1. Dec 2025
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    Emergence of endothelial subtypes and role of cell cycle control in arterial-venous specification during embryonic vascular development
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    Sensitized mast cells for targeted drug delivery and augmented cancer immunotherapy
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    Functional differences between CLL‐ and ALL‐derived CAR T cells in a 3D tumor microenvironment highlight CXCR4 and IL‐10 as potential modulatory targets
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    The inhibitory SAPS3–AMPK interaction detected in HEK293 cells is not detectable in muscle or liver from humans or mice
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    Imeglimin attenuates liver fibrosis by inhibiting vesicular ATP release from hepatic stellate cells
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    Gold nanoparticle transport across tumour-associated biological barriers: in vitro models, imaging, and quantification
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    Gamma
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    1. Drwf 10 Dec 2025
      Building Effective Asynchronous Online CoursesA comprehensive guide for educators and instructional designers creating engaging, well-structured asynchronous learning experiences that support student success.

      WEEK 7 PRESENTATION

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    An unfinished Pompeian construction site reveals ancient Roman building technology
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    1. tonz 10 Dec 2025

      https://web.archive.org/web/20251210080852/https://www.nature.com/articles/s41467-025-66634-7

      Cool! Hotmixing turns out to be the secret behind the durable Roman 'concrete'. A 2023 theory now confirmed by a Pompei construction site find.

      Researchers think this new insight may be applied in current building, and impact the climate footprint of modern day concrete.

      An unfinished Pompeian construction site reveals ancient Roman building technology in Zotero

      concrete construction romanempire pompei
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    b09252e85e37e5818b25f421b675b5673e9b3a8c97c44b784f8aa2d5edd6f106.PDF
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  12. www.biorxiv.org www.biorxiv.org
    Evolution of a fuzzy ribonucleoprotein complex in viral assembly
    5
    1. Public_Reviews 10 Dec 2025

      eLife Assessment

      This is a valuable study that combines biophysical and evolutionary approaches to understand why particular mutations in the SARS-CoV-2 protein N arose during the COVID-19 pandemic. The evidence is solid and supports the conclusions.

      Summary
    2. Public_Reviews 10 Dec 2025

      Reviewer #1 (Public review):

      Summary:

      The authors attempted to clarify the impact of N protein mutations on ribonucleoprotein (RNP) assembly and stability using analytical ultracentrifugation (AUC) and mass photometry (MP). These complementary approaches provide a more comprehensive understanding of the underlying processes. Both SV-AUC and MP results consistently showed enhanced RNP assembly and stability due to N protein mutations.<br /> The overall research design appears well planned, and the experiments were carefully executed.

      Strengths:

      SV-AUC, performed at higher concentrations (3 µM), captured the hydrodynamic properties of bulk assembled complexes, while MP provided crucial information on dissociation rates and complex lifetimes at nanomolar concentrations. Together, the methods offered detailed insights into association states and dissociation kinetics across a broad concentration range. This represents a thorough application of solution physicochemistry.

      Weaknesses:

      Unlike AUC, MP observes only a part of solution. In MP, bound molecules are accumulated on the glass surface (not dissociated) thus concentration in solution should change as time develops. How does such concentration change impact the result shown here?

      Comments on revisions:

      The response from the authors is appropriate and reasonable.

      Review 1
    3. Public_Reviews 10 Dec 2025

      Reviewer #2 (Public review):

      Summary:

      In this manuscript, the authors apply a variety of biophysical and computational techniques to characterize the effects of mutations in the SARS-CoV-2 N protein on the formation of ribonucleoprotein particles (RNPs). They find convergent evolution in multiple repeated independent mutations strengthening binding interfaces, compensating for other mutations that reduce RNP stability but which enhance viral replication.

      Strengths:

      The authors assay the effects of a variety of mutations found in SARS-CoV-2 variants of concern using a variety of approaches, including biophysical characterization of assembly properties of RNPs, combined with computational prediction of the effects of mutations on molecular structures and interactions. The findings of the paper contribute to our increasing understanding of the principles driving viral self-assembly, and increases the foundation for potential future design of therapeutics such as assembly inhibitors.

      Weaknesses:

      For the most part, the paper is well-written, the data presented support the claims made, and the arguments made easy to follow. However, I believe that parts of the presentation could be substantially improved. I found portions of the text to be overly long and verbose and likely could be substantially edited; the use of acronyms and initialisms is pervasive, making parts of the exposition laborious to follow; and portions of the figures are too small and difficult to read/understand.

      Comments on revisions:

      The authors have adequately addressed all of my concerns.

      Review 2
    4. Public_Reviews 10 Dec 2025

      Reviewer #3 (Public review):

      Summary:

      This manuscript investigates how mutations in the SARS-CoV-2 nucleocapsid protein (N) alter ribonucleoprotein (RNP) assembly, stability, and viral fitness. The authors focus on mutations such as P13L, G214C, G215C combining biophysical assays (SV-AUC, mass photometry, CD spectroscopy, EM), VLP formation, and reverse genetics. They propose that SARS-CoV-2 exploits "fuzzy complex" principles, where distributed weak interfaces in disordered regions allow both stability and plasticity, with measurable consequences for viral replication.

      Strengths:

      * The paper demonstrates a comprehensive integration of structural biophysics, peptide/protein assays, VLP systems, and reverse genetics.

      * Identification of both de novo (P13L) and stabilizing (G214C/G215C) interfaces provides a mechanistic insight into RNP formation.

      * Strong application of the "fuzzy complex" framework to viral assembly, showing how weak/disordered interactions support evolvability, is a significant conceptual advance in viral capsid assembly.

      * Overall, the study provides a mechanistic context for mutations that have arisen in major SARS-CoV-2 variants (Omicron, Delta, Lambda) and a mechanistic basis for how mutations influence phenotype via altered biomolecular interactions.

      Weaknesses:

      The weaknesses are shared via detailed comments to follow.

      Comments on revisions:

      The authors have addressed the criticisms of the original manuscript satisfactorily.

      Review 3
    5. Public_Reviews 10 Dec 2025

      Author response:

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

      Reviewer #1 (Public Review):

      Summary:

      The authors attempted to clarify the impact of N protein mutations on ribonucleoprotein (RNP) assembly and stability using analytical ultracentrifugation (AUC) and mass photometry (MP). These complementary approaches provide a more comprehensive understanding of the underlying processes. Both SV-AUC and MP results consistently showed enhanced RNP assembly and stability due to N protein mutations.

      The overall research design appears well planned, and the experiments were carefully executed.

      Strengths:

      SV-AUC, performed at higher concentrations (3 µM), captured the hydrodynamic properties of bulk assembled complexes, while MP provided crucial information on dissociation rates and complex lifetimes at nanomolar concentrations. Together, the methods offered detailed insights into association states and dissociation kinetics across a broad concentration range. This represents a thorough application of solution physicochemistry.

      We thank the Reviewer for this positive assessment. 

      Weaknesses:

      Unlike AUC, MP observes only a part of the solution. In MP, bound molecules are accumulated on the glass surface (not dissociated), thus the concentration in solution should change as time develops. How does such concentration change impact the result shown here?

      We agree with the Reviewer that the concentration in solution above the surface will change with time; however, the impact of surface adsorption turns out to be negligible. To show this we have added a calculation as Supplementary Methods that is based on the number of imaged adsorption events, the fraction of imaged area to total surface area, and the initial sample volume and concentration. Under our experimental conditions the reduction is less than 1%, which is well within the range of experimental concentration errors.

      This is in line with the observation that surface adsorption of proteins to glass is critical and needs to be prevented when working at picomolar concentrations (Zhao H, Mayer ML, Schuck P. 2014. Analysis of protein interactions with picomolar binding affinity by fluorescence-detected sedimentation velocity. Anal Chem 86:3181–3187. doi:10.1021/ac500093m), but is ordinarily negligible when working at the mid nanomolar concentration range. The difference in the MP experiments is that where usually the surface adsorption to glass and plastic is invisible, it is being imaged and quantified in MP. The negligible impact of surface adsorption on solution concentration in typical MP experiments is also in line with the results of several studies that have successfully measured dissociation constants of binding equilibria by MP (Young G et al., Science 360 (2018) 432; Wu & Piszczeck, Anal Biochem 592 (2020) 113575; Solterman et al. Angewandte Chemie 59 (2020) 10774) with samples in the 5-50 nM range and similar experimental setup. It should be noted that in the MP experiments no surface functionalization is employed, in contrast to optical biosensors that utilize surface-immobilized ligands and polymeric matrices and thereby enhance the surface binding capacity.

      Even though this depletion effect is negligible under ordinary MP conditions, the Reviewer raises a good point and readers may have a similar question with this novel technique. For this reason, we have added in the MP section of the Methods the sentence “In either configuration, the impact of surface binding on the sample concentration is < 1% and negligible, as described in the Supplementary Methods S1.” and added the detailed calculations in the Supplement accordingly. The use of SV as a traditional, orthogonal technique and the observation of consistent results with those of MP should further dispel readers’ methodological concerns in this point.

      Reviewer #2 (Public Review):

      Summary:

      In this manuscript, the authors apply a variety of biophysical and computational techniques to characterize the effects of mutations in the SARS-CoV-2 N protein on the formation of ribonucleoprotein particles (RNPs). They find convergent evolution in multiple repeated independent mutations strengthening binding interfaces, compensating for other mutations that reduce RNP stability but which enhance viral replication.

      Strengths:

      The authors assay the effects of a variety of mutations found in SARS-CoV-2 variants of concern using a variety of approaches, including biophysical characterization of assembly properties of RNPs, combined with computational prediction of the effects of mutations on molecular structures and interactions. The findings of the paper contribute to our increasing understanding of the principles driving viral self-assembly, and increase the foundation for potential future design of therapeutics such as assembly inhibitors.

      Thank you for highlighting the strengths of our paper and the potential impact on future design of therapeutics.

      Weaknesses:

      For the most part, the paper is well-written, the data presented support the claims made, and the arguments are easy to follow. However, I believe that parts of the presentation could be substantially improved. I found portions of the text to be overly long and verbose and likely could be substantially edited; the use of acronyms and initialisms is pervasive, making parts of the exposition laborious to follow; and portions of the figures are too small and difficult to read/understand.

      We are glad the Reviewer concurs the data support our conclusions, and finds the arguments easy to follow.  We appreciate the comment that the work was not optimally presented. To address this point, we have identified multiple opportunities to streamline the text without jeopardizing the clarity. We have also rewritten the end of the Introduction.

      As recommended, we have reduced and harmonized the use of acronyms and abbreviations throughout the text to improve readability. Specifically, we have now spelled out nucleic acid (NA), intrinsically disordered regions (IDR), full-length (FL), AlphaFold (AF3), and variants of concern (VOC).

      Finally, we have improved the presentation of most figures, adding labels and new panels, and increased the label font sizes to facilitate more detailed inspections of the data.

      Reviewer #3 (Public Review):

      This manuscript investigates how mutations in the SARS-CoV-2 nucleocapsid protein (N) alter ribonucleoprotein (RNP) assembly, stability, and viral fitness. The authors focus on mutations such as P13L, G214C, and G215C, combining biophysical assays (SV-AUC, mass photometry, CD spectroscopy, EM), VLP formation, and reverse genetics. They propose that SARS-CoV-2 exploits "fuzzy complex" principles, where distributed weak interfaces in disordered regions allow both stability and plasticity, with measurable consequences for viral replication.

      Strengths:

      (1) The paper demonstrates a comprehensive integration of structural biophysics, peptide/protein assays, VLP systems, and reverse genetics.

      (2) Identification of both de novo (P13L) and stabilizing (G214C/G215C) interfaces provides a mechanistic insight into RNP formation.

      (3) Strong application of the "fuzzy complex" framework to viral assembly, showing how weak/disordered interactions support evolvability, is a significant conceptual advance in viral capsid assembly.

      (4) Overall, the study provides a mechanistic context for mutations that have arisen in major SARS-CoV-2 variants (Omicron, Delta, Lambda) and a mechanistic basis for how mutations influence phenotype via altered biomolecular interactions.

      We are grateful for these comments highlighting this work as a significant conceptual advance.

      Weaknesses:

      (1) The arrangement of N dimers around LRS helices is presented in Figure 1C, but the text concedes that "the arrangement sketched in Figure 1C is not unique" (lines 144-146) and that AF3 modeling attempts yielded "only inconsistent results" (line 149).

      The authors should therefore present the models more cautiously as hypotheses instead. Additional alternative arrangements should be included in the Supplementary Information, so the readers do not over-interpret a single schematic model.

      We agree that in the absence of high-resolution structures the RNP models are hypothetical, and have now emphasized this in the Results, following the Reviewer’s recommendation. To present alternative arrangements that satisfy the biophysical constraints upfront, we have promoted the previous Supplementary Figure 11 showing different models to the first Supplementary Figure, and expanded it with examples of different oligomers. In this way it is referenced early on in the Results and in the legend to Figure 1C. We agree this strengthens the manuscript, as one of the take-home messages is the inherent polydispersity of the RNPs.

      The fact that AF3 can only provide inconsistent results will not come as a surprise, given the substantial disordered regions of the complex, and is a drawback of AF3 rather than our structural model. We slightly emphasized this point so as to clarify that the presentation of the AF3-based RNP structure serves solely as supporting evidence that our hypothetical model is sterically reasonable.

      The new Results paragraph reads:

      “As suggested in the cartoon of Figure 1C, this supports the hypothesis of a three-dimensional arrangement with a central LRS oligomer with symmetry properties and dimensions similar to low resolution EM images of model RNPs (Carlson et al., 2022, 2020) and cryo-ET of RNPs in virions (Klein et al., 2020; Yao et al., 2020).  It should be noted, however, that the arrangement sketched in Figure 1C is not unique and other subunit orientations could be envisioned that satisfy all constraints from experimentally observed binding interfaces, including different oligomers and anti-parallel subunits as illustrated in Supplementary Figure S1. Extending previous ColabFold structural predictions that show multiple N-protein dimers self-assembled via the LRS coiled-coils (Zhao et al., 2023), we attempted the AlphaFold modeling of RNPs combining multiple N dimers with SL7 RNA ligands, mimicking our biophysical assembly model. Current AlphaFold restrictions limit the prediction to pentamers of N-protein dimers with 10 copies of SL7 RNA. While only inconsistent results were obtained – which is not surprising given the large intrinsically disordered regions exceed the predictive power of AlphaFold – some models did produce an overall RNP organization similar to Figure 1C, suggesting such an arrangement is at least sterically reasonable with regard to possible N-protein subunit orientations in an RNP (Supplementary Figure S2)”

      (2) Negative-stained EM fibrils (Figure 2A) and CD spectra (Figure 2B) are presented to argue that P13L promotes β-sheet self-association. However, the claim could benefit from more orthogonal validation of β-sheet self-association. Additional confirmation via FTIR spectra or ThT fluorescence could be used to further distinguish structured β-sheets from amorphous aggregation.

      We completely agree that the application of multiple orthogonal biophysical methods can strengthen the conclusions. In addition to EM fibrils and CD spectra (a classical gold standard technique for protein secondary structure in solution), we already have support from ColabFold modeling, as well as NMR results from the Zweckstetter lab showing the potential for for β-sheet-like conformations.

      Furthermore, we believe the evidence for the absence of ‘amorphous aggregates’ is very strong, as this would be inconsistent with the long-range order required to create the visibly fibrillar morphology in EM, and amorphous aggregates would be inconsistent with the increased solution viscosity. In this context, it is also highly relevant that the β-sheet-like secondary structure recorded by CD is concentration-dependent and reversible upon dilution. The long-range spatial order of fibrils is consistent with the formation of secondary structure in solution.

      In addition, it must be kept in mind that what we see is specific to N-arm peptides carrying the P13L mutation (in EM, CD, and structural prediction) and does not occur in the other two N-arm peptides (ancestral N-arm and N-arm with deletion of 31-33), linker peptides, or C-arm peptides.

      Most importantly, as elaborated in more detail below, we do not claim that fibril formation is physiologically relevant. At the heart of this – in the context of the evolution of fuzzy complexes – is that the P13L mutation creates additional weak protein-protein interactions. Indeed, the assembly of fibrils geometrically requires at least two interfaces for each subunit. These weak interactions are at play physiologically in the context of the disordered RNP particles, and in macromolecular condensates, but not in the formation of fibrils. Therefore, while we appreciate the suggestion for FTIR spectra ThT staining, we are afraid further emphasis on the fibril structure might confuse the reader, and therefore we would rather clarify upfront that these fibrillar assemblies are not thought to form in vivo from full-length protein, but merely demonstrate the presence of N-arm self-association interfaces in the model of truncated peptides.

      Accordingly, we have amended the Results paragraph reporting the fibrils:

      “Thus, the N-arm mutation P13L is responsible for the formation of fibrils in N-arm peptides after prolonged storage. Some of these N-arm fibrils exhibit a twisted morphology with width of »5 nm (Figure 2A), in some instances exhibiting patterns of strand breaks. Such fibrils are frequently encountered in proteins that can stack β-sheets, such as in amyloids (Paravastu et al., 2008). While we have not observed fibril formation in the context of full-length N, and have no evidence such fibrils are physiologically relevant, their occurrence in solutions of truncated N-arm peptide nonetheless demonstrates the introduction of ordered N-arm self-association interfaces in conformations of P13L mutants.”

      And more completely summarized experimental evidence prior to describing the ColabFold prediction results (which previously did not include mention of the NMR):

      “Finally, confirming the interpretation of the EM images and the CD data, as well as the b-structure propensity reported from NMR data (Zachrdla et al., 2022), the structural prediction of N[10-20]:P13L in ColabFold displayed oligomers with stacking b-sheets …”

      (3) In the main text, the authors alternate between emphasizing non-covalent effects ("a major effect of the cysteines already arises in reduced conditions without any covalent bonds," line 576) and highlighting "oxidized tetrameric N-proteins of N:G214C and N:G215C can be incorporated into RNPs". Therefore, the biological relevance of disulfide redox chemistry in viral assembly in vivo remains unclear. Discussing cellular redox plausibility and whether the authors' oxidizing conditions are meant as a mechanistic stress test rather than physiological mimicry could improve the interpretation of these results.

      The paper could benefit if the authors provide a summary figure or table contrasting reduced vs. oxidized conditions for G214C/G215C mutants (self-association, oligomerization state, RNP stability). Explicitly discuss whether disulfides are likely to form in infected cells.

      We thank the Reviewer for raising this most interesting point.  The reason why the biological relevance of N dilsulfides remains unclear is simply that this is still unknown, unfortunately. Recently, Kubinski et al. have strongly argued for the formation of disulfides in infected cells, but in our view the evidence remains weak since the majority of disulfide bonds in that work presented as post-lysis artifacts, and it appears the non-covalent effects alone could explain the physiological observations. We aimed for a balanced presentation and wrote in the relevant Results section:

      “Covalent disulfide bonds in the LRS in non-reducing conditions were found to further promote LRS oligomerization. However, there is no conclusive data yet whether covalent bonds in the LRS occur in vivo, or any G215C effect is entirely non-covalent due to the significant strengthening of LRS helix oligomerization (see Discussion).”

      Despite the uncertainty regarding physiological disulfide bond formation, we believe it is useful to ask whether covalently crosslinked N dimers would aid or constrain RNP assembly in our biophysical model. We have now better explained this motivation in the Results section describing the RNP experiments:

      “Even though it is still unclear whether disulfide bonds of N cysteine mutants form in vivo, we were curious about the impact of disulfide-linked oligomers of the cysteine mutants on their RNP structure and stability in our biophysical assembly model.”

      The referenced paragraph from the Discussion reads:

      “Regarding the cysteine mutations that have been repeatedly introduced in the LRS prior to the rise of the Omicron VOCs, it is an open question whether they lead to covalent bonds in vivo or in the VLP assay. While examples of disulfide-linked viral nucleocapsid proteins have been reported (Kubinski et al., 2024; Prokudina et al., 2004; Wootton and Yoo, 2003), a methodological difficulty in their detection is artifactual disulfide bond formation post-lysis of infected cells (Kubinski et al., 2024; Wootton and Yoo, 2003).  However, our results clearly show that a major effect of the cysteines already arises in reduced conditions without any covalent bonds, through extension of the LRS helices, and concomitant redirection of the disordered N-terminal sequence. While oxidized tetrameric N-proteins of N:G214C and N:G215C can be incorporated into RNPs, the covalent bonds provided only marginally improved RNP stability.  Interestingly, the introduction of cysteines imposes preferences of RNP oligomeric states dependent on oxidation state, consistent with our MD simulations highlighting the impact of cysteine orientation of 214C versus 215C relative to the hydrophobic surface of the LRS helices. Overall, considering potentially detrimental structural constraints from covalent bonds on LRS clusters seeding RNPs, energetic penalties on RNP disassembly, as well as the required monomeric state of the LRS helix for interaction with the NSP3 Ubl domain (Bessa et al., 2022), at present it is unclear to what extent the formation of disulfide linkages between LRS helices would be beneficial or detrimental in the viral life cycle.”

      We feel that this text addresses the Reviewer’s comment, and that expanding the existing discussion further would conflict with other recommendations to shorten and focus the text.

      Finally, we have addressed the valuable suggestion of a new table summarizing the oligomeric state and self-association of the different cysteine mutants by inserting a new column in the existing Table 1 reporting all species’ oligomeric state at low micromolar concentrations. In this way they can be compared at a glance with the other mutants as well. A more detailed comparison of the concentration-dependent size-distribution is provided in Figure 4.

      (4) VLP assays (Figure 7) show little enhancement for P13L or G215C alone, whereas Figure 8 shows that P13L provides clear fitness advantages. This discrepancy is acknowledged but not reconciled with any mechanistic or systematic rationale. The authors should consider emphasizing the limitations of VLP assays and the sources of the discrepancy with respect to Figure 8.

      We thank the Reviewer for this comment, which highlights a very important point. 

