10,000 Matching Annotations
  1. Jul 2025
  2. 52.53.155.43 52.53.155.43
    41598_2019_38663_MOESM1_ESM-html.html
    134
    Visit annotations in context

    Tags

    Annotators

    URL

    52.53.155.43/9daa870b1bca8d8022e93e84971b51a673dd3574/41598_2019_38663_MOESM1_ESM-html.html
  3. 52.53.155.43 52.53.155.43
    1-s2.0-S2589004225013914-main-html.html
    1
    Visit annotations in context

    Tags

    Annotators

    URL

    52.53.155.43/90007a778f462697c048df1da24432bb37ed810f/1-s2.0-S2589004225013914-main-html.html
  4. pmc.ncbi.nlm.nih.gov pmc.ncbi.nlm.nih.gov
    Parent-of-origin regulation by maternal auts2 shapes neurodevelopment and behavior in fish
    2
    Visit annotations in context

    Tags

    Annotators

    URL

    pmc.ncbi.nlm.nih.gov/articles/PMC12063280/
  5. pmc.ncbi.nlm.nih.gov pmc.ncbi.nlm.nih.gov
    SOX10-Mediated Regulation of Enteric Glial Phenotype in vitro and its Relevance for Neuroinflammatory Disorders
    1
    Visit annotations in context

    Tags

    Annotators

    URL

    pmc.ncbi.nlm.nih.gov/articles/PMC11845537/
  6. pmc.ncbi.nlm.nih.gov pmc.ncbi.nlm.nih.gov
    Characterization of the adaptive immune response in a mouse model for HPV-positive head and neck squamous cell carcinoma with implications to human disease
    1
    Visit annotations in context

    Tags

    Annotators

    URL

    pmc.ncbi.nlm.nih.gov/articles/PMC11698698/
  7. www.biorxiv.org www.biorxiv.org
    Conserved and Unique Features of Terminal Telomeric Sequences in ALT-Positive Cancer Cells
    1
    1. Public_Reviews 16 Jul 2025

      Author Response:

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

      Reviewer #1 (Recommendations for the authors): 

      One minor question would be whether the authors could expand more on the application of END-Seq to examine the processive steps of the ALT mechanism? Can they speculate if the ssDNA detected in ALT cells might be an intermediate generated during BIR (i.e., is the ssDNA displaced strand during BIR) or a lesion? Furthermore, have the authors assessed whether ssDNA lesions are due to the loss of ATRX or DAXX, either of which can be mutated in the ALT setting?

      We appreciate the reviewer’s insightful questions regarding the application of our assays to investigate the nature of the ssDNA detected in ALT telomeres. Our primary aim in this study was to establish the utility of END-seq and S1-END-seq in telomere biology and to demonstrate their applicability across both ALT-positive and -negative contexts. We agree that exploring the mechanistic origins of ssDNA would be highly informative, and we anticipate that END-seq–based approaches will be well suited for such future studies. However, it remains unclear whether the resolution of S1-END-seq is sufficient to capture transient intermediates such as those generated during BIR. We have now included a brief speculative statement in the revised discussion addressing the potential nature of ssDNA at telomeres in ALT cells.

      Reviewer #2 (Recommendations for the authors):

      How can we be sure that all telomeres are equally represented? The authors seem to assume that END-seq captures all chromosome ends equally, but can we be certain of this? While I do not see an obvious way to resolve this experimentally, I recommend discussing this potential bias more extensively in the manuscript.

      We thank the reviewer for raising this important point. END-seq and S1-END-seq are unbiased methods designed to capture either double-stranded or single-stranded DNA that can be converted into blunt-ended double-stranded DNA and ligated to a capture oligo. As such, if a subset of telomeres cannot be processed using this approach, it is possible that these telomeres may be underrepresented or lost. However, to our knowledge, there are no proposed telomeric structures that would prevent capture using this method. For example, even if a subset of telomeres possesses a 5′ overhang, it would still be captured by END-seq. Indeed, we observed the consistent presence of the 5′-ATC motif across multiple cell lines and species (human, mouse, and dog). More importantly, we detected predictable and significant changes in sequence composition when telomere ends were experimentally altered, either in vivo (via POT1 depletion) or in vitro (via T7 exonuclease treatment). Together, these findings support the robustness of the method in capturing a representative and dynamic view of telomeres across different systems.

      That said, we have now included a brief statement in the revised discussion acknowledging that we cannot fully exclude the possibility that a subset of telomeres may be missed due to unusual or uncharacterized structures

      I believe Figures 1 and 2 should be merged.

      We appreciate the reviewer’s suggestion to merge Figures 1 and 2. However, we feel that keeping them as separate figures better preserves the logical flow of the manuscript and allows the validation of END-seq and its application to be presented with appropriate clarity and focus. We hope the reviewer agrees that this layout enhances the clarity and interpretability of the data.

      Scale bars should be added to all microscopy figures.

      We thank the reviewer for pointing this out. We have now added scale bars to all the microscopy panels in the figures and included the scale details in the figure legends.

      Reviewer #3 (Recommendations for the authors):

      Overall, the discussion section is lacking depth and should be expanded and a few additional experiments should be performed to clarify the results.

      We thank the reviewer for the suggestions. Based on this reviewer’s comments and comments for the other reviewers, we incorporated several points into the discussion. As a result, we hope that we provide additional depth to our conclusions.

      (1) The finding that the abundance of variant telomeric repeats (VTRs) within the final 30 nucleotides of the telomeric 5' ends is similar in both telomerase-expressing and ALT cells is intriguing, but the authors do not address this result. Could the authors provide more insight into this observation and suggest potential explanations? As the frequency of VTRs does not seem to be upregulated in POT1-depleted cells, what then drives the appearance of VTRs on the C-strand at the very end of telomeres? Is CST-Pola complex responsible?

