation was:

      𝑅𝐸𝑃𝐸𝐴𝑇~ 𝐹𝐸𝐸𝐷𝐵𝐴𝐶𝐾 ∗ (𝐶𝑂𝑁𝑇𝐸𝑋𝑇<sub>2-star</sub> + 𝐶𝑂𝑁𝑇𝐸𝑋𝑇<sub>3-star</sub>) + 𝐵𝐸𝑇𝑇𝐸𝑅 + (1|𝑝𝑎𝑟𝑡𝑖𝑐𝑖𝑝𝑎𝑛𝑡) ( 13 )

      In figure 4c, we independently calculate the repeat probability difference for the better (mostly rewarding) and worse (mostly non-rewarding) bandits and averaged across them. This calculation was done at the participants level, and finally averaged across participants.”

    1. Además, este padecimiento quizá contribuya a las alteraciones del sueño, la fatiga y, de particular importancia en niños, a los problemas de aprendizaje. +++

      Hacer diagnóstico diferencia con TDAH o desnutrición

    1. These nasal CPAP (NCPAP) devices deliver airflow that is continuously regulated to produce a set pressure, usually 4 to 7 cm H 2 O. NCPAP provides distending pressure to the airways and alveoli throughout the respiratory cycle.

      When researching the impact of CPAP on incidence of BPD did this study include CPAP on higher pressures? Will infants on a CPAP of 8 to 9cm experience BPD at similar rates of infants on invasive ventilation?

    1. If a computer system has multiple users and allows the concurrent execution of multiple processes, then access to data must be regulated. For that purpose, mechanisms ensure that files, memory segments, CPU, and other resources can be operated on by only those processes that have gained proper authorization from the operating system. For example, memory-addressing hardware ensures that a process can execute only within its own address space. The timer ensures that no process can gain control of the CPU without eventually relinquishing control. Device-control registers are not accessible to users, so the integrity of the various peripheral devices is protected.

      This passage highlights the importance of the one key role of the operating system is that of the hardware abstraction—hiding the low-level differences among the devices from both the users and the higher-level system components. In UNIX, this is achieved using the I/O subsystem, which is normalizes the communication with its various kinds of devices. The operating system offers a consistent interface, enabling programs to execute I/O operations without requiring knowledge of the specific characteristics of each device

    2. One of the purposes of an operating system is to hide the peculiarities of specific hardware devices from the user. For example, in UNIX, the peculiarities of I/O devices are hidden from the bulk of the operating system itself by the I/O subsystem. The I/O subsystem consists of several components:

      This passage highlights the importance of the one key role of the operating system is that of the hardware abstraction—hiding the low-level differences among the devices from both the users and the higher-level system components. In UNIX, this is achieved using the I/O subsystem, which is normalizes the communication with its various kinds of devices. The operating system offers a consistent interface, enabling programs to execute I/O operations without requiring knowledge of the specific characteristics of each device

    3. In a hierarchical storage structure, the same data may appear in different levels of the storage system. For example, suppose that an integer A that is to be incremented by 1 is located in file B, and file B resides on hard disk. The increment operation proceeds by first issuing an I/O operation to copy the disk block on which A resides to main memory. This operation is followed by copying A to the cache and to an internal register. Thus, the copy of A appears in several places: on the hard disk, in main memory, in the cache, and in an internal register (see Figure 1.15). Once the increment takes place in the internal register, the value of A differs in the various storage systems. The value of A becomes the same only after the new value of A is written from the internal register back to the hard disk.

      This passage illustrates data replication across the storage hierarchy. An individual data element, like the integer A, can be considered to exist at the same time across various storage levels: on the disk, in the main memory, in the cache, and within the CPU register. Changes happen initially in the quickest storage (register) before moving back to slower layers. The final value of A is consistent across all levels only after it is written back from the register to memory and disk. This demonstrates the principle of temporal locality and consistency management in hierarchical storage systems.

    4. Logging. We want to keep track of which programs use how much and what kinds of computer resources. This record keeping may be used for accounting (so that users can be billed) o

      Logging is the process of keeping a record of which programs or users are using computer resources, how much they are using, and what kinds of resources they access. These logs can be used for accounting, where users may be billed for their usage, as well as for monitoring system performance, detecting errors, and ensuring security. In simple terms, logging helps track activities in a system so administrators can analyze usage, identify problems, and maintain accountability.

    5. A process needs certain resources—including CPU time, memory, files, and I/O devices—to accomplish its task. These resources are typically allocated to the process while it is running. In addition to the various physical and logical resources that a process obtains when it is created, various initialization data (input) may be passed along. For example, consider a process running a web browser whose function is to display the contents of a web page on a screen. The process will be given the URL as an input and will execute the appropriate instructions and system calls to obtain and display the desired information on the screen. When the process terminates, the operating system will reclaim any reusable resources.

      A process needs the resources such as the CPU time, memory, files, and the I/O devices to execute its functions. These resources are assigned when the process executes, and it may also obtain the input data to direct its operation—for example, a web browser process receives the URL to show the web page. Once the process is complete, the OS retrieves its resources for use by other processes

    6. Multitasking is a logical extension of multiprogramming. In multitasking systems, the CPU executes multiple processes by switching among them, but the switches occur frequently, providing the user with a fast response time. Consider that when a process executes, it typically executes for only a short time before it either finishes or needs to perform I/O. I/O may be interactive; that is, output goes to a display for the user, and input comes from a user keyboard, mouse, or touch screen. Since interactive I/O typically runs at “people speeds,” it may take a long time to complete. Input, for example, may be bounded by the user's typing speed; seven characters per second is fast for people but incredibly slow for computers. Rather than let the CPU sit idle as this interactive input takes place, the operating system will rapidly switch the CPU to another process.

      Multitasking builds on the multiprogramming by making the process switching much faster and also more frequent. This gives the illusion such that the multiple programs are running at the same time, even though the CPU is just switching rapidly betweenthem. For example, while one program is waiting for the slow input from the keyboard or the mouse (at human speed), the CPU quickly moves to the another program instead of just sitting idle.

    7. One of the most important aspects of operating systems is the ability to run multiple programs, as a single program cannot, in general, keep either the CPU or the I/O devices busy at all times. Furthermore, users typically want to run more than one program at a time as well. Multiprogramming increases CPU utilization, as well as keeping users satisfied, by organizing programs so that the CPU always has one to execute. In a multiprogrammed system, a program in execution is termed a process.

      A major task of the operating system is to ensure the computer stays active and doesn't remain idle. One program cannot constantly engage both the CPU and input/output devices. This is why contemporary systems support multiprogramming—executing multiple programs simultaneously. In this manner, while one program is idle (for instance, awaiting data from the disk), the CPU can process another. This maintains system efficiency and ensures user satisfaction

    8. If there are no processes to execute, no I/O devices to service, and no users to whom to respond, an operating system will sit quietly, waiting for something to happen. Events are almost always signaled by the occurrence of an interrupt. In Section 1.2.1 we described hardware interrupts. Another form of interrupt is a trap (or an exception), which is a software-generated interrupt caused either by an error (for example, division by zero or invalid memory access) or by a specific request from a user program that an operating-system service be performed by executing a special operation called a system call.

      If nothing is happening—no programs to run, no input/output to handle, no user activity—the operating system just waits. Something new usually starts with an interrupt. Hardware interrupts come from the devices (like a keyboard press), but there are also software interrupts, called the traps (or exceptions). Traps are said to happen when the program causes an error (like dividing by zero or accessing the memory it shouldn’t) or when the program requests the help from an operating system. That request is called a system call, which is like the program raising its hand and asking the OS to step in.

    9. Finally, blade servers are systems in which multiple processor boards, I/O boards, and networking boards are placed in the same chassis. The difference between these and traditional multiprocessor systems is that each blade-processor board boots independently and runs its own operating system. Some blade-server boards are multiprocessor as well, which blurs the lines between types of computers. In essence, these servers consist of multiple independent multiprocessor systems.

      This passage explains blade servers, where multiple processor, I/O, and networking boards reside in a single chassis. Each blade boots independently and runs its own OS. Some blades are multiprocessor systems, effectively creating multiple independent multiprocessor systems within one chassis, blurring traditional system classifications.

    10. Recall from the beginning of this section that a general-purpose computer system consists of multiple devices, all of which exchange data via a common bus. The form of interrupt-driven I/O described in Section 1.2.1 is fine for moving small amounts of data but can produce high overhead when used for bulk data movement such as NVS I/O. To solve this problem, direct memory access (DMA) is used. After setting up buffers, pointers, and counters for the I/O device, the device controller transfers an entire block of data directly to or from the device and main memory, with no intervention by the CPU. Only one interrupt is generated per block, to tell the device driver that the operation has completed, rather than the one interrupt per byte generated for low-speed devices. While the device controller is performing these operations, the CPU is available to accomplish other work.

      This text describes Direct Memory Access (DMA), enhancing efficiency for large data transfers. Rather than having the CPU manage each byte (causing numerous interrupts), the device controller moves whole blocks of data straight between the device and memory. This minimizes CPU load and enables it to handle additional tasks during the transfer.

    11. A large portion of operating system code is dedicated to managing I/O, both because of its importance to the reliability and performance of a system and because of the varying nature of the devices.

      This passage highlights that a significant part of an operating system focuses on I/O management. The diversity of devices and the critical role of I/O in system performance and reliability make it a major responsibility of the OS.

    12. The basic interrupt mechanism works as follows. The CPU hardware has a wire called the interrupt-request line that the CPU senses after executing every instruction. When the CPU detects that a controller has asserted a signal on the interrupt-request line, it reads the interrupt number and jumps to the interrupt-handler routine by using that interrupt number as an index into the interrupt vector. It then starts execution at the address associated with that index. The interrupt handler saves any state it will be changing during its operation, determines the cause of the interrupt, performs the necessary processing, performs a state restore, and executes a return_from_interrupt instruction to return the CPU to the execution state prior to the interrupt. We say that the device controller raises an interrupt by asserting a signal on the interrupt request line, the CPU catches the interrupt and dispatches it to the interrupt handler, and the handler clears the interrupt by servicing the device. Figure 1.4 summarizes the interrupt-driven I/O cycle.

      This section describes the I/O cycle driven by interrupts. The CPU tracks an interrupt-request line; when a device sends a signal, the CPU refers to the interrupt number to find the matching interrupt handler in the interrupt vector. The handler preserves the existing state, manages the interrupt, reinstates the state, and hands control back to the interrupted program, ensuring smooth functionality.

    13. Consider a typical computer operation: a program performing I/O. To start an I/O operation, the device driver loads the appropriate registers in the device controller. The device controller, in turn, examines the contents of these registers to determine what action to take (such as “read a character from the keyboard”). The controller starts the transfer of data from the device to its local buffer. Once the transfer of data is complete, the device controller informs the device driver that it has finished its operation. The device driver then gives control to other parts of the operating system, possibly returning the data or a pointer to the data if the operation was a read. For other operations, the device driver returns status information such as “write completed successfully” or “device busy”. But how does the controller inform the device driver that it has finished its operation? This is accomplished via an interrupt.

      This passage describes the sequence of a typical I/O operation. The device driver sets up the device controller, which carries out the action and uses a buffer for data transfer. Once finished, the controller notifies the driver through an interrupt, allowing the OS to process the data or status information. This illustrates how the OS and hardware communicate efficiently.

    14. In the following subsections, we describe some basics of how such a system operates, focusing on three key aspects of the system. We start with interrupts, which alert the CPU to events that require attention. We then discuss storage structure and I/O structure.

      This passage introduces the three key aspects of system operation: interrupts, which notify the CPU of events needing attention, storage structure, and I/O structure. These fundamentals provide the foundation for understanding how the operating system coordinates hardware and software activities.

    15. How, then, can we define what an operating system is? In general, we have no completely adequate definition of an operating system. Operating systems exist because they offer a reasonable way to solve the problem of creating a usable computing system. The fundamental goal of computer systems is to execute programs and to make solving user problems easier. Computer hardware is constructed toward this goal. Since bare hardware alone is not particularly easy to use, application programs are developed. These programs require certain common operations, such as those controlling the I/O devices. The common functions of controlling and allocating resources are then brought together into one piece of software: the operating system.

      This passage explains that while there’s no single perfect definition of an operating system, it exists to make computing systems usable and efficient. By combining common functions like resource allocation and I/O control into one software layer, the OS simplifies program execution and helps users solve problems effectively.

    16. A slightly different view of an operating system emphasizes the need to control the various I/O devices and user programs. An operating system is a control program. A control program manages the execution of user programs to prevent errors and improper use of the computer. It is especially concerned with the operation and control of I/O devices.

      This section presents the operating system as a control program. Beyond just allocating resources, it ensures programs run safely and correctly, preventing misuse or conflicts. A major focus here is managing I/O devices, since they are often shared and prone to errors if not properly controlled.

    17. From the computer's point of view, the operating system is the program most intimately involved with the hardware. In this context, we can view an operating system as a resource allocator. A computer system has many resources that may be required to solve a problem: CPU time, memory space, storage space, I/O devices, and so on. The operating system acts as the manager of these resources. Facing numerous and possibly conflicting requests for resources, the operating system must decide how to allocate them to specific programs and users so that it can operate the computer system efficiently and fairly.

      This passage displys how the operating system as the hardware’s closest partner, serving as a resource allocator. Since programs and the users do constantly compete for the limited resources like the CPU time, memory, storage, and the I/O devices, the OS must make the fair and the efficient decisions about distribution. This role ensures smooth functioning of the entire computer system.

    18. The hardware—the central processing unit (CPU), the memory, and the input/output (I/O) devices—provides the basic computing resources for the system. The application programs—such as word processors, spreadsheets, compilers, and web browsers—define the ways in which these resources are used to solve users' computing problems. The operating system controls the hardware and coordinates its use among the various application programs for the various users.

      This section explains the relationship between hardware, applications, and the operating system. Hardware provides the raw resources, applications give users practical tools, and the operating system acts as the manager that controls and coordinates how those resources are shared among programs and users.

    19. In order to explore the role of an operating system in a modern computing environment, it is important first to understand the organization and architecture of computer hardware. This includes the CPU, memory, and I/O devices, as well as storage. A fundamental responsibility of an operating system is to allocate these resources to programs.

      This section highlights the importance that the operating system acts like the manager for the core hardware components—CPU, memory, the I/O devices, and the storage. By allocating these resources to the different programs, the OS will ensure the fairness, efficiency, and the stability in the modern computing environment. It reminds us that without this resource management,the multiple programs running at the same time would constantly conflict with each other.

    20. This output tells us the timestamp when the I/O operation occurred, whether the I/O was a Read or Write operation, and how many bytes were involved in the I/O. The final column reflects the duration (expressed as latency or LAT) in milliseconds of the I/O.

      This explanation makes it clear how BCC tools like disksnoop provide detailed insights into disk performance. I find it interesting that latency is included, as it directly shows performance bottlenecks. I wonder how this information could be used to optimize system performance.

    21. The specifics of writing custom BCC tools are beyond the scope of this text, but the BCC package (which is installed on the Linux virtual machine we provide) provides a number of existing tools that monitor several areas of activity in a running Linux kernel. As an example, the BCC disksnoop tool traces disk I/O activity. Entering the command

      It’s helpful that BCC comes with prebuilt tools like disksnoop, so I can monitor disk I/O without having to write custom eBPF programs. I wonder what other areas of the kernel can be traced with existing BCC tools and how much detail they provide.

    22. BCC (BPF Compiler Collection) is a rich toolkit that provides tracing features for Linux systems. BCC is a front-end interface to the eBPF (extended Berkeley Packet Filter) tool. The BPF technology was developed in the early 1990s for filtering traffic across a computer network. The “extended” BPF (eBPF) added various features to BPF. eBPF programs are written in a subset of C and are compiled into eBPF instructions, which can be dynamically inserted into a running Linux system. The eBPF instructions can be used to capture specific events (such as a certain system call being invoked) or to monitor system performance (such as the time required to perform disk I/O). To ensure that eBPF instructions are well behaved, they are passed through a verifier before being inserted into the running Linux kernel. The verifier checks to make sure that the instructions do not affect system performance or security.

      I find it interesting that the BCC uses an eBPF to dynamically insert the instructions into the running Linux system for tracing and the performance monitoring. The verifier seems like a crucial safeguard, ensuring that these types of instructions don’t compromise the security or the system performance. I wonder how complex it is to write the eBPF programs in practice, given the limited C subset and the need to pass the verification.This keeps it reflective, humanized, and shows the curiosity about the technical aspects.

    23. At a slightly less tailored level, the system description can lead to the selection of precompiled object modules from an existing library. These modules are linked together to form the generated operating system. This process allows the library to contain the device drivers for all supported I/O devices, but only those needed are selected and linked into the operating system. Because the system is not recompiled, system generation is faster, but the resulting system may be overly general and may not support different hardware configurations.

      This explains how precompiled modules help build an OS quickly without full recompilation. It makes sense for speed, but I’m curious—how often does this ‘overly general’ approach cause compatibility problems with less common hardware?

    24. In Section 2.8.3, we described how the overhead of message passing between different services running in user space compromises the performance of microkernels. To address such performance problems, Darwin combines Mach, BSD, the I/O kit, and any kernel extensions into a single address space. Thus, Mach is not a pure microkernel in the sense that various subsystems run in user space. Message passing within Mach still does occur, but no copying is necessary, as the services have access to the same address space.

      Darwin improves the microkernel performance by combining the mac, BSD, and the I/O Kit, and the kernel extensions into the single address space. This design is used for reducing the overhead of message passing between the services because they share the memory, so no copying is needed—showing the practical way to balance the microkernel modularity with its efficiency.

    25. As is true in other systems, major performance improvements in operating systems are more likely to be the result of better data structures and algorithms than of excellent assembly-language code. In addition, although operating systems are large, only a small amount of the code is critical to high performance; the interrupt handlers, I/O manager, memory manager, and CPU scheduler are probably the most critical routines. After the system is written and is working correctly, bottlenecks can be identified and can be refactored to operate more efficiently.

      As is true in other systems, major performance improvements in operating systems are more likely to be the result of better data structures and algorithms than of excellent assembly-language code. In addition, although operating systems are large, only a small amount of the code is critical to high performance; the interrupt handlers, I/O manager, memory manager, and CPU scheduler are probably the most critical routines. After the system is written and is working correctly, bottlenecks can be identified and can be refactored to operate more efficiently.

    26. The separation of policy and mechanism is important for flexibility. Policies are likely to change across places or over time. In the worst case, each change in policy would require a change in the underlying mechanism. A general mechanism flexible enough to work across a range of policies is preferable. A change in policy would then require redefinition of only certain parameters of the system. For instance, consider a mechanism for giving priority to certain types of programs over others. If the mechanism is properly separated from policy, it can be used either to support a policy decision that I/O-intensive programs should have priority over CPU-intensive ones or to support the opposite policy.

      This passage highlights why separating policy from mechanism increases flexibility in an operating system. Policies often change depending on context or over time, and if mechanisms were tightly coupled to policies, any change would require redesigning the mechanism. By keeping the mechanisms general and flexible, only the policy parameters needs to be adjusted. For example, the priority mechanism can be used to support the different policies, such as giving the preference to the I/O-intensive programs or the CPU-intensive programs, without modifying any of the underlying mechanism itself.

    27. Object files and executable files typically have standard formats that include the compiled machine code and a symbol table containing metadata about functions and variables that are referenced in the program. For UNIX and Linux systems, this standard format is known as ELF (for Executable and Linkable Format). There are separate ELF formats for relocatable and executable files. One piece of information in the ELF file for executable files is the program's entry point, which contains the address of the first instruction to be executed when the program runs. Windows systems use the Portable Executable (PE) format, and macOS uses the Mach-O format.

      Executable and the object files follow the standard formats which include both the actual machine code and the metadata (like details about functions and variables). On UNIX and the Linux systems, this format is called ELF (Executable and Linkable Format), with the different versions for the relocatable and the executable files. ELF files also are used specify the entry point, which is the first instruction to run when the program starts. Other operating systems use different formats—Windows uses PE (Portable Executable), and macOS uses Mach-O.

    28. Once the device has been requested (and allocated to us), we can read(), write(), and (possibly) reposition() the device, just as we can with files. In fact, the similarity between I/O devices and files is so great that many operating systems, including UNIX, merge the two into a combined file–device structure. In this case, a set of system calls is used on both files and devices. Sometimes, I/O devices are identified by special file names, directory placement, or file attributes.

      Once the device has been requested (and allocated to us), we can read(), write(), and (possibly) reposition() the device, just as we can with files. In fact, the similarity between I/O devices and files is so great that many operating systems, including UNIX, merge the two into a combined file–device structure. In this case, a set of system calls is used on both files and devices. Sometimes, I/O devices are identified by special file names, directory placement, or file attributes.

    1. procede de la intención de alterar la naturaleza tripartita delsigno para hacer de la notación el puro encuentro de un objeto yde su expresión.

      Nuevo verosímil para Barthes: destrucción del signo de manera regresiva, para cuestionar la representación desde el estudio de lo bello y las experiencias artísticas desde una perspectiva que no se basa en principios religiosos o espirituales, sino en la razón, la experiencia humana y el mundo terrenal.

    2. lo «real» estaba del lado de la Historia;pero era para oponerse mejor a lo verosímil, es decir, al ordenmismo del relato (de la imitación o «poesía»

      La supremacía de la Historia, de lo real, lo verdadero sobre lo verosímil, de la imitación de la poesía.