      For clarification and to improve the cohesion of the manuscript we have inserted a reference to the Discussion after the presentation of the VLP results, which provides a natural transition to the following description of the reverse genetics experiments:

      “As expanded on in the Discussion, the failure to observe enhancement by P13L alone may be related to limitations of the VLP assay in sensitivity, including the restriction to a single round of infection, and protein expression levels.”

      This references a paragraph in the Discussion about the limitations of the VLP assay in general and the reasons we believe the enhancement by P13L alone was not picked up:

      “…While this assay has been widely used for rapid assessment of spike protein and N variants (Syed et al., 2021), it has limitations due to the addition of non-genomic RNA and the lack of double membrane vesicles from which gRNA emerges through the NSP3/NSP4 pore complex potentially poised for packaging (Bessa et al., 2022; Ke et al., 2024; Ni et al., 2023). It should also be recognized that the results do not directly reflect the relative efficiency of RNP assembly only, since protein expression levels, their localization, and their posttranslational modifications are not controlled for. Susceptibility for such factors might be exacerbated with mutations that modulate weak protein interactions. For example, as shown previously (Syed et al., 2024; Zhao et al., 2024), a GSK3 inhibitor inhibiting N-protein phosphorylation significantly enhances VLP formation and eliminates the advantage provided for by the N:G215C mutation relative to the ancestral N – presumably due to an increase in assembly-competent, non-phosphorylated N-protein erasing an affinity advantage. A similar process may be underlying the absent or marginal improvement in VLP readout from the cysteine LRS mutants and P13L at the achieved transfection level in the present work, and the enhanced signal from R203K/G204R and R203M (the latter being consistent with previous reports (Li et al., 2025; Syed et al., 2021)) modulating protein phosphorylation. Nonetheless, mirroring the results of the biophysical in vitro experiments, the addition of RNP-stabilizing P13L and G214C mutations on top of R203K/G204R led to a significantly larger VLP signal.

      The VLP assay may be limited in sensitivity to mutation effects due to its restriction to a single round of infection. To avoid this and other potential limitations of the VLP assay for the study of viral packaging, for the key mutation N:P13L we carried out reverse genetics experiments. These showed the sole N:P13L mutation significantly increases viral fitness (Figure 8).”

      (5) Figures 5 and 6 are dense, and the several overlays make it hard to read. The authors should consider picking the most extreme results to make a point in the main Figure 5 and move the other overlays to the Supplementary. Additionally, annotating MP peaks directly with "2×, 4×, 6× subunits" can help non-experts.

      We completely agree with the Reviewer – these figures were very dense.  To mitigate this problem without having the reader to switch back-and-forth to the supplement, we subdivided the panels of Figure 5 and showed only a subset of curves in each.  In this way the data are easier to read while still readily compared. It is a large figure, but it contains the key data for the present work and is therefore worthwhile to have in one place. For the MP histogram data we also have inserted the suggested peak labels. Similarly, we have split Figure 6A into two panels for clarity.

      (6) The paper has several names and shorthand notations for the mutants, making it hard to keep up. The authors could include a table that contains mutation keys, with each shorthand (Ancestral, Nο/No, Nλ, etc.) mapped onto exact N mutations (P13L, Δ31-33, R203K/G204R, G214C/G215C, etc.). They could then use the same glyphs (Latin vs Greek) consistently in text and figure labels.

      Yes, we agree this is a problem and we apologize for the confusion. However, it is not possible to refer exclusively to either Latin or Greek terminology, which we feel would be even more detrimental to readability (the former being exhaustively lengthy and the latter being imprecise). But we have used a rational system: If the complete set of mutations of a variant are present, then its Greek letter will be used as an abbreviation, and otherwise we use Latin amino acid/position indicators for individual mutations or combinations thereof. Unfortunately, previously we inadvertently failed to explicitly mention this, and we are most grateful for the Reviewer to point this out.

      We have now rectified this by including upfront the sentence:

      “We will adopt a nomenclature where the complete set of defining mutations of a variant will be referred to by its Greek letter, i.e., N:P13L/R203K/G204R/G214C is N<sub>­­λ</sub>, and analogously the set of Omicron mutations N:P13L/Δ31-33/R203K/G204R are referred to as N<sub>ο</sub>; see Table 1”

      This will define the two shorthands N<sub>λ</sub> and N<sub>ο</sub> used. Furthermore, as suggested and pointed to in the text, Table 1 does provide the keys to mutation and variants, including the information in which variant any of the other mutations studied here occur.

      (7) The EM fibrils (Figure 2A) and CD spectra (Figure 2B) were collected at mM peptide concentrations. These are far above physiological levels and may encourage non-specific aggregation. Similarly, the authors mention" ultra-weak binding energies that require mM concentrations to significantly populate oligomers". On the other hand, the experiments with full-length protein were performed at concentrations closer to biologically relevant concentrations in the micromolar range. While I appreciate the need to work at high concentrations to detect weak interactions, this raises questions about physiological relevance.

      This is indeed an important point to clarify. We agree that much lower nucleocapsid protein concentrations are present in the cytosol on average, and these were used in our RNP assembly experiments. However, there are at least two important physiologically relevant cases where high local N concentrations do occur:

      (1) Once assembled in RNPs, the disordered N-terminal extensions are locally at a very high concentration within the volume they can explore while tethered to the NTD. A back-of-the-envelope calculation assuming 12 N-protein subunits confining 12 N-terminal extensions to the volume of a single RNP (≈14x14x14 nm<sup>3</sup> by cryoEM; Klein et al 2020) leads to an effective concentration of 7.4 mM. Obviously the N-arm peptides are not completely free and there will be constraints that would hinder or promote encounter complex probability, but interfaces with mM Kd are clearly strong enough to populate Narm-Narm contacts extending from N-protein in the RNP.

      Additionally, any interaction where N-proteins are brought in close proximity could allow weak N-arm interactions to provide additional stability. Besides the RNP, we demonstrate this in our Results for nucleic-acid liganded N tetramers (Figure 4B), but this might similarly occur in complexes with NSP3 or host proteins. Generally, it is quite common that small additional binding energies play important roles in the modulation of multivalent protein complexes.

      (2) Within the macromolecular condensate the local concentration will be substantially higher than on average within the infected cell.  While we do not know its precise concentration, it is well-established that the sum of many ultra-weak interactions is driving the formation of this dense liquid phase. In our previous eLife paper (Nguyen et al., 2024) we have shown LLPS is suppressed with the R203K/G204R mutation, but it is ‘rescued’ with the additional P13L/del31-33 mutation of the Omicron variant showing strong LLPS. Similarly, LLPS is suppressed by the LRS mutant L222P, but rescued in conjunction with P13L. This is another biologically relevant scenario where weak interactions are critical.

      We have emphasized these points in the revised manuscript as described below.

      Specifically:

      (a) Could some of the fibril/β-sheet features attributed to P13L (Figure 2A-C) reflect non-specific aggregation at high concentrations rather than bona fide self-association motifs that could play out in biologically relevant scenarios?

      We understand this concern from the experience with proteins that often have limited solubility and tendencies to aggregate, sometimes accompanied by unfolding and driven by hydrophobic interactions, or clustering on the path to LLPS. However, we are struggling to reconcile the picture of non-specific aggregation with the context of our P13L N-arm peptides. The term ‘non-specific aggregation’ implies the idea of amorphous aggregates, which we would contend is inconsistent with the observed geometry of fibrils, which exhibit long-range order. In addition, non-specific aggregation does not lead to increased solution viscosity, which we describe, but fibril formation does. Another connotation of ‘aggregates’ is irreversibility.  However, we find the beta-sheet-like conformation seen at 1 mM becomes significantly more disordered when the same sample is diluted to 0.4 mM peptide. This is consistent with a reversible self-association driven by a conformational change toward ordered secondary structure.

      To highlight the reversibility, we have clarified the description: “Interestingly, diluting the 1 mM sample (solid) to a concentration of 0.4 mM (dashed) reveals a large shift in the far-UV spectra … both indicative of a significant increase of disorder upon dilution. This is consistent with the stabilization of b-sheets in a reversible, strongly cooperative self-association process with an effective K<sub>D</sub> in the high mM to low mM range.”

      We have also inserted a concentration conversion to mg/ml units, which shows even 1 mM of peptides is only ~5 mg/ml, i.e. not excessively high. “While the ancestral N-arm at »1 mM (» 4.6 mg/ml) concentrations exhibits CD spectra with a minimum at »200 nm typical of disordered conformations (black)”

      With regard to the question of specificity, we have studied similar N-arm peptides without P13L mutations and with the 31-33 deletion under equivalent conditions. But we observe the reversible self-association, conformational change, and fibril formation only for those containing the P13L mutation, consistent with ColabFold predictions. Neither did we observe fibrils with disordered C-arm peptides.

      How these weak self-association motifs in the N-arm can be physiologically relevant in the context of full-length protein modulating the stability of multi-molecular complexes and enhancing LLPS was outlined above, and further clarified in the manuscript as detailed below.

      (b) How do the authors justify extrapolating from the mM-range peptide behaviors to the crowded but far lower effective concentrations in cells?

      As pointed out above, the key to this question is the local preconcentration as the N-arm peptides are tethered to the rest of protein in the context of flexible multi-molecular assemblies. Another mechanism to consider is the formation of condensates. The response to the next comment will expand on this.

      The authors should consider adding a dedicated section (either in Methods or Discussion) justifying the use of high concentrations, with estimation of local concentrations in RNPs and how they compare to the in vitro ranges used here. For concentration-dependent phenomena discussed here, it is vital to ensure that the findings are not artefacts of non-physiological peptide aggregation..

      The use of high concentration in biophysical experiments is quite common, for example, in NMR or crystallography, insofar as they elucidate molecular properties. We believe this is obvious; the Reviewer will certainly agree with us, and this does not require further elaboration. The property observed in this case is the existence of specific, weak protein self-association interfaces in the N-arm.

      Our response to the Reviewer’s point 7(a) addresses the distinction between artefactual aggregation and self-association of N-arm peptides. The relevance of these weak protein self-association interfaces in the context of the full-length protein is the second underlying question.

      As we have previously stated in a dedicated Results paragraph:

      “In contrast to the modulation of the coiled-coil LRS interfaces, the de novo creation of the N-arm self-association interface through beta-sheet interactions enabled by P13L cannot be readily observed in full-length N-protein at low M concentrations. Similar to the ancestral LRS interface, it provides only ultra-weak binding energies that require mM concentrations to significantly populate oligomers. This is fully consistent with the previous observation by SV-AUC that neither N:P13L,31-33 nor N<sub>o</sub> with the full set of Omicron mutations show any significant higher-order self-association at low M concentrations, whereas at high local concentrations – as observed in phase-separated droplets – they can modulate and cooperatively enhance self-association processes (Nguyen et al., 2024). (If fact, P13L can substitute for the LRS promoting LLPS, as observed in the rescue of LLPS by N:P13L,31-33/L222P mutants whereas N:L222P LRS-abrogating mutants are deficient in LLPS.) Another process that increases the local concentration of N-arm chains is the tetramerization of full-length N-protein. As described earlier, occupancy of the NA-binding site in the NTD allosterically promotes self-assembly of the LRS into higher oligomers (Zhao et al., 2021). We hypothesized that these oligomers may be cooperatively stabilized by additional N-arm interactions in P13L mutants.”

      To state completely unambiguously why weak interfaces are important, we have followed the Reviewer’s suggestion and added an additional clarification already earlier, at the end of the P13L Results section:

      “While this self-association interface in the P13L N-arm is weak and its direct observation in biophysical experiments requires mM concentrations, which far exceed average intracellular concentration of N, such  weak interactions can become highly relevant physiologically when high local concentrations are prevailing, for example, when the disordered extension is preconcentrated while tethered within macromolecular assemblies as in the RNP, or in macromolecular condensates.”

      Furthermore, we have added early in the Discussion:

      “Even though the solution affinity of the N-arm P13L interface is ultra-weak, the average local concentration of N-arm chains across the RNP volume (in a back-of-the-envelope calculation assuming a ≈14 nm cube (Klein et al., 2020) with a dodecameric N cluster) is ≈7.4 mM, such that disordered N-arm peptides could well create populations of N-arm clusters stabilizing RNPs through this interface.  However, besides the RNP-stabilizing mutants we have also observed unexpected RNP destabilization by the ubiquitous R203K/G204R double mutation, which may be caused by the introduction of additional charges close to the self-association interface in the LRS. In our experiments, this destabilization is more than compensated for by the P13L mutation. (Another scenario where ultra-weak interactions can have a critical impact is in molecular condensates. We previously reported the suppression of LLPS by the R203K/G204R mutation, which is rescued by the additional P13L/Δ31-33 mutation (Nguyen et al., 2024). This is consistent with compensatory weak stabilizing and destabilizing impacts of weak interactions on the RNP observed here.)”

      Reviewer #1 (Recommendations for the Authors):

      In Figure 1B, it is unclear what the orange lines connecting polypeptides represent, as well as the zig-zag orange lines in the N-arm.

      We thank the Reviewer for this comment. We intended this to represent regions of self-association but recognize the patterned background is confusing. We have changed this now to solid-colored backgrounds, and indicated this in the figure legend:

      “Regions of self-association are indicated by shaded backgrounds.”

      Regarding presentation, in Figure 5 (MP), the relationship between mass and oligomer size should be shown more clearly.

      We agree. To this end we have labeled the peaks in the MP histograms in Figure 5 with the oligomeric state of the 2N/2SL7 subunits.

      Reviewer #2 (Recommendations for the Authors):

      I find the science of the paper to be convincing and compellingly supported.

      Thank you for this positive statement.

      My primary complaints are with presentation or minor technical questions that, honestly, primarily arise due to my own ignorance and unfamiliarity with some of the techniques employed.

      My primary issue is with the figures. I find, generally, the text in axes labels, ticks, and legends to be too small to comfortably read. This is particularly true in the CD spectra and

      other data presented in Figures 1D, 2B, 4, 5, 6, and 8.

      We agree and have increased the font size of all text and labels of the plots in Figure 1, 2, 4, 5, 6, and 8.

      I also found the use of initialisms to be a bit overbearing and inconsistent. For example, the authors repeatedly switch between spelling out "nucleic acid" and the initialism "NA" (which is also never explicitly spelled out in the text). With the already substantial length of the text, my own personal opinion would be to suggest spelling out all initialisms in the interest of making the reading easier.

      This is a valid criticism. To improve the readability, we have followed this advice and systematically spelled out “nucleic acid” instead of using “NA”.  Similarly, we have now written out full-length instead of the abbreviation FL, and omitted the abbreviation IDR for intrinsically disordered regions, as well as VOC for variant of concern, and AF3 for AlphaFold.

      Regarding the reference to mutants, we have now explained upfront the system of Latin and Greek nomenclature we consistently applied.

      “We will adopt a nomenclature where the complete set of defining mutations of a variant will be referred to by its Greek letter, i.e., N:P13L/R203K/G204R/G214C is N­­<sub>l</sub>, and analogously the set of Omicron mutations N:P13L/Δ31-33/R203K/G204R are referred to as N<sub>ο</sub>; see Table 1”

      I found the text to be verbose, bordering on overly so; the Introduction is more than two pages long. The section "Enhanced oligomerization of the leucine-rich sequence through cysteine mutations" has two long paragraphs of introduction before the present results are discussed, et cetera. An (admittedly, very rough) estimation of the length of the paper places it at ~9,000 -10,000 words long, and I think that the presentation might benefit from significant editing and

      shortening.

      We agree the manuscript is longer than would be desirable, and we generally prefer not to insert mini-introductions into Results sections. On the other hand, in order to make a solid contribution to understanding the big picture of fuzzy complexes in molecular evolution of RNA virus proteins it is indispensable to go into the details of RNP assembly and several of the interfaces. Therefore, we feel the length is in the range that it needs to be without losing clarity. In addition, other Reviewer suggestions to extend the discussion, for example, of limitations of VLP assays and the in vivo state of cysteines, conflict with significant shortening.

      In the particular case of the cysteine mutations, cited by the Reviewer, we believe it is important to add detailed background on G215C, because the Results proceed in a comparison of the self-association mode between G215C and G214C. This is of significant interest in the present context not only for the independent introduction of interface-enhancing mutations highlighting the evolution of fuzzy complexes, but also because it illustrates the pleomorphic ability of RNPs.

      Nonetheless, we have slightly shortened this text and merged the background into a single paragraph. More generally, we have critically reread the text to remove tangential sentences where possible and to make it more concise.

      I have a few more specific comments.

      In Figure 1A, I suggest explicitly labeling the location of the LRS, as it comes up repeatedly.

      Yes, we thank the Reviewer for this suggestion and have introduced this label in Figure 1A.

      In Figure 1B, the legend indicates that the red lines indicate "new inter-dimer interactions." However, these red lines are overlayed on a vertical stripe of red squiggles; it is unclear to me and not explicitly described in the legend what these squiggles are meant to illustrate.

      We agree this background was confusing. As mentioned in our Response to Reviewer #1 we have replaced the structured background with a solid background and explained in the figure legend that these areas depict regions of self-association.

      On lines 44-45, the authors state, "The IDRs amount to 45%, ..." 45% of what?

      Thank you, this was unclear.  We have now clarified “The IDRs amount to ≈45% of total residues”

      In lines 244 - 246, the authors compare the sizes of complexes in reducing versus non- reducing conditions as measured by dynamic light scattering, stating, "However, dynamic light scattering (DLS) revealed the presence of N210-246:G214C complexes with hydrodynamic radii 244 ranging from 6 to 40 nm (in comparison to 1-2 nm for N210- 246:G215C(Zhao et al., 2022)) in reducing conditions, and slightly larger in non-reducing conditions (Supplementary Figure S4)." Using this single statistic seems to me to be a less-than-ideal way of characterizing what seems to me to be happening here. In Supplementary Figure 4, it appears to me that what is happening is that in non-reduced conditions, the sample is monodisperse, whereas in reducing conditions, the distribution becomes polydisperse/bimodal, with two clearly separate populations. I feel that this could use a more

      thorough description rather than just stating the overall range of particle sizes.

      Yes, the Reviewer is correct – it is indeed a good idea to be more precise here. To this end we have carried out cumulant analyses on the autocorrelation functions, as a time-honored method to quantify the polydispersity.  Both samples are polydisperse, but more so in reducing conditions. We have now added “For N210-246:G214C a cumulant analysis results in radii of 8.8 nm and 10.6 nm and polydispersity indices of 0.40 and 0.35 for reducing and non-reducing conditions, respectively”

      Finally, I have one remaining comment that is a result of my own inexperience with circular dichroism and interpreting the spectra. For me personally, I would appreciate a more thoroughdescription/illustration of the statistics involved in the CD spectra, but perhaps this is not necessary for people who are more familiar with interpreting these kinds of data. For example, in Figure 1D, it is not clear to me what the error bars/confidence intervals for the CD data look like. I see many squiggles, some of which the authors claim are significant (e.g., the differences between ~215 - 230 nm), and others are not worthy of comment. Let's say, for example, that I fit a smoothed spline through these data and then measure the magnitude of the fluctuations from that spline to define/quantify confidence intervals. What does that distribution look like? Or maybe the confidence intervals are so small that all squiggles are significant?

      Thank you, this is a good question. As mentioned in the methods section, the CD spectra shown are averages of triplicate scans. Therefore, it is straightforward to extract the standard deviation at each wavelength from the three measurements (although a spline would probably work just as well). The values are what one would expect for the squiggles to be random noise. In the region 215 – 220 nm characteristic for helical secondary structure the standard deviations are small relative to the separation between curves, which indicates that the differences are highly significant. Naturally, the curves do overlap in other spectral regions, which would make a plot including the wavelength-dependent error bars or confidence bands too crowded. Therefore, we have kept the plot of the averaged triplicate scans, but have now provided the average standard deviations for all species in the figure legend and mentioned their significant separation:

      “Triplicate scans yield average standard deviations of 0.13 (N), 0.17 (N+SL7), 0.16 (N<sub>l</sub>), and 0.21 (N<sub>l</sub> +SL7) 10<sup>3</sup> deg cm<sup>2</sup>/dmol, respectively, with non-overlapping confidence bands for the different species, for example, between 215-220 nm.”

      Reviewer #3 (Recommendations for the Authors):

      (1) The Discussion reiterates much of the background (mutational tolerance, fuzziness, SLiMs) already covered in the Introduction, diluting focus on the key new findings. The authors should consider shortening and refocusing the discussion on the main contributions in light of existing knowledge of viral assembly.

      In the Introduction we have provided background on intrinsically disordered proteins in general and their mutational tolerance, as well as the concept of fuzzy complexes. The first several paragraphs of the Discussion have a different focus, which is protein binding interfaces between viral proteins (obviously key in fuzzy complexes), specifically their modulation and the remarkable de novo introduction of binding interfaces. We believe this deserves emphasis, since this highlights a novel aspect of fuzziness, for the mutant spectrum of RNA viruses to encode a range and of assembly stabilities and architectures. 

      To reduce redundancy between the end of the Introduction and the beginning of the Discussion, we have shortened the last paragraph of the Introduction and removed its preview of the conclusions, as described in the response to the next comment of the Reviewer (see below).

      Unfortunately, the length of the Discussion is dictated in part also by the need to discuss methodological aspects, among them the limitations of VLP assays, and the redox state of the cysteine in the LRS mutants, which were important points recommended by other suggestions of the Reviewers. Similarly, we believe the discussion of other potential functions of Omicron N-arm mutations is warranted, as well as the background of the R203K/G204R double mutation that has attracted significant attention in the field due to its effects on phosphorylation and expression of truncated N species that also form RNPs. Our goal was to integrate the results by us and other laboratories regarding specific mutation effects into a comprehensive picture of molecular evolution of N, which we believe the framework of fuzzy complexes can provide.

      (2) The Abstract and early Introduction set a broad stage (IDPs, fuzziness), but don't explicitly state the concrete hypotheses that the experiments test. Please add 2-3 sentences in the Introduction that enumerate testable hypotheses, e.g.:

      (a) P13L creates a new N-arm interface that increases RNP stability.