      The reviewer raises a very interesting and relevant point. We are hesitant at this point to speculate on why we do not see a difference in variant repeats in ALT versus non-ALT cells, since additional data would be needed. One possibility is that variant repeats in ALT cells accumulate stochastically within telomeres but are selected against when they are present at the terminal portion of chromosome ends. However, to prove this hypothesis, we would need error-free long-read technology combined with END-seq. We feel that developing this approach would be beyond the scope of this manuscript.

      (2) The authors also note that, in ALT cells, the frequency of VTRs in the first 30 nucleotides of the S1-END-SEQ reads is higher compared to END-SEQ, but this finding is not discussed either. Do the authors think that the presence of ssDNA regions is associated with the VTRs? Along this line, what is the frequency of VTRs in the END-SEQ analysis of TRF1-FokI-expressing ALT cells? Is it also increased? Has TRF1-FokI been applied to telomerase-expressing cells to compare VTR frequencies at internal sites between ALT and telomerase-expressing cells?

      Similarly to what is discussed above, short reads have the advantage of being very accurate but do not provide sufficient length to establish the relative frequency of VTRs across the whole telomere sequence. The TRF1-FokI experiment is a good suggestion, but it would still be biased toward non-variant repeats due to the TRF1-binding properties. We plan to address these questions in a future study involving long-read sequencing and END-seq capture of telomeres.

      Finally, in these experiments (S1-END-SEQ or END-SEQ in TRF1-Fok1), is the frequency of VTRs the same on both the C- and the G-rich strands? It is possible that the sequences are not fully complementary in regions where G4 structures form.

      We thank the reviewer for this observation. While we do observe a higher frequency of variant telomeric repeats (VTRs) in the first 30 nucleotides of S1-END-seq reads compared to END-seq in ALT cells, we are currently unable to determine whether this difference is significant, as an appropriate control or matched normalization strategy for this comparison is lacking. Therefore, we refrain from overinterpreting the biological relevance of this observation.

      The reviewer is absolutely correct. Our calculation did not exclude the possibility of extrachromosomal DNA as a source of telomeric ssDNA. We have now addressed this point in our discussion.

      The reviewer is correct in pointing out that we still do not know what causes ssDNA at telomeres in ALT cells. Replication stress seems the most logical explanation based on the work of many labs in the field. However, our data did not reveal any significant difference in the levels of ssDNA at telomeres in non-ALT cells based on telomere length. We used the HeLa1.2.11 cell line (now clarified in the Materials section), which is the parental line of HeLa1.3 and has similarly long telomeres (~20 kb vs. ~23 kb). Despite their long telomeres and potential for replication-associated challenges such as G-quadruplex formation, HeLa1.2.11 cells did not exhibit the elevated levels of telomeric ssDNA that we observed in ALT cells (Figure 4B). Additional experiments are needed to map the occurrence of ssDNA at telomeres in relation to progression toward ALT.

      (3) Based on the ratio of C-rich to G-rich reads in the S1-END-SEQ experiment, the authors estimate that ALT cells contain at least 3-5 ssDNA regions per chromosome end. While the calculation is understandable, this number could be discussed further to consider the possibility that the observed ratios (of roughly 0.5) might result from the presence of extrachromosomal DNA species, such as C-circles. The observed increase in the ratio of C-rich to G-rich reads in BLM-depleted cells supports this hypothesis, as BLM depletion suppresses C-circle formation in U2OS cells. To test this, the authors should examine the impact of POLD3 depletion on the C-rich/G-rich read ratio. Alternatively, they could separate high-molecular-weight (HMW) DNA from low-molecular-weight DNA in ALT cells and repeat the S1-END-SEQ in the HMW fraction.

      The reviewer is absolutely correct. Our calculation did not exclude the possibility of extrachromosomal DNA as a source of telomeric ssDNA. We have now addressed this point in our discussion.

      (4) What is the authors' perspective on the presence of ssDNA at ALT telomeres? Do they attribute this to replication stress? It would be helpful for the authors to repeat the S1-END-SEQ in telomerase-expressing cells with very long telomeres, such as HeLa1.3 cells, to determine if ssDNA is a specific feature of ALT cells or a result of replication stress. The increased abundance of G4 structures at telomeres in HeLa1.3 cells (as shown in J. Wong's lab) may indicate that replication stress is a factor. Similar to Wong's work, it would be valuable to compare the C-rich/G-rich read ratios in HeLa1.3 cells to those in ALT cells with similar telomeric DNA content.

      The reviewer is correct in pointing out that we still do not know what causes ssDNA at telomeres in ALT cells. Replication stress seems the most logical explanation based on the work of many labs in the field. However, our data did not reveal any significant difference in the levels of ssDNA at telomeres in non-ALT cells based on telomere length. We used the HeLa1.2.11 cell line (now clarified in the Materials section), which is the parental line of HeLa1.3 and has similarly long telomeres (~20 kb vs. ~23 kb). Despite their long telomeres and potential for replication-associated challenges such as G-quadruplex formation, HeLa1.2.11 cells did not exhibit the elevated levels of telomeric ssDNA that we observed in ALT cells (Figure 4B). Additional experiments are needed to map the occurrence of ssDNA at telomeres in relation to progression toward ALT.

      Finally, Reviewer #3 raises a list of minor points:

      (1) The Y-axes of Figure 4 have been relabeled to account for the G-strand reads.

      (2) Statistical analyses have been added to the figures where applicable.

      (3) The manuscript has been carefully proofread to improve clarity and consistency throughout the text and figure legends

      (4) We have revised the text to address issues related to the lack of cross-referencing between the supplementary figures and their corresponding legends.