    3. vértigo de la notación

      Sensación de inestabilidad o desorientación que produce el texto en el lector al desestabilizar el sentido único y monolítico. Este vértigo surge al confrontar un texto que es lo opuesto a un tratado de un solo significado, ya que lo no codificado, lo que queda fuera del discurso normativo, genera esta sensación de profundidad y multiplicidad de interpretaciones.

    4. la finalidad estética de la descripción flaubertianaestá totalmente impregnada de imperativos «realistas», como si enapariencia la exactitud del referente, superior o indiferente a todaotra función, gobernara y justificara, ella sola, el describirlo o—en el caso de las descripciones reducidas a una palabra— eldenotarlo: las exigencias estéticas se impregnan aquí —al menosa título de coartada—: de exipencias referenciales

      Finalidad estética de la descripción en Flaubert

    5. el análisis estructural, ordinariamenteocupado hasta hoy en separar y sistematizar las grandes articu-laciones del relato, deja de lado, sea porque excluyen del inventa-rio (no hablando de ellos) todos los detalles «superfluos» (enrelación con la estructura), sea porque se tratan a estos mismosdetalles (el propio autor de estas líneas lo ha intentado)8 como«rellenos» (catálisis), afectados de un valor funcional indirecto, enla medida en que, al sumarse, constituyen algún indicio de carác-ter o de atmósfera y pueden ser así finalmente recuperados por laestructura.

      Omisiones del análisis estructural sobre lo que se considera relleno de valor funcional indirecto

    6. ipotiposis

      Figura retórica que consiste en una descripción realista, viva y detallada de algo o alguien, tan vívida que parece ser experimentada en el momento. El término, proveniente del griego hupotúpōsis (que significa "boceto" o "esbozo").

    1. necesitamos limpiar los cristales de nuestras ventanas para que las cosas que alcanzamos a ver queden libres de la monotonía del empañado cotidiano o familiar; y de nuestro afán de posesión.

      La renovación por parte de la fantasía desde esa subcreación tiene que ver con deshacer esa fatiga de la realidad y comodidad visual de su cotidianidad.

    2. la intención de combinar su uso más tradicional y elevado (equivalente a Imaginación) con las nociones derivadas de “irrealidad” (o sea, disimilitud con el Mundo Primario) y liberación de la esclavitud del “hecho” observado

      Noción de lo fantástico.

    3. En mi opinión, se tiene muy poco en cuenta este aspecto de la “mitología”: subcreación más que representación o que interpretación simbólica de las bellezas y los terrores del mundo.

      La mitología como subcreación más que representación. Se trata más de la posibilidad de moldear el mundo que de volver a los temas míticos de origen.

    4. La definición de un cuento de hadas -qué es o qué debiera ser- no depende, pues, de ninguna definición ni de ningún relato histórico de elfos o de hadas, sino de la naturaleza de Fantasía: el Reino Peligroso mismo y que sopla en ese país.

      Contexto de la definición. Características para entender los cuentos de hadas.

    1. Cuando este proceso se lleva a cabo en la épocamoderna, cualquier posibilidad de distinguir entrepoiesis ypraxis se desvanece. El «hacer» del hombre se determinacomo actividad productora de un efecto real (el opus deloperari, el factum del facere, el actus del agere), cuyo valor seaprecia en función de la voluntad que en ella se expresa, esdecir, en relación con su libertad y su creatividad. La experiencia central de la poiesis, la pro-ducción hacia la presencia, cede ahora su sitio a la consideración del «cómo», o sea,del proceso a través del que se ha producido el objeto.

      Poiesis en la época moderna

    1. Partiendo deundespojamientoqueigualaeldelamuerte,deunahumildadqueexcedeladeladerrotaylaple-garia,memaravillodeverrestablecersecadavez lacompleyidaddelasnegativas,lasresponsabilidades,losdones,las tristescon-fesiones,lasfragilesmenuras,l

      ⚰️ Despojamiento como la muerte

      El inicio compara el acto del amor (o de la entrega espiritual) con la muerte: desnudez absoluta, pérdida de todo poder, abandono del yo.

      🙇 Humildad más que derrota o plegaria

      En la derrota uno es humillado por otro.

      En la plegaria uno se humilla ante los dioses.

      Pero en el amor, la humildad es todavía más radical: es voluntaria. Uno se vacía por decisión propia.

      🔄 El retorno de lo humano

      Después de ese instante de anulación, vuelve la vida con toda su carga:

      Negativas: límites, rechazos.

      Responsabilidades: lo que uno debe asumir.

      Dones: lo que se recibe y se da.

      Tristes confesiones: la vulnerabilidad.

      Frágiles ternuras: la delicadeza que sobrevive en lo cotidiano.

      🧭 Sentido

      El pasaje muestra cómo el amor o la entrega íntima tiene un movimiento doble:

      Vaciamiento absoluto (como la muerte).

      Retorno a lo complejo y humano (responsabilidad, ternura, confesiones).

      Es casi una dialéctica: el amor como aniquilación momentánea seguida por la reconstrucción del tejido vital.

    2. Partiendo deundespojamientoqueigualaeldelamuerte,deunahumildadqueexcedeladeladerrotaylaple-garia,memaravillodeverrestablecersecadavez lacompleyidaddelasnegativas,lasresponsabilidades,losdones,las tristescon-fesiones,lasfragilesmenuras,l

      ⚰️ Despojamiento como la muerte

      El inicio compara el acto del amor (o de la entrega espiritual) con la muerte: desnudez absoluta, pérdida de todo poder, abandono del yo.

      🙇 Humildad más que derrota o plegaria

      En la derrota uno es humillado por otro.

      En la plegaria uno se humilla ante los dioses.

      Pero en el amor, la humildad es todavía más radical: es voluntaria. Uno se vacía por decisión propia.

      🔄 El retorno de lo humano

      Después de ese instante de anulación, vuelve la vida con toda su carga:

      Negativas: límites, rechazos.

      Responsabilidades: lo que uno debe asumir.

      Dones: lo que se recibe y se da.

      Tristes confesiones: la vulnerabilidad.

      Frágiles ternuras: la delicadeza que sobrevive en lo cotidiano.

      🧭 Sentido

      El pasaje muestra cómo el amor o la entrega íntima tiene un movimiento doble:

      Vaciamiento absoluto (como la muerte).

      Retorno a lo complejo y humano (responsabilidad, ternura, confesiones).

      Es casi una dialéctica: el amor como aniquilación momentánea seguida por la reconstrucción del tejido vital.

    Annotators

    1. A partir disso, o WebChat considera que, enquanto o Atendente está salvando informações da conversa com um cliente ainda não está disponível para receber novos atendimentos.

      Quando está salvando, ele já considera que o a gente está disponível para receber novos cards (o limite de card é considerado contando cards ativos).

  6. learn-us-east-1-prod-fleet01-beaker-xythos.content.blackboardcdn.com learn-us-east-1-prod-fleet01-beaker-xythos.content.blackboardcdn.com
    1. Trade and Trust” addresses the misconception amongsome libertarians that the institutional infrastructure neededto support specialization and trade is minimal.

      Stanford focuses on class divisions, while Kling focuses on specialization and trust. Both look at the big structures that make economies work, just in differently.

    2. iscovering new patterns of sustainablespecialization and trade is more complex and subtle and lessmechanical than what is assumed by the Keynesian and mon-etarist traditions.

      Finance is basically extended trust. Crises happen when that trust disappears Stanford focuses on class and inequality, but Kling focuses on trust and specialization. Both show important parts of how the economy works, just from different angles.

    3. If trade entails trust among strangers, then financial inter-mediation entails trust over time. If people lose trust infinancial intermediaries, then financial intermediation candecline precipitously. That sharp decline can have a broadeffect on the structure of production in the economy

      Trust is key. Without it, people can’t rely on each other’s work. What happens to economies when trust breaks down, like during financial crises...?

    4. Unfortunately, once they have taught the simple exam-ple of comparative advantage, with two producers and twotasks, most economists are done with the issue of specializa-tion. Instead, textbooks want to focus on scarcity and choice.Often, the student will read that economics is about the allo-cation of scarce resources given our unlimited wants. Or heor she may be told that economics is the study of how peoplemake rational choices among competing priorities.Scarcity and choice are certainly important concepts, butmaking them the central focus can lead to economic analysisthat is simplistic and mechanistic. In fact, the approach to eco-nomics that took hold after World War II treats the economyas a machine governed by equations.

      Kling says specialization and cooperation are more important than just scarcity for understanding how economies grow.

    5. Look at the list of ingredients in the cereal. Those ingre-dients had to be refined and shipped to the cereal manu-facturer. Again, those processes required many machines,which in turn had to be manufactured. The cereal grainsand other ingredients had to be grown, harvested, and pro-cessed. Machines were involved in those processes, and thosemachines had to be manufactured.

      Even simple things depend on lots of people working together. Does this mean understanding how we all rely on each other could change how we see value in the economy?

    6. hat econo-mists have lost the art of critical thinking.

      Kling thinks economists focus too much on models and not enough on real-world problems. Makes me wonder what ways of thinking could make economics more useful for regular people.

    7. He knows that his breakfast depends upon workerson the coffee plantations of Brazil, the citrus groves ofFlorida, the sugar fields of Cuba, the wheat farms ofthe Dakotas, the dairies of New York; that it has beenassembled by ships, railroads, and trucks, has beencooked with coal from Pennsylvania in utensils madeof aluminum, china, steel, and glass.

      To me this symbolizes how one nation's economic growth depends on many other nations through trade.

    8. “Filling in Frameworks” wrestles with the misconceptionthat economics is a science. This section looks at the difficul-ties that economists face in trying to adopt scientific methods. Isuggest that economics differs from the natural sciences in thatwe have to rely much less on verifiable hypotheses and muchmore on hard-to-verify interpretative frameworks. Economicanalysis is a challenge, because judging interpretive frame-works is actually harder than verifying scientific hypotheses.

      I find it interesting that Kling has pointed out in this section the difference between natural sciences and the complexity of economics. Noting that economics is largely interpretative and not always subject to the same verifiable scientific methods of study.

  7. learn-us-east-1-prod-fleet01-beaker-xythos.content.blackboardcdn.com learn-us-east-1-prod-fleet01-beaker-xythos.content.blackboardcdn.com
    1. Of course, capitalism can change its “look” a lot, while still preserving its core,underlying features. Indeed, one of the most impressive features of capitalism is itsflexibility: its capacity to change and adapt. Many economists and commentatorshave argued that capitalism today is not at all like capitalism in its early days (backin the soot and grime of the Industrial Revolution). Here are some of the terms usedto describe modern capitalism, implying (falsely) that it’s a whole “new” system:

      Capitalism can change how it looks over time, but its core hasn’t really changed. Even though services are bigger than goods now, information moves faster, private companies still dominate, most workers don’t own the businesses they work for, and inequality is still huge. So while the system adapts and evolves, the basic rules of capitalism stay the same.

    2. land and driven into cities, where they suffered horrendous exploitation andconditions that would be considered intolerable today: seven-day working weeks,twelve-hour working days, child labour, frequent injury, early death. Vast profitswere earned by the new class of capitalists, most of which they ploughed backinto new investment, technology, and growth – but some of which they used tofinance their own luxurious consumption. The early capitalist societies were not atall democratic: the right to vote was limited to property owners, and basic rights tospeak out and organize (including to organize unions) were routinely (and oftenviolently) trampled.

      The start of capitalism during the Industrial Revolution was harsh and unfair. Workers were forced into cities and faced terrible conditions while capitalists made huge profits. Do you think capitalism could have grown the same way if workers had better protections back then...;?

    3. Instead, economists refer simply to “the economy” – as if there is only one kindof economy, and hence no need to name or define it. This is wrong. As we havealready seen, “the economy” is simply where people work to produce the things weneed and want. There are different ways to organize that work. Capitalism is justone of them

      Stanford is saying that capitalism hasn’t always been here and might not stick around forever. It makes me think if something new takes its place, what would it look like, and would it deal with inequality any better?

    4. Conventionally trained economists take it as a proven fact that free tradebetween two countries always makes both sides better off. People who questionor oppose free trade – trade unionists, social activists, nationalists – must eitherbe acting from ignorance, or else are pursuing some narrow vested interest thatconflicts with the broader good. These troublesome people should be lectured to(and economists love nothing better than expounding their beautiful theory ofcomparative advantage*), or simply ignored. And that’s exactly what mostgovernments do. (Ironically, even some conventional economists now recognizethat traditional free trade theory is wrong, for many reasons – some of whichwe’ll discuss in Chapter 22 of this book. But that hasn’t affected the profession’snear-religious devotion to free trade policies.)

      He’s pointing out that economists act like free trade is more of a belief than a fact. I take this to mean that economics sometimes feels more like an ideology than an actual science. If free trade ends up hurting workers, shouldn’t economists question the ideas they’re basing it on?

  8. Aug 2025
    1. Resource allocation. When there are multiple processes running at the same time, resources must be allocated to each of them. The operating system manages many different types of resources. Some (such as CPU cycles, main memory, and file storage) may have special allocation code, whereas others (such as I/O devices) may have much more general request and release code.

      This passage highlights that the operating system is responsible for resource allocation, distributing CPU time, memory, file storage, and I/O devices among multiple running processes to ensure fair and efficient usage.

    2. Error detection. The operating system needs to be detecting and correcting errors constantly. Errors may occur in the CPU and memory hardware (such as a memory error or a power failure), in I/O devices (such as a parity error on disk, a connection failure on a network, or lack of paper in the printer), and in the user program (such as an arithmetic overflow or an attempt to access an illegal memory location).

      This passage explains that the operating system continuously detects and handles errors. These errors can arise in hardware (CPU, memory, or I/O devices) or in user programs, such as illegal memory access or arithmetic overflow, ensuring system stability.

    3. A hash function takes data as its input, performs a numeric operation on the data, and returns a numeric value. This numeric value can then be used as an index into a table (typically an array) to quickly retrieve the data. Whereas searching for a data item through a list of size n can require up to O(n) comparisons, using a hash function for retrieving data from a table can be as good as O(1), depending on implementation details. Because of this performance, hash functions are used extensively in operating systems.

      This passage explains how that a hash function converts the data into the numeric value, which can be used as an index to quickly access the data in the table. Unlike searching a list, which can take O(n) time, a hash table can often retrieve data in O(1) time. This efficiency is why operating systems frequently use hash functions for tasks like indexing and also the quick lookups.

    4. A tree is a data structure that can be used to represent data hierarchically. Data values in a tree structure are linked through parent–child relationships. In a general tree, a parent may have an unlimited number of children. In a binary tree, a parent may have at most two children, which we term the left child and the right child. A binary search tree additionally requires an ordering between the parent's two children in which left_child <= right_child. Figure 1.20 provides an example of a binary search tree. When we search for an item in a binary search tree, the worst-case performance is O(n) (consider how this can occur). To remedy this situation, we can use an algorithm to create a balanced binary search tree. Here, a tree containing n items has at most lg n levels, thus ensuring worst-case performance of O(lg n). We shall see in Section 5.7.1 that Linux uses a balanced binary search tree (known as a red-black tree) as part its CPU-scheduling algorithm.

      This passage explains how a tree is a hierarchical data structure with the parent–child relationships. Binary trees limit the parents to two children, and the binary search trees (BSTs) impose that an order for efficient searching. In the worst case, a BST can have O(n) search time, but while balancing the tree reduces this to O(log n). Linux uses the balanced trees, such as the red-black trees, in the CPU scheduling to improve the performance.

    5. Linked lists accommodate items of varying sizes and allow easy insertion and deletion of items. One potential disadvantage of using a list is that performance for retrieving a specified item in a list of size n is linear—O(n), as it requires potentially traversing all n elements in the worst case. Lists are sometimes used directly by kernel algorithms. Frequently, though, they are used for constructing more powerful data structures, such as stacks and queues.

      This passage highlights how that the linked lists are flexible, supporting the variable-sized items and easy insertion or the deletion. However, searching for a specific element can be slow (O(n)). Lists are often used directly in kernel algorithms or as building blocks for other structures like stacks and queues.

    6. Defining Operating Systems

      I know it’s tricky to even pin down the definition of an operating system (OS), because computers themselves can be used in so many ways, from small devices such as toasters to massive server machines. So it goes way way back to a time when hardware was practically unusable unless you wrapped up the same few common things like I/O control and resource allocation together in a piece of (software) system.

      A major point of confusion is there is no one definition Some people think the OS is everything that is shipped with the computer, whereas others only consider the kernel which is the thing that’s always running.

    7. What Operating Systems Do We begin our discussion by looking at the operating system's role in the overall computer system. A computer system can be divided roughly into four components: the hardware, the operating system, the application programs, and a user (Figure 1.1). Figure 1.1 Abstract view of the components of a computer system. The hardware—the central processing unit (CPU), the memory, and the input/output (I/O) devices—provides the basic computing resources for the system. The application programs—such as word processors, spreadsheets, compilers, and web browsers—define the ways in which these resources are used to solve users' computing problems. The operating system controls the hardware and coordinates its use among the various application programs for the various users. We can also view a computer system as consisting of hardware, software, and data. The operating system provides the means for proper use of these resources in the operation of the computer system. An operating system is similar to a government. Like a government, it performs no useful function by itself. It simply provides an environment within which other programs can do useful work. To understand more fully the operating system's role, we next explore operating systems from two viewpoints: that of the user and that of the system.

      Hardware provides resources, applications use them to solve problems, and the OS ensures everything runs smoothly between them and the users.

    1. An emergency need arose for someone to write 300 words o

      something that i'm thinking about is a parallel between restaurants -- so "junk words" and "fine dining words". Chat/LLM speaks to me as fluff, filler, words that come out just for convenience. Shakespeare, Mary Oliver, etc are the words that pack a punch. And that makes me think about art's irreplaceability - once someone creates authentic art, it loses its value if it's replicated, even down to the specifics.

    1.  Vှ Ø  ှ ှ ှ $ှ$ှ Y?ှ  ှ ှ v  Ą ှએă ?ှ  ှ ှ 'ှ $#  řှ  ှ #ှ #cμှ

      This is the author's definition of what play is, but I feel that play does not necessarily have to be social. Sometimes people play games by themselves to pass the time - is that still considered social?

    2. ှ Òှ $ 'ှ 

      I find this statement interesting. Is it possible to measure play using the empirical evidence we usually think of when we think of research? How do historians approach researching abstract concepts such as play? Are there standard procedures to study concepts such as play?

    3. ှ ''ှ ှ$Eှ ှ EÒှ ှ ƴှ Eှ !ှ E੃ÒQှ Òှ Òှ ,"ȥ'ܕှ ှ Òှ Eှ !ှ Eှ ¿Ò ှ $ှ |Ò'QÒ'ှ ှ

      I find this interesting because I also assumed that play is meant to serve some evolutionary function, such as building skills needed to survive in the world. I'm curious to know what everyone's hypotheses are about the function of play. Furthermore, is there a general consensus people have reached about the function of play or is the community divided across theories about the function of play?

    4.  ÒQှ ှ ှ Òှ !ှှ ှ ¤Òှ ှÒှ  $'ှ ှ ှ Ò Ò!Ò 

      This makes me wonder if play is a form of training that socializes a being to survive in the world, similar to how play can be used to educate kids.

    5. {Òှ ÒÒှ $+ှ  ှ |ÒှÒ+ှ 'ှ ƺ ှ ှ ¤'Òှ Òှ |Ò'QÒ'ှ 'Ògှ

      With today's scientific knowledge, I would equate this to the release of dopamine and other neurotransmitters in our brains during play. It is part of the reason we “enjoy” play and also incentivizes the next instance of play.

    6. Ƙှ t ÒQှ ှ ှ ှ 'ှ Òှှ ÒÒQှ $ှ ှQှ ှ $ှ ှ Òှ ¿ှှ 'Ò$ှ Ò''ှE ှ 'ှƘှ

      This theory was really interesting to me because it could have very different meanings for humans and other animals. For animals, roughhousing as a youth could prepare them and help build experience for fights over resources as an adult. For humans, our early childhood toys consist of things like matching shapes, completing simple puzzles, music makers, etc. This seems more suited to learning logic and fine motor skills--important skills for human adults.

    7. v“ှ $ှ ှ Q +ှ |ှ ှ ''ှ Òှ ှ ` ,ȥ $ှ '٦Ò଒ʤှ ˺ှ ှ ှ ||ှ ှ Òှ 'ʤှ ˺ှ ှ ှQE|'ှ 'ှ ÒE'$ှ Òှ Òှ Òʤှ ˺ှ Òှ ှ Qှ  ှ  ှှ $³ှ |ှှ $|''ှ Eʤ]ှ

      These three questions happen to contain three very different examples of play that show us a wide range from harmless and innocent to extremely harmful and maladaptive. A baby enjoying a game is both normal and great for her positive development, but the gambler who is "losing himself" in his passion is quite literally ruining his life. It's interesting how different forms of play can have very different effects on the player's life

    1. Nie wolno też zapominać o instalacjach chroniących– przede wszystkim odgromowej, która zabezpieczamieszkanie i całą elektronikę przed skutkami burzy.Profesjonalnie wykonany system przejmuje energięwyładowania i bezpiecznie odprowadza ją do ziemi

      to informacja dla spółdzielni - delete

      W przypadku mieszkań kluczową rolę przy planowaniu instalacji elektrycznej odgrywa materiał, z którego wykonany jest budynek. Jeżeli mamy do czynienia z blokiem z tzw. wielkiej płyty, podejście do rozprowadzenia przewodów musi być inne niż w nowym budownictwie. Najczęściej najlepszym rozwiązaniem okazuje się poprowadzenie instalacji po ścianach działowych, w podłodze lub w suficie podwieszanym – to nie tylko praktyczne, ale też korzystne cenowo dla inwestora.