      (b) G214C/G215C strengthens LRS oligomerization to stabilize higher-order N assemblies.

      We agree the introduction can be improved.  However, it seems to us that it cannot be neatly framed in the hypothesis – answer dichotomy, without losing a lot of nuances and without requiring an even longer and more detailed introduction.

      One of the main questions is to test whether the framework of fuzzy complexes can be applied to understand molecular evolution of N, and we feel the introduction is already flowing well towards this:

      “ … In fuzzy complexes the total binding energy is distributed into multiple distinct ultra-weak interaction sites (Olsen et al., 2017). Similar to individual RNA virus proteins with loose or absent structure, maintaining disorder and a spatial distribution of low-energy interactions in the protein complexes may increase the tolerance for mutations and improve evolvability of protein complexes.\

      The unprecedented worldwide sequencing effort of SARS-CoV-2 genomes during its rapid evolution in humans provides a unique opportunity to examine these concepts. ...”

      To bring this to a more concrete set of questions in the end, we have shortened and rewritten the last paragraph in the Introduction:

      “To examine how architecture and energetics of RNP assemblies can be impacted by N-protein mutations we study a panel of N-proteins derived from ancestral Wuhan-Hu-1 and different VOCs, including Alpha, Delta, Lambda, and Omicron (see Table 1), in biophysical experiments, VLP assays, and mutant virus. Specifically, we ask how the RNP size distribution and life-time is modulated by: (1) the novel binding interface created by the P13L mutation of Omicron; (2) enhancements of other weak self-association interfaces through G215C of Delta and G214C of Lambda; (3) the ubiquitous R203K/G204R double mutation of Alpha, Lambda, and Omicron.  We also test whether the P13L mutation improves viral fitness, similar to G215C and R203K/G204R. The results are discussed in the framework of fuzzy complexes and molecular evolution of N in the course of viral adaptation to the human host. Understanding the salient features of the binding interfaces in viral assembly and their evolution expands our foundation for the design of therapeutics such as assembly inhibitors.”

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    Mitochondrial Protein Carboxyl-Terminal Alanine-Threonine Tailing Promotes Glioblastoma Tumor Growth by Regulating Mitochondrial Function
    4
    1. Public_Reviews 10 Dec 2025

      eLife Assessment

      Glioblastoma is among the most aggressive cancers without a cure, and its cells are characterized by high mitochondrial membrane potential. This manuscript provides convincing evidence that glioblastoma tumorigenesis is closely linked to mitochondrial stress. The study makes a valuable contribution to the field by advancing our understanding of the metabolic mechanisms driving glioblastoma and highlighting potential therapeutic targets.

      Summary
    2. Public_Reviews 10 Dec 2025

      Reviewer #1 (Public review):

      Summary:

      Cai et al have investigated the role of msiCAT-tailed mitochondrial proteins that frequently exist in glioblastoma stem cells. Overexpression of msiCAT-tailed mitochondrial ATP synthase F1 subunit alpha (ATP5) protein increases the mitochondrial membrane potential and blocks mitochondrial permeability transition pore formation/opening. These changes in mitochondrial properties provide resistance to staurosporine (STS)-induced apoptosis in GBM cells. Therefore, msiCAT-tailing can promote cell survival and migration, while genetic and pharmacological inhibition of msiCAT-tailing can prevent the overgrowth of GBM cells.

      Strengths:

      The CATailing concept has not been explored in cancer settings. Therefore, the present provides new insights for widening the therapeutic avenue.

      Review 1
    3. Public_Reviews 10 Dec 2025

      Reviewer #2 (Public Review):

      This work explores the connection between glioblastoma, mito-RQC, and msiCAT-tailing. They build upon previous work concluding that ATP5alpha is CAT-tailed and explore how CAT-tailing may affect cell physiology and sensitivity to chemotherapy. The authors conclude that when ATP5alpha is CAT-tailed, it either incorporates into the proton pump or aggregates and that these events dysregulate MPTP opening and mitochondrial membrane potential and that this regulates drug sensitivity. This work includes several intriguing and novel observations connecting cell physiology, RQC, and drug sensitivity. This is also the first time this reviewer has seen an investigation of how a CAT tail may specifically affect the function of a protein.

      Comment from the Reviewing Editor:

      The revisions made the work more valuable and convincing. The authors adequately made point-by-point response to the reviewers comments by providing new data. Image acquisition and data analysis were further clarified. NEMF knockdown experiments and additional control data for ATP5α featuring a poly-glycine-serine (GS) tail support their conclusion.

      Review 2
    4. Public_Reviews 10 Dec 2025

      Author response:

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

      eLife Assessment:

      Glioblastoma is one of the most aggressive cancers without a cure. Glioblastoma cells are known to have high mitochondrial potential. This useful study demonstrates the critical role of the ribosome-associated quality control (RQC) pathway in regulating mitochondrial membrane potential and glioblastoma growth. Some assays are incomplete; further revision will improve the significance of this study.

      For clarity, we propose revising the second sentence to: "It is well-established that certain cancer cells, such as glioblastoma cells, exhibit elevated mitochondrial membrane potential."

      Reviewer #1 (Public Review):

      Summary:

      Cai et al have investigated the role of msiCAT-tailed mitochondrial proteins that frequently exist in glioblastoma stem cells. Overexpression of msiCAT-tailed mitochondrial ATP synthase F1 subunit alpha (ATP5) protein increases the mitochondrial membrane potential and blocks mitochondrial permeability transition pore formation/opening. These changes in mitochondrial properties provide resistance to staurosporine (STS)-induced apoptosis in GBM cells. Therefore, msiCAT-tailing can promote cell survival and migration, while genetic and pharmacological inhibition of msiCAT-tailing can prevent the overgrowth of GBM cells.

      Strengths:

      The CAT-tailing concept has not been explored in cancer settings. Therefore, the present provides new insights for widening the therapeutic avenue. 

      Your acknowledgment of our study's pioneering elements is greatly appreciated.

      Weaknesses:

      Although the paper does have strengths in principle, the weaknesses of the paper are that these strengths are not directly demonstrated. The conclusions of this paper are mostly well-supported by data, but some aspects of image acquisition and data analysis need to be clarified and extended.

      We are grateful for your acknowledgment of our study’s innovative approach and its possible influence on cancer therapy. We sincerely appreciate your valuable feedback. In response, this updated manuscript presents substantial new findings that reinforce our central argument. Moreover, we have broadened our data analysis and interpretation, as well as refined our methodological descriptions.

      Reviewer #2 (Public Review):

      This work explores the connection between glioblastoma, mito-RQC, and msiCAT-tailing. They build upon previous work concluding that ATP5alpha is CAT-tailed and explore how CAT-tailing may affect cell physiology and sensitivity to chemotherapy. The authors conclude that when ATP5alpha is CAT-tailed, it either incorporates into the proton pump or aggregates and that these events dysregulate MPTP opening and mitochondrial membrane potential and that this regulates drug sensitivity. This work includes several intriguing and novel observations connecting cell physiology, RQC, and drug sensitivity. This is also the first time this reviewer has seen an investigation of how a CAT tail may specifically affect the function of a protein. However, some of the conclusions in this work are not well supported. This significantly weakens the work but can be addressed through further experiments or by weakening the text.

      We appreciate the recognition of our study's novelty. To address your concerns about our conclusions, we have revised the manuscript. This revision includes new data and corrections of identified issues. Our detailed responses to your specific points are outlined below.

      Reviewer #1 (Recommendations For The Authors):

      (1) In Figure 1B, please replace the high-exposure blots of ATP5 and COX with representative results. The current results are difficult to interpret clearly. Additionally, it would be helpful if the author could explain the nature of the two different bands in NEMF and ANKZF1. Did the authors also examine other RQC factors and mitochondrial ETC proteins? I'm also curious to understand why CAT-tailing is specific to C-I30, ATP5, and COX-V, and why the authors did not show the significance of COX-V.

      We appreciate your inquiry regarding the data.  Additional attempts were made using new patient-derived samples; however, these results did not improve upon the existing ATP5⍺, (NDUS3)C-I30, and COX4 signals presented in the figure.  This is possibly due to the fact that CAT-tail modified mitochondrial proteins represent only a small fraction of the total proteins in these cells.  It is acknowledged that the small tails visible above the prominent main bands are not particularly distinct. To address this, the revised version includes updated images to better illustrate the differences. We believe the assertion that GBM/GSCs possess CAT-tailed proteins is substantiated by a combination of subsequent experimental findings. The figure (refer to new Fig. 1B) serves primarily as an introduction. It is important to note that the CAT-tailed ATP5⍺ plays a vital role in modulating mitochondrial potential and glioma phenotypes, a function which has been demonstrated through subsequent experiments.

      It is acknowledged that the CAT-tail modification is not exclusive to the ATP5⍺protein.  ATP5⍺ was selected as the primary focus of this study due to its prevalence in mitochondria and its specific involvement in cancer development, as noted by Chang YW et al.  Future research will explore the possibility of CAT tails on other mitochondrial ETC proteins. Currently, NDUS3 (C-I30), ATP5⍺, and COX4 serve as examples confirming the existence of these modifications. It remains challenging to detect endogenous CAT-tailing, and bulk proteomics is not yet feasible for this purpose. COX4 is considered significant.  We hypothesize that CAT-tailed COX4 may function similarly to the previously studied C-I30 (Wu Z, et al), potentially causing substantial mitochondrial proteostasis stress.  

      Concerning RQC proteins, our blotting analysis of GBM cell lines now includes additional RQC-related factors. The primary, more prominent bands (indicated by arrowheads) are, in our assessment, the intended bands for NEMF and ANKZF1.  Subsequent blotting analyses showed only single bands for both ANKZF1 and NEMF, respectively. The additional, larger molecular weight band of NEMF, which was initially considered for property analysis (phosphorylation, ubiquitination, etc.), was not examined further as it did not appear in subsequent experiments (refer to new Fig. S1C).

      References:

      Chang YW, et al. Spatial and temporal dynamics of ATP synthase from mitochondria toward the cell surface. Communications biology. 2023;6(1).

      Wu Z, et al. MISTERMINATE Mechanistically Links Mitochondrial Dysfunction With Proteostasis Failure. Molecular cell. 2019;75(4).

      (2) In addition to Figure 1B, it would be interesting to explore CAT-tailed mETC proteins in cancer tissue samples.

      This is an excellent point, and we appreciate the question. We conducted staining for ATP5⍺ and key RQC proteins in both tumor and normal mouse tissues. Notably, ATP5⍺ in GBM exhibited a greater tendency to form clustered punctate patterns compared to normal brain tissue, and not all of it co-localized with the mitochondrial marker TOM20 (refer to new Fig. S3C-E). Crucially, we observed a significant increase in NEMF expression within mouse xenograft tumor tissues, alongside a decrease in ANKZF1 expression (refer to new Fig. S1A, B). These findings align with our observations in human samples.

      (3) Please knock down ATP5 in the patient's cells and check whether both the upper band and lower band of ATP5 have disappeared or not.

      This control was essential and has been executed now. To validate the antibody's specificity, siRNA knockdown was performed. The simultaneous elimination of both upper and lower bands upon siRNA treatment (refer to new Fig. S2A) confirms they represent genuine signals recognized by the antibody.

      (4) In Figure 1C and ID, add long exposure to spot aggregation and oligomer. Figure 1D, please add the blots where control and ATP5 are also shown in NHA and SF (similar to SVG and GSC827).

      New data are included in the revised manuscript to address the queries. Specifically, the new Fig 1D now displays the full queue as requested, featuring blots for Control, ATP5α, AT3, and AT20. Our analysis reveals that AT20 aggregates exhibit higher expression and accumulation rates in GSC and SF cells.

      Fig. 1C has been updated to include experimental groups treated with cycloheximide and sgNEMF. Our results show that sgNEMF effectively inhibits CAT-tailing in GBM cell lines, whereas cycloheximide has no impact. After consulting with the Reporter's original creator and optimizing expression conditions, we observed no significant aggregates with β-globin-non-stop protein, potentially due to the length of endogenous CAT-tail formation (as noted by Inada, 2020, in Cell Reports). Our analysis focused on the ratio of CAT-tailed (red box blots) and non-CAT-tailed proteins (green box blots). Comparing these ratios revealed that both anisomycin treatment and sgNEMF effectively hinder the CAT-tailing process, while cycloheximide has no effect.

      (5) In Figure 1E, please double-check the results with the figure legend. ATP5A aggregated should be shown endogenously. The number of aggregates shown in the bar graph is not represented in micrographs. Please replace the images. For Figure 1E, to confirm the ATP5-specific aggregates, it would be better if the authors would show endogenous immunostaining of C-130 and Cox-IV.

      Labels in Fig. 1E were corrected to reflect that the bar graph in Fig. 1F indicates the number of cells with aggregates, not the quantity of aggregates per cell. The presence

      (6) Figure 3A. Please add representative images in the anisomycin sections. It is difficult to address the difference.

      We appreciate your feedback. Upon re-examining the Calcein fluorescence intensity data in Fig. 3A, we believe the images accurately represent the statistical variations presented in Fig. 3B. To address your concerns more effectively, please specify which signals in Fig. 3A you find potentially misleading. We are prepared to revise or substitute those images accordingly.

      (7) Figure 3D. If NEMF is overexpressed, is the CAT-tailing of ATP 5 reversed?

      Thank you. Your prediction aligns with our findings. We've added data to the revised Fig. S6A, B, which demonstrates that both NEMF overexpression and ANKZF1 knockdown lead to elevated levels of CRC. This increase, however, was not statistically significant in GSC cells. A plausible explanation for this discrepancy is that the MPTP of GSC cells is already closed, thus any additional increase in CAT-tailing activity does not result in further amplification.

      (8) Figure 3G. Why on the BN page are AT20 aggregates not the same as shown in Figure 2E?

      We appreciate your inquiry regarding the ATP5⍺ blots, specifically those in the original Fig. 3G (left) and 2E (right). Careful observation of the ATP5⍺ band placement in these figures reveals a high degree of similarity. Notably, there are aggregates present at the top, and the diffuse signals extend downwards. Given that this is a gradient polyacrylamide native PAGE, the concentration diminishes towards the top. Consequently, the non-rigid nature of the Blue Native PAGE gel may lead to slight variations in the aggregate signals; however, the overall patterns are very much alike. To mitigate potential misinterpretations, we have rearranged the blot order in the new Fig. 3M.

      (9) Figure 4D. The amount of aggregation mediated by AT20 is more compared to AT3. Why are there no such drastic effects observed between AT3 and AT20 in the Tunnel assay?

      The previous Figure 4D presents the quantification of cell migration from the experiment depicted in Figure 4C. But this is a good point. TUNEL staining results are directly influenced by mitochondrial membrane potential and the state of mitochondrial permeability transition pores

      (MPTP), not by the degree of protein aggregation. Our previous experiments showed comparable effects of AT3 and AT20 on mitochondria (Fig. 2E, 3K), which aligns with the expected similar outcomes on TUNEL staining. As for its biological nature, this could be very complicated. We hope to explore it in future studies.

      (10) Figure 5C: The role of NEMF and ANKZF1 can be further clarified by conducting Annexin-PI assays using FACS. The inclusion of these additional data points will provide more robust evidence for CAT-tailing's role in cancer cells.

      In response to your suggestion, we have incorporated additional data into the revised version.Using the Annexin-PI kit, we labeled apoptotic cells and detected them using flow cytometry (FACS). Our findings indicate that anisomycin pretreatment, NEMF knockdown (sgNEMF), and ANZKF1 upregulation (oeANKZF1) significantly increase the rate of STS-induced apoptosis compared to the control group (refer to new Fig. S9D-G).

      (11) Figure 5F: STS is a known apoptosis inhibitor. Why it is not showing PARP cleavage? Also, cell death analysis would be more pronounced, if it could be shown at a later time point. What is the STS and Anisomycin at 24h or 48h time-point? Since PARP is cleaved, it would also be better if the authors could include caspase blots.

      I guess what you meant to say here is "Staurosporine is a protein kinase inhibitor that can induce apoptosis in multiple mammalian cell lines." Our study observed PARP cleavage even in GSCs, which are typically more resistant to staurosporine-induced apoptosis (C-PARP in Fig. S9B). The ratio of C-PARP to total PARP increased. We selected a 180-minute treatment duration because longer treatments with STS + anisomycin led to a late stage of apoptosis and non-specific protein degradation (e.g., at 24 or 48 hours), making PARP comparisons less meaningful. Following your suggestion, we also examined caspase 3/7 activity in GSC cells treated with DMSO, CHX, and anisomycin. We found that anisomycin treatment also activated caspases (Fig. S9A).

      (12) In Figure 5, the addition of an explanation, how CAT-tailing can induce cell death, would add more information such as BAX-BCL2 ratio, and cytochrome-c release from the mitochondria.

      Thank you for your suggestion. In this study, we state that specific CAT-tails inhibit GSC cell death/apoptosis rather than inducing it. Therefore, we do not expect that examining BAX-BCL2 and mitochondrial cytochrome c release would offer additional insights.

      (13) To confirm the STS resistance, it would be better if the author could do the experiments in the STS-resistant cell line and then perform the Anisomycin experiments.

      Thank you. We should emphasize that our data primarily originates from GSC cells. These cells already exhibit STS-resistance when compared to the control cells (Fig. S8A-C).

      (14) It would be more advantageous if the author could show ATP5 CATailed status under standard chemotherapy conditions in either cell lines or in vivo conditions.

      This is an interesting question. It's worth exploring this question; however, GSC cells exhibit strong resistance to standard chemotherapy treatments like temozolomide (TMZ).

      Additionally, we couldn't detect changes in CAT-tailed ATP5⍺ and thus did not include that data.

      (15) In vivo (cancer mouse model or cancer fly model) data will add more weight to the story.

      We appreciate your intriguing question. An effective approach would be to test the RQC pathway's function using the Drosophila Notch overexpression-induced brain tumor model. However, Khaket et al. have conducted similar studies, stating, "The RNAi of Clbn, VCP, and Listerin (Ltn), homologs of key components of the yeast RQC machinery, all attenuated NSC over-proliferation induced by Notch OE (Figs. 5A and S5A–D, G)." This data supports our theory, and we have incorporated it into the Discussion. While the mouse model more closely resembles the clinical setting, it is not covered by our current IACUC proposal. We intend to verify this hypothesis in a future study.

      Reference:

      Khaket TP, Rimal S, Wang X, Bhurtel S, Wu YC, Lu B. Ribosome stalling during c-myc translation presents actionable cancer cell vulnerability. PNAS Nexus. 2024 Aug 13;3(8):pgae321.

      Reviewer #2 (Recommendations For The Authors):

      Figure 1B, C: To demonstrate that Globin, ATP5alpha, and C-130 are CAT-tailed, it is necessary to show that the high mobility band disappears after NEMF deletion or mutagenesis of the NFACT domain of NEMF. This can be done in a cell line. The anisomycin experiment is not convincing because the intensity of the bands drops and because no control is done to show that the effects are not due to translation inhibition (e.g. cycloheximide, which inhibits translation but not CAT tailing). Establishing ATP5alpha as a bonafide RQC substrate and CAT-tailed protein is critical to the relevance of the rest of the paper.

      Thank you for suggesting this crucial control experiment. To confirm the observed signal is indeed a bona fide CAT-tail, it's essential to demonstrate that NEMF is necessary for the CAT-tailing process. We have incorporated data from NEMF knockdown (sgNEMF) and cycloheximide treatment into the revised manuscript. Our findings show that both sgNEMF and anisomycin treatment effectively inhibit the formation of CAT-tailing signals on the reporter protein (Fig. 1C). Similarly, NEMF knockdown in a GSC cell line also effectively eliminated CAT-tails on overexpressed ATP5⍺ (Fig. S2B).

      In general, the text should be weakened to reflect that conclusions were largely gleaned from artificial CAT tails made of AT repeats rather than endogenously CAT-tailed ATP5alpha. CAT tails could have other sequences or be made of pure alanine, as has been suggested by some studies.

      Thank you for your reminder. We have reviewed the recent studies by Khan et al. and Chang et al., and we found their analysis of CAT tail components to be highly insightful. We concur with your suggestion regarding the design of the CAT tail sequence. We aimed to design a tail that maintained stability and resisted rapid degradation, regardless of its length. In the revised version, we clarify that our conclusions are based on artificial CAT tails, specifically those composed of AT repeat sequences (p. 9). We acknowledge that the presence of other sequence components may lead to different outcomes (p. 19).

      Reference:

      Khan D, Vinayak AA, Sitron CS, Brandman O. Mechanochemical forces regulate the composition and fate of stalled nascent chains. bioRxiv [Preprint]. 2024 Oct 14:2024.08.02.606406. Chang WD, Yoon MJ, Yeo KH, Choe YJ. Threonine-rich carboxyl-terminal extension drives aggregation of stalled polypeptides. Mol Cell. 2024 Nov 21;84(22):4334-4349.e7. 

      Throughout the work (e.g. 3B, C), anisomycin effects should be compared to those with cycloheximide to observe if the effects are specific to a CAT tail inhibitor rather than a translation inhibitor.

      We agree that including cycloheximide control experiments is crucial. The revised version now incorporates new data, as depicted in Fig. S5A, B, illustrating alterations in the on/off state of MPTP following cycloheximide treatment. Furthermore, Fig. S6A, B present changes in Calcium Retention Capacity (CRC) under cycloheximide treatment. The consistency of results across these experiments, despite cycloheximide treatment, suggests that anisomycin's role is specifically as a CAT tail inhibitor, rather than a translation inhibitor.

      Line 110, it is unclear what "short-tailed ATP5" is. Do you mean ATP5alpha-AT3? If so this needs to be introduced properly. Line 132: should say "may indicate accumulation of CAT-tailed protein" rather than "imply".

      We acknowledge your points. We have clarified that the "short-tailed ATP5α" refers to ATP5α-AT3 and incorporated the requested changes into the revised manuscript.

      Figure 1C: how big are those potential CAT-tails (need to be verified as mentioned earlier)?They look gigantic. Include a ladder.