      AuthorResponse
    Visit annotations in context

    Tags

    Annotators

    URL

    biorxiv.org/content/10.1101/2025.02.27.640565v2
  8. www.biorxiv.org www.biorxiv.org
    Molecular and Mechanical Signatures Contributing to Epidermal Differentiation and Barrier Formation
    1
    1. Public_Reviews 16 Jul 2025

      Author Response:

      The following is the authors’ response to the original reviews

      Reviewer #1 (Public review): 

      Summary: 

      The authors address a fundamental question for cell and tissue biology using the skin epidermis as a paradigm and ask how stratifying self-renewing epithelia induce differentiation and upward migration in basal dividing progenitor cells to generate suprabasal barrier-forming cells that are essential for a functional barrier formed by such an epithelium. The authors show for the first time that an increase in intracellular actomyosin contractility, a hallmark of barrier-forming keratinocytes, is sufficient to trigger terminal differentiation. Hence the data provide in vivo evidence of the more general interdependency of cell mechanics and differentiation. The data appear to be of high quality and the evidences are strengthened through a combination of different genetic mouse models, RNA sequencing, and immunofluorescence analysis. 

      To generate and maintain the multilayered, barrier-forming epidermis, keratinocytes of the basal stem cell layer differentiate and move suprabasally accompanied by stepwise changes not only in gene expression but also in cell morphology, mechanics, and cell position. Whether any of these changes is instructive for differentiation itself and whether consecutive changes in differentiation are required remains unclear. Also, there are few comprehensive data sets on the exact changes in gene expression between different states of keratinocyte differentiation. In this study, through genetic fluorescence labeling of cell states at different developmental time points the authors were able to analyze gene expression of basal stem cells and suprabasal differentiated cells at two different stages of maturation: E14 (embryonic day 14) when the epidermis comprises mostly two functional compartments (basal stem cells and suprabasal socalled intermediate cells) and E16 when the epidermis comprise three (living) compartments where the spinous layer separates basal stem cells from the barrier-forming granular layer, as is the case in adult epidermis. Using RNA bulk sequencing, the authors developed useful new markers for suprabasal stages of differentiation like MafB and Cox1. The transcription factor MafB was then shown to inhibit suprabasal proliferation in a MafB transgenic model. 

      The data indicate that early in development at E14 the suprabasal intermediate cells resemble in terms of RNA expression, the barrier-forming granular layer at E16, suggesting that keratinocytes can undergo either stepwise (E16) or more direct (E14) terminal differentiation. 

      Previous studies by several groups found an increased actomyosin contractility in the barrierforming granular layer and showed that this increase in tension is important for epidermal barrier formation and function. However, it was not clear whether contractility itself serves as an instructive signal for differentiation. To address this question, the authors use a previously published model to induce premature hypercontractility in the spinous layer by using spastin overexpression (K10-Spastin) to disrupt microtubules (MT) thereby indirectly inducing actomyosin contractility. A second model activates myosin contractility more directly through overexpression of a constitutively active RhoA GEF (K10-Arhgef11CA). Both models induce late differentiation of suprabasal keratinocytes regardless of the suprabasal position in either spinous or granular layer indicating that increased contractility is key to induce late differentiation of granular cells. A potential weakness of the K10-spastin model is the disruption of MT as the primary effect which secondarily causes hypercontractility. However, their previous publications provided some evidence that the effect on differentiation is driven by the increase in contractility (Ning et al. cell stem cell 2021). Moreover, the data are confirmed by the second model directly activating myosin through RhoA. These previous publications already indicated a role for contractility in differentiation but were focused on early differentiation. The data in this manuscript focus on the regulation of late differentiation in barrier-forming cells. These important data help to unravel the interdependencies of cell position, mechanical state, and differentiation in the epidermis, suggesting that an increase in cellular contractility in most apical positions within the epidermis can induce terminal differentiation. Importantly the authors show that despite contractility-induced nuclear localization of the mechanoresponsive transcription factor YAP in the barrier-forming granular layer, YAP nuclear localization is not sufficient to drive premature differentiation when forced to the nucleus in the spinous layer. 

      Overall, this is a well-written manuscript and a comprehensive dataset. Only the RNA sequencing result should be presented more transparently providing the full lists of regulated genes instead of presenting just the GO analysis and selected target genes so that this analysis can serve as a useful repository. The authors themselves have profited from and used published datasets of gene expression of the granular cells. Moreover, some of the previous data should be better discussed though. The authors state that forced suprabasal contractility in their mouse models induces the expression of some genes of the epidermal differentiation complex (EDC). However, in their previous publication, the authors showed that major classical EDC genes are actually not regulated like filaggrin and loricrin (Muroyama and Lechler eLife 2017). This should be discussed better and necessitates including the full list of regulated genes to show what exactly is regulated. 

      We thank the reviewers for their suggestions and comments.

      Thank you for the suggestion to include gene lists. We had an excel document with all this data but neglected to upload it with the initial manuscript. This includes all the gene signatures for the different cell compartments across development. We also include a tab that lists all EDC genes and whether they were up-regulated in intermediate cells and cells in which contractility was induced. Further, we note that all the RNA-Seq datasets are available for use on GEO (GSE295753).  

      In our previous publication, we indeed included images showing that loricrin and filaggrin were both still expressed in the differentiated epidermis in the spastin mutant. Both Flg and Lor mRNA were up in the RNA-Seq (although only Flg was statistically significant), though we didn’t see a notable change in protein levels. It is unclear whether this is just difficult to see on top of the normal expression, or whether there are additional levels of regulation where mRNA levels are increased but protein isn’t. That said, our data clearly show that other genes associated with granular fate were increased in the contractile skin. 