      Warto jednak pamiętać, że dobrze zaprojektowana instalacja to nie tylko oszczędność na etapie wykonania, ale też wygoda i bezpieczeństwo na lata. Dlatego już na samym początku warto przemyśleć liczbę gniazd, punktów świetlnych oraz ewentualne miejsce na dodatkowe przewody (np. do internetu, automatyki czy klimatyzacji), aby uniknąć kosztownych przeróbek w przyszłości.

    2. Co ważne – całość działa lokalnie, bez chmury i Wi-Fi, więcnie musisz martwić się o awarie internetu. To rozwiązaniedla osób, które chcą bezpieczeństwa i wygody w jednym

      usunąć

    3. Coraz częściej stosuje się oświetlenie LED, które możnawkomponować w stopnie lub balustradę. W tym przypadkuważne jest odpowiednie zaplanowanie miejsca montażuzasilacza – powinien być łatwo dostępny, a jednocześnieukryty, aby nie zakłócał estetyki wnętrza. Do wyboru mamytaśmy jednokolorowe, RGB oraz RGBW, pozwalające zmieniaćbarwę światła i dopasować je do nastroju czy aranżacji

      Każdy wariant oświetlenia schodów można obsługiwać tradycyjnym włącznikiem lub czujnikiem ruchu. Przy zastosowaniu taśm LED istnieje możliwość wzbogacenia sterowania o automatykę, która otwiera drogę do tworzenia nowoczesnych i efektownych aranżacji świetlnych.

    4. Jeśli planujesz lustro z podświetleniem LED,często potrzebujesz dedykowanego obwodu lub gniazdkaz zabezpieczeniem RCD/GFCI, szczególnie w strefie "mokrej"łazienki

      Jeśli w łazience przewidujesz jakiekolwiek gniazdko – np. do podłączenia lustra z podświetleniem LED czy innych urządzeń – zadbaj o to, by było ono chronione wyłącznikiem różnicowoprądowym RCD 30 mA typu A.

    Annotators

    1. shared scene/motion formats,

      Que las apps hablen el mismo idioma. Ejemplos: un formato estándar para la escena (geometría, materiales, cámaras, luces, capas) y otro para el movimiento (keyframes, curvas, esqueletos). Así, si generas un personaje en la herramienta A, lo animas en la B y lo compones en la C, no pierdes nada al exportar/importar (piensa en cosas tipo USD/glTF para escena o BVH/retargeting para movimiento).

    1. Even when a certain color is specified which all listeners have seeninnumerable times•· such as the red o[ the Coca-Cola signs which isthe same red ;tll over the country-· they will still think ofmany different reds

      Green - passages to remember

    Annotators

  9. drive.google.com drive.google.com
    1. Page, M. J., McKenzie, J. E., Bossuyt, P. M., Boutron, I., Hoffmann, T. C.,Mulrow, C. D., ... & Moher, D. (2021). The PRISMA 2020 statement: An updatedguideline for reporting systematic reviews. BMJ, 372, n71.https://doi.org/10.1136/bmj.n71101. Secomb, J. (2008). A systematic review of peer teaching and learning in clinicaleducation. Journal of Clinical Nursing, 17(6), 703–716.https://doi.org/10.1111/j.1365-2702.2007.01954.x102. Glynn, L. G., MacFarlane, A., Kelly, M., Cantillon, P., & Murphy, A. W. (2006).Helping each other to learn – a process evaluation of peer assisted learning. BMCMedical Education, 6(1).https://doi.org/10.1186/1472-6920-6-18103. Boud, D. (2014). Peer Learning in Higher Education (R. Cohen & J. (all of the U.of T. Sampson, Eds.). Routledge.https://doi.org/10.4324/9781315042565boud104. Kinchin, I. M. (2014). Concept Mapping as a Learning Tool in Higher Education:A Critical Analysis of Recent Reviews. The Journal of Continuing Higher Education,62(1), 39–49.https://doi.org/10.1080/07377363.2014.872011105. Topping, K. J. (2005). Trends in Peer Learning. Educational Psychology, 25(6),631–645.https://doi.org/10.1080/01443410500345172106. Ten Cate, O., & Durning, S. (2007). Peer teaching in medical education: Twelvereasons to move from theory to practice. Medical Teacher, 29(6), 591–599.https://doi.org/10.1080/01421590701606799

      alphabetical order + possible duplicates

    1. Unlike shallow learning methods that required humans to carefully engineer features, deep learning networks can automatically discover useful features directly from raw data

      difference between shallow and deep learning lied in teh amount o fdata. the shallow learning methods rely on humans to state what features to "notice"?

    1. Enzimas biotransformadoras inducibles.

      Los principales sistemas que se encargan del metabolismo de los fármacos son las enzimas, y estas pueden ser activadas o inducidas, así mismo, otros fármacos pueden influenciar la actividad de dicha enzima, ya sea mejorándola o disminuyendo su capacidad enzimática si otro fármaco usa la misma enzima para metabolizarse

    2. Cinética de primer orden

      Esto significa que, si hay mucho fármaco, se metaboliza mucho; si hay poco, se metaboliza poco, pero la fracción o porcentaje del fármaco que se elimina por metabolismo es constante

    3. cuando un fármaco muy liposoluble que actúa sobre el cerebro o el aparato cardiovascular se administra con rapidez por vía IV o por inhalación.

      La concentración del fármaco en su sitio de acción llega a saturarse, lo que hace que el resto del fármaco suspendido en la sangre se redistribuye en los tejidos a manera de "reserva" y una vez las concentraciones en el sitio diana disminuyen de nuevo, se liberan del tejido a la circulación para poder ser absorbido en el sitio diana, lo que prolonga el efecto a lo largo del tiempo.

    1. To m o v e f r o m r e a d i n g t o w r i t i n g , y o u n e e d t o r e a d a c t i v e l y, i n a t h o u g h t -ful spirit, and with an alert, inquiring mind. Reading actively means learning how to analyze what you read.

      Active reading is more than just going through the words. It is about paying attention to what is being said and how it is being said. This conscious effort to pay attention helps you notice the patterns in the text and the choices the writer makes, helping you discern what they mean when making such choices. It definitely takes focus. For example, when you are tired and reading the same paragraph over and over again, you are not taking in the text and reading actively. This is a mostly universal experience that stresses the importance of active reading.

    1. asynchronous I/O primitives

      I/O primitives = العمليات الأساسية الخاصة بالإدخال والإخراج (زي قراءة/كتابة فايل، الاتصال بقاعدة بيانات، الاتصال بالإنترنت…).

    1. 65.080 kg

      cái này khác với câu "d" ở trên là vì cái này cò dấu "." là dấu thập phân, mà thập phân thì giữ luôn số 0

    1. julgamento
      • Informativo 1142
      • ADI 7496 MC-Ref / GO
      • Órgão julgador: Tribunal Pleno
      • Relator(a): Min. DIAS TOFFOLI
      • Julgamento: 21/06/2024 (Virtual)
      • Ramo do Direito: Processual Penal, Constitucional Matéria: Foro por prerrogativa de função; medidas cautelares; autorização prévia para investigação criminal/ Repartição de competências; direito penal e processual penal; direitos e garantias fundamentais

      Tribunal de justiça e foro por prerrogativa de função: apreciação de medidas cautelares de natureza criminal

      Resumo

      • É inconstitucional — por violar a competência privativa da União para legislar sobre direito penal e processual penal (CF/1988, art. 22, I), o sistema acusatório e o princípio da isonomia (CF/1988, art. 5º, caput e LIII) — norma de Constituição estadual que condiciona à prévia autorização judicial, mediante decisão fundamentada da maioria absoluta do órgão especial do respectivo tribunal de justiça, o pedido de medida cautelar para fins de investigação criminal ou instrução processual penal em desfavor de autoridades com foro por prerrogativa de função.

      • A norma impugnada, ao regular o foro por prerrogativa de função, não poderia dispor diversamente ou desbordar dos limites estabelecidos no modelo federal que, no caso, estão contidos no próprio Regimento Interno do STF (art. 21, XV). Conforme disposto na referida norma, que possui status de lei ordinária, a competência para supervisão judicial dos atos investigatórios de autoridades com prerrogativa de foro deve ser conferida ao relator, não havendo, portanto, necessidade de deliberação colegiada (1).

      • A razão jurídica que justifica a necessidade de supervisão judicial dos atos investigatórios de autoridades com prerrogativa de foro no STF aplica-se, por simetria, às autoridades com prerrogativa de foro nos tribunais de segundo grau de jurisdição. Ademais, conforme jurisprudência desta Corte, a competência do respectivo tribunal para a supervisão judicial nesses casos não torna obrigatória a deliberação do respectivo órgão colegiado, sendo <u>suficiente</u> decisão do ministro ou desembargador relator (2).

      • Nesse contexto, a exigência de controle judicial prévio por deliberação de órgão colegiado do tribunal de justiça local, além de conferir tratamento diferenciado aos seus detentores de foro por prerrogativa de função, destoa da lógica estabelecida por outras importantes disposições do RISTF (art. 21, IV e V, §§ 5º e 8º, e art. 230-C, § 2º).

      • Com base nesses e em outros entendimentos, o Plenário, por unanimidade, converteu o referendo da medida cautelar em julgamento definitivo de mérito e, confirmando-a, julgou a ação parcialmente procedente para (i) declarar a inconstitucionalidade da expressão “mediante decisão fundamentada tomada pela maioria absoluta do órgão especial previsto no inciso VI do art. 93 da Constituição da República”, contida na alínea “p” do inciso VIII do art. 46 da Constituição do Estado de Goiás, com redação dada pela EC estadual nº 77/2023 (3); e (ii) dar à parte remanescente do referido dispositivo interpretação conforme a Constituição, a fim de esclarecer que “o Desembargador Relator pode apreciar monocraticamente as medidas cautelares penais requeridas durante a fase de investigação ou no decorrer da instrução processual nos casos de urgência e, ainda, quando a sigilosidade se mostrar necessária para assegurar a efetivação da diligência pretendida, ressalvada a obrigatoriedade de referendo pelo órgão colegiado competente, em momento oportuno, sobretudo quando resultar em prisão cautelar, mas sempre sem comprometer ou lhe frustrar a execução”.

      (1) Regimento Interno do STF/1980: “Art.21. São atribuições do Relator: (...) XV – determinar a instauração de inquérito a pedido do Procurador-Geral da República, da autoridade policial ou do ofendido, bem como o seu arquivamento, quando o requerer o Procurador-Geral da República, ou quando verificar: (...)”

      (2) Precedentes citados: ADI 6.732, ADI 7.083 e ADI 5.331.

      (3) Constituição do Estado de Goiás: “Art. 46. Compete privativamente ao Tribunal de Justiça: (...) VIII - processar e julgar originariamente: (...) p) o pedido de medida cautelar para fins de investigação criminal ou instrução processual penal, quando o investigado ou o processado for autoridade cujos atos estejam sujeitos diretamente à sua jurisdição, mediante decisão tomada pelo voto da maioria absoluta do órgão especial previsto no inciso XI do art. 93 da Constituição da República;”

    2. vantagens
      <div style="font-family: Arial, sans-serif; font-size: 13px; line-height: 1.35;"> <div style="font-weight: bold; margin-bottom: 6px;"> Quadro Comparativo – <span style="white-space:nowrap;">Adicional × Gratificação</span> </div> <table cellpadding="6" cellspacing="0" style="border-collapse: collapse; width: 100%; table-layout: fixed;"> <thead> <tr style="background: #f5f5f5; border-top: 2px solid #000; border-bottom: 2px solid #000;"> <th style="text-align:left; width:22%; border-left: 1px solid #ccc; border-right: 1px solid #ccc; word-break: keep-all; white-space: normal;"> Aspecto </th> <th style="text-align:left; width:39%; border-left: 1px solid #ccc; border-right: 1px solid #ccc; word-break: keep-all; white-space: normal;"> Direito Administrativo (Servidores) </th> <th style="text-align:left; width:39%; border-left: 1px solid #ccc; border-right: 1px solid #ccc; word-break: keep-all; white-space: normal;"> Direito do Trabalho (Celetistas) </th> </tr> </thead> <tbody> <tr style="border-bottom: 2px solid #000;"> <td style="border-left: 1px solid #ccc; border-right: 1px solid #ccc;">Fundamen-to</td> <td style="border-left: 1px solid #ccc; border-right: 1px solid #ccc;"> Vantagens pecuniárias ligadas ao tempo de serviço (ex facto temporis) ou à função/regime especial (ex facto officii). </td> <td style="border-left: 1px solid #ccc; border-right: 1px solid #ccc;"> Vantagens salariais por condições mais gravosas do trabalho ou por evento/norma (lei, ACT/ CCT). </td> </tr> <tr style="border-bottom: 2px solid #000;"> <td style="border-left: 1px solid #ccc; border-right: 1px solid #ccc;">Adicionais</td> <td style="border-left: 1px solid #ccc; border-right: 1px solid #ccc;"> Por tempo de serviço (p.ex., quinquênio, sexta-parte – CF/SP, art. 129).<br> De função (p.ex., nível universitário, dedicação exclusiva). </td> <td style="border-left: 1px solid #ccc; border-right: 1px solid #ccc;"> Salário-condição ligado a gravosidade: horas extras, insalubridade, periculosidade, adicional noturno, transferência, etc. </td> </tr> <tr style="border-bottom: 2px solid #000;"> <td style="border-left: 1px solid #ccc; border-right: 1px solid #ccc;">Gratifica-ções</td> <td style="border-left: 1px solid #ccc; border-right: 1px solid #ccc;"> De serviço (condições anormais: risco, insalubridade, representação).<br> Pessoais (propter personam: salário-família, salário-esposa). </td> <td style="border-left: 1px solid #ccc; border-right: 1px solid #ccc;"> Legais (p.ex., gratificação natalina/13º).<br> Convencionais/contratuais (p.ex., semestral, quebra de caixa), com natureza definida pela norma que as institui. </td> </tr> <tr style="border-bottom: 2px solid #000;"> <td style="border-left: 1px solid #ccc; border-right: 1px solid #ccc;">Natureza e incorpo-ração</td> <td style="border-left: 1px solid #ccc; border-right: 1px solid #ccc;"> Em regra, adicionais tendem a incorporar ao vencimento; gratificações são condicionais e cessam com o fato gerador, salvo lei que autorize incorporação. </td> <td style="border-left: 1px solid #ccc; border-right: 1px solid #ccc;"> Adicionais: integram o salário enquanto percebidos e cessam com a condição (Súm. TST 80, 139, 248, 265).<br> Gratificações: após a Lei 13.467/2017, apenas as legais têm natureza salarial; as demais não integram, salvo previsão expressa. </td> </tr> <tr style="border-bottom: 2px solid #000;"> <td style="border-left: 1px solid #ccc; border-right: 1px solid #ccc;">Exemplos clássicos</td> <td style="border-left: 1px solid #ccc; border-right: 1px solid #ccc;"> Quinquênio, sexta-parte (tempo); gratificação de risco/insalubridade (serviço); salário-família (pessoal). </td> <td style="border-left: 1px solid #ccc; border-right: 1px solid #ccc;"> Adicional de insalubridade/periculosidade/noturno/horas extras; gratificação natalina (13º); PLR (não salarial). </td> </tr> <tr style="border-bottom: 2px solid #000;"> <td style="border-left: 1px solid #ccc; border-right: 1px solid #ccc;">Síntese</td> <td style="border-left: 1px solid #ccc; border-right: 1px solid #ccc;"> “Adicional” costuma ser estável/incorporável; “gratificação” é condicional (salvo lei). </td> <td style="border-left: 1px solid #ccc; border-right: 1px solid #ccc;"> “Adicional” é condicional (salário-condição); “gratificação” depende da fonte normativa para natureza salarial. </td> </tr> </tbody> </table> <div style="margin-top:6px; font-size:12px; color:#444;"> Fontes doutrinárias: Meirelles; Maria Sylvia Zanella Di Pietro (Direito Administrativo).<br> Trabalhista: Godinho Delgado; Resende; Súmulas TST 80, 139, 248, 265. </div> </div>
    3. parecer prévio
      • Informativo STF
      • Edição 1185/2025 19 de agosto de 2025

      • Emissão de parecer prévio como condição para a Assembleia Legislativa apreciar as contas prestadas pelo governador - ADPF 434/AL

      • RESUMO: A ausência de parecer prévio do Tribunal de Contas estadual não impede o julgamento das contas do governador pela Assembleia Legislativa. Entendimento contrário configuraria restrição desproporcional à autonomia do Poder Legislativo.

      Conforme jurisprudência desta Corte (1), o referido parecer possui caráter meramente opinativo, tendo em vista a exclusividade da prerrogativa do Poder Legislativo para apreciar as contas em todas as esferas da Federação. Trata-se de opinião não vinculativa e cuja falta não enseja aprovação tácita das contas.

      Na espécie, ainda que a Assembleia Legislativa alagoana sustente haver reiterada inércia do Tribunal de Contas local, com a passagem de anos sem o envio do parecer técnico, não há qualquer óbice para que o Poder Legislativo estadual exerça sua competência constitucional (CF/1988, arts. 49, IX; 71, I; e 75). Por outro lado, a competência legislativa para disciplinar sobre direito penal e processual penal é privativa da União (CF/1988, arts. 22, I; e 85, parágrafo único) (2) (3). O texto constitucional não prevê penalidade para essa omissão. Apesar disso, a Constituição do Estado de Alagoas tipifica a omissão do Presidente do Tribunal de Contas como crime de responsabilidade (art. 97, I), medida que viola o princípio da simetria (4).

      Com base nesses entendimentos, o Plenário, por unanimidade, julgou improcedente a arguição e, por maioria – ante a abertura da causa de pedir – declarou a inconstitucionalidade da expressão “sob pena de crime de responsabilidade do Presidente do Tribunal” contida no art. 97, I, da Constituição do Estado de Alagoas (5).

    4. vedada
      • Informativo STF

      • Edição 1185/2025 19 de agosto de 2025

      • Vinculação remuneratória no âmbito estadual: equiparação do salário de empregados públicos ao vencimento de titulares de cargo efetivo – ADI 7.746/GO Relator: Ministro Cristiano Zanin

      • RESUMO: É inconstitucional – por desobedecer ao disposto no art. 37, XIII, da Constituição Federal – a vinculação da remuneração de empregados públicos aos vencimentos de servidores efetivos, pois resultaria em equiparação remuneratória entre agentes públicos pertencentes a categorias diferentes.

      O texto constitucional impede que determinadas categorias de servidores tenham seus vencimentos automaticamente majorados em decorrência do aumento concedido a carreiras diversas, já que cada uma deve ter estrutura remuneratória própria, estabelecida em lei específica, para que não haja aumentos salariais sem o devido processo legislativo.

      Conforme jurisprudência desta Corte (1), a atual redação do dispositivo acima citado estabelece ampla vedação quanto à vinculação ou equiparação da remuneração de servidores públicos, de forma a evitar que o aumento na remuneração concedida a determinados servidores públicos aplique-se de forma automática a outras categorias.

      Na espécie, o dispositivo impugnado dispõe que os empregados públicos que pertenceram aos quadros da Agência Goiana de Transportes e Obras (AGETOP – atual GOINFRA) fariam jus ao salário correspondente ao valor do vencimento fixado para o cargo efetivo equivalente. Essa medida permite que o aumento de vencimentos gere reflexos automáticos no vencimento dos empregados públicos da GOINFRA, de forma inconstitucional.

      Com base nesses entendimentos, o Plenário, por maioria, julgou parcialmente procedente a ação para conferir interpretação conforme a Constituição Federal ao art. 7º, § 3º, I, a, da Lei nº 15.665/2006 do Estado de Goiás (2), de modo a preservar o valor nominal da remuneração vigente na data da publicação da ata deste julgamento, vedados reajustes automáticos futuros decorrentes da vinculação remuneratória declarada inconstitucional.

    5. III
      • Informativo STF
      • Edição 1185/2025, 19 de agosto de 2025
      • Programa de auxílio aos desempregados e contratação temporária no âmbito municipal – RE 1.551.780/SP
      • RESUMO: É constitucional – pois concretiza o princípio da dignidade da pessoa humana e não ofende o princípio do concurso público (CF/1988, arts. 1º, III, e 37, II) – lei municipal que autoriza o chefe do Poder Executivo a criar programa de auxílio ao desempregado, de caráter assistencial, com o objetivo de dar ocupação, renda e qualidade profissional aos desempregados residentes no município.

      Esta Corte já reconheceu a constitucionalidade de normas que instituem programas sociais com a finalidade de integrar pessoas em situação de vulnerabilidade ao mercado de trabalho (1). Na espécie, a pretensão da lei municipal impugnada é conferir dignidade a pessoas em estado de vulnerabilidade (2), inexistindo falta de razoabilidade no programa questionado, pois o caráter assistencial em favor de pessoas desempregadas prevalece em relação ao interesse da Administração Pública municipal em reorganizar os quadros de seus servidores públicos.

      Ademais, não há que se falar em aplicação do que foi decidido no julgamento do processo paradigma do Tema 612 da repercussão geral, em que se fixaram os parâmetros de validade da contratação temporária de servidores públicos, a fim de impedir que ela seja utilizada como subterfúgio para o poder público se evadir da obrigação de realizar concurso público.

      Com base nesse entendimento, o Plenário, por unanimidade, negou provimento ao recurso para manter o acórdão recorrido, o qual julgou improcedente a ação direta de inconstitucionalidade estadual movida contra a Lei nº 1.937/2023 do Município de Rubiácea/SP.