      In the revised Fig. 1D, molecular weight markers have been included to denote signal sizes. The aggregates in the previous Fig. 1C, also present in the control plasmid, are likely a result of signal overexposure. The CAT-tailed protein is observed just above the intended band in these blots. These aggregates have been re-presented in the updated figures, and their signal intensities quantified.

      Line 170: "indicating that GBM cells have more capability to deal with protein aggregation". This logic is unclear. Please explain.

      We appreciate your question and have thoroughly re-evaluated our conclusion. We offer several potential explanations for the data presented in Fig. 1D: (1) ATP5α-AT20 may demonstrate superior stability. (2) GSC (GBM) cells might lack adequate mechanisms to monitor protein accumulation. (3) GSC (GBM) cells could possess an increased adaptive capacity to the toxicity arising from protein accumulation. This discussion has been incorporated into the revised manuscript (lines 166-169).

      Line 177: how do you know the endogenous ATP5alpha forms aggregates due to CAT-tailing? Need to measure in a NEMF hypomorph.

      We understand your concern and have addressed it. Revised Fig. 3G, H demonstrates that a reduction in NEMF levels, achieved through sgNEMF in GSC cells, significantly diminishes ATP5α aggregation. This, in conjunction with the Anisomycin treatment data presented in revised Fig. 3E, F, confirms the substantial impact of the CAT-tailing process on this aggregation.

      Line 218: really need a cycloheximide or NEMF hypomorph control to show this specific to CAT-tailing.

      We have revised the manuscript to include data from sgNEMF and cycloheximide treatments, specifically Fig. 3G, H, and Fig. S5C, D, as detailed in our response above.

      Lines 249,266, Figure 5A: The mentioned experiments would benefit from controls including an extension of ATP5alpha that was not alanine and threonine, perhaps a gly-ser linker, as well as an NEMF hypomorph.

      We sincerely appreciate your insightful comments. In response, the revised manuscript now incorporates control data for ATP5α featuring a poly-glycine-serine (GS) tail. This data is specifically presented in Figs. S2E-G, S4E, S7A, D, E, and S8F, G. Our experimental findings consistently demonstrate that the overexpression of ATP5α, when modified with GS tails, had no discernible impact on protein aggregation, mitochondrial membrane potential, GSC cell mobility, or any other indicators assessed in our study.

      Figure S5A should be part of the main figures and not in the supplement.

      This has been moved to the main figure (Fig. 5C).

      AuthorResponse
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    biorxiv.org/content/10.1101/2024.05.15.594447v4
  14. www.compactmag.com www.compactmag.com
    The Great Feminization
    32
    1. dentropy 10 Dec 2025
      Make it legal to have a masculine office culture again.

      This requires an Adult type character in the room, sad thing is we don't have that. There are no "Adult" gen Z characters. I haven't even seen any Millenial "Adults".

      When I say Adult imagine a competent school principal that everyone respects and listens to. The kids are sort of scared from him but they know they can talk to him in a friendly way. When there is a dispute he is the arbitrator that people trust.

      There is no "Arbitrator people trust" in organizations any more. There is just HR.

    2. dentropy 10 Dec 2025
      Let’s make hiring meritocratic in substance and not just name, and we will see how it shakes out.

      There's also a race problem here, not just gender. The Indian in group preference is very noticeable.

    3. dentropy 10 Dec 2025

      Quote from the Article

      Many people think wokeness is over, slain by the vibe shift, but if wokeness is the result of demographic feminization, then it will never be over as long as the demographics remain unchanged.

    4. dentropy 10 Dec 2025
      Many people think wokeness is over, slain by the vibe shift, but if wokeness is the result of demographic feminization, then it will never be over as long as the demographics remain unchanged.

      Most important quote of the article

    5. dentropy 10 Dec 2025
      What man wants to work in a field where his traits are not welcome? What self-respecting male graduate student would pursue a career in academia when his peers will ostracize him for stating his disagreements too bluntly or espousing a controversial opinion?
      Questions
    7. dentropy 10 Dec 2025
      Feminization is not an organic result of women outcompeting men. It is an artificial result of social engineering, and if we take our thumb off the scale it will collapse within a generation.

      Wow that's a strong statement

    8. dentropy 10 Dec 2025
      Ross Douthat described this line of thinking in an interview this year with Jonathan Keeperman, a.k.a. “L0m3z,” a right-wing publisher who helped popularize the term “the longhouse”

      Nice to see "The Longhouse" mentioned in here

      Definition
    11. dentropy 10 Dec 2025
      Lithwick lauds women for their irreverent attitude to the law’s formalities, which, after all, originated in an era of oppression and white supremacy. “The American legal system was fundamentally a machine built to privilege propertied white men,” Lithwick writes. “But it’s the only thing going, and you work with what you have.” Those who view the law as a patriarchal relic can be expected to treat it instrumentally. If that ethos comes to prevail throughout our legal system, then the trappings will look the same, but a revolution will have occurred.

      The Lawyers, and the way law is interpreted, of the 2050's are going to be very different from the 1950's

    12. dentropy 10 Dec 2025
      But they lacked many of the safeguards that our legal system holds sacred, such as the right to confront your accuser, the right to know what crime you are accused of, and the fundamental concept that guilt should depend on objective circumstances knowable by both parties, not in how one party feels about an act in retrospect. These protections were abolished because the people who made these rules sympathized with the accusers, who were mostly women, and not with the accused, who were mostly men.

      I feel like this would be used to bully people, like if "Mean Girls" politics resonates with real life this is a dangerous president.

    13. dentropy 10 Dec 2025
      The field that frightens me most is the law. All of us depend on a functioning legal system, and, to be blunt, the rule of law will not survive the legal profession becoming majority female.

      Wow there buddy, I may need to come back and read this later, that's an intense statement

      Is there are presidence for this?

    14. dentropy 10 Dec 2025
      The most relevant differences are not about individuals but about groups. In my experience, individuals are unique and you come across outliers who defy stereotypes every day, but groups of men and women display consistent differences. Which makes sense, if you think about it statistically. A random woman might be taller than a random man, but a group of ten random women is very unlikely to have an average height greater than that of a group of ten men. The larger the group of people, the more likely it is to conform to statistical averages

      There is a meme for this I saw on Twitter, "But not all X are Y"

    15. dentropy 10 Dec 2025
      but you live in a country where what gets written in The New York Times determines what is publicly accepted as the truth. If the Times becomes a place where in-group consensus can suppress unpopular facts (more so than it already does), that affects every citizen.

      A knowledge garden of contested NYT facts and biases would be interesting, I wonder if an AI could do it

    17. dentropy 10 Dec 2025
      That is because women’s conflicts were traditionally within the tribe over scarce resources, to be resolved not by open conflict but by covert competition with rivals, with no clear terminus.

      Possibly like fighting over a mate

    20. dentropy 10 Dec 2025
      Female group dynamics favor consensus and cooperation. Men order each other around, but women can only suggest and persuade. Any criticism or negative sentiment, if it absolutely must be expressed, needs to be buried in layers of compliments. The outcome of a discussion is less important than the fact that a discussion was held and everyone participated in it. The most important sex difference in group dynamics is attitude to conflict. In short, men wage conflict openly while women covertly undermine or ostracize their enemies.

      This is very well articulated, I think about this all the time but this really get's to the point and makes it clear

    21. dentropy 10 Dec 2025
      71 percent of men said protecting free speech was more important than preserving a cohesive society, and 59 percent of women said the opposite.

      I would like to know more about the attributes of this 29% of men verses the 71% of men. Do they go to the gym? What do they eat? What was their father like growing up?

    22. dentropy 10 Dec 2025
      survey data showing sex differences in political values.

      The Political Parties are Gender Sex Based now,

      I like the idea of a Man get's a vote, if he get's married he get's two, and if he has over 3+ children then has three votes. Get divorced, only gets one now

    24. dentropy 10 Dec 2025
      Possibly because, like most people, I think of feminization as something that happened in the past before I was born. When we think about women in the legal profession, for example, we think of the first woman to attend law school (1869), the first woman to argue a case before the Supreme Court (1880), or the first female Supreme Court Justice (1981).

      Those are some good dates to remember

    25. dentropy 10 Dec 2025
      Wokeness is not a new ideology, an outgrowth of Marxism, or a result of post-Obama disillusionment. It is simply feminine patterns of behavior applied to institutions where women were few in number until recently. How did I not see it before?
      Based
    26. dentropy 10 Dec 2025
      “wokeness” is simply an epiphenomenon of demographic feminization.

      Okay let me process this,

      So if women attain positions of power, then power starts to operate in a feminized way

      Wokeness is just the byproduct of people wielding Feminized Power

      Ah gotcha, makes sense now

    28. dentropy 10 Dec 2025
      Experts chimed in to declare that everything Summers had said about sex differences was within the scientific mainstream. These rational appeals had no effect on the mob hysteria.

      Reminds me of the theme in Wicked: For Good where "The Wizard" of says if Alphaba tells the people of Oz the truth they will not believe her because they will not want to

    29. dentropy 10 Dec 2025
      “When he started talking about innate differences in aptitude between men and women, I just couldn’t breathe because this kind of bias makes me physically ill,”

      Ideology producing a physiological response is fascinating

    31. dentropy 10 Dec 2025
      “Diversifying the Science and Engineering Workforce,” Larry Summers gave a talk that was supposed to be off the record. In it, he said that female underrepresentation in hard sciences was partly due to “different availability of aptitude at the high end” as well as taste differences between men and women “not attributable to socialization.”

      Google's Ideological Echo Chamber - Wikipedia

    32. dentropy 10 Dec 2025
      The entire “woke” era could be extrapolated from that moment, from the details of how Summers was cancelled and, most of all, who did the cancelling: women.

      The gender dynamic in "Woke", "Leftist" culture is facinating. Like what does leadership look like in those communities, how much mob mentality is there, how does one attain power (Social Reputation + Audience) in those communities

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  15. www.biorxiv.org www.biorxiv.org
    Decision-making components and times revealed by the single-trial electro-encephalogram
    5
    1. Public_Reviews 10 Dec 2025

      eLife Assessment

      Weindel et al examine behavioural and EEG data in an innovative contrast comparison paradigm where they vary mean contrast widely while keeping contrast difference constant. As intended, this allowed an elegant decomposition of processing stages: while sensory encoding shortened with increasing contrast in keeping with Pieron's law, the period of decision formation lengthened, in keeping with Fechner's law, which was applied to drift rates in a diffusion model of that period. This is an important demonstration of how these two laws apply in concert, to two distinct processing levels, and the multivariate topography parsing, mixed effect models and diffusion models are convincing.

      Summary
    2. Public_Reviews 10 Dec 2025

      Reviewer #1 (Public review):

      This study uses a new 'hidden multivariate pattern method' to parse in time and space the neural events intervening between stimulus and response in an immediately-reported perceptual decision, and use the resultant neural event timing information to show quite convincingly that Pieron's and Fechner's laws can apply in concert at distinct processing levels.

      They designed a clever contrast comparison paradigm in which the contrast difference is kept constant while widely manipulating mean contrast, so that sensory encoding of the overall stimulus would be boosted with increasing mean contrast, whereas decision difficulty and hence duration would increase. With this, they found that the time intervening between early sensory-evoked components, up to an 'N200'-type component associated with launching the decision process, varies inversely with contrast according to Pieron's law. Meanwhile, the time intervals running up to neural events peaking near the time of response, consistent with decision termination, increases with contrast, fitting Fechner's law. Further, a diffusion model whose drift rates are scaled by Fechner's law, fit to RT, predicts the observed proportion of correct responses very well.

      In the process of review and revision it was highlighted that presumably the full sequence of neural events intervening between stimulus and response is massively task dependent, but;

      (1) The method is intended to capture all key components that specifically covary with RT, as opposed to each and every component in general, and

      (2) The main conclusions of the study mentioned above do not change whether the method is set up to track three neural events, or five, as was done in the final analysis.

      The propensity for topographic parsing algorithms to potentially lump-together distinct processes that partially co-evolve was acknowledged, but a key clarification in review was that even though the method entails a specification of neural event duration - which was changed from 50 to 25 ms - the success of the method is not strongly contingent on the actual underlying neural events in question having that very duration - indeed, the components extracted using that short template duration can be observed to evolve over a longer time frame associated with the Fechner diffusion process.

      Notably, standard average event-related potential analysis was able to show expected amplitude effects - where sensory signals increased with contrast but decision signals decreased - but assessment of the by-trial distribution of their timings was grealy aided by the HMP method.

      One of the stages of processing implicated in the parsing analysis was linked to attention orientation, and the authors speculate on whether this might reflect a spatially-selective deployment of attention or a resource allocation, but sensibly refrain from speculating too far since the focus here was on the sensory and decision process durations and their respective adherence to Pieron and Fechner's laws.

      Review 1
    3. Public_Reviews 10 Dec 2025

      Reviewer #2 (Public review):

      Summary:

      The authors decomposed response times into component processes and manipulated the duration of these processes in opposing directions by varying contrast, and overall by manipulating speed-accuracy tradeoffs. They identify different processes and their durations by identifying neural states in time and validate their functional significance by showing that their properties vary selectively as expected with predicted effects of the contrast manipulation. They identify 4 processes: stimulus encoding, attention orienting, decision and motor execution. These map onto 5 classical event related potentials. The decision-making component matched the CPP and its properties varied with contrast and predicted decision-accuracy.

      Strengths:

      The design of the experiment is remarkable and offers crucial insights. The analyses techniques are beyond-state-of-the art and the analyses are well motivated and offer clear insights.

      Weaknesses:

      The number of identified events depends on the parameter setting of the analysis. While the authors discuss weaknesses of the approach this needs to be made explicit as well. It is also unclear to what extent topographies map onto processes since e.g., different combinations of sources can lead to the same scalp topography.

      Review 2
    4. Public_Reviews 10 Dec 2025

      Reviewer #3 (Public review):

      Summary:

      In this manuscript the authors examine the processing stages involved in perceptual decision-making using a new approach to analysing EEG data, combined with a critical stimulus manipulation. This new EEG analysis method enables single-trial estimates of the timing and amplitude of transient changes in EEG time-series recurrent across trials in a behavioural task. The authors find evidence for five events between stimulus onset and the response in a two-spatial-interval visual discrimination task. By analysing the timing and amplitude of these events in relation to behaviour and the stimulus manipulation, the authors interpret these events as related to separable processing stages for stimulus encoding (first two events), attention orientation (second event), motor planning (fourth event) and decision (deliberation, final event). This is largely consistent with previous findings from both event-related potentials (across trials) and single-trial estimates using decoding techniques and neural network approaches. However, by taking a data-driven approach (as opposed to theory-driven decoding analyses) a more nuanced picture emerges: there are several stimulus encoding steps which may contribute differently to behaviour, and decision processes extend beyond the planning of the motor response.

      Strengths:

      This work is not only important for the conceptual advance, but also in promoting this new analysis technique, which will likely prove useful in future research. For the broader picture, this work is an excellent example of the utility of neural measures for mental chronometry.

      Weaknesses:

      Though beyond the scope of this manuscript, these results should be considered within the broader decision-making literature, where task or domain-specific processes may not generalise (for example, in value-based decision-making).

      Review 3
    5. Public_Reviews 10 Dec 2025

      Author response:

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

      Reviewer #1 (Public review):  

      From my reading, this study aimed to achieve two things:  

      (1) A neurally-informed account of how Pieron's and Fechner's laws can apply in concert at distinct processing levels.  

      (2) A comprehensive map in time and space of all neural events intervening between stimulus and response in an immediately-reported perceptual decision.  

      I believe that the authors achieved the first point, mainly owing to a clever contrast comparison paradigm, but with good help also from a new topographic parsing algorithm they created. With this, they found that the time intervening between an early initial sensory evoked potential and an "N2" type process associated with launching the decision process varies inversely with contrast according to Pieron's law. Meanwhile, the interval from that second event up to a neural event peaking just before response increases with contrast, fitting Fechner's law, and a very nice finding is that a diffusion model whose drift rates are scaled by Fechner's law, fit to RT, predicts the observed proportion of correct responses very well. These are all strengths of the study.   

      We thank the reviewer for their comments that added context to the events we detected in relation to previous findings. We also believe that the change in the HMP algorithm suggested by the reviewer improved the precision of our analyses and the manuscript. We respond to the reviewer’s specific comments below.

      (1) The second, generally stated aim above is, in the opinion of this reviewer, unconvincing and ill-defined. Presumably, the full sequence of neural events is massively task-dependent, and surely it is more in number than just three. Even the sensory evoked potential typically observed for average ERPs, even for passive viewing, would include a series of 3 or more components - C1, P1, N1, etc. So are some events being missed? Perhaps the authors are identifying key events that impressively demarcate Pieron- and Fechner-adherent sections of the RT, but they might want to temper the claim that they are finding ALL events. In addition, the propensity for topographic parsing algorithms to potentially lump together distinct processes that partially co-evolve should be acknowledged.  

      We agree with the reviewer that the topographical solutions found by HMP will be dependent on the task and the quality and type of data. We address this point in the last section of the discussion (see also response to R3.5). We would also like to add that the events detected by HMP are, by construction, those that contribute to the RT and not necessarily all ERPs elicited by a stimulus.

      In addition to the new last section of the discussion we also make these points clear in the revised manuscript at the discussion start: 

      “By modeling the recorded single-trial EEG signal between stimulus onset and response as a sequence of multivariate events with varying by-trial peak times, we  aimed to detect recurrent events that contribute to the duration of the reaction time in the present perceptual decision-making task”.

      Regarding the typical visual ERPs, in response to this comment but also comments R1.2, R1.3 and R2.1, we aimed for a more precise description of the topographies and thus reduced the width of the HMP expected events to 25ms. This ensures that we do not miss events shorter than the initial expectations of 50ms (see Appendix B of Weindel et al., 2024 and also response to  R1.3). This new estimation provides evidence for at least two of the visual ERPs that, based on their timings and topographies (in relation with the spatial frequency of the stimulus), we interpret as the N40 and the P100 (see response to R1.5 for the justification of this categorization). We provide a description and justification of the interpretations in the result section “Five trial-recurrent sequential events occur in the EEG during decisions” and the discussion section “Visual encoding time”.

      (2) To take a salient example, the last neural event seems to blend the centroparietal positivity with a more frontal midline negativity, some of which would capture the CNV and some motor-execution related components that are more tightly time-locked to, of course, the response. If the authors plotted the traditional single-electrode ERP at the frontal focus and centroparietal focus separately, they are likely to see very different dynamics and contrast- and SAT-dependency. What does this mean for the validity of the multivariate method? If two or more components are being lumped into one neural event, wouldn't it mean that properties of one (e.g., frontal burstiness at response) are being misattributed to the other (centroparietal signal that also peaks but less sharply at response)?

      Using the new HMP parameterization described above we show that the reviewer's intuition was correct. Using an expected pattern duration of 25ms the last event in the original manuscript splits in two events. The before-last event, now referred to the lateralized readiness potential (LRP) presents a strong lateralization (Figure 3) with an increased negativity over the motor cortex contralateral to the right hand. The effect of contrast is mostly on the last event that we interpret as the CPP (Figure 5). Despite the improved precision of the topographies of the identified events, it is however to be noted that some components will overlap. If the LRP is generated when a certain amount of evidence is accumulated (e.g. that the CPP crosses a certain value) then a time-based topography will necessarily include that CPP activity in addition to the lateralized potential. We discuss this in the section “Motor execution” of the discussion:

      “Adding the abrupt onset of this potential, we believe that this event is the start of motor execution, engaged after a certain amount of evidence. The evidence for this interpretation is manifest in the fact that the event's topography shares some activity with the CPP event that follows, an expected result if the LRP is triggered at a certain amount of evidence, indexed by the CPP”.

      (3) Also related to the method, why must the neural events all be 50 ms wide, and what happens if that is changed? Is it realistic that these neural events would be the same duration on every trial, even if their duration was a free parameter? This might be reasonable for sensory and motor components, but unlikely for cognitive.  

      The HMP method is sensitive to the event's duration as shown in the manuscript about the method (Appendix B of Weindel et al., 2024). Nevertheless as long as the topography in the real data is longer than the expected one it shouldn't be missed (i.e. same goes for by-trial variations in the event width). For this reason we halved the expected event width of 50ms (introduced by the original HsMM-MVPA paper by Anderson and colleagues) in the revision. This new estimation with 25ms thus is much less likely to miss events as evidenced by the new visual and motor events. In the revised manuscript this is addressed at the start of the Results section:

      “Contrary to previous applications (Anderson et al.,2016; Berberyan et al., 2021; Zhang et al., 2018; Krause et al., 2024) we assumed that the multivariate pattern was represented by a 25ms half-sine as our previous research showed that a shorter expected pattern width increases the likelihood of detecting cognitive events (see Appendix B of Weindel et al., 2024)”.

      Regarding the event width as a free parameter this is both technically and statistically difficult to implement as the amount of computing capacity, flexibility and trade-offs among the HMP parameters would, given the current implementation, render the model unfit for most computers and statistically unidentifiable.

      (4) In general, I wonder about the analytic advantage of the parsing method - the paradigm itself is so well-designed that the story may be clear from standard average event-related potential analysis, and this might sidestep the doubts around whether the algorithm is correctly parsing all neural events.  

      Average ERP analysis suffers from an impossibility to differentiate between an effect of an experimental factor on the amplitude vs. on the timing of the underlying components (Luck, 2005). Furthermore the overlap of components across trials bluries the distinction between them. For both reasons we would not be able to reach the same level of certainty and precision using ERP analyses. Furthermore the relatively low number of trials per experimental cell (contrast level X SAT X participant = 6 trials) makes the analyses hard to perform on ERP which typically require more trials per modality. From the reviewer’s comment we understand that this point was not clear. We therefore discuss this in the revision, Section “Functional interpretation of the events” of the results:

      “Nevertheless identifying neural dynamics on these ERPs centered on stimulus is complicated by the time variation of the underlying single-trial events (see probabilities displayed in Figure 3 for an illustration and Burle et al., 2008, for a discussion). The likely impact of contrast on both amplitude and time on the underlying single-trial event does not allow one to interpret the average ERP traces as showing an effect in one or the other dimension without strong assumptions (Luck, 2005)”.