      Reviewer #2 (Public review): 

      Summary: 

      The manuscript from Prado-Mantilla and co-workers addresses mechanisms of embryonic epidermis development, focusing on the intermediate layer cells, a transient population of suprabasal cells that contributes to the expansion of the epidermis through proliferation. Using bulk-RNA they show that these cells are transcriptionally distinct from the suprabasal spinous cells and identify specific marker genes for these populations. They then use transgenesis to demonstrate that one of these selected spinous layer-specific markers, the transcription factor MafB is capable of suppressing proliferation in the intermediate layers, providing a potential explanation for the shift of suprabasal cells into a non-proliferative state during development. Further, lineage tracing experiments show that the intermediate cells become granular cells without a spinous layer intermediate. Finally, the authors show that the intermediate layer cells express higher levels of contractility-related genes than spinous layers and overexpression of cytoskeletal regulators accelerates the differentiation of spinous layer cells into granular cells. 

      Overall the manuscript presents a number of interesting observations on the developmental stage-specific identities of suprabasal cells and their differentiation trajectories and points to a potential role of contractility in promoting differentiation of suprabasal cells into granular cells. The precise mechanisms by which MafB suppresses proliferation, how the intermediate cells bypass the spinous layer stage to differentiate into granular cells, and how contractility feeds into these mechanisms remain open. Interestingly, while the mechanosensitive transcription factor YAP appears deferentially active in the two states, it is shown to be downstream rather than upstream of the observed differences in mechanics. 

      Strengths: 

      The authors use a nice combination of RNA sequencing, imaging, lineage tracing, and transgenesis to address the suprabasal to granular layer transition. The imaging is convincing and the biological effects appear robust. The manuscript is clearly written and logical to follow. 

      Weaknesses: 

      While the data overall supports the authors' claims, there are a few minor weaknesses that pertain to the aspect of the role of contractility, The choice of spastin overexpression to modulate contractility is not ideal as spastin has multiple roles in regulating microtubule dynamics and membrane transport which could also be potential mechanisms explaining some of the phenotypes. Use of Arghap11 overexpression mitigates this effect to some extent but overall it would have been more convincing to manipulate myosin activity directly. It would also be important to show that these manipulations increase the levels of F-actin and myosin II as shown for the intermediate layer. It would also be logical to address if further increasing contractility in the intermediate layer would enhance the differentiation of these cells. 

      We agree with the reviewer that the development of additional tools to precisely control myosin activity will be of great use to the field. That said, our series of publications has clearly demonstrated that ablating microtubules results in increased contractility and that this phenocopies the effects of Arhgef11 induced contractility. Further, we showed that these phenotypes were rescued by myosin inhibition with blebbistatin. Our prior publications also showed a clear increase in junctional acto-myosin through expression of either spastin or Arhgef11, as well as increased staining for the tension sensitive epitope of alpha-catenin (alpha18).  We are not aware of tools that allow direct manipulation of myosin activity that currently exist in mouse models.  

      The gene expression analyses are relatively superficial and rely heavily on GO term analyses which are of course informative but do not give the reader a good sense of what kind of genes and transcriptional programs are regulated. It would be useful to show volcano plots or heatmaps of actual gene expression changes as well as to perform additional analyses of for example gene set enrichment and/or transcription factor enrichment analyses to better describe the transcriptional programs 

      We have included an excel document that lists all the gene signatures. In addition, a volcano plot is included in the new Fig 2, Supplement 1. All our NGS data are deposited in GEO for others to perform these analyses. As the paper does not delve further into transcriptional regulation, we do not specifically present this information in the paper.  

      Claims of changes in cell division/proliferation changes are made exclusively by quantifying EdU incorporation. It would be useful to more directly look at mitosis. At minimum Y-axis labels should be changed from "% Dividing cells" to % EdU+ cells to more accurately represent findings 

      We changed the axis label to precisely match our analysis. We note that Figure 1, Supplement 1 also contains data on mitosis.  

      Despite these minor weaknesses the manuscript is overall of high quality, sheds new light on the fundamental mechanisms of epidermal stratification during embryogenesis, and will likely be of interest to the skin research community. 

      Reviewer #3 (Public review): 

      Summary: 

      This is an interesting paper by Lechler and colleagues describing the transcriptomic signature and fate of intermediate cells (ICs), a transient and poorly defined embryonic cell type in the skin. ICs are the first suprabasal cells in the stratifying skin and unlike later-developing suprabasal cells, ICs continue to divide. Using bulk RNA seq to compare ICs to spinous and granular transcriptomes, the authors find that IC-specific gene signatures include hallmarks of granular cells, such as genes involved in lipid metabolism and skin barrier function that are not expressed in spinous cells. ICs were assumed to differentiate into spinous cells, but lineage tracing convincingly shows ICs differentiate directly into granular cells without passing through a spinous intermediate. Rather, basal cells give rise to the first spinous cells. They further show that transcripts associated with contractility are also shared signatures of ICs and granular cells, and overexpression of two contractility inducers (Spastin and ArhGEF-CA) can induce granular and repress spinous gene expression. This contractility-induced granular gene expression does not appear to be mediated by the mechanosensitive transcription factor, Yap. The paper also identifies new markers that distinguish IC and spinous layers and shows the spinous signature gene, MafB, is sufficient to repress proliferation when prematurely expressed in ICs. 

      Strengths: 

      Overall this is a well-executed study, and the data are clearly presented and the findings convincing. It provides an important contribution to the skin field by characterizing the features and fate of ICs, a much-understudied cell type, at high levels of spatial and transcriptomic detail. The conclusions challenge the assumption that ICs are spinous precursors through compelling lineage tracing data. The demonstration that differentiation can be induced by cell contractility is an intriguing finding and adds a growing list of examples where cell mechanics influence gene expression and differentiation. 