    6. Municípios

      Aposentadoria especial de guardas municipais – ADPF 1.095/DF - Relatores: Ministro Gilmar Mendes - DIREITO ADMINISTRATIVO SERVIDOR PÚBLICO; GUARDA MUNICIPAL; APOSENTADORIA ESPECIAL - RESUMO: As guardas municipais fazem parte do Sistema Único de Segurança Pública (SUSP), mas não possuem direito à aposentadoria especial, visto que o rol constitucional de categorias com direito a esse benefício é taxativo e não as contempla.

      Esta Corte, embora tenha reconhecido que as guardas municipais fazem parte do SUSP, não lhes conferiu integral isonomia com os demais órgãos de segurança pública, na medida em que há peculiaridades relevantes quanto ao regime jurídico desses órgãos (1).

      A EC nº 103/2019, por sua vez, estabeleceu rol taxativo das categorias em que se pode instituir idade e tempo de contribuição diferenciados mediante lei complementar (2). Como as guardas municipais não f iguram de modo expresso nessa listagem, os respectivos entes federados ficam impedidos de conceder aposentadoria especial para essas carreiras (3).

      Também é inaplicável a regra de aposentadoria especial do art. 40, § 4º-C do texto constitucional (4). Isso porque não se admite presunção de exposição a agentes nocivos à saúde pelo mero enquadramento profissional ou ocupacional. Ao contrário, é indispensável que se comprove a efetiva exposição a agentes químicos, físicos e/ou biológicos (5).

      Por fim, eventual concessão da aposentadoria especial às guardas municipais sem a elaboração de plano próprio que contenha a devida indicação de fonte de custeio e de medidas compensatórias configura desobediência ao art. 195, § 5º da CF/1988 (6). Com base nesses e em outros entendimentos, o Plenário, por maioria, julgou improcedente a arguição.

    1. será proposta
      • Informativo nº 859
      • 26 de agosto de 2025.
      • PRIMEIRA TURMA Processo: Processo em segredo de justiça, Rel. Ministro Gurgel de Faria, Primeira Turma, por maioria, julgado em 19/8/2025.

      Ramo do Direito DIREITO ADMINISTRATIVO, DIREITO PROCESSUAL CIVIL

      TemaPaz, Justiça e Instituições Eficazes <br /> Ação de Improbidade administrativa. Defensoria pública. Ilegitimidade ativa.

      Destaque - A Defensoria Pública não possui legitimidade para propor a ação de improbidade administrativa.

      Informações do Inteiro Teor - A controvérsia volta-se ao debate acerca da legitimidade ativa da Defensoria Pública para a ação de improbidade administrativa.

      • A Lei n. 11.448/2007 alterou o art. 5º da Lei n. 7.347/1985 para incluir a Defensoria Pública como legitimada ativa para a propositura da ação civil pública em sentido largo; mas, podendo, não alterou a legitimidade para a propositura de ação civil pública regida pela Lei n. 8.429/1992 (Lei de Improbidade Administrativa - LIA), cujo objeto específico é a condenação pela prática de atos ímprobos.

      • Isto é, a escolha do legislador operou-se mediante "silêncio eloquente", excluindo da Defensoria Pública a legitimidade para propor ação civil pública cujo pedido seja de aplicar as sanções previstas no art. 12 da Lei n. 8.429/1992.

      • Note-se que, embora ambas as ações civis públicas (a geral da Lei n. 7.347/1985 e a de improbidade administrativa da Lei n. 8.429/1992) tenham algum ponto de aproximação, notadamente por serem instrumentos de proteção a direito transindividual, pelo que integram, em caráter global, o microssistema da tutela coletiva, elas diferenciam-se bastante no aspecto ontológico. É que as ações de improbidade são revestidas de caráter punitivo/sancionador próprio, sem equivalente na ação civil pública geral, e, por isso, aquela é regida por regras especiais, inclusive no que concerne à legitimidade ativa.

      • Compreende-se que essa distinção entre a ação civil pública geral e a ação voltada a condenação por atos ímprobos também se extrai da opção do legislador ordinário, que resolveu concentrar exclusivamente no Ministério Público a legitimidade para propor esta última (art. 17, caput, da LIA, com a redação atual).

      • Não se desconhece que o STF, após a ADI 7042, declarou a inconstitucionalidade parcial, com interpretação conforme sem redução de texto, do caput e dos §§ 6º-A e 10-C do art. 17 da Lei n. 8.429/1992, na redação dada pela Lei n. 14.230/2021, de modo a restabelecer a existência de legitimidade ativa concorrente e disjuntiva entre o Ministério Público e as pessoas jurídicas interessadas para a propositura da ação por ato de improbidade administrativa e para a celebração de acordos de não persecução civil.

      • Contudo, no que se refere à ação de improbidade, esse julgamento somente admitiu a legitimidade ativa concorrente entre o Ministério Público e a pessoa jurídica supostamente lesada pelo ato ímprobo, sem que tenha sido estendida a ampliação da legitimidade à Defensoria Pública.

      • Destarte, a legitimidade para propor a ação civil com fundamento na Lei n. 7.347/1985 não confere, em absoluto, a mesma legitimidade para propor a ação de improbidade da Lei n. 8.429/1992, sendo, portanto, a Defensoria Pública parte ilegítima para propor a ação de improbidade administrativa.

  10. www.planalto.gov.br www.planalto.gov.br
    1. § 3º
      • Informativo nº 859
      • 26 de agosto de 2025.
      • RECURSOS REPETITIVOS
      • Processo: REsp 2.148.059-MA, Rel. Ministro Luis Felipe Salomão, Corte Especial, por unanimidade, julgado em 20/8/2025. (Tema 1306).

      REsp 2.148.580-MA, Rel. Ministro Luis Felipe Salomão, Corte Especial, por unanimidade, julgado em 20/8/2025 (Tema 1306).

      REsp 2.150.218-MA, Rel. Ministro Luis Felipe Salomão, Corte Especial, por unanimidade, julgado em 20/8/2025 (Tema 1306).

      Ramo do Direito DIREITO PROCESSUAL CIVIL

      TemaPaz, Justiça e Instituições Eficazes <br /> Fundamentação por referência (per relationem ou por remissão). Ato decisório. Técnica de fundamentação. Cabimento. Tema 1306.

      Destaque - 1) A técnica da fundamentação por referência (per relationem) é permitida desde que o julgador, ao reproduzir trechos de decisão anterior, documento e/ou parecer como razões de decidir, enfrente, ainda que de forma sucinta, as novas questões relevantes para o julgamento do processo, dispensada a análise pormenorizada de cada uma das alegações ou provas.

      • 2) O § 3º do artigo 1.021, do CPC não impede a reprodução dos fundamentos da decisão agravada como razões de decidir pela negativa de provimento de agravo interno quando a parte deixa de apresentar argumento novo para ser apreciado pelo colegiado.

      Informações do Inteiro Teor - Cinge-se a controvérsia a definir se a fundamentação por referência (per relationem ou por remissão) - na qual são reproduzidas as motivações contidas em decisão judicial anterior como razões de decidir - resulta na nulidade do ato decisório, à luz do disposto nos artigos 489, § 1º, e 1.022, parágrafo único, inciso II, do Código de Processo Civil de 2015.

      • Segundo a doutrina, a obrigatoriedade da fundamentação das decisões judiciais, sob pena de nulidade, consubstancia, "a um só tempo, princípio processual, dever do juiz, direito individual da parte e garantia da Administração Pública".

      • Tal obrigatoriedade - de justificação da convicção do magistrado em decisões judiciais - encontra-se prevista na Constituição Federal de 1988 (art. 93, inciso IX), tendo relação intrínseca com a definição da República Federativa do Brasil como Estado Democrático de Direito.

      • Cuida-se de direito fundamental do jurisdicionado - consectário da garantia do devido processo legal - que subordina todos os integrantes do Poder Judiciário, aos quais é vedado proferir decisões arbitrárias, ou seja, pronunciamentos jurisdicionais que não se coadunem com o conceito democrático do exercício do poder, que exige a justificação - dialógica, racional e inteligível - do ato decisório de modo a viabilizar o seu "controle interno" pela parte e pelas instâncias judiciais subsequentes, bem como o seu "controle externo e difuso" pela sociedade, o que revela uma dupla função dessa obrigatoriedade.

      • O Código de Processo Civil de 2015 inseriu o dever de fundamentação das decisões judiciais entre as "normas fundamentais do processo civil" (artigo 11), determinando ainda que: (i) em regra, "não se proferirá decisão contra uma das partes sem que ela seja previamente ouvida" (artigo 9º, caput); e (ii) "o juiz não pode decidir, em grau algum de jurisdição, com base em fundamento a respeito do qual não se tenha dado às partes oportunidade de se manifestar, ainda que se trate de matéria sobre a qual deva decidir de ofício" (artigo 10).

      • No capítulo que versa sobre a "sentença" (lato sensu), o artigo 489 do CPC enumera os elementos essenciais do ato decisório, bem como hipóteses - exemplificativas - de "decisões não fundamentadas". Do referido dispositivo se extrai que o dever de fundamentação da decisão judicial considera-se adequadamente atendido quando o magistrado explicita as razões fáticas e jurídicas consideradas determinantes para a resposta oferecida no processo dentre outras conclusões possíveis.

      • Ademais, nos termos do rol previsto no § 1º do artigo 489 do CPC, a fundamentação da decisão judicial deve ainda conter: a) explicação sobre o vínculo entre a norma jurídica - considerada aplicável à espécie - e a causa ou a questão decidida nos autos (inciso I); b) especificação do motivo concreto para o emprego de conceito jurídico indeterminado (inciso II); c) exame da situação concreta submetida ao crivo do Judiciário, revelando-se insuficiente a invocação de motivos que se prestariam a justificar qualquer outra decisão (inciso III); d) enfrentamento de "todos os argumentos deduzidos no processo capazes de, em tese, infirmar a conclusão adotada pelo julgador" (inciso IV); e) juízo de conformação entre a ratio decidendi de precedente - ou de enunciado de súmula - aplicado na decisão e o caso concreto (inciso V); f) indicação das diferenças fáticas que justificam a não aplicação de precedente obrigatório ao caso concreto (inciso VI, primeira parte); e g) informação sobre a superação de precedente obrigatório invocado nos autos (inciso VI, parte final).

      • Assim, à luz do disposto no parágrafo único do artigo 1.022 do CPC, considera-se omissa - e, portanto, impugnável por embargos de declaração - a decisão que: (i) deixa de se manifestar sobre tese firmada em julgamento de casos repetitivos ou em incidente de assunção de competência aplicável à espécie; ou (ii) incorre em qualquer das hipóteses de ausência de fundamentação descritas no § 1º do artigo 489.

      • Com as alterações promovidas em 2010 no Decreto-Lei n. 4.657/1942 (Lei de Introdução às Normas do Direito Brasileiro - LINDB), também passou a ser exigido que as consequências práticas - postas no debate judicial e que tenham lastro probatório nos autos - constem da fundamentação da decisão que, nas esferas administrativa, controladora ou judicial, decretar a invalidação de ato, contrato, ajuste, processo ou norma administrativa (artigos 20 e 21).

      • Diante desse cenário normativo, discute-se se a utilização da técnica da fundamentação por referência - por remissão ou per relationem - é compatível com o dever de fundamentação imposto a todos os órgãos do Poder Judiciário, cuja inobservância resulta na nulidade do ato decisório. Trata-se de técnica discursiva na qual são reproduzidas as motivações contidas em decisão judicial anterior - ou em documento outro, a exemplo de parecer do Ministério Público - como razões de decidir.

      • De acordo com a doutrina, a fundamentação por referência apresenta duas formas habituais: (i) a exclusiva (ou pura); e (ii) a integrativa (ou moderada). A utilização da "fundamentação por referência exclusiva ou pura" - ou seja, aquela consubstanciada na mera remissão ou transcrição integral dos fundamentos de outra peça processual sem análise específica dos argumentos trazidos pela parte - <u>implica violação ao direito fundamental ao contraditório</u> e vai de encontro às disposições contidas no § 1º do artigo 489 do CPC. Por outro lado, é válida a "fundamentação por referência integrativa ou moderada", na qual a transcrição de decisão ou parecer anterior é acompanhada de análise própria (do julgador) que dialoga com os argumentos levantados pela parte em sua impugnação.

      • Ao tratar da matéria (sob o enfoque constitucional), o Supremo Tribunal Federal reconheceu a validade da fundamentação por referência - como técnica de motivação da decisão judicial - quando verificada "a compatibilidade entre o que alegado e o entendimento fixado pelo órgão julgador", ficando dispensado "o exame detalhado de cada argumento suscitado" (RE 1.397.056 ED-AgR/MA, Ministra Rosa Weber, Tribunal Pleno, DJe de 28/3/2023).

      • Essa mesma exegese encontra-se retratada na jurisprudência do Superior Tribunal de Justiça (inclusive das Turmas de Direito Penal).

      • Revela-se importante destacar, outrossim, que, em relação à norma inserta no § 3º do artigo 1.021 do CPC - segundo a qual "é vedado ao relator limitar-se à reprodução dos fundamentos da decisão agravada para julgar improcedente o agravo interno" - a jurisprudência do STJ firmou-se no sentido da necessidade de interpretação do referido comando em conjunto com a regra do inciso IV do § 1º do artigo 489, que somente reputa nula a decisão judicial que deixa de "enfrentar todos os argumentos deduzidos no processo capazes de, em tese, infirmar a conclusão adotada pelo julgador".

      • Não obstante, é certo que já foram constatadas, por esta Corte, hipóteses de utilização da técnica de fundamentação por referência com flagrante violação dos artigos 489, § 1º, 1.021, § 3º, e 1.022, parágrafo único, inciso II, do CPC. Nesses casos, em virtude do uso inadequado da referida técnica discursiva, determinou-se o retorno dos autos à origem para rejulgamento de embargos de declaração das partes.

      • Nesse contexto doutrinário e jurisprudencial, sendo pacífica a possibilidade de utilização da técnica de fundamentação da decisão por remissão, mas com cautela para garantir o contraditório e o direito à defesa, fixam-se as seguintes teses para fins dos artigos 1.036 a 1.041 do CPC:

      • 1) A técnica da fundamentação por referência (per relationem) é permitida desde que o julgador, ao reproduzir trechos de decisão anterior, documento e/ou parecer como razões de decidir, enfrente, ainda que de forma sucinta, as novas questões relevantes para o julgamento do processo, dispensada a análise pormenorizada de cada uma das alegações ou provas.

      • 2) O § 3º do artigo 1.021, do CPC não impede a reprodução dos fundamentos da decisão agravada como razões de decidir pela negativa de provimento de agravo interno quando a parte deixa de apresentar argumento novo para ser apreciado pelo colegiado.

    2. obrigação de fazer ou de não fazer
      • Informativo nº 859
      • 26 de agosto de 2025.
      • SEGUNDA TURMA
      • Processo: REsp 2.218.969-SP, Rel. Ministro Afrânio Vilela, Segunda Turma, por unanimidade, julgado em 19/8/2025.

      Ramo do Direito DIREITO ADMINISTRATIVO, DIREITO PROCESSUAL CIVIL

      Tutela do patrimônio histórico-cultural. Imóvel tombado. Restauração. Meras intenções e atos administrativos convergentes com a pretensão judicial. Perda de objeto. Inocorrência. Necessidade de atendimento integral do pedido. Condução estrutural da fase executória.

      Destaque - A mera intenção ou mesmo o início das obras de restauração de bem tombado não caracteriza por si só a perda de interesse processual, uma vez que o cumprimento integral da obrigação judicial é necessário para a extinção do processo por perda do objeto.

      Informações do Inteiro Teor - A questão em discussão consiste em saber se o início das obras de restauração do bem tombado pelo município caracteriza perda de interesse processual, tornando desnecessária a continuidade da demanda.

      • No caso, trata-se de ação civil pública ajuizada pelo Ministério Público contra um município, visando à restauração do Galpão da Oficina de Locomotivas, patrimônio tombado por Lei Municipal. A sentença condenou o ente federativo a executar as obras no prazo de seis meses, sob pena de multa e a apelação foi desprovida.

      • A deterioração do bem é registrada desde a década de 1980, o tombamento ocorreu nos anos 1990, o imóvel está interditado desde 2009 e a municipalidade manifesta reiteradamente, ao longo de décadas, suas melhores intenções de devolver o bem à coletividade, sem efetivá-lo.

      • No que tange ao interesse de agir do Ministério Público, o município recorrente defende sua inexistência porque teria conduzido a matéria administrativa de maneira adequada, com licitação e início das obras para restaurar o bem tombado.

      • A pretensão de que seja reconhecida a perda de objeto dita unilateral depende de que a parte ré entregue ao autor o bem da vida integralmente demandado em juízo. No caso, a parte apenas manifesta a intenção de entregar parte do bem da vida demandado.

      • Nas situações envolvendo o Poder Público, essa pretensão de reconhecimento da perda de objeto deve ser tratada com ainda maior critério. Isso porque, nos termos da doutrina, "Atores governamentais com frequência usam a perda de objeto para evadirem-se de precedentes desfavoráveis.".

      • Por isso, a mera intenção ou mesmo início das obras de restauração não caracteriza perda de objeto, pois o cumprimento <u>integral</u> da obrigação judicial é necessário para a extinção do interesse processual.

      • O cumprimento da obrigação disposta na sentença, portanto, somente poderá ser verificado na fase executória do provimento. É o juízo da execução que poderá considerar de modo efetivo os atos e esforços concretos da municipalidade que atendem de forma mais eficiente ao provimento judicial, inclusive com eventual modulação de prazos e multas, que não devem ser afastados de plano.

      • Além disso, do cenário descrito, depreende-se a <u>natureza estrutural da demanda</u>. Assim, a abordagem da causa, ainda que tardiamente, já em sua fase executória, pode e deve ser feita pela lente dos processos estruturais.

      • É certo que o caso trata de uma "nanoinstitucionalidade", uma situação de violação sistemática dos direitos da coletividade à cultura e à memória bastante delimitada, com um provimento jurisdicional bem específico, condição que nem sempre é entendida como matéria estrutural, dado seu limitado alcance.

      • Mas isso não impede que, no momento da execução da sentença, práticas, métodos e princípios típicos do processo estrutural sejam adotados pelo juízo exequente, conforme necessários e adequados. Nesse sentido, recentemente o Conselho Nacional de Justiça - CNJ aprovou a Recomendação n. 163 de 16/6/2025, norma incentivadora da condução estrutural de processos judiciais.

      • Ademais, considerando que se trata de patrimônio histórico e cultural de uma municipalidade, a adoção de técnicas estruturais de condução do feito atende, ainda, à concretização do princípio da participação comunitária, conforme reconhecido desde 1967 pelos Estados membros da Organização dos Estados Americanos - OEA,

      • Nessa linha, e conforme disposto pelo Fórum Permanente de Processualistas Civis, como boa prática na condução do feito estrutural em instância recursal, afigura-se adequado dar indicações concretas ao juízo de execução sobre os parâmetros de atuação nessa circunstância (Boa Prática n. 22/FPPC), nos termos já adotados, inclusive como paradigmas desse agir, por esta Corte (REsp n. 1.854.842/CE, relatora Ministra Nancy Andrighi, Terceira Turma, julgado em 2/6/2020, DJe de 4/6/2020).

      • Assim, recomenda-se ao magistrado encarregado da execução, resguardada sua independência funcional, a adoção de, entre outras, de medidas de natureza estruturante, tais como: i) estabelecimento de comitê de condução e monitoramento do projeto de restauração, inclusive com a participação de entidades da sociedade civil representantes do setor de cultura e memória, órgãos especializados de suporte, como o CREA, e representantes do Legislativo, além das partes e representante do juízo; ii) a eventual dilação do prazo de conclusão das obras, inclusive com suspensão temporária das multas condicionada ao cumprimento de eventual cronograma acordado pelas partes; iii) determinação de publicação no portal do Poder Executivo Municipal de relatórios periódicos, em intervalos de não mais que 45 dias, de execução do projeto de restauração, com os itens mínimos que entender necessários; e iv) realização de audiência pública prévia ao encerramento da obra, na sua iminência, para coleta de manifestações da sociedade sobre o alcance dos objetivos da sentença de conhecimento e prestação de contas pelos réus.

      • Recomenda-se, ainda, ao Tribunal respectivo que providencie o apoio institucional necessário ao magistrado singular na implementação dessas medidas, tudo orientado pelo princípio maior de cooperação.

      • Por fim, ao contrário do que alega a municipalidade, se a sentença confirma sua intenção administrativa, a imposição judicial pode destravar as diversas amarras burocráticas e políticas impostas a seus próprios gestores.

      • A sentença não será vazia, mas catalisadora dos efeitos concretos da política pública de proteção ao patrimônio histórico-cultural que a própria Administração não só é obrigada por lei a implementar, como assim também o deseja há tantas décadas.

      • Será, ainda, obrigação específica, decorrente de título judicial executivo, que vinculará não só a gestão atual como as futuras, de forma impessoal e para além de voluntarismos, como exige a situação degradante enfrentada pelo bem municipal tombado.

      • Por isso, o provimento judicial resguardará tanto a pretensão do autor como as intenções do réu, de modo a concretizá-las a ambas as partes. Desse modo, o objeto jurídico, que deve ser entendido como a devolução à coletividade do bem histórico-cultural que verdadeiramente lhe pertence, permanece íntegro.