      (5) In particular, would the authors consider plotting CPP waveforms in the traditional way, across contrast levels? The elegant design is such that the C1 component (which has similar topography) will show up negative and early, giving way to the CPP, and these two components will show opposite amplitude variations (not just temporal intervals as is this paper's main focus), because the brighter the two gratings, the stronger the aggregate early sensory response but the weaker the decision evidence due to Fechner. I believe this would provide a simple, helpful corroborating analysis to back up the main functional interpretation in the paper.  

      We agree with the suggestion and have introduced the representation on top of Figure 5 for sets of three electrodes in the occipital, posterior and frontal regions. The new panels clearly show an inversion of the contrast effect dependent on the time and locus of the electrodes. We discuss this in Section “Functional interpretation of the events” of the results:

      “This representation shows that there is an inversion of the contrast effect with higher contrasts having a higher amplitude on the electrodes associated with visual potentials in the first couple of deciseconds (left panel of Figure 5A) while parietal and frontal electrodes shows a higher amplitude for lower contrasts in later portions of the ERPs (middle and right panel of Figure 5A)”.

      To us, this crucially shows that we cannot achieve the same decomposition using traditional ERP analyses. In these plots it appears that while, as described by the reviewer, there is an inversion, the timing and amplitude of the changes due to contrast can hardly be interpreted.

      (6) The first component is picking up on the C1 component (which is negative for these stimulus locations), not a "P100". Please consult any visual evoked potential study (e.g., Luck, Hillyard, etc). It is unexpected that this does not vary in latency with contrast - see, for example. Gebodh et al (2017, Brain Topography) - and there is little discussion of this. Could it be that nonlinear trends were not correctly tested for?  

      We disagree with the reviewer on the interpretation of the ERP. The timing of the detected component is later than the one usually associated with a C1. Furthermore the central display does not create optimal conditions to detect a C1

      We do agree that the topography raises the confusion but we believe that this is due to the spatial frequency of the stimulus that generates a high posterior positivity (see references in the following extract). The new HMP solution also now happens to show an effect of contrast on the P100 latencies, we believe this is due to the increased precision in the time location of the component. We discuss this in the “Visual encoding time” section of the discussion:

      “The following event, the P100, is expressed around 70ms after the N40, its topography is congruent with reports for stimuli with low spatial frequencies as used in the current study (Kenemans et al., 2002, 2000; Proverbio et al., 1996). The timing of this P100 component is changed by the contrast of the stimulus in the direction expected by the Piéron law (Figure 4A)”. 

      (7) There is very little analysis or discussion of the second stage linked to attention orientation - what would the role of attention orientation be in this task? Is it spatial attention directed to the higher contrast grating (and if so, should it lateralise accordingly?), or is it more of an alerting function the authors have in mind here?  

      We agree that we were not specific enough on the interpretation of this attention stage. We now discuss our hypothesis in the section “Attention orientation” of the discussion:  

      “We do however observe an asymmetry in the topographical map Figure 3. This asymmetry might point to an attentional bias with participants (or at least some participants) allocating attention to one side over the other in the same way as the N2pc component (Luck and Hillyard, 1994, Luck et al., 1997). Based on this collection of observations, we conclude that this third event represents an attention orientation process. In line with the finding of Philiastides et al. (2006), this attention orientation event might also relate to the allocation of resources. Other designs varying the expected cognitive load or spatial attention could help in further interpreting the functional role of this third event”.

      We would like to add that it is unlikely that the asymmetry we mention in the discussion cannot stem from the redirection towards higher contrast as the experimental design balanced the side of presentation. We therefore believe that this is a behavioral bias rather than a bias toward the highest contrast stimulus as suggested by the reviewer. We hope that, while more could be tested and discussed, this discussion is sufficient given the current manuscript's goal.

      Reviewer #2 (Public review):  

      Summary:  

      The authors decomposed response times into component processes and manipulated the duration of these processes in opposing directions by varying contrast, and overall by manipulating speed-accuracy tradeoffs. They identify different processes and their durations by identifying neural states in time and validate their functional significance by showing that their properties vary selectively as expected with the predicted effects of the contrast manipulation. They identify 3 processes: stimulus encoding, attention orienting, and decision. These map onto classical event-related potentials. The decision-making component matched the CPP, and its properties varied with contrast and predicted decision-accuracy, while also exhibiting a burst not characteristic of evidence accumulation.  

      Strengths:  

      The design of the experiment is remarkable and offers crucial insights. The analysis techniques are beyond state-of-the-art, and the analyses are well motivated and offer clear insights.  

      Weaknesses:  

      It is not clear to me that the results confirm that there are only 3 processes, since e.g., motor preparation and execution were not captured. While the authors discuss this, this is a clear weakness of the approach, as other components may also have been missed. It is also unclear to what extent topographies map onto processes, since, e.g., different combinations of sources can lead to the same scalp topography.  

      We thank the reviewer for their kind words and for the attention they brought on the question of the missing motor preparation event. In light of this comment (and also R1.1, R3.3) the revised manuscript uses a finer grained approach for the multivariate event detection. This preciser estimation comes from the use of a shorter expected pattern in which the initial expectation of a 50ms half-sine was halved, therefore ensuring that we do not miss events shorter than the initial expectations (see Appendix B of Weindel et al., 2024 and also response to  R1.3). In the new solution the motor component that the reviewer expected is found as evidenced by the topography of the event, its lateralization and a time-to-response congruent with a response execution event. This is now described in the section “Motor execution” of the revised manuscript: 

      “The before last event, identified as the LRP, shows a strong hemispheric asymmetry congruent with a right hand response. The peak of this event is approximately 100 ms before the response which is congruent with reports that the LRP peaks at the onset of electromyographical activity in the effector muscle (Burle et al., 2004), typically happening 100ms before the response in such decision-making tasks (Weindel et al., 2021). Furthermore, while its peak time is dependent on contrast, its expression in the EEG is less clearly related to the contrast manipulation than the following CPP event”.

      Reviewer #3 (Public review):  

      Summary:  

      In this manuscript, the authors examine the processing stages involved in perceptual decision-making using a new approach to analysing EEG data, combined with a critical stimulus manipulation. This new EEG analysis method enables single-trial estimates of the timing and amplitude of transient changes in EEG time-series, recurrent across trials in a behavioural task. The authors find evidence for three events between stimulus onset and the response in a two-spatial-interval visual discrimination task. By analysing the timing and amplitude of these events in relation to behaviour and the stimulus manipulation, the authors interpret these events as related to separable processing stages for stimulus encoding, attention orientation, and decision (deliberation). This is largely consistent with previous findings from both event-related potentials (across trials) and single-trial estimates using decoding techniques and neural network approaches.  

      Strengths:  

      This work is not only important for the conceptual advance, but also in promoting this new analysis technique, which will likely prove useful in future research. For the broader picture, this work is an excellent example of the utility of neural measures for mental chronometry.  

      We appreciate the very positive review and thank the reviewer for pointing out important weaknesses in our original manuscript and also providing resources to address them in the recommendations to authors. Below we comment on each identified weakness and how we addressed them.   

      Weaknesses:  

      (1) The manuscript would benefit from some conceptual clarifications, which are important for readers to understand this manuscript as a stand-alone work. This includes clearer definitions of Piéron's and Fechner's laws, and a fuller description of the EEG analysis technique.

      We agree that the description of both laws were insufficient, we therefore added the following text in the last paragraph of the introduction:

      “Piéron’s law predicts that the time to perceive the two stimuli (and thus the choice situation) should follow a negative power law with the stimulus intensity (Figure 1, green curve). In contradistinction, Fechner’s law states that the perceived difference between the two patches follows the logarithm of the absolute contrast of the two patches (Figure 1, yellow curve). As the task of our participants is to judge the contrast difference, Piéron’s law should predict the time at which the comparison starts (i.e. the stimuli become perceptible), while Fechner’s law should implement the comparison, and thus decision, difficulty”.

      Regarding the EEG analysis technique we added a few elements at the start of the result:

      “The hidden multivariate pattern model (HMP) implemented assumed that a task-related multivariate pattern event is represented by a half-sine whose timing varies from trial to trial based on a gamma distribution with a shape parameter of 2 and a scale, controlling the average latency of the event, free-to-vary per event (Weindel et al., 2024)”.

      We also made the technique clearer at the start of the discussion:

      “By modeling the recorded single-trial EEG signal between stimulus onset and response as a sequence of multivariate events with varying by-trial peak times, we aimed to detect recurrent events that contribute to the duration of the reaction time in the present perceptual decision-making task. In addition to the number of events, using this hidden multivariate pattern approach (Weindel et al., 2024) we estimated the trial-by-trial probability of each event’s peak, therefore accessing at which time sample each event was the most likely to occur”.

      Additionally, we added a proper description in the method section (see the new first paragraph of the “Hidden multivariate pattern” subsection). 

      (2) The manuscript, broadly, but the introduction especially, may be improved by clearly delineating the multiple aims of this project: examining the processes for decision-making, obtaining single-trial estimates of meaningful EEG-events, and whether central parietal positivity reflects ramping activity or steps averaged across trials.

      For the sake of clarity we removed the question of the ramping activity vs steps in the introduction and focused on the processes in decision-making and their single-trial measurement as this is the main topic of the paper. Furthermore the references provided by the reviewer allowed us to write a more comprehensive review of previous studies and how the current study is in line with those. These changes are mainly manifested in these new sentences:

      “As an example Philiastides et al. (2006) used a classifier on the EEG activity of several conditions to show that the strength of an early EEG component was proportional to the strength of the stimulus while a later component was related to decision difficulty and behavioral performance (see also Salvador et al., 2022; Philiastides and Sajda, 2006). Furthermore the authors interpreted that a third EEG component was indicative of the resource allocated to the upcoming decision given the perceived decision difficulty. In their study, they showed that it is possible to use single-trial information to separate cognitive processes within decision-making. Nevertheless, their method requires a decoding approach, which requires separate classifiers for each component of interest and restrains the detection of the components to those with decodable discriminating features (e.g. stimuli with strong neural generators such as face stimuli, see Philiastides et al., 2006)”.

      (3) A fuller discussion of the limitations of the work, in particular, the absence of motor contributions to reaction time, would also be appreciated. 

      As laid out in responses to comments R1.1 and R2 the new estimates now include evidence for a motor preparation component. We discuss this in the new “motor execution” paragraph in the discussion section. Additionally we discuss the limitation of the study and the method in the two last paragraphs of the discussion (in the new Section “Generalization and limitation”).

      (4) At times, the novelty of the work is perhaps overstated. Rather, readers may appreciate a more comprehensive discussion of the distinctions between the current work and previous techniques to gauge single-trial estimates of decision-related activity, as well as previous findings concerning distinct processing stages in decision-making. Moreover, a discussion of how the events described in this study might generalise to different decision-making tasks in different contexts (for example, in auditory perception, or even value-based decision-making) would also be appreciated.  

      We agree that the original text could be read as overstating. In addition to the changes linked to R3.2 we also now discuss the link with the previous studies in the before-last paragraph of the discussion before the conclusion in the new “Generalization and limitations” section:

      “The present study showed what cognitive processes are contributing to the reaction time and estimated single-trial times of these processes for this specific perceptual decision-making task. The identified processes and topographies ought to be dependent on the task and even the stimuli (e.g. sensory events will change with the sensory modality). More complex designs might generate a higher number of cognitive processes (e.g. memory retrieval from a cue, Anderson et al., 2016) and so could more natural stimuli which might trigger other processes in the EEG (e.g. appraisal vs. choice as shown by Frömer et al., 2024). Nevertheless, the observation of early sensory vs. late decision EEG components is likely to generalize across many stimuli and tasks as it has been observed in other designs and methods (Philiastides et al., 2006; Salvador et al., 2022). To these studies we add that we can evaluate the trial-level contribution, as already done for specific processes (e.g. Si et al., 2020; Sturm et al., 2016), for the collection of events detected in the current study”.

      Reviewing Editor Comments:  

      As you will see, all three reviewers agree that the paper makes a valuable contribution and has many strengths. You will also see that they have provided a range of constructive comments highlighting potential issues with the interpretation of the outcomes of your signal decomposition method. In particular, all three reviewers point out that your results do not identify separate motor preparation signals, which we know must be operating on this type of task. The reviewers suggest further discussion of this issue and the potential limitations of your analysis approach, as well as suggesting some additional analyses that could be run to explore this further. While making these changes would undoubtedly enhance the paper and the final public reviews, I should note that my sense is that they are unlikely to change the reviewers' ratings of the significance of the findings and the strength of evidence in the final eLife assessment  

      Reviewer #1 (Recommendations for the authors):  

      (1) Abstract: "choice onset" is ill-defined and not the label most would give the start of the RT interval. Do you mean stimulus onset?  

      We replaced with "choice onset" with "stimulus onset" in the abstract

      (2) Similarly "choice elements" in the introduction seem to refer to sensory attributes/objects being decided about?  

      We replaced "choice-elements" with "choice-relevant features of the stimuli"

      (3) "how the RT emerges from these putative components" - it would be helpful to specify more what level of answer you're looking for, as one could simply answer "when they're done."  

      We replaced with "how the variability in RTs emerges from these putative components"

      (4) Line 61-62: I'm not sure this is a fully correct characterisation of Frömer et al. It was not similar in invoking a step function - it did not invoke any particular mechanism or function, and in that respect does not compare well to Latimer et al. Also, I believe it was the overlap of stimulus-locked components, not response-locked, that they argued could falsely generate accumulator-like buildup in the response-locked ERP.  

      We indeed wrongly described Frömer et al. The sentence is now "In human EEG data, the classical observation of a slowly evolving centro-parietal positivity, scaling with evidence accumulation, was suggested to result from the overlap of time-varying stimulus-related activity in the response-locked event related potential"

      (5) Line 78: Should this be single-trial *latency*?  

      This referred to location in time but we agree that the term is confusing and thus replaced it with latencies.

      (6) The caption of Figure 1 should state what is meant by the y-axis "time"  

      We added the sentence "The y-axis refers the time predicted by each law given a contrast value (x-axis) and the chosen set of parameters." in the caption of Figure 1

      (7) Line 107: Is this the correct description of Fechner's law? If the perceived difference follows the log of the physical difference, then a constant physical difference should mean a constant perceived difference. Perhaps a typo here.  

      This was indeed a typo we replaced the corresponding part of the sentence with "the perceived difference between the two patches follows the logarithm of the absolute contrast of the two patches"

      (8) Line 128: By scale, do you mean magnitude/amplitude?  

      No, this refers to the parameter of a gamma distribution. To clarify we edited the sentence:  "based on a gamma distribution with a shape parameter of 2 and a scale parameter, controlling the average latency of the event, free-to-vary per event"

      (9) The caption of Figure 3 is insufficient to make sense of the top panel. What does the inter-event interval mean, and why is it important to show? What is the "response" event?  

      We agree that the top panel was insufficiently described. To keep the length of the paper short and because of the relatively low amount of information provided by these panels we replaced them for a figure only showing the average topographies as well as the asymmetry tests for each event.

      (10) Figure 4: caption should say what the top vs bottom row represents (presumably, accuracy vs speed emphasis?), and what the individual dots represent, given the caption says these are "trial and participant averaged". A legend should be provided for the rightmost panels.  

      We agree and therefore edited Figure 4. The beginning of the caption mentioned by the reviewer now reads: “A) The panels represent the average duration between events for each contrast level, averaged across participants and trials (stimulus and response respectively as first and last events) for accuracy (top) and speed instructions (bottom).”. Additionally we added legends for the SAT instructions and the model fits.

      (11) Line 189: argued for a decision-making role of what?  

      Stafford and Gurney (2004) proposed that Pieron’s law could reflect a non-linear transformation from sensory input to action outcomes, which they argued reflected a response mechanism. We (Van Maanen et al., 2012) specified this result by showing that a Bayesian Observer Model in which evidence for two alternative options was accumulated following Bayes Rule indeed predicted a power relation between the difference in sensory input of the two alternatives, and mean RT. However, the current data suggest that such an explanation cannot be the full story, as also noted by R3. To clarify this point we replaced the comment by the following sentence:

      “Note that this observation is not necessarily incongruent with theoretical work that argued that Piéron’s law could also be a result of a response selection mechanism (Stafford and Gurney, 2004; Van Maanen et al., 2012; Palmer et al., 2005). It could be that differences in stimulus intensity between the two options also contribute to a Piéron-like relationship in the later intervals, that is convoluted with Fechner’s law (see Donkin and Van Maanen, 2014 for a similar argument). Unfortunately, our data do not allow us to discriminate between a pure logarithmic growth function and one that is mediated by a decreasing power function”.

      (12) Table 2: There is an SAT effect even on the first interval, which is quite remarkable and could be discussed more - does this mean that the C1 component occurs earlier under speed pressure? This would be the first such finding.  

      The original event we qualified as a P100 was sensitive to SAT but the earliest event is now the N40 and isn’t statistically sensitive to speed pressure in this data. We believe that the fact that the P100 is still sensitive to SAT is not a surprise and therefore do not outline it.

      (13) Line 221: "decrease of activation when contrast (and thus difficulty) increases" - is this shown somewhere in the paper?  

      The whole section for this analysis was rewritten (see comment below)

      (14) I find the analysis of Figure 5 interesting, but the interpretation odd. What is found is that the peak of the decision signal aligns with the response, consistent with previous work, but the authors choose to interpret this as the decision signal "occurring as a short-lived burst." Where is the quantitative analysis of its duration across trials? It can at least be visually appraised in the surface plot, and this shows that the signal has a stimulus-locked onset and, apart from the slowest RTs, remains present and for the most part building, until response. What about this is burst-like? A peak is not a burst.  

      This was the residue of a previous version of the paper where an analysis reported that no evidence accumulation trace was found. But after proper simulations this analysis turned out to be false because of a poor statistical test. Thus we removed this paragraph in the revised manuscript and Figure 5 has now been extended to include surface plots for all the events.

      Reviewer #2 (Recommendations for the authors):  

      Overall, I really enjoyed reading this paper. However, in some places the approach is a bit opaque or the results are difficult to follow. As I read the paper, I noted:  

      Did you do a simple DDM, or did you do a collapsing bound for speed?  

      The fitted DDM was an adaptation of the proportional rate diffusion model. We make this clearer at the end of the introduction: "Given that Fechner’s law is expected to capture decision difficulty we connected this law to the classical diffusion decision models by replacing the rate of accumulation with Fechner’s law in the proportional rate diffusion model of Palmer et al.(2005).”

      It is confusing that the order of intervals in the text doesn't match the order in the table. It might be better to say what events the interval is between rather than assuming that the reader reconstructs.  

      We agree and adapted the order in both the text and the table. The table is now also more explicit (e.g. RT instead of S-R)

      Otherwise, I do wonder to what extent the method is able to differentiate processes that yield similar scalp topographies and find it a bit concerning that no motor component was identified.  

      We believe that the new version with the LRP/CPP is a demonstration that the method can handle similar topographies. The method can handle events with close topographies as long as they are separate in time, however if they are not sequential to one another the method cannot capture both events. We now discuss this, in relation with the C1/P100 overlap, in the discussion section “Visual encoding time”:

      “Nevertheless this event, seemingly overlapping with the P100 even at the trial level (Figure 5C), cannot be recovered by the method we applied. The fact that the P100 was recovered instead of the C1 could indicate that only the timing of the P100 contributes to the RT (see Section 3 of Weindel et al., 2024)”.

      And we more generally address the question of overlap in the new section “Generalization and limitation”.

      Reviewer #3 (Recommendations for the authors):  

      Major Comments:  

      (1) If we agree on one thing, it is that motor processes contribute to response time. Line 364: "In the case of decision-making, these discrete neural events are visual encoding, attention-orientation, and decision commitment, and their latency make up the reaction time." Does the third event, "decision commitment", capture both central parietal positivity (decision deliberation) and motor components? If so, how can the authors attribute the effects to decision deliberation as opposed to motor preparation?  

      Thanks to the suggestions also in the public part. This main problem is now addressed as we do capture both a motor component and a decision commitment.

      Line 351 suggests that the third event may contain two components.  

      This was indeed our initial, badly written, hypothesis. Nevertheless the new solution again addresses this problem.

      The time series in Figure 6 shows an additional peak that is not evident in the simulated ramp of Appendix 1.  

      This was probably due to the overlap of both the CPP and the LRP. It is now much clearer that the CPP looks mostly like a ramp while the LRP looks much more like a burst-like/peaked activity. We make this clear in the “Decision event” paragraph of the discussion section:

      “Regarding the build-up of this component, the CPP is seen as originating from single-trial ramping EEG activities but other work (Latimer et al., 2015; Zoltowski et al., 2019) have found support for a discrete event at the trial-level. The ERPs on the trial-by-trial centered event in Figure 5 show support for both accounts. As outlined above, the LRP is indeed a short burst-like activity but the build-up of the CPP between high vs low contrast diverges much earlier than its peak”.

      Previous analyses (Weindel et al., 2024) found motor-related activity from central parietal topographies close to the response by comparing the difference in single-trial events on left- vs right-hand response trials. The authors suggest at line 315 that the use of only the right hand for responding prevented them from identifying a motor event.  

      The use of only the right hand should have made the event more identifiable because the topography would be consistent across trials (rather than inverting on left vs right hand response trials).  

      The reviewer is correct, in the original manuscript we didn’t test for lateralization, but the comment of the reviewer gave us the idea to explicitly test for the asymmetry (Figure 3). This test now clearly shows what would be expected for a motor event with a strong negativity over the left motor cortex.

      The authors state on line 422 that the EEG data were truncated at the time of the response.  

      Could this have prevented the authors from identifying a motor event that might overlap with the timing of the response?  