      Weaknesses: 

      A weakness of the study is an over-reliance on overexpression and sufficiency experiments to test the contributions of MafB, Yap, and contractility in differentiation. The inclusion of loss-offunction approaches would enable one to determine if, for example, contractility is required for the transition of ICs to granular fate, and whether MafB is required for spinous fate. Second, whether the induction of contractility-associated genes is accompanied by measurable changes in the physical properties or mechanics of the IC and granular layers is not directly shown. The inclusion of physical measurements would bolster the conclusion that mechanics lies upstream of differentiation. 

      We agree that loss of function studies would be useful. For MafB, these have been performed in cultured human keratinocytes, where loss of MafB and its ortholog cMaf results in a phenotype consistent with loss of spinous differentiation (Pajares-Lopez et al, 2015). Due to the complex genetics involved, generating these double mutant mice is beyond the scope of this study. Loss of function studies of myosin are also complicated by genetic redundancy of the non-muscle type II myosin genes, as well as the role for these myosins in cell division and in actin cross linking in addition to contractility. In addition, we have found that these myosins are quite stable in the embryonic intestine, with loss of protein delayed by several days from the induction of recombination. Therefore, elimination of myosins by embryonic day e14.5 with our current drivers is not likely possible. Generation of inducible inhibitors of contractility is therefore a valuable future goal. 

      Several recent papers have used AFM of skin sections to probe tissue stiffness. We have not attempted these studies and are unclear about the spatial resolution and whether, in the very thin epidermis at these stages, we could spatially resolve differences. That said, we previously assessed the macro-contractility of tissues in which myosin activity was induced and demonstrated that there was a significant increase in this over a tissue-wide scale (Ning et al, Cell Stem Cell, 2021).  

      Finally, whether the expression of granular-associated genes in ICs provides them with some sort of barrier function in the embryo is not addressed, so the role of ICs in epidermal development remains unclear. Although not essential to support the conclusions of this study, insights into the function of this transient cell layer would strengthen the overall impact.  

      By traditional dye penetration assays, there is no epidermal barrier at the time that intermediate cells exist. One interpretation of the data is that cells are beginning to express mRNAs (and in some cases, proteins) so that they are able to rapidly generate a barrier as they become granular cells. In addition, many EDC genes, important for keratinocyte cornification and barrier formation, are not upregulated in ICs at E14.5. We have attempted experiments to ablate intermediate cells with DTA expression - these resulted in inefficient and delayed death and thus did not yield strong conclusions about the role of intermediate cells. Our findings that transcriptional regulators of granular differentiation (such as Grhl3 and Hopx) are also present in intermediate cells, should allow future analysis of the effects of their ablation on the earliest stages of granular differentiation from intermediate cells. In fact, previous studies have shown that Grhl3 null mice have disrupted barrier function at embryonic stages (Ting et al, 2005), supporting the role of ICs in being important for barrier formation. (?)

      Recommendations for the authors:

      Reviewer #1 (Recommendations for the authors): 

      Overall, this is a well-written manuscript and a comprehensive dataset. Only the RNA sequencing result should be presented more transparently providing the full lists of regulated genes instead of presenting just the GO analysis and selected target genes so that this analysis can serve as a useful repository. The authors themselves have profited from and used the published dataset of gene expression of the granular cells. Moreover, some of the previous data should be better discussed though. The authors state that forced suprabasal contractility in their mouse models induces the expression of some genes of the epidermal differentiation complex (EDC). However, in their previous publication, the authors showed that major classical EDC genes are actually not regulated like filaggrin and loricrin (Muroyama and Lechler eLife 2017). This should be discussed better and necessitates including the full list of regulated genes to show what exactly is regulated. 

      A general point regarding statistics throughout the manuscript. It seems like regular T-tests or ANOVAs have been used assuming Gaussian distribution for sample sizes below N=5 which is technically not correct. Instead, non-parametric tests like e.g. the Mann-Whitney test should be used. Since Graph-Pad was used for statistics according to the methods this is easy to change. 

      Figure 1: It would be good to show the FACS plot of the analyzed and sorted population in the supplementary figures. 

      If granular cells can be analyzed and detected by FACS, why were they not included in the RNA sequencing analysis? 

      Figure 1 supplement 1c: cell division numbers are analyzed from only 2 mice and the combined 5 or 4 fields of view are used for statistics using a test assuming normal distribution which is not really appropriate. Means per mice should be used or if accumulated field of views are used, the number should be increased using more stringent tests. Otherwise, the p-values here clearly overstate the significance. 

      Granular cells could not be specifically isolated in the approach we used. The lectin binds to both upper spinous and granular cells. For this reason, we relied on a separate granular gene list as described.

      For Figure 1 Supplement 1, we removed the statistical analysis and use it simply as a validation of the data in Figure 1.  

      Figure 2: It is not completely clear on which basis the candidate genes were picked. They are described to be the most enriched but how do they compare to the rest of the enriched genes. The full list of regulated genes should be provided. 

      Some markers for IC or granular layer are verified either by RNA scope or immunofluorescence. Is there a technical reason for that? It would be good to compare protein levels for all markers.  Figure 2-Supplement 1: There is no statement about the number of animals that these images are representative for. 

      We have included a volcano plot to show where the genes picked reside. We have also included the full gene lists for interested readers. 

      When validated antibodies were available, we used them. When they were not, we performed RNA-Scope to validate the RNA-Seq dataset. 

      We have included animal numbers in the revised Fig 2-Supplement 2 legend (previously Fig 2Supplement 1).  

      Figure 4b: It would be good to include the E16 spinous cells to get an idea of how much closer ICs are to the granular population. 