    3. condenará o agravante a pagar ao agravado multa

      Observe que o destinatário desta multa é o agravado, isto é, a parte contrária, e não ao fundo de aparelhamento do judiciário, como reiterado pelo REsp 1.846.734-RS (Info 666)


      Jurisprudência em teses - EDIÇÃO 182 - AGRAVO INTERNO

      1) O agravo interposto contra decisão monocrática do Tribunal de origem, com o objetivo de exaurir a instância recursal ordinária, a fim de permitir a interposição de recurso especial e do extraordinário, não é manifestamente inadmissível ou infundado, o que torna inaplicável a multa prevista no art. 557, § 2º, do Código de Processo Civil. (Tese julgada sob o rito do art. 543-C do CPC/73 - Tema n. 434)

      2) Em regra, descabe a imposição da multa (art. 1.021, § 4º, do CPC) em razão do não provimento do agravo interno em votação unânime, pois é necessária a configuração da manifesta inadmissibilidade ou improcedência do recurso para autorizar sua incidência.

      3) Dever ser paga à parte contrária a multa do art. 1.021, § 4º, do Código de Processo Civil aplicada na hipótese de agravo interno manifestamente inadmissível ou improcedente.


      • Informativo nº 857
      • 12 de agosto de 2025.
      • RECURSOS REPETITIVOS
      • Processo: REsp 2.043.826-SC, Rel. Ministro Mauro Campbell Marques, Corte Especial, por unanimidade, julgado em 6/8/2025 (Tema 1201).

      Multa prevista no art. 1.021, § 4º, do CPC. Critérios de aplicação. Manifesta improcedência ou inadmissibilidade. Agravo interno. Precedentes qualificados. Demonstração da distinção. Possibilidade. Revisão do Tema 434/STJ.

      Destaque - 1) O agravo interposto contra decisão do Tribunal de origem, ainda que com o objetivo de exaurir a instância recursal ordinária, a fim de permitir a interposição de recurso especial e/ou extraordinário, quando apresentado contra decisão baseada em precedente qualificado oriundo do STJ ou do STF, autoriza a aplicação da multa prevista no art. 1.021, § 4º, do CPC (revisão do Tema 434/STJ);

      • 2) A multa prevista no art. 1.021, § 4º, CPC, não é cabível quando (i) alegada fundamentadamente a distinção ou a superação do precedente qualificado oriundo do STJ ou do STF ou (ii) a decisão agravada estiver amparada em julgado de tribunal de segundo grau;

      • 3) Excetuadas as hipóteses supra, caberá ao órgão colegiado verificar a aplicação da multa, considerando-se as peculiaridades do caso concreto.

      Informações do Inteiro Teor - A primeira questão afetada para julgamento tem o seguinte teor: aplicabilidade da multa prevista no § 4º do art. 1.021 do CPC quando a decisão recorrida se baseia em precedente qualificado (art. 927, III, do CPC).

      • No regime do CPC/73, o Superior Tribunal de Justiça pacificou orientação no sentido de que o agravo interposto contra decisão monocrática do Tribunal de origem, com o objetivo de exaurir a instância recursal ordinária, a fim de permitir a interposição de recurso especial e do extraordinário, não é manifestamente inadmissível ou infundado, motivo pelo qual inaplicável a multa que era prevista no art. 557, § 2º, do Código revogado. Essa orientação foi adotada em sede de recurso especial submetido ao regime dos recursos repetitivos (Tema 434/STJ).

      • Ainda que o escopo do agravo interno seja viabilizar a interposição de recurso aos Tribunais Superiores (exaurimento de instância), não se pode olvidar que os recursos especial ou extraordinário terão seguimento negado quando o acórdão recorrido coincidir com a orientação do tribunal superior, proferida em sede de recurso extraordinário ou especial submetido ao regime dos recursos repetitivos.

      • Consequentemente, o simples argumento de que se pretende o exaurimento de instância, por si só, não é suficiente para que seja reconhecida a ilegalidade da multa. Diante desse quadro, revela-se necessária a revisão do referido Tema Repetitivo.

      • Por outro lado, o precedente qualificado autoriza tanto a improcedência liminar do pedido (pelo juízo singular) quanto o julgamento monocrático pelo relator (no âmbito dos tribunais). Admitir-se a interposição de recurso em face de decisão baseada em precedente qualificado - especialmente quando não há nenhuma sinalização de alteração do precedente - implica negar a própria finalidade da sua criação. Assim, o respeito ao precedente qualificado é regra de observância obrigatória.

      • Entretanto, reconhecer que inexiste ilegalidade ao se declarar manifestamente improcedente agravo interno que impugna decisão baseada em precedente qualificado, para fins de aplicação da multa prevista no § 4º do art. 1.021 do CPC, não impõe que o órgão colegiado sempre declare manifestamente improcedente agravo interno interposto contra esse tipo de decisão. Em se tratando de penalidade, a aplicação deve ser proporcional.

      • A título de exemplo, é comum em questões tributárias o questionamento de acórdão proferido sob o regime dos recursos repetitivos, no Superior Tribunal de Justiça, perante o Supremo Tribunal Federal. Trata-se de matéria amplamente regulamentada pela Constituição Federal, admitindo, ao menos em tese, a viabilização do recurso extraordinário. Nesse cenário, reconhecida a existência de repercussão geral, não é recomendável a imposição da penalidade em sede de agravo interno, ainda que a decisão recorrida esteja amparada em acórdão vinculante do Superior Tribunal de Justiça.

      • Nesse cenário, pode-se afirmar que inexiste ilegalidade ao se declarar manifestamente improcedente agravo interno que impugna decisão baseada em precedente qualificado, para fins de aplicação da multa prevista no § 4º do art. 1.021 do CPC, ressalvados os casos em que a aplicação da multa não é recomendada (v.g. acórdão vinculante proferido pelo Tribunal local e recurso especial repetitivo pendente no STJ; acórdão vinculante proferido pelo STJ e pendência de julgamento de repercussão geral no STF).

      • Por sua vez, a segunda questão afetada para julgamento tem o seguinte teor: possibilidade de se considerar manifestamente inadmissível ou improcedente (ainda que em votação unânime) agravo interno cujas razões apontam a indevida ou incorreta aplicação de tese firmada em sede de precedente qualificado.

      • Como se constata, a questão guarda relação com o instituto da distinção, que é tratado especialmente nos arts. 489, V e VI e 1.037, §§ 9º a 13, do CPC.

      • Segundo a doutrina, "o distinguishing expressa a distinção entre os casos para o efeito de se subordinar, ou não, o caso sob julgamento ao precedente", ou seja, "o distinguishing revela a demonstração entre as diferenças fáticas entre os casos ou a demonstração de que a ratio do precedente não se amolda ao caso sob julgamento, uma vez que os fatos de um e outro são diversos".

      • Acerca do tema, é esclarecedor o Enunciado 306 do Fórum Permanente de Processualistas Civis (FPPC): "O precedente vinculante não será seguido quando o juiz ou tribunal distinguir o caso sob julgamento, demonstrando, fundamentalmente, tratar-se de situação particularizada por hipótese fática distinta, a impor solução jurídica diversa".

      • A Segunda Turma do STJ, em recente precedente, afirmou que "o pedido (ou requerimento) de distinção deve ser apresentado na forma do art. 1.037, § 8º e seguintes do CPC. Nesse regime, tal pedido deve ser interposto na primeira oportunidade, após a determinação de sobrestamento, quando este ocorre em Tribunal Superior" (PDist no AgInt nos EDcl no AREsp n. 2.360.573/PE, relator Ministro Mauro Campbell Marques, Segunda Turma, julgado em 14/5/2024, DJe de 17/5/2024).

      • Destarte, em se tratando de decisão do relator baseada em precedente qualificado, a alegação de distinção deve ser formulada na primeira oportunidade, assim como ocorre com pedido de distinção previsto no art. 1.037, § 8º e seguintes do CPC.

      • Cabe ressaltar que é ônus da parte demonstrar a existência de distinção, em consonância com o Enunciado 9 da ENFAM. Essa demonstração deve ocorrer de forma fundamentada, de modo que é descabida a simples alegação de indevida ou incorreta aplicação de tese firmada em sede de precedente qualificado.

      • Assim, o agravo interposto contra decisão do Tribunal de origem, ainda que com o objetivo de exaurir a instância recursal ordinária, a fim de permitir a interposição de recurso especial e/ou extraordinário, quando apresentado contra decisão baseada em precedente qualificado oriundo do STJ ou do STF, autoriza a aplicação da multa prevista no art. 1.021, § 4º, do CPC (revisão do TR 434/STJ).

      • Além disso, a multa prevista no art. 1.021, §4º, CPC não é cabível quando (i) alegada de forma fundamentada a distinção ou superação do precedente qualificado oriundo do STJ ou do STF ou (ii) a decisão agravada estiver amparada em julgado de tribunal de segundo grau.

      • Por fim, excetuadas as hipóteses supra, caberá ao órgão colegiado verificar a aplicação da multa, considerando-se as peculiaridades do caso concreto.

    1. Art. 113
      • Informativo nº 859
      • 26 de agosto de 2025.
      • TERCEIRA TURMA
      • Processo: * Processo em segredo de justiça, Rel. Ministra Nancy Andrighi, Terceira Turma, por unanimidade, julgado em 17/6/2025, DJEN 27/6/2025.

      Ação de exoneração de alimentos. Alimentos entre ex-cônjuges. Pagamento de pensão alimentícia por mais de duas décadas após o termo final da obrigação. Liberalidade. Expectativa legítima de continuidade da prestação. Supressio configurada.

      Destaque - É possível a manutenção do pagamento de pensão alimentícia por prazo indeterminado, na hipótese em que o ex-marido, mesmo exonerado, optou voluntariamente por continuar realizando o pagamento de alimentos por duas décadas, em razão da configuração dos institutos da supressio para o alimentante, que deixou de exercer seu direito de cessar os pagamentos, e da surrectio para a alimentanda diante da expectativa de que o direito de exoneração dos alimentos não mais seria reivindicado pelo ex-cônjuge.

      Informações do Inteiro Teor - Cinge-se a controvérsia em decidir se o pagamento de pensão alimentícia pelo ex-marido, por mais de duas décadas após o termo final da obrigação, configura a incidência do instituto da supressio, fazendo nascer para a ex-esposa a expectativa legítima de continuidade da prestação, em homenagem à boa-fé objetiva.

      • A confiança, no contexto das relações privadas, desempenha papel fundamental ao assegurar proteção qualificada ao comportamento humano, sendo expressão concreta da solidariedade social constitucionalmente albergada. Essa confiança impõe a todos o dever jurídico de não frustrar, injustificadamente, as legítimas expectativas de terceiros. No âmbito das relações familiares, a noção de confiança deve ser especialmente protegida, de forma que as condutas contrárias à confiança serão, em regra, também contrárias à boa-fé objetiva.

      • A tutela da confiança assume relevância ética nas relações privadas ao proibir comportamentos contraditórios (venire contra factum proprium) e ao reconhecer efeitos decorrentes da inércia prolongada (supressio) ou da prática constante (surrectio). Tais figuras jurídicas operam como mecanismos de estabilização das expectativas, impedindo mudanças abruptas de conduta que contrariem a confiança anteriormente depositada.

      • Identifica-se a supressio como a perda de determinada faculdade jurídica em razão do não exercício prolongado desse direito, o que leva ao seu esvaziamento. Em contrapartida, a surrectio consiste no surgimento de uma vantagem para determinada pessoa, justamente porque a outra parte deixou de exercer o direito ao qual faria jus, criando, assim, a expectativa de que esse direito não mais seria reivindicado futuramente.

      • A supressio aproxima-se, sem dúvida, do venire contra factum proprium, pois ambas as figuras atuam como fatores de preservação da confiança alheia. Mas dele se diferencia primordialmente pois, enquanto no venire, a expectativa do outro decorre de uma conduta ativa anterior, que não pode ser desmentida posteriormente; na supressio, a expectativa nasce da omissão prolongada do titular do direito, cuja inércia, associada a elementos objetivos que indiquem o desuso, conduz à convicção de que tal direito não será mais exercido.

      • Assim, a inércia prolongada do credor de alimentos em promover a execução da pensão em débito pode gerar, no devedor, a legítima expectativa de que a prestação não é mais necessária, conduzindo à estabilização da situação de inadimplemento. Em sentido inverso, o alimentante que, mesmo exonerado, opta voluntariamente por continuar realizando os pagamentos, conduz ao alimentando a expectativa de continuidade da prestação, a qual pode tornar-se juridicamente relevante, especialmente diante da reiterada e sistemática manifestação de vontade.

      • A aplicação da boa-fé no âmbito do Direito de Família reforça a dimensão ética e funcional da confiança, reafirmando seu papel como vetor interpretativo e integrativo. A eventual violação de justa expectativa deverá ser verificada na situação em concreto, devendo o julgador buscar a melhor forma de concretização das expectativas e esperanças criadas no ambiente familiar.

      • O caráter de transitoriedade dos alimentos entre ex-cônjuges parece traduzir o conteúdo da boa-fé objetiva, uma vez que deve a obrigação alimentar garantir o fornecimento de auxílio material ao cônjuge depreciado em razão de sua vulnerabilidade social e econômica, até que possa retomar sua autonomia financeira.

      • Os alimentos transitórios não serão cabíveis, entretanto, quando as necessidades são permanentes, em decorrência da incapacidade perene do alimentando de promover seu próprio sustento.

      • A jurisprudência do Superior Tribunal de Justiça tem admitido a perenidade da obrigação de prestar alimentos entre ex-cônjuges em situações excepcionais, como na impossibilidade prática de reinserção do alimentando no mercado de trabalho; em hipótese de idade avançada do alimentando; ou de condição de saúde fragilizada.

      • Dessa forma, constatando-se, na espécie, a incapacidade laboral do alimentando, saúde fragilizada, idade avançada ou qualquer impossibilidade prática de inserção no mercado de trabalho, ou de adquirir autonomia financeira, a pensão alimentícia entre ex-cônjuges poderá ser fixada por prazo indeterminado.

      • No caso, é incontroverso que as partes se encontram divorciadas há mais de 30 (trinta anos), tendo firmado acordo para pagamento de pensão alimentícia pelo ex-marido à ex-esposa, correspondente a 5% dos seus rendimentos líquidos, além de pagamento de plano de saúde, pelo prazo de um ano. Referido acordo fora homologado judicialmente em 1993.

      • Dois anos depois, as partes peticionaram nos autos da ação de divórcio requerendo a alteração do acordo, para que o pagamento da pensão alimentícia fosse prorrogado por prazo indeterminado. Embora não tenha o juízo conhecido do pedido, em razão da necessidade de ajuizamento de ação própria, o ex-marido permaneceu alcançando a pensão alimentícia à ex-esposa por mais de 25 (vinte e cinco) anos, até o ajuizamento da ação de exoneração, em julho de 2018.

      • O fato de a ex-esposa ter recebido pensão alimentícia por mais de 25 (vinte e cinco) anos, no entanto, não demonstra sua inércia em retomar a independência financeira. Do contrário, a inércia do ex-marido em permanecer realizando os pagamentos mensais acordados por longo período, mesmo que exonerado, provocou na alimentanda a expectativa de que o direito de exoneração não seria mais por ele exercida.

      • Portanto, evidencia-se, da conduta do alimentante, o instituto da supressio, visto que deixou de exercer seu direito de cessar o pagamento dos alimentos por mais de duas décadas, conduzindo à estabilização da situação de fato. Lado outro, surge para a alimentanda a surrectio, diante da expectativa de que o direito de exoneração dos alimentos não mais seria reivindicado pelo ex-marido.

      • Com efeito, o alimentante que, mesmo exonerado, opta voluntariamente por continuar realizando os pagamentos, conduz ao alimentando a expectativa de continuidade da prestação, a qual pode tornar-se juridicamente relevante, especialmente diante da reiterada e sistemática manifestação de vontade.

      • Some-se a isso o fato de que a ex-esposa teve de abdicar de seu trabalho em razão de mudança da família para a cidade de Petrópolis, em função do emprego do ex-marido. A realidade vivenciada pelo casal ao tempo da constância da sociedade conjugal deve ser considerada quando da fixação da pensão alimentícia.

      • Ademais, tendo em vista que a alimentanda é pessoa idosa, possui doença grave e se encontra impossibilitada de se reinserir no mercado de trabalho; e o alimentante aufere renda suficiente para permanecer cumprindo a obrigação constituída; deve-se manter o pagamento da pensão alimentícia por prazo indeterminado.

  11. www.planalto.gov.br www.planalto.gov.br
    1. § 4º

      Trata-se de presunção relativa e não absoluta, cabendo à Administração executar as diligências previstas no § 2º, conforme acórdão do TCU:

      1. O critério definido no art. 59, § 4º, da Lei 14.133/2021 conduz a uma presunção <u>relativa</u> de inexequibilidade de preços, devendo a Administração, nos termos do art. 59, § 2º, da referida lei, dar à licitante a oportunidade de demonstrar a exequibilidade de sua proposta.

      (TCU, Acórdão 465/2024 Plenário, Representação, Relator Ministro-Substituto Augusto Sherman - Boletim nº 478)

    2. território
        1. O critério de desempate por localidade (art. 60, § 1º, inciso I, da Lei 14.133/2021) não se aplica a licitações realizadas no âmbito da Administração Pública Federal, por ausência de expressa previsão legal. A preferência por empresas estabelecidas no território do promotor do certame é restrita às licitações realizadas por órgãos e entidades dos entes subnacionais.

      (TCU, Acórdão 1733/2025, Plenário, Representação, Relator Ministro Benjamin Zymler)

      Obs.: Ou seja, propostas empatadas poderão ser decididas, se em igualdade de condições, por critério territorial. No entanto, observe que somente é aplicável tal critério aos entes subnacionais.

      Com isso, o TCU entendeu que é indevido o desempate por critérios territoriais em licitações federais por expressa falta de previsão legal, considerando a expressa delimitação da abrangência da norma. Ou seja, aplicar-se critérios territoriais em licitações federais viola o princípio da legalidade e da isonomia.

    1. deshielded proton

      When a highly electronegative atom (like N, O, or F) bonds with a hydrogen atom, it pulls electron density away from hydrogen, creating a partial positive charge on the hydrogen atom. This 'deshields' the hydrogen's proton, making it more attractive to lone pairs of electrons on other electronegative atoms, thus facilitating hydrogen bonding.

    1. Author response:

      General Statements:

      The formation of three-dimensional tubes is a fundamental process in the development of organs and aberrant tube size leads to common diseases and congenital disorders, such as polycystic kidney disease, asthma, and lung hypoplasia. The apical (luminal) extracellular matrix (ECM) plays a critical role in epithelial tube morphogenesis during organ formation, but its composition and organization remain poorly understood. Using the Drosophila embryonic salivary gland as a model, we reveal a critical role for the PAPS Synthetase (Papss), an enzyme that synthesizes the universal sulfate donor PAPS, as a critical regulator of tube lumen expansion. Additionally, we identify two zona pellucida (ZP) domain proteins, Piopio (Pio) and Dumpy (Dpy) as key apical ECM components that provide mechanical support to maintain a uniform tube diameter.

      The apical ECM has a distinct composition compared to the basal ECM, featuring a diverse array of components. Many studies of the apical ECM have focused on the role of chitin and its modification, but the composition of the non-chitinous apical ECM and its role, and how modification of the apical ECM affects organogenesis remain elusive. The main findings of this manuscript are listed below.

      (1) Through a deficiency screen targeting ECM-modifying enzymes, we identify Papss as a key enzyme regulating luminal expansion during salivary gland morphogenesis. 

      (2) Our confocal and transmission electron microscopy analyses reveal that Papss mutants exhibit a disorganized apical membrane and condensed aECM, which are at least partially linked to disruptions in Golgi structures and intracellular trafficking. Papss is also essential for cell survival and basal ECM integrity, highlighting the role of sulfation in regulating both apical and basal ECM.

      (3) Salivary gland-specific overexpression of wild-type Papss rescues all defects in Papss mutants, but the catalytically inactive mutant form does not, suggesting that defects in sulfation are the underlying cause of the phenotypes.

      (4) We identify two ZP domain proteins, Piopio (Pio) and Dumpy (Dpy), as key components of the salivary gland aECM. In the absence of Papss, Pio is progressively lost from the aECM, while the Dpy-positive aECM structure is condensed and detaches from the apical membrane, resulting in a narrowed lumen. 

      (5) Mutations in pio or dpy, or in Notopleural (Np), which encodes a matriptase that cleaves Pio, cause the salivary gland lumen to develop alternating bulges and constrictions. Additionally, loss of pio results in loss of Dpy in the salivary gland lumen, suggesting that the Dpycontaining filamentous structures of the aECM is critical for maintaining luminal diameter, with Pio playing an essential role in organizing this structure.

      (6) We further reveal that the cleavage of the ZP domain of Pio by Np is critical for the role of Pio in organizing the aECM structure.

      Overall, our findings underscore the essential role of sulfation in organizing the aECM during tubular organ formation and highlight the mechanical support provided by ZP domain proteins in maintaining tube diameter. Mammals have two isoforms of Papss, Papss1 and Papss2. Papss1 shows ubiquitous expression, with higher levels in glandular cells and salivary duct cells, suggesting a high requirement for sulfation in these cell types. Papss2 shows a more restricted expression, such as in cartilage, and mutations in Papss2 have been associated with skeletal dysplasia in humans. Our analysis of the Drosophila Papss gene, a single ortholog of human Papss1 and Papss2, reveals its multiple roles during salivary gland development. We expect that these findings will provide valuable insights into the function of these enzymes in normal development and disease in humans. Our findings on the key role of two ZP proteins, Pio and Dpy, as major components of the salivary gland aECM also provide valuable information on the organization of the non-chitinous aECM during organ formation.