      We thank the reviewer for this suggestion. This would have been a possibility but the problem is that adding samples after the response also adds the post-response processes (error monitoring, button release, stimulus disappearance, etc.). While increasing the samples after the response is definitely something that we need to inspect, we think that the separation we achieved in this revision doesn’t call for this supplementary analysis.

      The largest effects of contrast on the third event amplitude appear around the peak as opposed to the ramp. If the peak is caused by the motor component, how does this affect the conclusions that this third event shows a decision-deliberation parietal processes as opposed to a motor process (a number of studies suggest a causal role for motor processes in decision-making e.g. Purcell et al., 2010 Psych Rev; Jun et al., 2021 Nat Neuro; Donner et al., 2009 Curr Bio).  

      This result now changed and it does look like the peak capturing most of the effect is no longer true. We do however think that there might be some link to theories of motor-related accumulation. We therefore added this to the discussion in the Motor execution section:

      “Based on all these observations, it is therefore very likely that this LRP event signs the first passage of a two-step decision process as suggested by recent decision-making models (Servant et al., 2021; Verdonck et al., 2021; Balsdon et al., 2023)”.

      I would suggest further investigation into the motor component (perhaps by extending the time window of analysed EEG to a few hundred ms after the response) and at least some discussion of the potential contribution of motor processes, in relation to the previous literature.  

      We believe that the absence of a motor component is sufficiently addressed in the revised manuscript and in the responses to the other comments.    

      (2) What do we learn from this work? Readers would appreciate more attention to previous findings and a clearer outline of how this work differs. Two points stand out, outlined below. I believe the authors can address these potential complaints in the introduction and discussion, and perhaps provide some clarification in the presentation of the results.  

      In the introduction, the authors state that "... to date, no study has been able to provide single-trial evidence of multiple EEG components involved in decision-making..." (line 64). Many readers would disagree with this. For example, Philiastides, Ratcliff, & Sadja (2006) use a single-trial analysis to unravel early and late EEG components relating to decision difficulty and accuracy (across different perceptual decisions), which could be related to the components in the current work. Other, network-based single-trial EEG analyses (e.g., Si et al., 2020, NeuroImage, Sturn et al., 2016 J Neurosci Methods) could also be related to the current component approach. Yet other approaches have used inverse encoding models to examine EEG components related to separable decision processes within trials (e.g., Salvador et al., 2022, Nat Comms). The results of the current work are consistent with this previous work - the two components from Philiastides et al., 2006 can be mapped onto the components in the current work, and Salvador et al., 2022 also uncover stimulus- and decision-deliberation related components.  

      We completely agree with the reviewer that the link to previous work was insufficient. We now include all references that the reviewer points out both in the introduction (see response R3.2) and in the discussion (see response R3.4). We wish to thank the reviewer for bringing these papers to our attention as they are important for the manuscript.

      The authors relate their components to ERPs. This prompts the question of whether we would get the same results with ERP analyses (and, on the whole, the results of the current work are consistent with conclusions based on ERP analyses, with the exception of the missing motor component). It's nice that this analysis is single-trial, but many of the follow-up analyses are based on grouping by condition anyway. Even the single-trial analysis presented in Figure 4 could be obtained by median splits (given the hypotheses propose opposite directions of effects, except for the linear model). 

      We do not agree with the reviewer in the sense that classical ERP analyses would require much more data-points. The performance of the method is here to use the information shared across all contrast levels to be able to model the processing time of a single contrast level (6 trials per participant). Furthermore, as stated in the response to R1.4 and R1.5, the aim of the paper is to have the time of information processing components which cannot be achieved with classical ERPs without strong, and likely false, assumptions.

      Medium Comments:  

      (1) The presentation of Piéron's law for the behavioural analysis is confusing. First, both laws should be clearly defined for readers who may be unfamiliar with this work. I found the proposal that Piéron's law predicts decreasing RT for increasing pedestal contrast in a contrast discrimination paradigm task surprising, especially given the last author's previous work. For example, Donkin and van Maanen (2014) write "However, the commonality ofPiéron's Law across so many paradigms has lead researchers (e.g., Stafford & Gurney, 2004; Van Maanen et al., 2012) to propose that Piéron's Law is unrelated to stimulus scaling, but is a result of the architecture of the response selection (or decision making) process." The pedestal contrast is unrelated to the difficulty of the contrast discrimination task (except for the consideration of Fechner's law). Instead, Piéron's law would apply to the subjective difference in contrast in this task, as opposed to the pedestal contrast. The EEG results are consistent with these intuitions about Piéron's law (or more generally, that contrast is accumulated over time, so a later EEG component for lower pedestal contrast makes sense): pedestal contrast should lead to faster detection, but not necessarily faster discrimination. Perhaps, given the complexity of the manuscript as a whole, the predictions for the behavioural results could be simplified?  

      We agree that the initial version was confusing. We now clarified the presentation of Piéron's law at the end of the introduction (see also response to R2).

      Once Fechner's law is applied, decision difficulty increases with increasing contrast, so Piéron's law on the decision-relevant intensity (perceived difference in contrast) would also predict increasing RT with increasing pedestal contrast. It is unlikely that the data are of sufficient resolution to distinguish a log function from a power of a log function, but perhaps the claim on line 189 could be weakened (the EEG results demonstrate Piéron's law for detection, but do not provide evidence against Piéron's law in discrimination decisions).  

      This is an excellent observation, thank you for bringing it to our attention. Indeed, the data support the notion that Pieron’s law is related to detection, but do not rule out that it is also related to decision or discrimination. In earlier work, we (Donkin & Van Maanen, 2014) addressed this question as well, and reached a similar conclusion. After fitting evidence accumulation models to data, we found no linear relationship between drift rates and stimulus difficulty, as would have been the case if Pieron's law could be fully explained by the decision process (as -indirectly- argued by Stafford & Gurney, 2004; Van Maanen et al., 2012). The fact that we observed evidence for a non-linear relationship between drift rates and stimulus difficulty led us to the same conclusion, that Pieron’s law could be reflected in both discrimination and decision processes. We added the following comment to the discussion about the functional locus of Pieron's law to clarify this point:

      “Note that this observation is not necessarily incongruent with theoretical work that argued that Piéron’s law could also be a result of a response selection mechanism (Stafford and Gurney, 2004; Van Maanen et al., 2012; Palmer et al., 2005). It could be that differences in stimulus intensity between the two options also contribute to a Piéron like relationship in the later intervals, that is convoluted with Fechner’s law (see Donkin and Van Maanen, 2014, for a similar argument). Unfortunately, our data do not allow us to discriminate between a pure logarithmic growth function and one that is mediated by a decreasing power function”.

      (2) Appendix 1 shows that the event detection of the HMP method will also pick up on ramping activity. The description of the problem in the introduction is that event-like activity could look like ramping when averaged across trials. To address this problem, the authors should simulate events (with some reasonable dispersion in timing such that they look like ramping when averaged) and show that the HMP method would not pull out something that looked like ramping. In other words, the evidence for ramping in this work is not affected by the previously identified confounds.  

      We agree that this demonstration was necessary and thus added the suggested simulation to Appendix 1. As can be seen in the Figure 1 of the appendix, when we simulate a half-sine the average ERP based on the timing of the event looks like a half-sine.

      (3) Some readers may be interested in a fuller discussion of the failure of the Fechner diffusion model in the speed condition.  

      We are unsure which failure the reviewer refers to but assumed it was in relation to the behavioral results and thus added: 

      It is unlikely that neither Piéron nor Fechner law impact the RT in the speed condition. Instead this result is likely due to the composite nature of the RT where both laws co-exist in the RT but cancel each other out due to their opposite prediction.

      Minor Comments:  

      (1) "By-trial" is used throughout. Normally, it is "trial-by-trial" or "single-trial" or "trial-wise".

      We replaced all occurrences of “by-trial”  with the three terms suggested were appropriate.

      (2) Line 22: "The sum of the times required for the completion of each of these precessing steps is the reaction time (RT)." The total time required. Processing.  

      Corrected for both.

      (3) Line 26/27: "Despite being an almost two century old problem (von Helmholtz, 2021)." Perhaps the citation with the original year would make this point clearer.  

      We agree and replaced the citation.

      (4) Line 73: "accounted by estimating". Accounted for by estimating.  

      Corrected.

      (5) Line 77 "provides an estimation on the." Of the.  

      Corrected.

      (6) Line 86: "The task of the participants was to answer which of two sinusoidal gratings." The picture looks like Gabor's? Is there a 2d Gaussian filter on top of the grating? Clarify in the methods, too.  

      We incorrectly described the stimuli as those were indeed just Gabor’s. This is now corrected both in the main text and the method section.

      (7) Figure 1 legend: "The Fechner diffusion law" Fechner's law or your Fechner diffusion model?  

      Law was incorrect so we changed to model as suggested.

      (8) Line 115: "further allows to connects the..." Allows connecting the.  

      Corrected.

      (9) Line 123: "lower than 100 ms or higher than..." Faster/slower.  

      Corrected.

      (10) Line 131: "To test what law." Which law.?  

      Corrected to model.

      (11) Figure 2 legend: "Left: Mean RT (dot) and average fit (line) over trials and participants for each contrast level used." The fit is over trials and participants? Each dot is? Average trials for each contrast level in each participant?  

      This sentence was corrected to “Mean RT (dot) for each contrast level and averaged predictions of the individual fits (line) with Accuracy (Top) and Speed (Bottom) instructions.”.

      (12) Line 231: "A comprehensive analysis of contrast effect on". The effect of contrast on.  

      This title was changed to “functional interpretation of the events”.

      (13) Line 23: "the three HMP event with". Three HMP events.

      The sentence no longer exists in the revised manuscript.

      (14) Line 270: "Secondly, we computed the Pearson correlation coefficient between the contrast averaged proportion of correct." Pearson is for continuous variables. Proportion correct is not continuous. Use Spearman, Kendall, or compute d'.  

      The reviewer rightly pointed out our error, we corrected this by computing Spearman correlation.

      (15)  Line 377: "trial 𝑛 + 1 was randomly sampled from a uniform distribution between 0.5 and 1.25 seconds." It's just confusing why post-response activity in Figure 5 does look so consistent. Throughout methods: "model was fitted" should be "was fit", and line 448, "were split".  

      We do not have a specific hypothesis of why the post-response activity in the previous Figure 5 was so consistent. Maybe the Gaussian window (same as in other manuscripts with a similar figure, e.g. O’Connell et al. 2012) generated this consistency. We also corrected the errors mentioned in the methods.

      (16) The linear mixed models paragraph is a bit confusing. Can it clearly state which data/ table is being referred to and then explain the model? "The general linear mixed model on proportion of correct responses was performed using a logit link. The linear mixed models were performed on the raw milliseconds scale for the interval durations and on the standardized values for the electrode match." We go directly from proportion correct to raw milliseconds...  

      The confusion was indeed due to the initial inclusion of a general linear mixed model on proportion correct which was removed as it was not very informative. The new revision should be clearer on the linear mixed models (see first sentence of subsection ‘linear mixed models' in the method section).

      (17) A fuller description of the HMP model would be appreciated.  

      We agree that this was necessary and added the description of the HMP model in the corresponding method section “Hidden multivariate pattern” in addition to a more comprehensive presentation of HMP in the first paragraph of the Result and Discussion sections.

      (18) Line 458: "Fechner's law (Fechner, 1860) states that the perceived difference (𝑝) between the two patches follows the logarithm of the difference in physical intensity between..." ratio of physical intensity.  

      Corrected.

      (19) P is defined in equations 2 and 4. I would include the beta in equation 4, like in equation 2, then remove the beta from equations 3 and 5 (makes it more readable). I would also just include the delta in equation 2, state that in this case, c1 = c+delta/2 or whatever.  

      This indeed makes the equation more readable so we applied the suggestions for equations 2, 3, 4 and 5. The delta was not added in equation 2 but instead in the text that follows:

      “Where 𝐶1 = 𝐶0 + 𝛿, again with a modality and individual specific adjustment slope (𝛽).” 

      (20) The appendix suggests comparing the amplitudes with those in Figure 3, but the colour bar legend is missing, so the reader can only assume the same scale is used?  

      We added the color bar as it was indeed missing. Note though that the previous version displayed the estimation for the simulated data while this plot in the revised manuscript shows the solution on real data obtained after downsampling the data (and therefore look for a larger pattern as in the main text). We believe that this representation is more useful given that the solution for the downsampled data is no longer the same as the one in the main text (due to the difference in pattern width).

      AuthorResponse
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  16. courses.ecu.edu.au courses.ecu.edu.au
    Digital Tools for Study : Curio
    1
    1. nicjohnston 10 Dec 2025
      ECU Digital Tools Checklist.pdf

      The checklist feels too extensive for what we would ask a student to do. Suggest pairing it back alot or sticking with the 3 S style of short evaluation. Just Part B comparison for example.

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  17. spitfirenews.com spitfirenews.com
    How should we talk about our thinnest celebrities?
    5
    1. dentropy 10 Dec 2025

      My thoughts on the article,

      The difference in appearance and body type between that of toys of Ariana Grande and the literal Aria Grande is notable.

      A part of me asks why Aria Grande wants to be so skinny. Like she's not even attractive anymore. It may have something to do with Women who are attractive interpreting their beauty as a curse because is attracts attention they would no longer have.

      I have never heard of "Stan Culture" before. It's basically fan accounts for people and fandoms. I wonder if the Aria Grande "Stan Accounts" interface with the memes I posted in this other part of the article

      https://hyp.is/OY-gdNWMEfCXyP8m6RghMA/spitfirenews.com/p/ariana-grande-eating-disorder-wicked-cynthia-erivo

    2. dentropy 10 Dec 2025
      “You people would ‘it’s not okay to comment on women’s bodies and she’s always been skinny’

      I believe that "Skinny" as a beauty standard is just a leftover effect of Gay Men of Power in the Designer Fashion industry choosing women that look like the men they want to fuck

    3. dentropy 10 Dec 2025
      of “stan culture” that

      Stan Culture seems to be a Twitter Subculture where people don't like AI and has something to do with running Fan Accounts for Celeberties and Fandoms

    4. dentropy 10 Dec 2025
      Her body is blown up to be 30 feet tall on the AMC Theatres screen where I saw the sequel to Wicked and shrunk to 11 inches as a Barbie doll bearing her likeness. Her frame, with corsets cinching her waist and gemstones adorning her collarbones, is plastered across billboards and buses and in between posts on my FYP on the most popular apps.

      I never thought about how Ariana Grande looks so skinny that she is sick in real life yet there are toys and lego of here. I wonder how they contrast

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  18. www.biorxiv.org www.biorxiv.org
    Identification of M-Sec as a unique cellular regulator of CSF-1 receptor activation
    1
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  19. www.youtube.com www.youtube.com
    Santé mentale et addictions : de l’intime au populationnel - Maria Melchior (2025)
    1
    1. tanemika 10 Dec 2025

      Santé Mentale et Addictions : De l'Intime au Populationnel

      Résumé Exécutif

      Ce document de synthèse analyse les thèmes centraux de la leçon inaugurale de Maria Melchior, épidémiologiste et titulaire de la chaire Santé Publique 2025-2026 au Collège de France.

      La santé mentale, désignée grande cause nationale pour 2025 et 2026, est présentée comme un défi majeur qui nécessite une double approche : une compréhension empathique de la souffrance intime et une analyse rigoureuse des dynamiques populationnelles.

      L'épidémiologie offre un regard distancié mais essentiel pour quantifier l'ampleur du phénomène, identifier les facteurs de risque et éclairer les politiques publiques.

      Les données révèlent une prévalence élevée en France : un adulte sur dix souffre de dépression ou d'anxiété, et une part significative de la population, y compris les jeunes, est touchée par des conduites addictives (tabac, alcool, cannabis, mais aussi jeux et internet).

      Un constat central est celui des inégalités sociales "massives" qui se manifestent dès l'enfance, creusant un fossé entre les populations défavorisées, plus à risque et ayant moins accès aux soins, et les plus privilégiées.

      L'étude de la santé mentale se heurte à des défis de taille, notamment une forte stigmatisation persistante dans la société et des difficultés métrologiques dues à l'absence de marqueurs biologiques objectifs.

      La stratégie de santé publique la plus efficace, selon le "paradoxe de la prévention" de Geoffrey Rose, ne consiste pas uniquement à cibler les individus les plus à risque, mais à améliorer la santé mentale de l'ensemble de la population en agissant sur les déterminants sociaux.

      Le concept d' "universalisme proportionné" affine cette approche en combinant des actions universelles avec un soutien renforcé pour les groupes les plus vulnérables.

      En conclusion, l'amélioration de la santé mentale collective passe par des interventions qui dépassent le système de soins pour s'attaquer aux racines du mal-être : l'isolement, les inégalités sociales, et les conditions de vie et de travail.

      --------------------------------------------------------------------------------

      1. Le Double Regard sur la Santé Mentale : Intime et Populationnel

      L'analyse de la santé mentale exige une articulation constante entre la souffrance individuelle et les dynamiques collectives. L'épidémiologie, bien que centrée sur l'étude des populations, ne peut ignorer la dimension subjective et intime du mal-être psychique.

      L'Impératif de l'Empathie : L'Intime Derrière les Chiffres

      Maria Melchior insiste sur la nécessité de ne jamais oublier que "derrière les concepts, les théories et les chiffres, il y a de vraies personnes et des histoires singulières".

      Cette prise de conscience, issue d'une expérience personnelle durant ses études de psychologie, souligne que toute démarche de recherche sur la santé mentale doit conserver une forme d'empathie et s'interroger sur le vécu des personnes concernées.

      S'intéresser à la santé mentale, même à grande échelle, requiert d'imaginer une personne réelle et ce qui se passe en elle.

      L'Approche Épidémiologique : Monter en Généralité

      L'épidémiologie se distingue par sa démarche observationnelle et intégrative.

      Elle ne se limite pas aux mécanismes biologiques, mais englobe une large gamme de facteurs de risque : psychologiques, médicaux, comportementaux, sociaux et économiques.

      • Objectif : Identifier les facteurs qui augmentent ou diminuent le risque de troubles psychiques et d'addictions à l'échelle d'une population.

      • Méthode : Mettre en place des enquêtes de grande ampleur pour dégager des tendances concernant les variations de risque dans le temps, l'espace et entre les sous-groupes.

      • Finalité : Passer de situations particulières à des points communs pour "monter en généralité" et identifier les forces qui régissent les comportements humains. Les chiffres produits peuvent ainsi éclairer les politiques publiques et, en retour, aider à mieux saisir des situations individuelles.

      2. Panorama de la Santé Mentale et des Addictions en France

      Les grandes enquêtes épidémiologiques menées en France, notamment par Santé publique France et l'Observatoire français des drogues et des tendances addictives (OFDT), permettent de dresser un tableau précis de la prévalence des troubles psychiques et des addictions.

      Population Cible

      Trouble / Addiction

      Statistique Clé et Source

      Adultes

      Épisode dépressif caractérisé

      1 personne sur 10 (Baromètre SPF, 2021)

      États anxieux

      1 personne sur 10 (Baromètre SPF, 2021)

      Consommation d'alcool à risque

      Plus d'1 personne sur 5

      Consommation de cannabis (année)

      1 personne sur 10

      Tabagisme quotidien

      1 personne sur 4 (taux en baisse)

      Toute population

      Addiction comportementale (jeux d'argent)

      1 personne sur 10 a un comportement problématique (OFDT, 2023)

      Adolescents

      Risque de dépression (modéré à sévère)

      14 % des collégiens, 15 % des lycéens

      (17 ans)

      Usage excessif des réseaux sociaux

      1 jeune sur 5 (ESCAPADE, 2017)

      (17 ans)

      Jeux d'argent et de hasard (année)

      1/3 des jeunes de 17 ans, bien qu'interdit aux mineurs (ESCAPADE)

      Enfants

      Trouble probable de la santé mentale

      13 % des enfants (Étude Enabee, 2002)

      Les addictions comportementales, notamment liées à l'usage d'internet (réseaux sociaux, jeux vidéo) et aux jeux d'argent en ligne, sont un phénomène en hausse, particulièrement chez les jeunes.

      3. Facteurs de Risque et Inégalités Sociales Massives

      L'épidémiologie permet d'identifier des groupes plus vulnérables et des facteurs de risque spécifiques.

      • Différences de genre : Les filles et les femmes présentent des niveaux plus élevés de dépression et d'anxiété, tandis que les garçons et les hommes sont plus touchés par les troubles du comportement, l'hyperactivité/inattention et les conduites addictives.

      • Inégalités sociales : Qualifiées de "massives", elles apparaissent dès l'enfance et se creusent avec le temps. Les enfants issus des familles et des quartiers les plus défavorisés ont les risques les plus élevés tout en ayant l'accès aux soins le plus faible.

      Un rapport de la Cour des comptes de 2023 illustre cette disparité : le recours aux soins en pédopsychiatrie est deux fois plus élevé à Paris qu'en Seine-Saint-Denis.

      • Facteurs environnementaux : De nouvelles recherches explorent l'impact de facteurs comme l'absence d'espaces verts ou l'exposition aux nuisances sonores sur la santé mentale.

      4. Les Défis de l'Étude de la Santé Mentale

      Étudier la santé mentale présente des obstacles uniques, tant sur le plan social qu'éthique et méthodologique.

      La Stigmatisation et la Peur

      Les troubles psychiques continuent de faire peur et d'être associés à des représentations négatives.

      • Dangerosité perçue : 74 % des personnes interrogées en 2014 estimaient que les "malades mentaux" sont dangereux.

      • Discrimination : Dans un sondage de 2023, 80 % des personnes estiment qu'avoir un trouble psychique réduit les opportunités de trouver un emploi ou un logement, et 63 % pensent que les personnes concernées sont moins bien traitées dans le système éducatif ou au travail.

      Les Enjeux Éthiques de la Recherche

      La nature intime de la santé mentale suscite des questionnements éthiques fréquents dans la recherche.

      La crainte principale est que poser des questions sur la souffrance psychique, et notamment sur les pensées suicidaires, pourrait inciter à un passage à l'acte.