      We have included a new Venn diagram showing the overlap between each of the IC and spinous signatures with the granular cell signature in Fig 4B. Overall, 36% of IC signature genes are in common with granular cells, while just 20% of spinous genes overlap.  

      Reviewer #2 (Recommendations for the authors): 

      (1)  Figure 6B is confusing as y-axis is labeled as EdU+ suprabasal cells whereas basal cells are also quantified. 

      We have altered the y-axis title to make it clearer.  

      (2)  Not clear why HA-control is sometimes included and sometimes not. 

      We include the HA when it did not disrupt visualization of the loss of fluorescence. As it was uniform in most cases, we excluded it for clarity in some images. HA staining is now included in Fig 3C.

      (3)  The authors might reconsider the title as it currently is somewhat vague, to more precisely represent the content of the manuscript. 

      We thank the reviewer for the suggestion. We considered other options but felt that this gave an overview of the breadth of the paper.  

      Reviewer #3 (Recommendations for the authors): 

      (1)  ICs are shown to express Tgm1 and Abca12, important for cornified envelope function and formation of lamellar bodies. Do ICs provide any barrier function at E14.5? 

      By traditional dye penetration assays, there is no epidermal barrier at the time that intermediate cells exist. One interpretation of the data is that cells are beginning to express mRNAs (and in some cases, proteins) so that they are able to rapidly generate a barrier as they become granular cells.  

      (2)  Genes associated with contractility are upregulated in ICs and granular cells. And ICs have higher levels of F-actin, MyoIIA, alpha-18, and nuclear Yap. Does this correspond to a measurable difference in stiffness? Can you use AFM to compare to physical properties of ICs, spinous, and granular cells? 

      Several recent papers have used AFM of skin sections to probe tissue stiDness. We have not attempted these studies and are unclear about the spatial resolution and whether in the very thin epidermis at these stages whether we could spatially resolve diDerences. It is also important to note that this tissue rigidity is influenced by factors other than contractility. That said, we previously assessed the macro-contractility of tissues in which myosin activity was induced and demonstrated that there was a significant increase in this over a tissue-wide scale (Ning et al, Cell Stem Cell, 2021).

      (3)  Overexpression of two contractility inducers (spastin and ArhGEF-CA) can induce granular gene expression and repress spinous gene expression, suggesting differentiation lies downstream of contractility. Is contractility required for granular differentiation? 

      This is an important question and one that we hope to directly address in the future. Published studies have shown defects in tight junction formation and barrier function in myosin II mutants. However, a thorough characterization of differentiation was not performed.  

      (4)  ICs are a transient cell type, and it would be important to know what is the consequence of the epidermis never developing this layer. Does it perform an important temporary structural/barrier role, or patterning information for the skin?

      We have attempted experiments to ablate intermediate cells with DTA expression - this resulted in ineDicient and delayed death and thus did not yield strong conclusions. Our findings that transcriptional regulators of granular diDerentiation (such as Grhl3 and Hopx) are also present in intermediate cells, should allow future analysis of the eDects of their ablation on the earliest stages of granular diDerentiation from intermediate cells.

      AuthorResponse
    Visit annotations in context

    Tags

    Annotators

    URL

    biorxiv.org/content/10.1101/2024.07.23.604736v2
  9. www.sciencedirect.com www.sciencedirect.com
    Rebalancing of mitochondrial homeostasis through an NAD+-SIRT1 pathway preserves intestinal barrier function in severe malnutrition
    1
    Visit annotations in context

    Tags

    Annotators

    URL

    sciencedirect.com/science/article/pii/S2352396423003754
  10. www.sciencedirect.com www.sciencedirect.com
    Ce6 derivative photodynamic therapy triggers PANoptosis and enhances antitumor immunity with LAG3 blockade in cutaneous squamous cell carcinoma
    1
    Visit annotations in context

    Tags

    Annotators

    URL

    sciencedirect.com/science/article/pii/S266637912500312X
  11. ucite-h12o.github.io ucite-h12o.github.io
    Pertenencias - UCITE Matriz
    2
    1. ucite_matriz 16 Jul 2025
      será de 72 horas tras el ingreso, si no ha mediado requerimiento judicial.

      si no está detenido, cuando se cumpla cualquiera de las siguientes condiciones: - 72 horas - hasta el primer contacto con fuerzas de seguridad y que sea indicado por las mismas - hasta que el paciente y la familia lo requiera

    Visit annotations in context

    Annotators

    URL

    ucite-h12o.github.io/matriz/pertenencias/
  12. www.biorxiv.org www.biorxiv.org
    Dimerization and dynamics of angiotensin-I converting enzyme revealed by cryo-EM and MD simulations
    1
    1. Public_Reviews 15 Jul 2025

      Author Response:

      The following is the authors response to the original reviews.

      Reviewer #1 (Public review): 

      Summary: 

      The authors report four cryoEM structures (2.99 to 3.65 Å resolution) of the 180 kDa, full-length, glycosylated, soluble Angiotensin-I converting enzyme (sACE) dimer, with two homologous catalytic domains at the N- and C-terminal ends (ACE-N and ACE-C). ACE is a protease capable of effectively degrading Aβ. The four structures are C2 pseudo-symmetric homodimers and provide insight into sACE dimerization. These structures were obtained using discrete classification in cryoSPARC and show different combinations of open, intermediate, and closed states of the catalytic domains, resulting in varying degrees of solvent accessibility to the active sites. 