      We believe that our results will be of broad interest to many cell and developmental biologists studying organogenesis and the ECM, as well as those investigating the mechanisms underlying human diseases associated with conserved mutations.

      Point-by-point description of the revisions:

      We are delighted that all three reviewers were enthusiastic about the work. Their comments and suggestions have improved the paper. The details of the changes we have made in response to each reviewer’s comments are included in italicized text below.

      Reviewer #1 (Evidence, reproducibility and clarity):

      PAPS is required for all sulfotransferase reactions in which a sulfate group is covalently attached to amino acid residues of proteins or to side chains of proteoglycans. This sulfation is crucial for properly organizing the apical extracellular matrix (aECM) and expanding the lumen in the Drosophila salivary gland. Loss of Papss potentially leads to decreased sulfation, disorganizing the aECM, and defects in lumen formation. In addition, Papss loss destabilizes the Golgi structures.

      In Papss mutants, several changes occur in the salivary gland lumen of Drosophila. The tube lumen is very thin and shows irregular apical protrusions. There is a disorganization of the apical membrane and a compaction of the apical extracellular matrix (aECM). The Golgi structures and intracellular transport are disturbed. In addition, the ZP domain proteins Piopio (Pio) and Dumpy (Dpy) lose their normal distribution in the lumen, which leads to condensation and dissociation of the Dpy-positive aECM structure from the apical membrane. This results in a thin and irregularly dilated lumen.

      (1) The authors describe various changes in the lumen in mutants, from thin lumen to irregular expansion. I would like to know the correct lumen diameter, and length, besides the total area, by which one can recognize thin and irregular.

      We have included quantification of the length and diameter of the salivary gland lumen in the stage 16 salivary glands of control, Papss mutant, and salivary gland-specific rescue embryos (Figure 1J, K). As described, Papss mutant embryos have two distinct phenotypes, one group with a thin lumen along the entire lumen and the other group with irregular lumen shapes. Therefore, we separated the two groups for quantification of lumen diameter. Additionally, we have analyzed the degree of variability for the lumen diameter to better capture the range of phenotypes observed (Figure 1K’). These quantifications enable a more precise assessment of lumen morphology, allowing readers to distinguish between thin and irregular lumen phenotypes.

      (2) The rescue is about 30%, which is not as good as expected. Maybe the wrong isoform was taken. Is it possible to find out which isoform is expressed in the salivary glands, e.g., by RNA in situ Hyb? This could then be used to analyze a more focused rescue beyond the paper.

      Thank you for this point, but we do not agree that the rescue is about 30%. In Papss mutants, about 50% of the embryos show the thin lumen phenotype whereas the other 50% show irregular lumen shapes. In the rescue embryos with a WT Papss, few embryos showed thin lumen phenotypes. About 40% of the rescue embryos showed “normal, fully expanded” lumen shapes, and the remaining 60% showed either irregular (thin+expanded) or slightly overexpanded lumen. It is not uncommon that rescue with the Gal4/UAS system results in a partial rescue because it is often not easy to achieve the balance of the proper amount of the protein with the overexpression system. 

      To address the possibility that the wrong isoform was used, we performed in situ hybridization to examine the expression of different Papss spice forms in the salivary gland. We used probes that detect subsets of splice forms: A/B/C/F/G, D/H, and E/F/H, and found that all probes showed expression in the salivary gland, with varying intensities. The original probe, which detects all splice forms, showed the strongest signals in the salivary gland compared to the new probes which detect only a subset. However, the difference in the signal intensity may be due to the longer length of the original probe (>800 bp) compared to other probes that were made with much smaller regions (~200 bp). Digoxigenin in the DIG labeling kit for mRNA detection labels the uridine nucleotide in the transcript, and the probes with weaker signals contain fewer uridines (all: 147; ABCFG, 29; D, 36; EFH, 66). We also used the Papss-PD isoform, for a salivary gland-specific rescue experiment and obtained similar results to those with Papss-PE (Figure 1I-L, Figure 4D and E). 

      Furthermore, we performed additional experiments to validate our findings. We performed a rescue experiment with a mutant form of Papss that has mutations in the critical rescues of the catalytic domains of the enzyme, which failed to rescue any phenotypes, including the thin lumen phenotype (Figure 1H, J-L), the number and intensity of WGA puncta (Figure 3I, I’), and cell death (Figure 4D, E). These results provide strong evidence that the defects observed in Papss mutants are due to the lack of sulfation.  

      (3) Crb is a transmembrane protein on the apicolateral side of the membrane. Accordingly, the apicolateral distribution can be seen in the control and the mutant. I believe there are no apparent differences here, not even in the amount of expression. However, the view of the cells (frame) shows possible differences. To be sure, a more in-depth analysis of the images is required. Confocal Z-stack images, with 3D visualization and orthogonal projections to analyze the membranes showing Crb staining together with a suitable membrane marker (e.g. SAS or Uif). This is the only way to show whether Crb is incorrectly distributed. Statistics of several papas mutants would also be desirable and not just a single representative image. When do the observed changes in Crb distribution occur in the development of the tubes, only during stage 16? Is papss only involved in the maintenance of the apical membrane? This is particularly important when considering the SJ and AJ, because the latter show no change in the mutants.

      We appreciate your suggestion more thoroughly analyze Crb distribution. We adapted a method from a previous study (Olivares-Castiñeira and Llimargas, 2017) to quantify Crb signals in the subapical region and apical free region of salivary gland cells. Using E-Cad signals as a reference, we marked the apical cell boundaries of individual cells and calculated the intensity of Crb signals in the subapical region (along the cell membrane) and in the apical free region. We focused on the expanded region of the SG lumen in Papss mutants for quantification, as the thin lumen region was challenging to analyze. This quantification is included in Figure 2D. Statistical analysis shows that Crb signals were more dispersed in SG cells in Papss mutants compared to WT.

      (4) A change in the ECM is only inferred based on the WGA localization. This is too few to make a clear statement. WGA is only an indirect marker of the cell surface and glycosylated proteins, but it does not indicate whether the ECM is altered in its composition and expression. Other important factors are missing here. In addition, only a single observation is shown, and statistics are missing.

      We understand your concern that WGA localization alone may not be sufficient to conclude changes in the ECM. However, we observed that luminal WGA signals colocalize with Dpy-YFP in the WT SG (Figure 5-figure supplement 2C), suggesting that WGA detects the aECM structure containing Dpy. The similar behavior of WGA and Dpy-YFP signals in multiple genotypes further supports this idea. In Papss mutants with a thin lumen phenotype, both WGA and Dpy-YFP signals are condensed (Figure 5E-H), and in pio mutants, both are absent from the lumen (Figure 6B, D). We analyzed WGA signals in over 25 samples of WT and Papss mutants, observing consistent phenotypes. We have included the number of samples in the text. While we acknowledge that WGA is an indirect marker, our data suggest that it is a reliable indicator of the aECM structure containing Dpy. 

      (5) Reduced WGA staining is seen in papss mutants, but this could be due to other circumstances. To be sure, a statistic with the number of dots must be shown, as well as an intensity blot on several independent samples. The images are from single confocal sections. It could be that the dots appear in a different Z-plane. Therefore, a 3D visualization of the voxels must be shown to identify and, at best, quantify the dots in the organ.

      We have quantified cytoplasmic punctate WGA signals. Using spinning disk microscopy with super-resolution technology (Olympus SpinSR10 Sora), we obtained high-resolution images of cytoplasmic punctate signals of WGA in WT, Papss mutant, and rescue SGs with the WT and mutant forms of Papss-PD. We then generated 3D reconstructed images of these signals using Imaris software (Figure 3E-H) and quantified the number and intensity of puncta. Statistical analysis of these data confirms the reduction of the number and intensity of WGA puncta in Papss mutants (Figure 3I, I’). The number of WGA puncta was restored by expressing WT Papss but not the mutant form. By using 3D visualization and quantification, we have ensured that our results are not limited to a single confocal section and account for potential variations in Z-plane localization of the dots.

      (6) A colocalization analysis (statistics) should be shown for the overlap of WGA with ManII-GFP.

      Since WGA labels multiple structures, including the nuclear envelope and ECM structures, we focused on assessing the colocalization of the cytoplasmic WGA punctate signals and ManIIGFP signals. Standard colocalization analysis methods, such as Pearson’s correlation coefficient or Mander’s overlap coefficient, would be confounded by WGA signals in other tissues. Therefore, we used a fluorescent intensity line profile to examine the spatial relationship between WGA and ManII-GFP signals in WT and Papss mutants (Figure 3L, L’). 

      (7) I do not understand how the authors describe "statistics of secretory vesicles" as an axis in Figure 3p. The TEM images do not show labeled secretory vesicles but empty structures that could be vesicles.

      Previous studies have analyzed “filled” electron-dense secretory vesicles in TEM images of SG cells (Myat and Andrew, 2002, Cell; Fox et al., 2010, J Cell Biol; Chung and Andrew, 2014, Development). Consistent with these studies, our WT TEM images show these vesicles. In contrast, Papss mutants show a mix of filled and empty structures. For quantification, we specifically counted the filled electron-dense vesicles (now Figure 3W). A clear description of our analysis is provided in the figure legend.

      (8) The quality of the presented TEM images is too low to judge any difference between control and mutants. Therefore, the supplement must present them in better detail (higher pixel number?).

      We disagree that the quality of the presented TEM images is too low. Our TEM images have sufficient resolution to reveal details of many subcellular structures, such as mitochondrial cisternae. The pdf file of the original submission may not have been high resolution. To address this concern, we have provided several original high-quality TEM images of both WT and Papss mutants at various magnifications in Figure 2-figure supplement 2. Additionally, we have included low-magnification TEM images of WT and Papss mutants in Figure 2H and I to provide a clearer view of the overall SG lumen morphology. 

      (9) Line 266: the conclusion that apical trafficking is "significantly impaired" does not hold. This implies that Papss is essential for apical trafficking, but the analyzed ECM proteins (Pio, Dumpy) are found apically enriched in the mutants, and Dumpy is even secreted. Moreover, they analyze only one marker, Sec15, and don't provide data about the quantification of the secretion of proteins.

      We agree and have revised our statement to “defective sulfation affects Golgi structures and multiple routes of intracellular trafficking”. 

      (10) DCP-1 was used to detect apoptosis in the glands to analyze acellular regions. However, the authors compare ST16 control with ST15 mutant salivary glands, which is problematic. Further, it is not commented on how many embryos were analyzed and how often they detect the dying cells in control and mutant embryos. This part must be improved.

      Thank you for the comment. We agree and have included quantification. We used stage 16 samples from WT and Papss mutants to quantify acellular regions. Since DCP-1 signals are only present at a specific stage of apoptosis, some acellular regions do not show DCP-1 signals. Therefore, we counted acellular regions regardless of DCP-1 signals. We also quantified this in rescue embryos with WT and mutant forms of Papss, which show complete rescue with WT and no rescue with the mutant form, respectively. The graph with a statistical analysis is included (Figure 4D, E).

      (11) WGA and Dumpy show similar condensed patterns within the tube lumen. The authors show that dumpy is enriched from stage 14 onwards. How is it with WGA? Does it show the same pattern from stage 14 to 16? Papss mutants can suffer from a developmental delay in organizing the ECM or lack of internalization of luminal proteins during/after tube expansion, which is the case in the trachea.

      Dpy-YFP and WGA show overlapping signals in the SG lumen throughout morphogenesis. DpyYFP is SG enriched in the lumen from stage 11, not stage 14 (Figure 5-figure supplement 2). WGA is also detected in the lumen throughout SG morphogenesis, similar to Dpy. In the original supplemental figure, only a stage 16 SG image was shown for co-localization of Dpy-YFP and WGA signals in the SG lumen. We have now included images from stage 14 and 15 in Figure 5figure supplement 2C. 

      Given that luminal Pio signals are lost at stage 16 only and that Dpy signals appear as condensed structures in the lumen of Papss mutants, it suggests that the internalization of luminal proteins is not impaired in Papss mutants. Rather, these proteins are secreted but fail to organize properly. 

      (12) Line 366. Luminal morphology is characterized by bulging and constrictions. In the trachea, bulges indicate the deformation of the apical membrane and the detachment from the aECM. I can see constrictions and the collapsed tube lumen in Fig. 6C, but I don't find the bulges of the apical membrane in pio and Np mutants. Maybe showing it more clearly and with better quality will be helpful.

      Since the bulging phenotype appears to vary from sample to sample, we have revised the description of the phenotype to “constrictions” to more accurately reflect the consistent observations. We quantified the number of constrictions along the entire lumen in pio and Np mutants and included the graph in Figure 6F.

      (13) The authors state that Papss controls luminal secretion of Pio and Dumpy, as they observe reduced luminal staining of both in papss mutants. However, the mCh-Pio and Dumpy-YFP are secreted towards the lumen. Does papss overexpression change Pio and Dumpy secretion towards the lumen, and could this be another explanation for the multiple phenotypes? 

      Thank you for the comment. To clarify, we did not observe reduced luminal staining of Pio and Dpy in Papss mutants, nor did we state that Papss controls luminal secretion of Pio and Dpy. In Papss mutants, Pio luminal signals are absent specifically at stage 16 (Figure 5H), whereas strong luminal Pio signals are present until stage 15 (Figure 5G). For Dpy-YFP, the signals are not reduced but condensed in Papss mutants from stages 14-16 (Figure 5D, H). 

      It remains unclear whether the apparent loss of Pio signals is due to a loss of Pio protein in the lumen or due to epitope masking resulting from protein aggregation or condensation. As noted in our response to Comment 11 internalization of luminal proteins seems unaffected in Papss mutants; proteins like Pio and Dpy are secreted into the lumen but fail to properly organize. Therefore, we have not tested whether Papss overexpression alters the secretion of Pio or Dpy.

      In our original submission, we incorrectly stated that uniform luminal mCh-Pio signals were unchanged in Papss mutants. Upon closer examination, we found these signals are absent in the expanded luminal region in stage 16 SG (where Dpy-YFP is also absent), and weak mCh-Pio signals colocalize with the condensed Dpy-YFP signals (Figure 5C, D). We have revised the text accordingly. 

      Regulation of luminal ZP protein level is essential to modulate the tube expansion; therefore, Np releases Pio and Dumpy in a controlled manner during st15/16. Thus, the analysis of Pio and Dumpy in NP overexpression embryos will be critical to this manuscript to understand more about the control of luminal ZP matrix proteins.

      Thanks for the insightful suggestion. We overexpressed both the WT and mutant form of Np using UAS-Np.WT and UAS-Np.S990A lines (Drees et al., 2019) and analyzed mCh-Pio, Pio antibody, and Dpy-YFP signals. It is important to note that these overexpression experiments were done in the presence of the endogenous WT Np. 

      Overexpression of Np.WT led to increased levels of mCh-Pio, Pio, and Dpy-YFP signals in the lumen and at the apical membrane. In contrast, overexpression of Np.S990A resulted in a near complete loss of luminal mCh-Pio signals. Pio antibody signals remained strong at the apical membrane but was weaker in the luminal filamentous structures compared to WT. 

      Due to the GFP tag present in the UAS-Np.S990A line, we could not reliably analyze Dpy-YFP signals because of overlapping fluorescent signals in the same channel. However, the filamentous Pio signals in the lumen co-localized with GFP signals, suggesting that these structures might also include Dpy-YFP, although this cannot be confirmed definitively. 

      These results suggest that overexpressed Np.S990A may act in a dominant-negative manner, competing with endogenous Np and impairing proper cleavage of Pio (and mCh-Pio). Nevertheless, some level of cleavage by endogenous Np still appears to occur, as indicated by the residual luminal filamentous Pio signals. These new findings have been incorporated into the revised manuscript and are shown in Figure 6H and 6I.

      (14) Minor:

      Fig. 5 C': mChe-Pio and Dumpy-YFP are mixed up at the top of the images.

      Thanks for catching this error.  It has been corrected.

      Sup. Fig7. A shows Pio in purple but B in green. Please indicate it correctly.

      It has been corrected.

      Reviewer #1 (Significance):

      In 2023, the functions of Pio, Dumpy, and Np in the tracheal tubes of Drosophila were published. The study here shows similar results, with the difference that the salivary glands do not possess chitin, but the two ZP proteins Pio and Dumpy take over its function. It is, therefore, a significant and exciting extension of the known function of the three proteins to another tube system. In addition, the authors identify papss as a new protein and show its essential function in forming the luminal matrix in the salivary glands. Considering the high degree of conservation of these proteins in other species, the results presented are crucial for future analyses and will have further implications for tubular development, including humans.

      Reviewer #2 (Evidence, reproducibility and clarity):

      Summary:

      There is growing appreciation for the important of luminal (apical) ECM in tube development, but such matrices are much less well understood than basal ECMs. Here the authors provide insights into the aECM that shapes the Drosophila salivary gland (SG) tube and the importance of PAPSS-dependent sulfation in its organization and function.

      The first part of the paper focuses on careful phenotypic characterization of papss mutants, using multiple markers and TEM. This revealed reduced markers of sulfation (Alcian Blue staining) and defects in both apical and basal ECM organization, Golgi (but not ER) morphology, number and localization of other endosomal compartments, plus increased cell death. The authors focus on the fact that papss mutants have an irregular SG lumen diameter, with both narrowed regions and bulged regions. They address the pleiotropy, showing that preventing the cell death and resultant gaps in the tube did not rescue the SG luminal shape defects and discussing similarities and differences between the papss mutant phenotype and those caused by more general trafficking defects. The analysis uses a papss nonsense mutant from an EMS screen - I appreciate the rigorous approach the authors took to analyze transheterozygotes (as well as homozygotes) plus rescued animals in order to rule out effects of linked mutations.

      The 2nd part of the paper focuses on the SG aECM, showing that Dpy and Pio ZP protein fusions localize abnormally in papss mutants and that these ZP mutants (and Np protease mutants) have similar SG lumen shaping defects to the papss mutants. A key conclusion is that SG lumen defects correlate with loss of a Pio+Dpy-dependent filamentous structure in the lumen. These data suggest that ZP protein misregulation could explain this part of the papss phenotype.

      Overall, the text is very well written and clear. Figures are clearly labeled. The methods involve rigorous genetic approaches, microscopy, and quantifications/statistics and are documented appropriately. The findings are convincing, with just a few things about the fusions needing clarification.

      Minor comments

      (1) Although the Dpy and Qsm fusions are published reagents, it would still be helpful to mention whether the tags are C-terminal as suggested by the nomenclature, and whether Westerns have been performed, since (as discussed for Pio) cleavage could also affect the appearance of these fusions.

      Thanks for the comment. Dpy-YFP is a knock-in line in which YFP is inserted into the middle of the dpy locus (Lye et al., 2014; the insertion site is available on Flybase). mCh-Qsm is also a knock-in line, with mCh inserted near the N-terminus of the qsm gene using phi-mediated recombination using the qsm<sup>MI07716</sup> line (Chu and Hayashi, 2021; insertion site available on Flybase). Based on this, we have updated the nomenclature from Qsm-mCh to mCh-Qsm throughout the manuscript to accurately reflect the tag position. To our knowledge, no western blot has been performed on Dpy-YFP or mCh-Qsm lines. We have mentioned this explicitly in the Discussion.  

      (2) The Dpy-YFP reagent is a non-functional fusion and therefore may not be a wholly reliable reporter of Dpy localization. There is no antibody confirmation. As other reagents are not available to my knowledge, this issue can be addressed with text acknowledgement of possible caveats.

      Thanks for raising this important point. We have added a caveat in the Discussion noting this limitation and the need for additional tools, such as an antibody or a functional fusion protein, to confirm the localization of Dpy.

      (3) TEM was done by standard chemical fixation, which is fine for viewing intracellular organelles, but high pressure freezing probably would do a better job of preserving aECM structure, which looks fairly bad in Fig. 2G WT, without evidence of the filamentous structures seen by light microscopy. Nevertheless, the images are sufficient for showing the extreme disorganization of aECM in papss mutants.

      We agree that HPF is a better method and intent to use the HPF system in future studies. We acknowledge that chemical fixation contributes to the appearance of a gap between the apical membrane and the aECM, which we did not observe in the HPF/FS method (Chung and Andrew, 2014). Despite this, the TEM images still clearly reveal that Papss mutants show a much thinner and more electron-dense aECM compared to WT (Figure 2H, I), consistent to the condensed WGA, Dpy, and Pio signals in our confocal analyses. As the reviewer mentioned, we believe that the current TEM data are sufficient to support the conclusion of severe aECM disorganization and Golgi defects in Papss mutants.

      (4) The authors may consider citing some of the work that has been done on sulfation in nematodes, e.g. as reviewed here: https://pubmed.ncbi.nlm.nih.gov/35223994/ Sulfation has been tied to multiple aspects of nematode aECM organization, though not specifically to ZP proteins.

      Thank you for the suggestion. Pioneering studies in C. elegans have highlighted the key role of sulfation in diverse developmental processes, including neuronal organization, reproductive tissue development, and phenotypic plasticity. We have now cited several works.  

      Reviewer #2 (Significance):

      This study will be of interest to researchers studying developmental morphogenesis in general and specifically tube biology or the aECM. It should be particularly of interest to those studying sulfation or ZP proteins (which are broadly present in aECMs across organisms, including humans).