      Cependant, la science invalide cette crainte :

      "De méta-analyses [...] montrent qu'interroger des personnes [...] sur leurs pensées ou sur leurs intentions suicidaires non seulement n'entraîne pas de passage à l'acte mais n'est pas non plus perçu de manière négative et pourrait même parfois être associé à une légère diminution des comportements suicidaires."

      L'Exemple de la Cohorte Tempo

      L'étude de cohorte Tempo, qui suit plus de 1000 personnes depuis l'enfance jusqu'à l'âge adulte, illustre la faisabilité et la richesse de la recherche longitudinale en santé mentale.

      • Originalité : C'est l'une des rares études au monde à disposer de données sur trois générations (les participants, leurs parents via la cohorte Gazel, et bientôt leurs propres enfants), permettant d'étudier la transmission intergénérationnelle.

      • Résultats clés :

      ◦ Le trouble de l'hyperactivité/inattention (TDAH) de l'enfance persiste sur près de 30 ans et est associé à des conduites addictives, des difficultés scolaires et un risque de chômage accru.   

      ◦ La consommation de cannabis à l'adolescence a des effets délétères sur le parcours scolaire et professionnel 20 ans plus tard.   

      ◦ La consommation ponctuelle importante d'alcool à l'adolescence prédit un trouble de l'usage à l'âge adulte dans 25 % des cas.

      5. La Mesure en Santé Mentale : De la Subjectivité à la Catégorisation

      L'un des plus grands défis de l'épidémiologie psychiatrique est la mesure des troubles.

      L'Absence de "Gold Standard" Biologique

      Contrairement à de nombreuses maladies, il n'existe pas de test biologique (sanguin, cérébral) pour diagnostiquer un trouble psychique.

      L'évaluation repose entièrement sur la parole et le comportement rapportés par les personnes, ce qui introduit une part d'incertitude.

      L'Évolution des Classifications (DSM/CIM)

      Pour standardiser l'évaluation, des classifications ont été développées.

      • Historique : Les premières nosographies (Pinel, Kraepelin) se concentraient sur les pathologies les plus sévères observées en asile.

      • Le tournant du DSM : La nécessité d'évaluer les conscrits américains lors des guerres mondiales a accéléré le développement de manuels standardisés.

      Une révolution a eu lieu dans les années 1970 sous l'égide de Robert Spitzer : le Diagnostic and Statistical Manual (DSM) est passé d'une approche basée sur les causes psychanalytiques (difficiles à observer) à une définition basée sur des symptômes observables et leurs répercussions sur la vie des personnes.

      • Conséquence : Cette approche a rendu possible la création de questionnaires standardisés, pierre angulaire de l'épidémiologie psychiatrique moderne.

      Définir le "Normal" et le "Pathologique"

      Selon la réflexion du philosophe Georges Canguilhem, un état n'est pas pathologique simplement parce qu'il est statistiquement rare ou jugé négativement par la société (l'exemple de l'homosexualité, autrefois listée comme un trouble mental, en est une illustration frappante).

      La définition moderne d'un état pathologique se centre sur la souffrance psychique exprimée par la personne et l'impact négatif des symptômes sur sa vie.

      6. La Perspective de Santé Publique : Stratégies et Paradoxes

      La santé publique considère que les caractéristiques d'une population influencent en retour la santé de chaque individu qui la compose.

      Le Paradoxe de la Prévention et l'Universalisme Proportionné

      • Le Paradoxe de Geoffrey Rose : Les maladies et leurs facteurs de risque se distribuent sur un continuum dans la population.

      Par conséquent, la stratégie de prévention la plus efficace ne consiste pas à cibler uniquement les quelques individus à très haut risque, mais à décaler légèrement la distribution de l'ensemble de la population.

      Autrement dit, une petite amélioration de la santé mentale de tous a un impact collectif plus grand qu'une grande amélioration pour quelques-uns.

      • L'Universalisme Proportionné de Michael Marmot : Cette approche moderne combine la vision populationnelle de Rose avec une attention particulière pour les plus vulnérables.

      Il s'agit de mettre en place des actions universelles bénéfiques à tous, tout en modulant l'intensité de l'aide en fonction des besoins. Le programme Improva de promotion de la santé mentale dans les collèges en est un exemple.

      L'Importance des Symptômes "Intermédiaires"

      Le fardeau sociétal le plus lourd n'est pas le fait des cas les plus sévères (qui sont peu nombreux), mais de la masse de personnes présentant des symptômes intermédiaires ou "infracliniques".

      Même sans correspondre à un diagnostic formel, ces symptômes causent de la souffrance et altèrent significativement la qualité de vie, la capacité à travailler ou à nouer des liens.

      7. Conclusion et Perspectives d'Action

      Pour améliorer la santé mentale de la population, il est impératif d'agir sur ses déterminants, qui se situent en grande partie en dehors du système de santé.

      • Agir sur les déterminants sociaux : Suivant les travaux d'Émile Durkheim sur l'isolement et de Lisa Berkman sur les réseaux sociaux, il est crucial d'améliorer la densité et la qualité des liens relationnels.

      Cela passe par une action sur leurs causes profondes : les inégalités sociales, les conditions de travail, l'accès au logement et les politiques de protection des familles.

      • La Grande Cause Nationale 2025-2026 : Cet engagement politique vise à améliorer les perceptions collectives des troubles psychiques pour faciliter l'accès aux soins et réduire la stigmatisation.

      • Améliorer la littératie en santé mentale : La diffusion à grande échelle des connaissances issues de la recherche épidémiologique est fondamentale pour que chacun puisse mieux reconnaître les signes de mal-être (chez soi ou chez les autres) et accepter les personnes qui souffrent.

      Collège de France webinaire conférence vidéo santé mentale 2025
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  20. courses.ecu.edu.au courses.ecu.edu.au
    Discussing AI Use Transparently : Curio
    1
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  21. courses.ecu.edu.au courses.ecu.edu.au
    Topic: Exploring bias in AI
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  22. polcompball.wiki polcompball.wiki
    Neoreactionaryism - Polcompball Wiki
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  23. Local file Local file
    Untitled document
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    1. simransen 10 Dec 2025
      As a result, I have much to unlearn as a biologist.

      This correlates with Sarah Ahmed' feminist killjoy, the homework to unlearn everything, including the sciences; this is the works of becoming a feminist (Ahmed 2016).

    2. simransen 10 Dec 2025
      Linnaean“marriage of plants” produced modern reproductive biology and its battle ofthe sexes.

      I came back to this after reading the disability and paragraph and the authors' prospective at the end. I realize that scientists like this are ignorant on understanding plants. He clearly lacked the initiative to study plants and instead plastered his perception of plants based on societal expectations.

    3. simransen 10 Dec 2025
      They cannot move, and yet they can do so much! Thelanguage of movement and ableism is striking in the plant literature,

      I notice a common pattern from sex to disability, where plants are always humanized when being studied.

    4. simransen 10 Dec 2025
      obility is a mindset of theable-bodied human as prototype, and in built worlds that restrict rather thaninclude.

      How can we correlate ableism to plants?

    5. simransen 10 Dec 2025
      In detailing why and how plants have sex, we mustask whether plants actually have sex. Is sex, modeled around human reproduc-tion and its embrangled histories, the best term for what plants do?

      I was always very confused about this as well. How do plants and animals without mammal genital get involved in "sex"? Why is their reproduction always sexualized?

    7. simransen 10 Dec 2025
      For example, howdid the tumbleweed, a foreign and indeed invasive plant, become an icon ofthe American West? Why are some plants reviled and others celebrated?

      This reminds me of the invasive species of the European Pine trees placed in Palestine during the Nakba. The Israeli forces planted pine trees while occupying Palestinian villages to replicate the European infrastructures, this is a biological warfare of colonialism (Josephson 2025). https://origins.osu.edu/read/environmental-nakba-israel-palestine-water

    8. simransen 10 Dec 2025
      Recent efforts of digitization and decolonization have done little toalleviate colonial legacies. Colonial-era practices endure

      With the rise of anti-South Asian sentiment, South Asian countries are posted on social media with negative criticism of the polluted rivers, and littered streets; media consumers use this as an excuse to dehumanize South Asian people. But South Asian countries are lack the studies and sources for environmental work, as well as the politics involved, lobbyed by the BJP right wing Indian party and the Trump administration.

    9. simransen 10 Dec 2025
      Incontrast, Africa and Asia herbaria house far fewer specimens than are collectedthere. Of the specimens with digital images, 80 percent are held by Europeanand North American institutions,

      The author explains that botany has only been properly studied at Europe and North American institutions, compared to the rest of the world such as Africa and Asia where specimens are barely discovered.

    10. simransen 10 Dec 2025
      The questions are central to our embrangled histories. We travel theLinnaean labyrinth in five pa

      Banu's introduction is very metaphorical to the term Labyrinth, making her book very enaging for readers, especially non-stem students like myself.

    11. simransen 10 Dec 2025
      But whatever the name, the same histories andissues persist.

      Science is often very exclusive to stem students, or its western epistemologies as Harding explains is only comprehensible from a anglo saxon male perspective. Banu encurages botany to be accessible to everyone, making learning and findings unlimted, and creating that change of feminist science.

    12. simransen 10 Dec 2025
      I have retained the term botany, but you caneasily substitute newer terms like plant sciences or plant biology

      Banu reassures to her readers that botany and this book is for everyone to read, which is why she is inclusive with these scientific terms.

    13. simransen 10 Dec 2025
      Both queer and disability studies have blossomed into ecological thought.Queer and trans ecologies have pushed for a more expansive understandingof the world in terms of rethinking ethics and multispecies entanglements.

      This challenges Linnaeus notion of the male and female genitalia of plants, and his concept and time of the sex reproduction of plants correlating with the nuclear family tradition. If scientists like Linnaeus could not comprehend the difference of time with the growth of plants, they would be labeled as weird, or the way human beings are labelled, "queer", a term now reclaimed by the queer community.

    14. simransen 10 Dec 2025
      After all, plants are forever forced intohuman time for science and commerce—botany, agriculture, horticulture, andplant biotechnologies.

      I appreciate how Banu correlates the growth and sciences of a plant to queer theory. She pulls apart the definition of queer as not only a homosexual term but something does not align the strict labels and frameworks that human beings apply. She explains in a way that nature and plants are queer in itself if humans wanted to label it.

    15. simransen 10 Dec 2025
      The bookis inspired by multiplicity, hybridity, interdisciplinarity—epistemologies andmethodologies drawn from many disciplines, multiple methods to engagewith the plant world, and multiple genres of writing.

      The author explains decolonization to not be a simple process, and they previously mention how colonization was a huge project. For that reason, we need to approach colonization with variaety of other resources also affected, creating a bigger alternative project.

    16. simransen 10 Dec 2025
      My main goals are threefold: explore how botany was shaped by colonial-ism; demonstrate how that history endures in contemporary botany; and askhow we might undo these legacies to imagine an interdisciplinary and coun-tercolonial botany that is less anthropocentric and more empirically attunedto plant worlds

      This is the author's thesis to challenging the colonial science and overturning it with feminist science and botany.

    17. simransen 10 Dec 2025
      Histories of care work remain deeply feminized and racialized

      This reminds me of a conversation I had in my sociology class about hate crime and racial discrimination in the healthcare systems. How the demographics of nurses are BIPOC and women, and have faced tremendous racism and sexism at workplace during the COVID-19 pandemic.

    18. simransen 10 Dec 2025
      Under“the medical model,” disabled and queer bodies were pathologized as lesser,deviant, and undesirable, with profound consequences.

      How the colonial mindset truly affected the kinship and families in South Asia, leaving countries like India truly displaced and underesourced. How this broke families apart by not believing in disabilities and making children feel less valuable in a competitized society, catching up to the first world countries.

    19. simransen 10 Dec 2025
      Lost, forgotten, and erased are the genealo-gies of women of color feminists, indigenous feminists, and postcolonial, dias-poric, crip, queer, and trans feminists, who have always written more syncreticsymbiotic stories that do not privilege the “human.

      These are the multitude of genres the author speaks on, to taking epistemology at a radical stance.

    20. simransen 10 Dec 2025
      I take an epistemologically radical stance.I offer a multitude of genres—from disciplinary forms of articles and essays, toautobiographical and biographical entries, memoir, manifesto, fables, fiction,and speculative fabulations

      The author encourages to expand epistemology across variety of metholodologies to find more findings, implications and overall increase the studies of the botany field.

    21. simransen 10 Dec 2025
      As Lorde remindsus, we must celebrate difference by attending to our shared histories

      The author challenges Linnaeus concept of labeling human beings and living things from a negative perception, the way that hethinks. Lorde is used here to explain that studying plants can involve celebrating their differences.

    22. simransen 10 Dec 2025
      I wantto create bodies and landscapes without centers and peripheries and withouthierarchical ordering

      The author answers their question so beautifully at the very end of this paragraph. Instead decentering the human out of plants, the author visualized a space where humans, living things and plants exist without a hierarchy, an eco-cosmipolitanism, the idea that all humans, animals, and living things are members of a single community.

    23. simransen 10 Dec 2025
      nature is consistently gendered feminine (for example, “mother nature”), bi-ology has persistently shaped the workings of nature as masculine and patri-archal—nature red in tooth and claw.

      In what way nature is considered feminine as mother nature? What are "the maternal instincts" of nature that are constructed by the patriarchy to call something mother nature?

    24. simransen 10 Dec 2025
      Botany wasin the forefront of debates on female education, and writings in the eighteenthcentury reveal an “ambivalence in the process of the feminization of botany.”5

      This is the kind of feminist epistemology that Hardings encourages in her reading about the feminist research method.

    25. simransen 10 Dec 2025
      Linnaeus’s nuptaiae plantarum (or the marriageof plants) opened up a polyandrous and polygynous sexual imagination wheremultiple husbands and wives were housed in flowers.

      I find it quite pathetic how easily people sexualize objects and living things and I cannot understand how that works, but I see the influence of scientists like Linnaeus encouraging this type of objectification in scientific studies. This reminds me of Paasnonen's concept of objectification, where people and things simply exist to be objectified, and that is due to the cultural dynamics and social constructions of a society.

    26. simransen 10 Dec 2025
      He organized plants and flowers around an anthropo-morphic imagery and in sexual binaries—male and female. In flowers, stamensbecame male and husbands, and pistils became female and wives; fertilizationwas likened to husbands and wives on their nuptial flower bed consummating asexual union and marriage.

      The author beings with a strong evidence of the sexism uprooted in the plant biology of the classification of species. This correlates with Mulvey's concept of phallocentrism, where the attraction of a woman is centred by the male genital. In the sense of this reading. Linnaeus has labeled plants based on human anatomy, aligned with social contructions of rigid gender roles.

    27. simransen 10 Dec 2025
      As I hope to show in this book, plant biology poorly captures the richness of

      The author's main point of this chapter and this book is to highlight the colonial epistemology and influence on plant biology and how it lacks accuracy on the study of plants. The author recommends different epistemologies, especially the field of botany and how it is beneficial for the study of plants, also encouraging social justice. (p.1-2).

    Annotators

  24. nmoer.pressbooks.pub nmoer.pressbooks.pub
    Chapter 10: Thesis Development
    3
    1. linda_perea 10 Dec 2025
      A thesis is not your paper’s topic, but rather your interpretation of the question or subject.

      This is your interpretation. It is up to you to support it with evidence provided to you by the author. This is your time to take a stance on your argument.

    2. linda_perea 10 Dec 2025
      Consider placing the thesis toward the bottom of your introduction. This allows you a few sentences to introduce the concept and prepare the reader for your purpose.

      make sure to place your thesis at the bottom of your first paragraph as well as restate your thesis near the end. Me personally this helps me keep track of the argument as I am writing my thesis.

    3. linda_perea 10 Dec 2025
      A thesis statement is an argumentative central claim in a paper; the entire paper is focused on demonstrating that claim as a valid perspective.

      A thesis statement is an argument that YOU must support with evidence was well as an explanation for how this evidence is relevant to your argument. This is a claim made by you with the help of an author. Make sure that your argument is valid as well as relevant to the prompt of your essay.

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    nmoer.pressbooks.pub/english1101/chapter/chapter-8-thesis-development-mytext-cnm/
  25. publish.obsidian.md publish.obsidian.md
    Welcome to World History 1! - World History 1 - The Ancient and Medieval World - Obsidian Publish
    3
    1. JosieTalaro2008 10 Dec 2025
      These texts were either freshly translated or distributed in Greek, in printed books. And for people who couldn't afford all the new books, the new availability of inexpensive paper spurred an explosion of notebooks called zibaldoni, in which regular people wrote down excerpts of books they had read, things they had heard, or discoveries they had made themselves

      It is cool how they either translated or distributed the books in Greek. Also, for the ones who could not afford those books they had less expensive options for them.

    2. JosieTalaro2008 10 Dec 2025
      Zheng He’s first expedition left China in July 1405 with 62 large ships, over 200 smaller ships, and 28,000 soldiers. The largest ships were 425 feet long, over six times the length of the 65-foot caravels the Spanish and Portuguese would use on their explorations nearly a century later.

      This is also very interesting. Even at this time, they had could make ships that were over 400 feet long. It is also intriguing Zheng left with over 200 small ships and 28,000 soldiers. This is a lot!

    3. JosieTalaro2008 10 Dec 2025
      After gaining control of the fabled wealth of the Delhi Sultans, Timur stripped the city of not only gold and jewels, but of architects, masons, and other artisans whom he took back to Samarkand to build monuments.

      This is interesting that Timur stripped the city of all jewels, gold, masons, etc. I feel like this is a huge act to do.

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    publish.obsidian.md/worldhistory1/Chapter+2/2.1+-+River+Valleys+and+Cultural+Change
  26. rocio-hernandez.quarto.pub rocio-hernandez.quarto.pub
    4  Métodos numéricos y optimización con restricciones tipo caja – Inundaciones
    5
    1. yofre 10 Dec 2025
      En la gráfica de log10∥∇Ωf(xk)∥ vs iteraciones se observa: GD con pendiente suave (decadencia lineal lenta), Newton–CG con caída abrupta tras pocas iteraciones, L-BFGS-B con comportamiento cuasi-superlineal desde etapas medias, y BFGS similar a L-BFGS-B en reducción teórica pero con violaciones de cotas en la trayectoria.

      misma situación

    2. yofre 10 Dec 2025
      Dado que el Hessiano contiene términos μ/(xi−1)2 que crecen cuando xi→1+, el problema es efectivamente mal condicionado cerca de la frontera. L-BFGS-B tolera bien esta condición gracias a la proyección y al escalado implícito. El método Newton–CG también es capaz de manejar esta situación, ya que no requiere formar explícitamente el Hessiano, sino únicamente productos del tipo ∇2f(x)v hessiano-vector, los cuales pueden evaluarse eficientemente incluso en problemas de gran escala. Sin embargo, su desempeño depende críticamente de una implementación robusta del producto hessiano-vector y, en problemas altamente mal condicionados, de un precondicionador adecuado

      misma situación

    3. yofre 10 Dec 2025
      L-BFGS-B y los métodos proyectados garantizan factibilidad en todas las iteraciones; BFGS sin restricciones produce iteraciones infactibles (p. ej. x3(5)=0.98<1) y por tanto es inadecuado en aplicaciones con restricciones físicas ineludibles.

      sobra el circulo porque solo es una frse no tres como antes

    5. yofre 10 Dec 2025
      matriz de precondicionamiento isotrópica, como la identidad, no refleja adecuadamente la geometría local del problema

      woow ... con calma ... que significa que una matriz es isotrópica? lo controatio a anisotrópica? que tiene que ver esta noción con la geometría local del problema?

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    rocio-hernandez.quarto.pub/inundaciones/chapters/metodos.html
  27. courses.ecu.edu.au courses.ecu.edu.au
    Topic: ECU systems and support
    2
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    courses.ecu.edu.au/courses/64678/discussion_topics/812674
  28. rocio-hernandez.quarto.pub rocio-hernandez.quarto.pub
    3  Fundamentación Teórica y Formulación Matemática del Modelo – Inundaciones
    8
    1. yofre 10 Dec 2025
      - ψ(0)=0, - ψ es estrictamente creciente y convexa, - ψ∈C1(R>0), - ψ′(0+)=1 (normalización que permite interpretar βC como el costo marginal inicial por unidad de faltante).

      coloca bien el entorno itemize

    2. yofre 10 Dec 2025
      Paso 1: Solución EOQ

      quita los pasos ... mejor coloca: el tamaño óptimo del pedido es. titulo paso 2 sobra, el paso 3 intégralo al inicio del párrafo

    3. yofre 10 Dec 2025
      - D~∼Normal(μD=1000,σD=300), - c=20 unidades monetarias/unidad, - h=2 unidades monetarias/unidad, - p=25 unidades monetarias/unidad (penalización lineal), - K=1000 unidades monetarias (costo fijo “administrativo” asignado arbitrariamente).

      mal entorno itemize

    7. yofre 10 Dec 2025
      - μD=(800+1600)/2=1200, - σD2=(1600−800)2/12=640000/12≈53333.33, - σD≈53333.33≈230.94.

      quita los guiones ... es muy de IA ... preenta estos valores de corrido

    8. yofre 10 Dec 2025
      Paso 1: Expresar Z(Q) por tramos

      quita lo de pasos ... se vé muy IA ... para rescatra los títulos, haz lo siguiente: Al final del paso 1 pones lo que permite expresar a Z(Q) por tramos ... los otros pasos sobran. quita el paso 4 y coloca: lo anterior nos permite interpretar ...