      To deepen the understanding of the gradient of heterogeneity (from closed to open states) observed with discrete classification, the authors performed all-atom MD simulations and continuous conformational analysis of cryo-EM data using cryoSPARC 3DVA, cryoDRGN, and RECOVAR. cryoDRGN and cryoSPARC 3DVA revealed coordinated open-closed transitions across four catalytic domains, whereas RECOVAR revealed independent motion of two ACE-N domains, also observed with cryoSPARC-focused classification. The authors suggest that the discrepancy in the results of the different methods for continuous conformational analysis in cryo-EM could result from different approaches used for dimensionality reduction and trajectory generation in these methods. 

      Strengths: 

      This is an important study that shows, for the first time, the structure and the snapshots of the dynamics of the full-length sACE dimer. Moreover, the study highlights the importance of combining insights from different cryo-EM methods that address questions difficult or impossible to tackle experimentally while lacking ground truth for validation. 

      Weaknesses: 

      The open, closed, and intermediate states of ACE-N and ACE-C in the four cryo-EM structures from discrete classification were designated quantitatively (based on measured atomic distances on the models fitted into cryo-EM maps, Figure 2D). Unfortunately, atomic models were not fitted into cryo-EM maps obtained with cryoSPARC 3DVA, cryoDRGN, and RECOVAR, and the open/closed states in these cases were designated based on qualitative analysis. As the authors clearly pointed out, there are many other methods for continuous conformational heterogeneity analysis in cryo-EM. Among these methods, some allow analyzing particle images in terms of atomic models, like MDSPACE (Vuillemot et al., J. Mol. Biol. 2023, 435:167951), which result in one atomic model per particle image and can help in analyzing cooperativity of domain motions through measuring atomic distances or angular differences between different domains (Valimehr et al., Int. J. Mol. Sci. 2024, 25: 3371). This could be discussed in the article. 

      Reviewer #2 (Public review): 

      Summary: 

      The manuscript presents a valuable contribution to the field of ACE structural biology and dynamics by providing the first complete full-length dimeric ACE structure in four distinct states. The study integrates cryo-EM and molecular dynamics simulations to offer important insights into ACE dynamics. The depth of analysis is commendable, and the combination of structural and computational approaches enhances our understanding of the protein's conformational landscape. However, the strength of evidence supporting the conclusions needs refinement, particularly in defining key terms, improving structural validation, and ensuring consistency in data analysis. Addressing these points through major revisions will significantly improve the clarity, rigor, and accessibility of the study to a broader audience, allowing it to make a stronger impact in the field. 

      Strengths: 

      The integration of cryo-EM and MD simulations provides valuable insights into ACE dynamics, showcasing the authors' commitment to exploring complex aspects of protein structure and function. This is a commendable effort, and the depth of analysis is appreciated. 

      Weaknesses: 

      Several aspects of the manuscript require further refinement to improve clarity and scientific rigor as detailed in my recommendations for the authors. 

      Reviewer #3 (Public review): 

      Summary: 

      Mancl et al. report four Cryo-EM structures of glycosylated and soluble Angiotensin-I converting enzyme (sACE) dimer. This moves forward the structural understanding of ACE, as previous analysis yielded partially denatured or individual ACE domains. By performing a heterogeneity analysis, the authors identify three structural conformations (open, intermediate open, and closed) that define the openness of the catalytic chamber and structural features governing the dimerization interface. They show that the dimer interface of soluble ACE consists of an N-terminal glycan and protein-protein interaction region, as well as C-terminal protein-protein interactions. Further heterogeneity mining and all-atom molecular dynamic simulations show structural rearrangements that lead to the opening and closing of the catalytic pocket, which could explain how ACE binds its substrate. These studies could contribute to future drug design targeting the active site or dimerization interface of ACE. 

      Strengths: 

      The authors make significant efforts to address ACE denaturation on cryo-EM grids, testing various buffers and grid preparation techniques. These strategies successfully reduce denaturation and greatly enhance the quality of the structural analysis. The integration of cryoDRGN, 3DVA, RECOVAR, and all-atom simulations for heterogeneity analysis proves to be a powerful approach, further strengthening the overall experimental methodology. 

      Weaknesses: 

      In general, the findings are supported by experimental data, but some experimental details and approaches could be improved. For example, CryoDRGN analysis is limited to the top 5 PCA components for ease of comparison with cryoSPARC 3DVA, but wouldn't an expansion to more components with CryoDRGN potentially identify further conformational states? The authors also say that they performed heterogeneity analysis on both datasets but only show data for one. The results for the first dataset should be shown and can be included in supplementary figures. In addition, the authors mention that they were not successful in performing cryoSPARC 3DFLex analysis, but they do not show their data or describe the conditions they used in the methods section. These data should be added and clearly described in the experimental section. 

      Some cryo-EM data processing details are missing. Please add local resolution maps, box sizes, and Euler angle distributions and reference the initial PDB model used for model building. 

      Reviewer #1 (Recommendations for the authors): <br /> Major point: 

      The authors could discuss the use of continuous conformational heterogeneity analysis methods that analyze particle images in terms of atomic models, based on MD simulations, like MDSPACE (Vuillemot et al., J. Mol. Biol. 2023, 435:167951). MDSPACE can be used on a dataset preprocessed with cryoSPARC or Relion by discrete classification to reduce compositional heterogeneity and obtain initial particle poses. It results in one atomic model per particle image and can help in analyzing the cooperativity of domain motions by measuring atomic distances or angular differences between different domains (Valimehr et al., Int. J. Mol. Sci. 2024, 25: 3371). 