      This study adds to the literature demonstrating the importance of luminal matrix in shaping tubular organs and greatly advances understanding of the luminal matrix in the Drosophila salivary gland, an important model of tubular organ development and one that has key matrix differences (such as no chitin) compared to other highly studied Drosophila tubes like the trachea.

      The detailed description of the defects resulting from papss loss suggests that there are multiple different sulfated targets, with a subset specifically relevant to aECM biology. A limitation is that specific sulfated substrates are not identified here (e.g. are these the ZP proteins themselves or other matrix glycoproteins or lipids?); therefore it's not clear how direct or indirect the effects of papss are on ZP proteins. However, this is clearly a direction for future work and does not detract from the excellent beginning made here.

      My expertise: I am a developmental geneticist with interests in apical ECM

      Reviewer #3 (Evidence, reproducibility and clarity):

      In this work Woodward et al focus on the apical extracellular matrix (aECM) in the tubular salivary gland (SG) of Drosophila. They provide new insights into the composition of this aECM, formed by ZP proteins, in particular Pio and Dumpy. They also describe the functional requirements of PAPSS, a critical enzyme involved in sulfation, in regulating the expansion of the lumen of the SG. A detailed cellular analysis of Papss mutants indicate defects in the apical membrane, the aECM and in Golgi organization. They also find that Papss control the proper organization of the Pio-Dpy matrix in the lumen. The work is well presented and the results are consistent.

      Main comments

      - This work provides a detailed description of the defects produced by the absence of Papss. In addition, it provides many interesting observations at the cellular and tissular level. However, this work lacks a clear connection between these observations and the role of sulfation. Thus, the mechanisms underlying the phenotypes observed are elusive. Efforts directed to strengthen this connection (ideally experimentally) would greatly increase the interest and relevance of this work.

      Thank you for this thoughtful comment. To directly test whether the phenotypes observed in Papss mutants are due to the loss of sulfation activity, we generated transgenic lines expressing catalytically inactive forms of Papss, UAS-PapssK193A, F593P, in which key residues in the APS kinase and ATP sulfurylase domains are mutated. Unlike WT UAS-Papss (both the Papss-PD or Papss-PE isoforms), the catalytically inactive UAS-Papssmut failed to rescue any of the phenotypes, including the thin lumen phenotype (Figure 1I-L), altered WGA signals (Figure I, I’) and the cell death phenotype (Figure 4D, E). These findings strongly support the conclusion that the enzymatic sulfation activity of Papss is essential for the developmental processes described in this study.  

      - A main issue that arises from this work is the role of Papss at the cellular level. The results presented convincingly indicate defects in Golgi organization in Papss mutants. Therefore, the defects observed could stem from general defects in the secretion pathway rather than from specific defects on sulfation. This could even underly general/catastrophic cellular defects and lead to cell death (as observed).

      This observation has different implications. Is this effect observed in SGs also observed in other cells in the embryo? If Papss has a general role in Golgi organization this would be expected, as Papss encodes the only PAPs synthatase in Drosophila.

      Can the authors test any other mutant that specifically affect Golgi organization and investigate whether this produces a similar phenotype to that of Papss?

      Thank you for the comment. To address whether the defects observed in Papss mutants stem from general disruption of the secretory pathway due to Golgi disorganization, we examined mutants of two key Golgi components: Grasp65 and GM130. 

      In Grasp65 mutants, we observed significant defects in SG lumen morpholgy, including highly irregular SG lumen shape and multiple constrictions (100%; n=10/10). However, the lumen was not uniformly thin as in Papss mutants. In contrast, GM130 mutants–although this line was very sick and difficult to grow–showed relatively normal salivary glands morphology in the few embryos that survived to stage 16 (n=5/5). It is possible that only embryos with mild phenotypes progressed to this stages, limiting interpretation. These data have now been included in Figure 3-figure supplement 2. Overall, while Golgi disruption can affect SG morphology, the specific phenotypes seen in Papss mutants are not fully recapitulated by Grasp65 or GM130 loss. 

      - A model that conveys the different observations and that proposes a function for Papss in sulfation and Golgi organization (independent or interdependent?) would help to better present the proposed conclusions. In particular, the paper would be more informative if it proposed a mechanism or hypothesis of how sulfation affects SG lumen expansion. Is sulfation regulating a factor that in turn regulates Pio-Dpy matrix? Is it regulating Pio-Dpy directly? Is it regulating a

      product recognized by WGA?

      For instance, investigating Alcian blue or sulfotyrosine staining in pio, dpy mutants could help to understand whether Pio, Dpy are targets of sulfation.

      Thank you for the comment. We’re also very interested in learning whether the regulation of the Pio-Dpy matrix is a direct or indirect consequence of the loss of sulfation on these proteins. One possible scenario is that sulfation directly regulates the Pio-Dpy matrix by regulating protein stability through the formation of disulfide bonds between the conserved Cys residues responsible for ZP module polymerization. Additionally, the Dpy protein contains hundreds of EGF modules that are highly susceptible to O-glycosylation. Sulfation of the glycan groups attached to Dpy may be critical for its ability to form a filamentous structure. Without sulfation, the glycan groups on Dpy may not interact properly with the surrounding materials in the lumen, resulting in an aggregated and condensed structure. These possibilities are discussed in the Discussion.

      We have not analyzed sulfation levels in pio or dpy mutants because sulfation levels in mutants of single ZP domain proteins may not provide much information. A substantial number of proteoglycans, glycoproteins, and proteins (with up to 1% of all tyrosine residues in an organism’s proteins estimated to be sulfated) are modified by sulfation, so changes in sulfation levels in a single mutant may be subtle. Especially, the existing dpy mutant line is an insertion mutant of a transposable element; therefore, the sulfation sites would still remain in this mutant. 

      - Interpretation of Papss effects on Pio and Dpy would be desired. The results presented indicate loss of Pio antibody staining but normal presence of cherry-Pio. This is difficult to interpret. How are these results of Pio antibody and cherry-Pio correlating with the results in the trachea described recently (Drees et al. 2023)?

      In our original submission, we stated that the uniform luminal mCh-Pio signals were not changed in Papss mutants, but after re-analysis, we found that these signals were actually absent from the expanded luminal region in stage 16 SG (where Dpy-YFP is also absent), and weak mCh-Pio signals colocalize with the condensed Dpy-YFP signals (Figure 5C, D). We have revised the text accordingly. 

      After cleavages by Np and furin, the Pio protein should have three fragments. The Nterminal region contains the N-terminal half of the ZP domain, and mCh-Pio signals show this fragment. The very C-terminal region should localize to the membrane as it contains the transmembrane domain. We think the middle piece, the C-terminal ZP domain, is recognized by the Pio antibody. The mCh-Pio and Pio antibody signals in the WT trachea (Drees et al., 2023) are similar to those in the SG. mCh-Pio signals are detected in the tracheal lumen as uniform signals, at the apical membrane, and in cytoplasmic puncta. Pio antibody signals are exclusively in the tracheal lumen and show more heterogenous filamentous signals. 

      In Papss mutants, the middle fragment (the C-terminal ZP domain) seems to be most affected because the Pio antibody signals are absent from the lumen. The loss of Pio antibody signals could be due to protein degradation or epitope masking caused by aECM condensation and protein misfolding. This fragment seems to be key for interacting with Dpy, since Pio antibody signals always colocalize with Dpy-YFP. The N-terminal mCh-Pio fragment does not appear to play a significant role in forming a complex with Dpy in WT (but still aggregated together in Papss mutants), and this can be tested in future studies.

      In response to Reviewer 1’s comment, we performed an additional experiment to test the role of Np in cleaving Pio to help organize the SG aECM. In this experiment, we overexpressed the WT and mutant form of Np using UAS-Np.WT and UAS-Np.S990A lines (Drees et al., 2019) and analyzed mCh-Pio, Pio antibody, and Dpy-YFP signals. Np.WT overexpression resulted in increased levels of mCh-Pio, Pio, and Dpy-YFP signals in the lumen and at the apical membrane. However, overexpression of Np.S990A resulted in the absence of luminal mCh-Pio signals. Pio antibody signals were strong at the apical membrane but rather weak in the luminal filamentous structures. Since the UAS-Np.S990A line has the GFP tag, we could not reliably analyze Dpy-YFP signals due to overlapping Np.S990A.GFP signals in the same channel. However, the luminal filamentous Pio signals co-localized with GFP signals, and we assume that these overlapping signals could be Dpy-YFP signals. 

      These results suggest that overexpressed Np.S990A may act in a dominant-negative manner, competing with endogenous Np and impairing proper cleavage of Pio (and mCh-Pio). Nevertheless, some level of cleavage by endogenous Np still appears to occur, as indicated by the residual luminal filamentous Pio signals. These new findings have been incorporated into the revised manuscript and are shown in Figure 6H and 6I. 

      A proposed model of the Pio-Dpy aECM in WT, Papss, pio, and Np mutants has now been included in Figure 7.

      -  What does the WGA staining in the lumen reveal? This staining seems to be affected differently in pio and dpy mutants: in pio mutants it disappears from the lumen (as dpy-YFP does), but in dpy mutants it seems to be maintained. How do the authors interpret these findings? How does the WGA matrix relate to sulfated products (using Alcian blue or sulfotyrosine)?

      WGA binds to sialic acid and N-acetylglucosamine (GlcNAc) residues on glycoproteins and glycolipids. GlcNAc is a key component of the glycosaminoglycan (GAG) chains that are covalently attached to the core protein of a proteoglycan, which is abundant in the ECM. We think WGA detects GlcNAc residues in the components of the aECM, including Dpy as a core component, based on the following data. 1) WGA and Dpy colocalize in the lumen, both in WT (as thin filamentous structures) and Papss mutant background (as condensed rod-like structures), and 2) are absent in pio mutants. WGA signals are still present in a highly condensed form in dpy mutants. That’s probably because the dpy mutant allele (dpyov1) has an insertion of a transposable element (blood element) into intron 11 and this insertion may have caused the Dpy protein to misfold and condense. We added the information about the dpy allele to the Results section and discussed it in the Discussion.

      Minor points:

      - The morphological phenotypic analysis of Papss mutants (homozygous and transheterozygous) is a bit confusing. The general defects are higher in Papss homozygous than in transheterozygotes over a deficiency. Maybe quantifying the defects in the heterozygote embryos in the Papss mutant collection could help to figure out whether these defects relate to Papss mutation.

      We analyzed the morphology of heterozygous Papss mutant embryos. They were all normal. The data and quantifications have now been added to Figure 1-figure supplement 3. 

      - The conclusion that the apical membrane is affected in Papss mutants is not strongly supported by the results presented with the pattern of Crb (Fig 2). Further evidences should be provided. Maybe the TEM analysis could help to support this conclusion

      We quantified Crb levels in the sub-apical and medial regions of the cell and included this new quantification in Figure 2D. TEM images showed variation in the irregularity of the apical membrane, even in WT, and we could not draw a solid conclusion from these images.

      - It is difficult to understand why in Papss mutants the levels of WGA increase. Can the authors elaborate on this?

      We think that when Dpy (and many other aECM components) are condensed and aggregated into the thin, rod-like structure in Papss mutants, the sugar residues attached to them must also be concentrated and shown as increased WGA signals.   

      - The explanation about why Pio antibody and mcherry-Pio show different patterns is not clear. If the antibody recognizes the C-t region, shouldn't it be clearly found at the membrane rather than the lumen?

      The Pio protein is also cleaved by furin protease (Figure 5B). We think the Pio fragment recognized by the antibody should be a “C-terminal ZP domain”, which is a middle piece after furin + Np cleavages. 

      - The qsm information does not seem to provide any relevant information to the aECM, or sulfation.

      Since Qsm has been shown to bind to Dpy and remodel Dpy filaments in the muscle tendon (Chu and Hayashi, 2021), we believe that the different behavior of Qsm in the SG is still informative. As mentioned briefly in the Discussion, the cleaved Qsm fragment may localize differently, like Pio, and future work will need to test this. We have shortened the description of the Qsm localization in the manuscript and moved the details to the figure legend of Figure 5-figure supplement 3.

      Reviewer #3 (Significance):

      Previous reports already indicated a role for Papss in sulfation in SG (Zhu et al 2005). Now this work provides a more detailed description of the defects produced by the absence of Papss. In addition, it provides relevant data related to the nature and requirements of the aECM in the SG. Understanding the composition and requirements of aECM during organ formation is an important question. Therefore, this work may be relevant in the fields of cell biology and morphogenesis.

    1. AbstractRice (Oryza sativa) is one of the most important staple food crops worldwide, and its wild relatives serve as an important gene pool in its breeding. Compared with cultivated rice species, African wild rice (Oryza longistaminata) has several advantageous traits, such as resistance to increased biomass production, clonal propagation via rhizomes, and biotic stresses. However, previous O. longistaminata genome assemblies have been hampered by gaps and incompleteness, restricting detailed investigations into their genomes. To streamline breeding endeavors and facilitate functional genomics studies, we generated a 343-Mb telomere-to-telomere (T2T) genome assembly for this species, covering all telomeres and centromeres across the 12 chromosomes. This newly assembled genome has markedly improved over previous versions. Comparative analysis revealed a high degree of synteny with previously published genomes. A large number of structural variations were identified between the O. longistaminata and O. sativa. A total of 2,466 segmentally duplicated genes were identified and enriched in cellular amino acid metabolic processes. We detected a slight expansion of some subfamilies of resistance genes and transcription factors. This newly assembled T2T genome of O. longistaminata provides a valuable resource for the exploration and exploitation of beneficial alleles present in wild relative species of cultivated rice.

      This work has been peer reviewed in GigaScience (see https://doi.org/10.1093/gigascience/giaf074), which carries out open, named peer-review. These reviews are published under a CC-BY 4.0 license and were as follows:

      Reviewer 2: Chengzhi Liang

      The authors generated a 343-Mb telomere-to-telomere (T2T) genome assembly for an African wild rice (Oryza longistaminata), covering all telomeres and centromeres across the 12 chromosomes, and performed genome annotation and analyses on structural variations and NLR genes. While the manuscript has provided a valuable genome sequence, several problems should be addressed before the manuscript can be published.

      Major issues 1. The authors estimated that the genome heterozygosity is 1.27%, which is quite high, so I am wondering how large the assembled genome size is using only HiFi data, which could reflect the actual heterozygosity rate of the genome, particularly by comparing it with the final genome size of 12 chromosomes. If there was only one gap in the initial assembly of Hifiasm (a total of 13 contigs), it is unlikely that the genome has such a high heterozygosity. In Table 1, the total size of assembled genome was 331,045,917bp. If this is the summed size of 12 chromosomes, it should be used as the final genome size in the main text. Please clarify. Also, what is the base accuracy of Ultra-long CycloneSEQ data? which is useful to readers for this is a new sequencing technology. 2. For SV detection, considering that the assembled genome in the manuscript (does it have a accession ID or name?) is an African wild rice, it is rather strange that the authors did not compare it with an O. glaberrima genome, but with an O. sativa genome. Meanwhile, the name of the genomes should be mentioned since there were so many different genomes in each species, all with different SV variations between them. 3. The conclusion that "This distribution suggests that chromosomes 1, 4, 3, and 2 might have contributed to the evolution of rice in previously unrecognized ways (Table S8)" is purely speculative, and thus should be removed from the manuscript, or the authors should provide more evidence to support it. 4. The author claimed that "Compared with other Oryza species, O. longistaminata has many fewer NBS-lRR domain genes, which reflects a contraction of resistance genes in this species." Please give specific gene numbers for each species. Meanwhile, the conclusion does not look right here since it looks that O. longistaminata had more NBS-LRR genes than other species.

      Minor issues 1. What is "quartets"? 2. The author used "11 Oryza species" which included O. indica, please clarify what this species is.Bold

    2. AbstractRice (Oryza sativa) is one of the most important staple food crops worldwide, and its wild relatives serve as an important gene pool in its breeding. Compared with cultivated rice species, African wild rice (Oryza longistaminata) has several advantageous traits, such as resistance to increased biomass production, clonal propagation via rhizomes, and biotic stresses. However, previous O. longistaminata genome assemblies have been hampered by gaps and incompleteness, restricting detailed investigations into their genomes. To streamline breeding endeavors and facilitate functional genomics studies, we generated a 343-Mb telomere-to-telomere (T2T) genome assembly for this species, covering all telomeres and centromeres across the 12 chromosomes. This newly assembled genome has markedly improved over previous versions. Comparative analysis revealed a high degree of synteny with previously published genomes. A large number of structural variations were identified between the O. longistaminata and O. sativa. A total of 2,466 segmentally duplicated genes were identified and enriched in cellular amino acid metabolic processes. We detected a slight expansion of some subfamilies of resistance genes and transcription factors. This newly assembled T2T genome of O. longistaminata provides a valuable resource for the exploration and exploitation of beneficial alleles present in wild relative species of cultivated rice.

      This work has been peer reviewed in GigaScience (see https://doi.org/10.1093/gigascience/giaf074), which carries out open, named peer-review. These reviews are published under a CC-BY 4.0 license and were as follows:

      Reviewer 1: Francois Sabot

      The manuscript from Guang et al deals with a T2T assembly for the wild perennial African rice Oryza longistaminata. Using last up to date technologies and approaches, authors provided a high quality assembly for this wild species, rending it a valuable ressource for understanding rice evolution. While the results as assembly are of high quality, the interpretation of some biological results, in particular about the NBS-LRR, are quite weird, in my opinion, and need to be more refined. That's why I think the manuscript should be published, but after major corrections.

      in details:

      -Introduction: not sure the exceptional biomass is a good idea from longistaminata, as this plant has avery high content in silicium, rendering its biomass complex to use. - Methods: We do not have access to most of the command options and command-lines. please provide them at least as a texte file in supp data. In addition, some of the references for tools are missing. Finally, please provide the accession number of the assembled plant. - Assembly in itself: O longistaminata is a outcrossing heterozygous organism. Did you obtained the two haplotypes ? - Comparison with the previous longistaminata genome: is the inversion in middle of Chr6 specific ? or due to an error of previous assembly ? - Table 1: what do you mean "Total size of assembled genomes (bp) 331,045,917" ? What is the residual percentage of N ? - Figure 1 and others: please show the legend in other way, here we may mix it with the main text. in addition, check the legends for spelling and the size of figure (3b eg) for lisibility - Syri/MUMmer analysis: you limit as min size at 1kb ? What was the order of query vs ref ? can we have a bed file with the positions ? - SD: is there a statistical link between chromosome size and number of SD ? It could explain why the first 4 ones have more SD. In general, the data are missing stats. - GO in SD: any statistical validation ? - Genomes comparison: please provide the acc number of the genome you used for comparison. - NBS-LRR: the longistaminata genome has 215 genes for 116 to 289 for other oryza so I cannot see any contraction or expansion. in addition, the text here is weird, starting speaking of onctraction then going to expansion ??? - TF analysis; the african assemblies are quite bad I think, explaining the discrepency. For glaberrima, did you check the one from Tranchant-Dubreuil et al, 2023 ?

    1. s

      Como orden, pondría: 1. Contexto (primer parrafo) 2. Barreras y desafios que contrae 3. Percepción ciudadana 4. Cómo se relaciona la migración con la cohesión horizontal (conceptual o literatura previa + datos elsoc)

    1. o common had thissubstitution become that by 1954 House Beautiful was presenting its readerswith “another example of how the TV set is taking the place of the fireplaceas the focal point

      Television displaced older icons like the fireplace and piano as the center of family life.

    2. e more melodramatic socialproblem films su as Come Back Little Sheba (1952) and A Hatful of Rain(1957) were aracter studies of emotionally unstable, oen drug-dependent,family men. S

      These movies showed men who were struggling emotionally or with addiction. It’s interesting that TV and film in the 1950s didn’t just show perfect families, they also showed serious problems inside the home.

    1. bland foods: broth, cake, cooked cereal, cookie, cracker, egg, Jell-O, marshmallows, milk, pasta, pie, plain rice, potato, pudding, soft banana

      Shreddies and Cornflakes being much better than toast

    1. rickshaws

      ​a small, light vehicle with two wheels used especially in some Asian countries to carry passengers. The rickshaw is pulled by somebody walking or riding a bicycle.

    1. Art. 499

      Súmula nº 269/TST - DIRETOR ELEITO. CÔMPUTO DO PERÍODO COMO TEMPO DE SERVIÇO - O empregado eleito para ocupar cargo de diretor tem o respectivo contrato de trabalho suspenso, não se computando o tempo de serviço desse período, salvo se permanecer a subordinação jurídica inerente à relação de emprego.

    2. Contrato

      Súmula nº 363/TST - CONTRATO NULO. EFEITOS - A contratação de servidor público, após a CF/1988, sem prévia aprovação em concurso público, encontra óbice no respectivo art. 37, II e § 2º, somente lhe conferindo direito ao pagamento da contraprestação pactuada, em relação ao número de horas trabalhadas, respeitado o valor da hora do salário mínimo, e dos valores referentes aos depósitos do FGTS.

      Obs.: Apesar da patente nulidade de contrato de servidor admitido sem concurso público, ao menos lhe garante a contraprestação e o FGTS


      Súmula nº 430/TST - ADMINISTRAÇÃO PÚBLICA INDIRETA. CONTRATAÇÃO. AUSÊNCIA DE CONCURSO PÚBLICO. NULIDADE. ULTERIOR PRIVATIZAÇÃO. CONVALIDAÇÃO. INSUBSISTÊNCIA DO VÍCIO - Convalidam-se os efeitos do contrato de trabalho que, considerado nulo por ausência de concurso público, quando celebrado originalmente com ente da Administração Pública Indireta, continua a existir após a sua privatização.