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    rocio-hernandez.quarto.pub/inundaciones/chapters/Teoria.html
  29. courses.ecu.edu.au courses.ecu.edu.au
    ECU Systems & Support : Curio
    1
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    courses.ecu.edu.au/courses/64678/pages/ecu-systems-and-support
  30. www.dzmm.io www.dzmm.io
    DZMM
    1
    1. oninashi 10 Dec 2025
      • artist:rhasta, best quality, realistic, from below, 1female, white and lace negligee, pussy, erect_clitoris, rosy_labia, pantyhose, nipple_slip, mature, housewife, shy, sweaty, futanari, squat, chastity cage
      • artist:rhasta, best quality, realistic, from below, 1female, solo, white and lace negligee, pussy, erect_clitoris, rosy_labia, pantyhose, nipple_slip, mature, housewife, shy, sweaty, futanari, squatting, (rosy areola:1.5), one-handed support, handjob, squirting
      • artist:rhasta, best quality, realistic, from below, 1female, white and lace nightdress, pantyhose, see-through pantyhose, puffy_nipples, nipple_slip, mature, sweat, (futanari:1.5), hair ornament, squatting, pussy, erect_clitoris, rosy_labia
      提示词 RST
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    dzmm.io/draw/generate/create
  31. 10.2.91.75:8002 10.2.91.75:8002
    OR2025 Developer Track: A year of Hybrid ML/AI cataloging aid in Archipelago Commons
    1
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    10.2.91.75:8002/ops/en/preprint/download/1/1/1
  32. courses.ecu.edu.au courses.ecu.edu.au
    Responsible Use of AI : Curio
    1
    1. nicjohnston 10 Dec 2025
      AI gives you a list of journal articles to use in your research. Two references look unfamiliar, but you include them without checking. What is the best description of this behaviour?

      It's more likely students will ask AI a question on a research topic and within the answer false references will emerge. Student then need to verify them. It's also unlikely students would look at a list of references and then not recognise a few. Likely they would not recognise any if they haven't done any searching of journals. All of this practice is dishonest, not just potentially. Add link to Library evaluating outputs page. https://ecu.au.libguides.com/generative-ai/critical-assessment

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    courses.ecu.edu.au/courses/64679/pages/responsible-use-of-ai
  33. mubi.com mubi.com
    The Last Samurai: A Conversation with Takeshi Kitano
    1
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    mubi.com/es/notebook/posts/the-last-samurai-a-conversation-with-takeshi-kitano
  34. www.forbes.com www.forbes.com
    The Next Frontier For AI Is The Human Brain
    1
    1. Jakemorrill 09 Dec 2025
      Whenever you use your brain to do anything—think a thought, read a book, speak a sentence, move your arm—detectable physical events take place inside your brain in certain patterns. Specifically, information flows through your brain’s neurons via tiny pulses of electricity: the same basic physical force that powers lightbulbs and kitchen appliances and iPhones. These tiny electrical signals trigger other physical activities in your brain as well, including changes in magnetic fields and blood flow.

      This seems like a poor caricature of how the brain works...

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    forbes.com/sites/robtoews/2025/12/07/the-next-frontier-for-ai-is-the-human-brain/
  35. www.biorxiv.org www.biorxiv.org
    Time-resolved phylogenomics analysis reveals patterns in biosphere nutrient limitation through Earth history
    1
    1. austinhpatton 09 Dec 2025
      Habitat reconstruction results were based on much larger trees within each phylum (beyond those shown on the tree), so habitat reconstruction results were superimposed onto this tree for visualization purposes.

      I suspect/hope you did these ancestral state reconstructions on time-calibrated phylogenies? If so, I would just clarify this here given these methods are only described in the supplement, and this methodological detail will have strong impacts on their outcome, as it does not make sense to conduct ASR on non-time-calibrated trees.

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    biorxiv.org/content/10.1101/2025.11.13.688358v1
  36. python-textbook.pythonhumanities.com python-textbook.pythonhumanities.com
    3.3. Creating Rules-Based Pipeline for Holocaust Documents — Introduction to Python for Humanists
    1
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    python-textbook.pythonhumanities.com/03_spacy/03_03_04_building_holocaust_pipeline_rules_based.html
  37. nmoer.pressbooks.pub nmoer.pressbooks.pub
    Chapter 18: Reflection Writing
    3
    1. Livialoya 09 Dec 2025
      he best way to begin a reflection is with an open mind so you can develop a thoughtful response

      make sure to always think about your reflection and have an open mind .

    2. Livialoya 09 Dec 2025
      Reflection writing, specifically reflecting on your own writing process, is a common assignment in English courses because it encourages you to think through and evaluate the strengths and weaknesses

      the reflection writing process lets you get better understanding of your strengths in writing and weaknesses.

    3. Livialoya 09 Dec 2025
      You can write a reflection to help you develop an idea, think about an experience, consider the impact of your actions or choices, illustrate your understanding of a concept, or reflect on a moment

      reflection lets you look at your writing and better get a full picture of what you wrote.

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    nmoer.pressbooks.pub/english1101/chapter/chapter-13-reflection-writing-mytext-cnm/
  38. www.youtube.com www.youtube.com
    Journée du climat - Lundi 8 décembre matin
    1
    1. tanemika 09 Dec 2025

      Document d'information : Enjeux et Perspectives de la Transition Climatique et Énergétique

      Résumé Exécutif

      Ce document synthétise les analyses et les perspectives issues de la Journée du Climat organisée à Le Mans Université, dix ans après les Accords de Paris.

      Il met en lumière une réalité complexe : si des progrès notables ont été accomplis, les grands objectifs climatiques mondiaux demeurent hors d'atteinte.

      Les émissions de CO2 continuent d'augmenter à l'échelle planétaire, et la consommation d'énergies fossiles atteint des niveaux records, principalement en raison de la croissance des marchés asiatiques.

      Dans ce contexte, la France représente un cas singulier, avec un mix électrique déjà largement décarboné grâce au nucléaire et aux énergies renouvelables.

      Cependant, le pays fait face à un paradoxe majeur : alors que la consommation réelle d'électricité est en baisse depuis 2017, la politique énergétique nationale prévoit une augmentation massive de la capacité de production. Cette divergence crée un risque de surproduction, de perturbation du marché et de tensions sur le réseau électrique et le parc nucléaire.

      La transition énergétique induit également de nouvelles dépendances stratégiques, notamment vis-à-vis des minéraux critiques pour les batteries, les panneaux solaires et les éoliennes, dont le raffinage est massivement contrôlé par la Chine.

      La technologie des batteries, pilier de la décarbonation des transports et du stockage des énergies renouvelables, est au cœur de ces enjeux.

      L'Europe peine à établir une chaîne de valeur souveraine, comme en témoigne l'échec de projets d'envergure.

      Des innovations de rupture, telles que les batteries sodium-ion développées en France, et l'intégration de diagnostics avancés ("batteries intelligentes") offrent des perspectives prometteuses pour améliorer la durabilité et la performance.

      Enfin, l'efficacité de la transition repose sur son ancrage territorial.

      Les stratégies doivent intégrer les services écosystémiques (comme le carbone bleu), encourager l'implication citoyenne (via les communautés énergétiques) et repenser la gouvernance.

      Les approches descendantes, qu'il s'agisse de réglementations européennes ou des négociations climatiques mondiales (COP), montrent leurs limites en peinant à intégrer les réalités et les aspirations locales, soulignant l'impératif d'une concertation plus juste et inclusive.

      --------------------------------------------------------------------------------

      1. La Transition Énergétique : Entre Progrès et Réalités Incontournables

      La transition énergétique constitue le défi central de la lutte contre le changement climatique.

      L'analyse présentée par Marc Fontecave, Professeur au Collège de France, dresse un tableau lucide de la situation, soulignant les écarts entre les ambitions affichées et les dynamiques réelles.

      1.1. Un Bilan Mondial Contrasté et des Objectifs Hors d'Atteinte

      La première observation est sans appel : les objectifs fixés lors des Accords de Paris ne seront pas atteints.

      • Objectifs manqués : L'ambition de limiter le réchauffement à 1,5°C d'ici 2100 et d'atteindre la neutralité carbone en 2050 est désormais considérée comme "relativement inatteignable".

      • Hausse des émissions : Les émissions mondiales de CO2 continuent leur progression.

      Le rythme d'augmentation en 2024 est comparable à celui des dix années précédentes. Cette hausse est principalement tirée par les marchés asiatiques en croissance rapide, notamment l'Inde.

      • Dépendance fossile record : Loin de diminuer, la consommation mondiale de charbon, de pétrole et de gaz naturel n'a jamais été aussi élevée.

      Les projections indiquent une augmentation continue des capacités mondiales de charbon et une demande record pour le pétrole en 2025.

      • Un fossé persistant : Un écart se creuse entre les connaissances scientifiques, les déclarations politiques et les actions concrètes.

      Bien que l'Europe et la France voient leurs émissions territoriales diminuer, ce chiffre doit être nuancé.

      Pour la France, une part importante de cette baisse est attribuée à une désindustrialisation continue.

      L'empreinte carbone du pays, qui inclut les émissions liées aux importations, ne baisse pratiquement pas.

      1.2. La Singularité du Cas Français

      La France se distingue par une situation énergétique particulière qui en fait un cas d'étude à part.

      • Forte électrification : Avec 25-27 % d'électricité dans sa consommation énergétique totale, la France est l'un des pays les plus électrifiés au monde.

      • Électricité très décarbonée : La production électrique française est à 95 % bas-carbone, ce qui place la dépendance du pays aux énergies fossiles juste en dessous de 60 %, une performance bien meilleure que la moyenne mondiale.

      • Facture fossile : Cette dépendance représente néanmoins une facture considérable, s'élevant en moyenne à 60 milliards d'euros par an pour l'importation d'hydrocarbures.

      Les trois piliers de la transition énergétique pour la France sont :

      1. La diminution de la consommation : Tous les scénarios, y compris la feuille de route gouvernementale, prévoient une baisse drastique de la consommation d'énergie, de 1500 TWh actuellement à moins de 1000 TWh.

      2. L'électrification des usages : Pour sortir des fossiles, il est nécessaire d'électrifier massivement les transports (véhicules électriques), le chauffage (pompes à chaleur) et l'industrie (fours électriques, hydrogène vert).

      L'électrification directe est privilégiée pour son efficacité énergétique supérieure.

      3. Le recours au carbone et à la chaleur non fossiles : Pour les usages non électrifiables, des alternatives comme la biomasse (bois, biocarburants), la géothermie et les biogaz sont nécessaires, bien qu'elles présentent des limites (gisements, compétition avec l'alimentaire, empreinte carbone).

      1.3. Le Paradoxe de la Consommation et de la Production Électrique

      L'analyse de la production et de la consommation électrique en France révèle une divergence préoccupante.

      État des lieux de la production électrique française (Données 2024)

      Indicateur

      Valeur

      Commentaire

      Production totale

      ~540 TWh

      La France est le premier pays exportateur d'électricité en Europe.

      Part du nucléaire

      ~360 TWh

      Socle du mix, assurant près de 70 % de la production.

      Production bas-carbone

      95 %

      Niveau le plus élevé depuis 1950.

      Part des fossiles

      3,6 %

      Niveau le plus bas depuis 1950.

      Intensité carbone

      21 g CO2/kWh

      Parmi les plus basses du monde (vs. ~360 g CO2/kWh en Allemagne).

      La politique nucléaire a connu un changement majeur, passant d'un projet de fermeture de réacteurs à la décision d'en construire 14 nouveaux (6 confirmés, 8 en option).

      La capacité des réacteurs français à moduler leur production ("pilotabilité") est un atout stratégique pour équilibrer le réseau.

      Le paradoxe identifié est le suivant :

      • Une consommation en baisse : Contrairement aux projections, la consommation d'électricité en France diminue depuis 2017 pour atteindre en 2024 son niveau de 2004.

      Cette baisse s'explique par l'efficacité énergétique, les prix élevés, la sobriété, la désindustrialisation et une électrification des usages plus lente que prévu.

      • Une production planifiée en forte hausse : La feuille de route du gouvernement, basée sur des scénarios de consommation désormais obsolètes (projections RTE 2021/2023), prévoit une augmentation de la production de près de 200 TWh, principalement via l'éolien et le solaire.

      • Les risques associés : Cette décorrélation pourrait mener à une surproduction structurelle, perturbant gravement le marché, nécessitant une modulation excessive et techniquement risquée du parc nucléaire, et créant des tensions sur les réseaux électriques.

      De nouveaux scénarios de consommation revus à la baisse par RTE sont attendus pour corriger cette trajectoire.

      1.4. Nouvelles Dépendances et Impératifs de Recherche

      La transition énergétique, si elle réduit la dépendance aux fossiles, en crée de nouvelles.

      • Dépendance aux minéraux : La production de batteries, d'éoliennes et de panneaux solaires nécessite une quantité croissante de ressources minérales (graphite, lithium, cobalt, cuivre, etc.).

      Le raffinage de ces matériaux est très largement dominé par la Chine, créant une nouvelle dépendance géopolitique.

      • Maturité technologique : De nombreuses technologies clés ne sont pas encore matures et nécessitent des efforts de recherche et d'innovation considérables.

      Cela inclut la production d'hydrogène vert, le recyclage des matériaux, l'amélioration des rendements photovoltaïques, le développement de mines responsables, la décarbonation de l'industrie lourde (acier), la valorisation de la biomasse, le nucléaire de 4ème génération, la modernisation des réseaux et le stockage d'énergie.

      --------------------------------------------------------------------------------

      2. Le Stockage Électrochimique : Pilier Technologique de la Décarbonation

      Les batteries sont au cœur de la transition, essentielles pour la mobilité électrique et pour stabiliser les réseaux face à l'intermittence des énergies renouvelables.

      La conférence de Jean-Marie Tarascon, Professeur au Collège de France, a mis en évidence les avancées, les défis et les innovations de ce secteur stratégique.

      2.1. L'Ascension des Batteries et le Défi de la Souveraineté Européenne

      Le stockage électrochimique est en passe de devenir la forme dominante de stockage d'énergie, dépassant le stockage hydroélectrique.

      • Marchés en plein essor : La demande est tirée par trois secteurs majeurs : le véhicule électrique (50 % des ventes mondiales prévues en 2030), le stockage stationnaire pour les énergies renouvelables, et les drones.

      • Les Gigafactories : Pour répondre à cette demande, des usines de très grande capacité se construisent dans le monde.

      L'Europe, avec plus de 20 projets dont 6 en France, tente d'acquérir sa souveraineté, visant 19 % de la production mondiale en 2029.

      • Le manque de chaîne de valeur : L'Europe reste massivement dépendante, important 98 % des machines d'assemblage et une part similaire des matériaux.

      L'échec du projet suédois Northvolt, qui visait une intégration verticale complète sans maîtriser toute la chaîne de valeur, illustre cette fragilité. La proposition de créer un "Airbus des batteries" pour fédérer les compétences se heurte aux réticences des acteurs à collaborer.

      2.2. Innovations et Matériaux d'Avenir

      La recherche scientifique est la clé pour surmonter les dépendances et améliorer les performances.

      • Du NMC au LFP : Dans le lithium-ion, la technologie dominante des véhicules électriques évolue.

      Les matériaux NMC (Nickel-Manganèse-Cobalt) à haute densité énergétique cèdent du terrain aux matériaux LFP (Lithium-Fer-Phosphate), qui sont moins chers, plus sûrs et ne contiennent pas de cobalt.

      Cependant, la production de LFP est contrôlée à 88 % par la Chine.

      • La technologie Sodium-ion : Portée en France par la start-up Tiamat, cette technologie représente une alternative stratégique.

      Le sodium est 10 000 fois plus abondant que le lithium.

      Bien que moins denses en énergie, les batteries sodium-ion offrent une puissance supérieure, une durée de vie exceptionnelle (jusqu'à 17 000 cycles) et un coût potentiellement plus faible.

      Elles sont idéales pour le stockage stationnaire (ex: data centers) et la mobilité légère.

      • Vers le tout-solide et les batteries intelligentes :

      La recherche s'oriente vers les batteries "tout-solide", qui remplacent l'électrolyte liquide par un solide pour plus de sécurité et de densité énergétique, bien que des défis d'interface persistent.

      Une autre innovation majeure est l'intégration de capteurs (fibres optiques) au cœur des batteries pour en suivre l'état de santé en temps réel (température, pression, chimie).

      Ce "passeport de santé" permettra d'optimiser leur usage, de faciliter leur seconde vie et de développer des systèmes d'auto-réparation.

      2.3. Enjeux de Durabilité : Écocompatibilité et Recyclage

      La durabilité des batteries est un enjeu aussi important que leur performance.

      • Pression sur les ressources :

      Un véhicule électrique utilise six fois plus de minéraux qu'un véhicule thermique.

      La demande en lithium, cobalt et nickel pourrait dépasser la production d'ici 2030.

      L'exploitation de nouvelles ressources, y compris en Europe (comme le lithium en France), et surtout le développement du recyclage ("mine urbaine") sont impératifs.

      • Réglementation européenne : L'UE met en place un cadre strict imposant la déclaration de l'empreinte carbone, des taux de matériaux recyclés obligatoires (dès 2030) et un passeport électronique pour chaque batterie.

      • Recherche sur le recyclage : Les méthodes actuelles (pyrométallurgie, hydrométallurgie) sont énergivores.

      L'un des objectifs de la recherche est de concevoir des batteries "de type Lego", facilement démontables pour un recyclage ciblé de leurs composants.

      --------------------------------------------------------------------------------

      3. L'Ancrage Territorial : Clé de Voûte d'une Transition Juste et Efficace

      La réussite de la transition climatique ne peut être décrétée d'en haut ; elle doit s'incarner dans les territoires, en tenant compte de leurs spécificités géographiques, sociales et économiques.

      3.1. Des Territoires aux Stratégies Plurielles

      Les approches locales varient considérablement, reflétant la diversité des enjeux.

      • Plans Climat-Air-Énergie Territoriaux (PCAET) : L'analyse des PCAET dans l'Ouest de la France montre un foisonnement d'initiatives.

      Si l'atténuation (mitigation) est un axe commun, les notions d'adaptation et de résilience sont traitées de manière inégale, la résilience étant plus prégnante dans les territoires littoraux directement menacés.

      • Rôle des écosystèmes : Les écosystèmes locaux sont des alliés pour la neutralité carbone.

      Les zones humides littorales, par exemple, stockent massivement du carbone ("carbone bleu") tout en fournissant d'autres services essentiels comme la protection contre les inondations.

      • Controverses du "Rewilding" : Les stratégies de restauration, comme le réensauvagement, peuvent générer des conflits.

      Laisser des écosystèmes évoluer librement ou réintroduire de grands animaux se heurte aux paysages culturels et agricoles européens, créant des tensions sur les usages et des chocs de valeurs.

      Le succès de telles approches dépend fondamentalement de l'inclusion et de la concertation avec les populations locales.

      3.2. L'Énergie Citoyenne et les Nouvelles Mobilités

      L'implication des citoyens est un levier puissant pour accélérer la transition.

      • Communautés énergétiques citoyennes : Des collectifs de citoyens émergent pour produire et consommer localement de l'énergie renouvelable.

      Ces initiatives favorisent l'appropriation locale des enjeux, contribuent à la justice énergétique et permettent de lutter contre la précarité.

      L'Ouest de la France est une région particulièrement dynamique, accueillant près d'un quart des projets citoyens nationaux.

      • Décarboner les mobilités : Le secteur des transports représente 31 % des émissions de CO2 en France, les trajets domicile-travail en voiture comptant pour une part significative (13 % du total national).

      Comprendre les facteurs (individuels, contextuels, normes sociales) qui influencent le choix du mode de transport est essentiel pour concevoir des politiques publiques efficaces favorisant les mobilités douces.

      3.3. Gouvernance Globale et Concertation : Les Limites du Modèle Actuel

      L'articulation entre les décisions locales, nationales et internationales reste un point de friction majeur.

      • Approches descendantes : Des réglementations comme celle de l'UE sur la déforestation importée, bien qu'intentionnées, peuvent être perçues comme unilatérales et impérialistes par les pays producteurs, qui se tournent vers d'autres marchés moins regardants.

      De même, dans certains pays comme Haïti, les plans climatiques sont souvent impulsés par des acteurs internationaux et déconnectés des réalités du terrain.

      • Le défi des COP : Les négociations climatiques mondiales, comme la COP30 au Brésil, peinent à intégrer de manière authentique la voix des populations locales et des peuples autochtones.

      Leurs préoccupations sont souvent diluées dans un langage diplomatique visant le consensus, ce qui conduit à une forme de décision à deux vitesses et pousse les groupes non entendus à s'auto-organiser en marge des processus officiels.

      L'enjeu est de traduire les aspirations des territoires en politiques internationales concrètes et justes.

      durable 2025 conférence Collège de France webinaire environnement écologie vidéo
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    Enhancer-driven random gene overexpression (ERGO): a method to study gene function in Chlamydomonas
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    1. ray.futia 09 Dec 2025
      Enhancer-driven random gene overexpression (ERGO): a method to study gene function in Chlamydomonas

      Your enhancer-insertion library is a useful tool for probing carotenoid regulation, and the CMRP1 follow-up is both convincing and compelling. The long-range activity of the enhancer in your top hit is intriguing and raises a few questions about how ERGO should be interpreted.

      If an insertion can influence genes across ~2 Mb, then many nearby loci are plausible targets. How confident are you that CMRP1 is the primary driver rather than one member of a broader set of co-activated genes? More generally, because NHEJ insertions favor open and insertion-tolerant regions, regulators positioned in less permissive chromatin may never be sampled. Insertions that disrupt essential genes, or essential neighboring genes, would also eliminate the corresponding clones before screening, also impacting sampling. Do you have a sense of how much of the genome is protected in this way? Along those lines, have you looked at whether enhancer effectiveness varies with chromatin context, and whether some genomic regions tend to dampen or block enhancer activity?

      Did you characterize the expression of neighboring genes at all to distinguish between CMRP1-driven changes and insertion-related ones? Given that many insertions are tandem or structurally complex, did you assess whether enhancer copy number, truncation, or orientation contributed to the expression patterns or phenotypes you observed?

      Finally, the use of ERGO here implements a pigment phenotype in the yellow-in-the-dark background. Do you envision pairing the enhancer library with non-colorimetric reporters or selectable screens to expand beyond carotenoid metabolism in the future?

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