      Obs.: Muito embora nulo o contrato por falta de concurso público, ele poderá ser convalidado na hipótese de privatização.


      Súmula nº 455/TST - EQUIPARAÇÃO SALARIAL. SOCIEDADE DE ECONOMIA MISTA. ART. 37, XIII, DA CF/1988. POSSIBILIDADE. - À sociedade de economia mista não se aplica a vedação à equiparação prevista no art. 37, XIII, da CF/1988, pois, ao admitir empregados sob o regime da CLT, equipara-se a empregador privado, conforme disposto no art. 173, § 1º, II, da CF/1988.

      Obs.: Logo, estatais por deverem ter regime jurídico próprio de empresas privadas, podem ter que equiparar salários por adotarem o regime celetista.


      OJ-SDI1-366 - ESTAGIÁRIO. DESVIRTUAMENTO DO CONTRATO DE ESTÁGIO. RECONHECIMENTO DO VÍNCULO EMPREGATÍCIO COM A ADMINISTRAÇÃO PÚBLICA DIRETA OU INDIRETA. PERÍODO POSTERIOR À CONSTI-TUIÇÃO FEDERAL DE 1988. IMPOSSIBILIDADE - Ainda que desvirtuada a finalidade do contrato de estágio celebrado na vigência da Constituição Federal de 1988, é inviável o reconhecimento do vínculo empregatício com ente da Administração Pública direta ou indireta, por força do art. 37, II, da CF/1988, bem como o deferimento de indenização pecuniária, exceto em relação às parcelas previstas na Súmula nº 363 do TST, se requeridas.

      Obs.: Via de regra, o desvirtuamento de estágio possa gerar o reconhecimento vínculo empregatício, não é admissível essa hipótese para a Administração Pública em decorrência do Princípio do Concurso Público. No entanto, isso não impede o reconhecimento, se o caso, do pagamento do previsto em contrato + FGTS (Sum 363)


      OJ-SDI2-128 - AÇÃO RESCISÓRIA. CONCURSO PÚBLICO ANULADO POSTERIORMENTE. APLICAÇÃO DA SÚMULA Nº 363 DO TST - O certame público posteriormente anulado equivale à contratação realizada sem a observância da exigência contida no art. 37, II, da Constituição Federal de 1988. Assim sendo, aplicam-se à hipótese os efeitos previstos na Súmula nº 363 do TST.

    1. Plantea es que la Psicología, tal como se ha configurado históricamente, tiene raíces conservadoras e individualistas. Eso significa que gran parte de la disciplina se ha centrado en el individuo aislado, en sus procesos internos, sin atender con suficiente fuerza a lo que Marx llamaría las condiciones materiales de existencia. El artículo propone recuperar la perspectiva crítica y liberadora que Ignacio Martín-Baró desarrolló, precisamente porque él entendía que la psicología debía situarse en la vida concreta de las personas y en su contexto histórico, social y político.

      Para llegar a ese punto, el texto dialoga con otros autores que también han trabajado desde marcos marxistas, como Reich, que analiza cómo la ideología penetra hasta en la vida psíquica y en la organización del deseo, o Ratner, que retoma a Vygotsky para insistir en que no hay subjetividad fuera de un entramado sociohistórico. Laraia, desde la antropología, recuerda que el ser humano es producto del medio cultural en el que se socializa. Todo esto converge en una misma dirección: si la Psicología se desentiende del contexto social y cultural, se convierte en una ciencia mutilada, incapaz de comprender plenamente a su objeto.

      El aporte central de Martín-Baró —que el artículo recupera— es la idea de una Psicología Social de la liberación. Esto supone considerar de forma seria las contradicciones de clase, los procesos de deshumanización que genera el capitalismo y, al mismo tiempo, las luchas sociales que abren horizontes de emancipación. El marxismo aquí no se presenta como un dogma, sino como una herramienta crítica para interrogar tanto la práctica científica como la praxis social.

    1. Juan Pablo Patriglia tiene la capacidad de situar el proyecto intelectual y político de García Linera en una clave que excede la pura reconstrucción bibliográfica. Comprende las publicaciones editadas desde la Vicepresidencia de Bolivia como un ejercicio de interpretación del marxismo, en el sentido gramsciano y benjaminiano del término, como un proceso de reapropiación, de actualización y de disputa de sentidos. El autor consigue mostrar cómo, bajo la gestión de García Linera, la edición y circulación de textos marxistas fueron parte de una estrategia de producción teórica que buscó enlazar la tradición crítica con las experiencias de lucha de Bolivia y de América Latina. Me parece especialmente interesante la manera en articula las tres dimensiones de su estudio: la obra propia de García Linera, la recuperación de intelectuales latinoamericanos como Echeverría, Veraza o Dussel, y la edición de inéditos de Marx. Cada uno de estos apartados es tratado como partes de un mismo esfuerzo de traducción que rompe con el eurocentrismo y contribuye a un marxismo vivo, situado y latinoamericano. Allí radica, creo, la virtud de este trabajo: hacer visible la densidad teórica y política que tuvo el proyecto editorial de la Vicepresidencia de Bolivia al mando de García Linera. El artículo da cuenta de un momento relevante de la historia intelectual reciente de América Latina. Ofrece un análisis sólido, bien documentado y con una clara apuesta interpretativa.

    1. Description

      A humorous celebration of the value of quality blogging within the Hive Blogger Ecosystem. Learn that true merit lies in the art of writing rather than superficial metrics or visual content. Content creation is not merely driven by algorithms or financial incentives. But by the integrity and passion of writers dedicated to sharing insightful narratives.

    1. cooperativa médica
      • Informativo nº 853
      • 10 de junho de 2025.
      • QUARTA TURMA
      • Processo: REsp 2.183.714-SP, Rel. Ministro Marco Buzzi, Quarta Turma, por unanimidade, julgado em 3/6/2025.

      Ramo do Direito RECUPERAÇÃO JUDICIAL

      TemaSaúde e Bem-Estar Paz, Justiça e Instituições Eficazes<br /> Recuperação judicial. Cooperativas médicas. Possibilidade. Alteração da Lei n. 11.101/2005 pela Lei n. 14.112/2020.

      Destaque As cooperativas médicas estão legitimadas, expressamente, por força de lei, a requerer o benefício da recuperação judicial.

      Informações do Inteiro Teor - Cinge-se a controvérsia em saber se as cooperativas médicas podem se submeter ao regime de recuperação judicial, conforme a alteração promovida pela Lei n. 14.112/2020 na Lei n. 11.101/2005.

      • Registra-se que não há, na hipótese, antinomia (conflito aparente) entre regras jurídicas. A Lei n. 5.764/1971 é norma geral que define, de forma ampla, a política nacional de cooperativismo. Por sua vez, a Lei n. 11.101/2005 (Lei de Recuperação Judicial e Falências ) estabelece um regime jurídico especial para as sociedades que desenvolvem atividades empresariais e enfrentam dificuldades financeiras, com o objetivo de manter a viabilidade econômica delas, para, assim, superar a crise econômica.

      • Sendo certo que o regramento e a aplicação da Lei de Recuperação Judicial e Falências - LRJF, por expressa dicção legal (art. 2º), são excepcionados em apenas duas hipóteses literais.

      • E no caso, observa-se claramente do texto legal que as cooperativas médicas não estão nominalmente excluídas do regime recuperacional, visto que a exceção contida no art. 4º da Lei n. 5.764/1971 afasta tão-somente a possibilidade de decretação de falência. Mesmo nesse particular, relativo à vedação de sua submissão ao regime falimentar, é importante que ao intérprete não é dado realizar uma análise recortada da lei, visto que, conforme o método do diálogo das fontes, há de ser compreendido o sentido sistêmico da legislação em exame, porquanto o ordenamento jurídico é harmônico entre si.

      • Assim, verifica-se que o artigo 6º, § 13, da Lei n. 11.101/2005, é particularmente relevante ao afirmar que as sociedades cooperativas médicas estão sujeitas ao disposto na lei em foco. Esse dispositivo, incluído pela Lei n. 14.112 de 2020, dispõe apenas sobre os efeitos da recuperação judicial em relação aos contratos e às obrigações decorrentes dos atos cooperativos praticados pelas sociedades cooperativas em relação aos seus cooperados. E, na parte final do § 13, excepciona da vedação ao regime da recuperação judicial, a cooperativa médica operadora de plano de assistência à saúde.

      • A inclusão expressa das sociedades cooperativas no âmbito da Lei n. 11.101/2005, demonstra que o legislador reconheceu a importância de garantir a essas entidades a possibilidade de reestruturação financeira por meio da recuperação judicial. Esse entendimento é reforçado pelo fato de que as cooperativas médicas desempenham um papel social relevante, contribuindo para o acesso à saúde e para a sustentabilidade do sistema de saúde como um todo.

      • É importante dizer que a redação final do art. 6º, § 13, da Lei n. 11.101/2005, foi objeto de análise, em sede de Ação Direta de Inconstitucionalidade pelo Supremo Tribunal Federal, ADI 7442/DF, em que foi declarada a constitucionalidade da inclusão das cooperativas médicas no regime de recuperação judicial, reforçando a legitimidade dessas entidades para requerer tal benefício.

    2. não poderá prever prazo superior a 1 (um) ano
      • Informativo nº 857
      • 12 de agosto de 2025.
      • QUARTA TURMA
      • Processo: REsp 1.875.820-SP, Rel. Ministro João Otávio de Noronha, Quarta Turma, por unanimidade, julgado em 5/8/2025.

      Ramo do Direito DIREITO EMPRESARIAL, RECUPERAÇÃO JUDICIAL

      TemaPaz, Justiça e Instituições Eficazes <br /> Pagamento de créditos trabalhistas. Prazo. Marco inicial. Data da concessão da recuperação.

      Destaque - O prazo para pagamento dos créditos trabalhistas deve ser contado a partir da concessão da recuperação judicial, e não da data do pedido.

      Informações do Inteiro Teor - Cinge-se a controvérsia acerca do termo inicial a ser fixado para o pagamento dos créditos trabalhistas em sede de recuperação judicial.

      • A questão relativa ao marco inicial para pagamento de credores trabalhistas em processos de recuperação judicial foi definida no Superior Tribunal de Justiça por ocasião do julgamento dos recursos especiais n. 1.924.164/SP (DJe 17/6/20211) e n. 947.732/SP (DJe 1º/10/2021).

      • O art. 54 da Lei 11.101/2005 estabelece que o plano de recuperação judicial não pode prever prazo superior a 1 (um) ano para o pagamento de créditos trabalhistas ou decorrentes de acidentes de trabalho vencidos até a data do pedido. Essa limitação visa proteger os trabalhadores, cujos créditos têm natureza alimentar e são, portanto, merecedores de tratamento especial.

      • A lei, no entanto, não especifica a data de início do prazo de um ano para o pagamento desses créditos.

      • Assim, a interpretação que a doutrina vem dando, corroborada pela jurisprudência do STJ, é que o prazo deve ser contado a partir da concessão da recuperação judicial, e não da data do pedido (RESP 1.960.888).

      • Esse entendimento advém de que: 1) A concessão da recuperação judicial é o marco que confere eficácia à novação dos créditos (art. 59 da Lei de Recuperação Judicial e Falência); 2) Antes dessa decisão, o plano ainda pode ser rejeitado, podendo haver a convolação em falência; e 3) A novação só se aperfeiçoa com a homologação judicial do plano, o que condiciona o início do cumprimento das obrigações.

    1. Art. 41
      • Informativo nº 854
      • 17 de junho de 2025.
      • RECURSOS REPETITIVOS
      • Compartilhe:
      • Processo: REsp 1.993.530-RS, Rel. Ministra Regina Helena Costa, Primeira Seção, por unanimidade, julgado em 11/6/2025. (Tema 1233).

      Ramo do Direito DIREITO ADMINISTRATIVO, DIREITO PREVIDENCIÁRIO

      TemaPaz, Justiça e Instituições Eficazes <br /> Servidor público. Abono de permanência. Natureza remuneratória e permanente. Adicional de férias e gratificação natalina (13º Salário). Verbas calculadas com base na remuneração do servidor. Inclusão. Legalidade. Tema 1233.

      Destaque - O abono de permanência, dada sua natureza remuneratória e permanente, integra a base de incidência das verbas calculadas sobre a remuneração do servidor público, tais como o adicional de férias e a gratificação natalina (13º salário).

      Informações do Inteiro Teor - O abono de permanência constitui estímulo pecuniário à permanência na ativa do servidor público que já reuniu as condições legais para se aposentar voluntariamente, sendo pago até o implemento dos requisitos para a aposentadoria compulsória, correspondente, no máximo, ao montante da contribuição previdenciária por ele devida.

      • Conforme entendimento firmando em sede de recurso especial submetido à sistemática repetitiva (Tema 424/STJ), o abono de permanência ostenta natureza remuneratória, porquanto se incorpora ao conjunto de vantagens percebidas pelo servidor em razão do exercício do cargo, sendo pago de forma regular enquanto a atividade laboral for mantida - vale dizer, como contraprestação/retribuição pelo trabalho -, sem denotar reparação ou recomposição patrimoniais.

      • A incidência de tal parcela na composição das bases de cálculo da gratificação natalina e do adicional de férias decorre, efetivamente, da própria definição de remuneração contida no art. 41 da Lei n. 8.112/1990, que compreende o vencimento básico acrescido de vantagens permanentes.

      • O valor correspondente a tal benefício integra permanentemente a remuneração do servidor enquanto perdurar a relação de trabalho.

      • Dessa forma, fixa-se a seguinte tese: o abono de permanência, dada sua natureza remuneratória e permanente, integra a base de incidência das verbas calculadas sobre a remuneração do servidor público, tais como o adicional de férias e a gratificação natalina (13º salário).

    1. aposentadoria
      • Informativo nº 855
      • 1º de julho de 2025.
      • PRIMEIRA TURMA
      • Processo: REsp 1.457.398-SE, Rel. Ministro Gurgel de Faria, Primeira Turma, por unanimidade, julgado em 13/5/2025, DJEN 21/5/2025.

      Ramo do Direito DIREITO PREVIDENCIÁRIO

      TemaPaz, Justiça e Instituições Eficazes <br /> Aposentadoria por tempo de contribução. Tutela antecipada. Revogação posterior. Valores recebidos. Tempo de serviço. Contagem. Impossibilidade.

      Destaque - O período de recebimento do benefício de aposentadoria por tempo de contribuição, em razão de tutela provisória posteriormente revogada, não pode ser somado ao seu tempo de contribuição com a finalidade de obter a aposentadoria.

      Informações do Inteiro Teor - A questão submetida a julgamento consiste em saber se o tempo em que o recorrente recebeu o benefício de aposentadoria por tempo de contribuição em razão de tutela provisória posteriormente revogada pode ser somado ao seu tempo de contribuição com a finalidade de obter a aposentadoria.

      • Em regra, a tutela antecipada ou de urgência figura como provimento judicial provisório e reversível (art. 273, § 2º, do CPC/1973 e artigos 296 e 300, § 3º, do CPC/2015). Ou seja, a rigor, a revogação da decisão que concede o mandamento provisório produz efeitos imediatos e retroativos, impondo o retorno à situação anterior ao deferimento da medida, cujo ônus deve ser suportado pelo beneficiário da tutela.

      • Com efeito, como o cumprimento provisório ocorre por iniciativa e responsabilidade do autor, cabe a este, em regra, suportar o ônus decorrente da reversão da decisão precária, visto que, a rigor, pode, de antemão, prever os resultados de eventual cassação da medida, escolher sujeitar-se a tais consequências e até mesmo trabalhar previamente para evitar ou mitigar o dano no caso de reversão.

      • Nesse sentido, no julgamento da Pet 12.482/DF, a Primeira Seção do STJ ratificou o entendimento anteriormente firmado no julgamento do Tema repetitivo n. 692 do STJ, segundo o qual a reforma da decisão que antecipa os efeitos da tutela obriga a parte beneficiária à restituição dos valores recebidos.

      • Desse modo, cassada a decisão que antecipa a tutela, a parte beneficiária obriga-se à devolução dos valores, uma vez que é da natureza do instituto a reversibilidade dos efeitos da decisão, conforme dispõe o § 3º do art. 300 do CPC.

      • Portanto, a tutela provisória, tendo natureza precária e provisória, uma vez cassada, deve restituir as partes à situação anterior ao seu deferimento.

      • Além disso, a legislação que rege a matéria (Lei n. 8.213/1991 e o Decreto n. 3.048/1999) estabelece expressamente qual é o período que deve ser considerado como tempo de contribuição do segurado que deixou de exercer atividade remunerada.

      • Verifica-se, pois, que o conceito normativo de tempo de contribuição é o tempo correspondente aos períodos para os quais tenha havido contribuição obrigatória ou facultativa para o Regime Geral de Previdência Social - RGPS.

      • Logo, conjugando-se a definição do que deve ser considerado como tempo de contribuição, nos termos da legislação de regência, com a natureza precária da antecipação de tutela, os valores recebidos a título de antecipação dos efeitos da tutela não podem ser equiparados aos benefícios de auxílio-acidente ou de aposentadoria por invalidez.

    1. § 2º
      • Informativo nº 855
      • 1º de julho de 2025.
      • QUARTA TURMA
      • Processo: REsp 2.188.689-RS, Rel. Ministro Marco Buzzi, Quarta Turma, por unanimidade, julgado em 17/6/2025.

      Ramo do Direito DIREITO DO CONSUMIDOR

      TemaPaz, Justiça e Instituições Eficazes <br /> Superendividamento. Audiência de conciliação. Ausência de aceitação do plano de pagamento sugerido pelo devedor e falta de apresentação de contraposta. Sanções do art. 104-A, § 2º, do CDC. Impossibilidade de aplicação analógica.

      Destaque - Na audiência preliminar referente à repactuação de dívidas por superendividamento, embora recomendável à luz dos princípios da boa-fé e da cooperação entre os litigantes, não há obrigação legal para o credor apresentar contraproposta ou aderir ao plano de pagamento formulado pelo devedor, sendo inaplicável as sanções do art. 104-A, § 2º, do CDC.

      Informações do Inteiro Teor - Cinge-se a controvérsia à possibilidade, ou não, de se impor ao credor as penalidades do artigo 104-A, § 2º do CDC quando, embora devidamente representado por preposto e advogado com poderes para transigir na audiência preliminar atinente à repactuação de dívidas por superendividamento, deixe de aderir ou oferecer contraproposta ao plano de pagamento apresentado pelo devedor.

      • O tema possui inegável relevância jurídica, espelhando importante desdobramento da aplicação prática da Lei n. 14.181/2021, que promoveu alterações no Código de Defesa do Consumidor e no Estatuto da Pessoa Idosa para aperfeiçoar a disciplina de concessão de crédito ao consumidor e, em especial, dispor sobre a prevenção e o tratamento do superendividamento.

      • Assim, entende-se por superendividamento a impossibilidade manifesta de o consumidor pessoa natural, de boa-fé, pagar a totalidade de suas dívidas de consumo, exigíveis e vincendas, às quais englobam quaisquer compromissos financeiros assumidos decorrentes de relação de consumo, inclusive operações de crédito, compras a prazo e serviços de prestação continuada, sem comprometer seu mínimo existencial, conforme artigo 54-A, § 1º e 2º do CDC.

      • A Lei n. 14.181/2021 inovou ao introduzir, no CDC, tratamento amplo acerca do superendividamento, não mais limitado a pretensões revisionais em demandas judiciais ou renegociações individuais, em mutirões de dívidas. Nesse sentido, a novatio legis oferece uma espécie de antídoto à crise financeira do consumidor, mediante a organização de um plano para viabilizar o pagamento dos seus débitos, restabelecer seu acesso ao mercado e voltar a consumir, além de preservar o mínimo existencial.

      • O procedimento estabelecido em lei prescreve uma fase conciliatória e preventiva à repactuação de dívidas, mediante realização de audiência preliminar com todos os credores, oportunidade na qual o consumidor apresentará um plano voluntário para o pagamento dos débitos.

      • Nessa primeira etapa foram fixadas sanções contra comportamentos do credor que inviabilizem ou retirem a utilidade da própria audiência, quais sejam: o não comparecimento injustificado, ou de seu procurador com poderes especiais e plenos para transigir (art. 104-A, § 2º, do CDC).

      • Nessas hipóteses específicas, que colidem com os princípios nos quais se baseia a lei, em especial, a cooperação e a solidariedade, ocorrerá a suspensão da exigibilidade do débito e a interrupção dos encargos da mora, bem como a sujeição compulsória ao plano de adimplemento da dívida, caso o montante devido ao ausente for certo e conhecido pelo consumidor, circunstância na qual o pagamento do respectivo crédito somente ocorrerá após saldado o débito junto aos credores presentes à audiência conciliatória.

      • Em que pese a importância da audiência e o prestígio dado pelo sistema à autocomposição, não há respaldo legal para a aplicação, por analogia, das penalidades acima referidas, isto é, caso não haja acordo entre as partes, ou na hipótese do credor não apresentar contraproposta.

      • Dessa forma, a ausência de aceitação do plano de pagamento sugerido pelo devedor e a falta de apresentação de contraposta não geram, como consequência, a aplicação dos efeitos do § 2º do artigo 104-A do CDC, ensejando, apenas, a eventual instauração da segunda fase do processo de superendividamento para a revisão e integração dos contratos e repactuação das dívidas, ficando a cargo do juiz a possibilidade de conceder tutelas cautelares, as quais podem incluir, entre outras, as medidas do § 2º do artigo 104-A do CDC.