      We agree that MDSPACE is a promising and useful tool for analysis, and are excited to implement such a method. Prior to manuscript submission, we have had discussions with the primary author, Slavica Jonic, about how we may employ her software in our analysis. Unfortunately, we were unable to overcome significant computational issues, notably MDSPACE’s lack of GPU functionality, which prevent us from employing MDSPACE in a reasonable manner for our dataset. We hope to employ MDSPACE in future work, once the computational issues have been addressed, and have added a section on MDSPACE to the discussion in an effort to increase the visibility of MDSPACE, as we feel it is an exciting approach that deserves more visibility. We have added a substantial discussion on this point, specifically on MDspace as follows:

      line 565-574

      Similarly, MDSPACE holds tremendous promise as a method for investigating conformational dynamics from cryo-EM data (61). MDSPACE integrates cryo-EM particle data with short MD simulations to fit atomic models into each particle image through an iterative process which extracts dynamic information. However, the lack of GPU-enabled processing for MDSPACE requires either a dedicated a computational setup that diverges from most other cryo-EM software, or access to a CPU-based supercomputer, which severely limits the accessibility of such software. Despite these challenges, both 3DFlex and MDSPACE use promising approaches to study protein conformational dynamics. We look forward to exploring effective methods to incorporate these strategies into our future research.

      Minor points: 

      (1) Lines 348-350: "The discrepancy in population size between these clusters is likely due to bias in the initial particle poses, rather than a subunit-specific preference for the open state." Which bias? The cluster size is related to conformations, not to poses. 

      We hope to emphasize that the assignment of particles to either the OC or CO cluster is likely due to the particle orientation within the complete dimer refinement, and the discrepancy in size between OC and CO clusters does not necessarily indicate a domain specific preference for one state or another, which would carry allosteric implications. This remains a possibility, but we hope to avoid over-interpretation of our results with the statement above.

      The statement was altered to now read:

      Line 418-423

      “The discrepancy in population size between these clusters is likely due to bias in the initial particle orientation, rather than a subunit-specific preference for the open state. As the O/C state and the C/O state are 180 degree rotations of each other, particle assignment to either cluster is likely influenced by the initial particle orientation of the complete dimer, and we currently lack the data to discern any allosteric implication to the orientation assignment.”

      (2) Line 519: "Micrographs with a max CTF value worse than 4Å were removed from the dataset,..." (also, lines 822-823 in supplementary material). <br /> Do you want to say that micrographs with a resolution worse than 4 A were removed? 

      Max CTF value was replaced with CTF fit resolution to properly match the parameter used in Cryosparc.

      (3) Figure 2C: The black lines are barely visible. Can you make them thicker and in red color? 

      The figure has been amended.

      (4) Figure 2D: The values for Chain A and Chain B in the second row (ACE-C) of sACE-3.05 columns are 17.9 (I) (Chain A) and 13.9 (C) (Chain B). Shouldn't they be reversed (13.9 (C) (Chain A) and 17.9 (I) (Chain B))? 

      The values are now correct. sACE-3.65 chains were flipped in the table, and the updated color scheme should make it easier to map the values from the table to their corresponding structure.

      Reviewer #2 (Recommendations for the authors): 

      The manuscript presents the first complete full-length dimeric ACE structure. The integration of cryo-EM and MD simulations provides valuable insights into ACE dynamics, showcasing the authors' commitment to exploring complex aspects of protein structure and function. This is a commendable effort, and the depth of analysis is appreciated. However, several aspects of the manuscript require further refinement to improve clarity and scientific rigor. In the view of this reviewer, a major revision is necessary. Please see the detailed comments below: 

      (1) Definition of "Conformational Heterogeneity": The term "conformational heterogeneity" should be clearly defined when citing references 27-29. <br /> References 27 and 29 use MD simulations, which reveal "conformational flexibility" rather than "conformational heterogeneity" as observed in cryo-EM data. A more precise distinction should be made. 

      We have changed the term “conformational heterogeneity” to the broader “conformational dynamics

      (2) Figure Adjustments for Clarity: <br /> Figure 1B: A scale bar is needed for accurate representation. 

      A 100 Angstrom scale bar was added to figure 1B.

      Figure 2A, B: Using a Cα trace representation would improve clarity and make structural differences more apparent. 

      We found using a Cα trace representation makes the figure too confusing and impossible to determine individual structural elements. Everything just becomes a jumble of lines.

      Additionally, a Cα displacement vs. residue index plot (with Figure 1A placed along the x-axis) should be included alongside Figures 2A and B to provide quantitative insight into structural variations. 

      This analysis has been combined with several other suggestions and now comprises a new figure 4.

      (3) Structural Resolution and Validation: <br /> Euler angle distribution and 3D-FSC analysis should be provided to help the audience assess how these factors influence the resolution of each structure. <br /> Local resolution analysis in Relion should be included to determine if there are dynamic differences among the four structures. <br /> To enhance structural interpretation, the manuscript would benefit from showcasing examples of bulky side-chain densities (e.g., Trp, Phe, Tyr) for each of the four structures. 

      Information is included in Figure S3 and S5.

      (4) Glycan Modeling Considerations: <br /> Since the resolution of cryo-EM does not allow for precise glycan composition determination, additional experimental validation (e.g., Glyco-MS) would strengthen the modeling. If experimental support is unavailable, appropriate references should be cited to justify the modeled glycans. 

      Minimal glycan modeling was performed with the goal of demonstrating that the protein is glycosylated. We have highlighted that we chose 12 N-linked glycosylation sites that have the observed extra density, an indication that glycan should be present and modeled them with complex glycans in the manuscript.  

      (5) Advanced Cryo-EM and MD Analyses: 3DFlex Analysis: <br /> It is recommended that the authors explore 3DFlex to better capture conformational variability. CryoSPARC's community support can assist in proper implementation. 

      We have incorporated our 3Dflex analysis in our discussion as follows:

      Line 553-565

      Surprisingly, we did not observe such motion using cryoSPARC 3DFlex, a neural network-based method analyzing our cryo-EM data of sACE (54). Central to the working of cryoSPARC 3DFlex is the generation of a tetrahedral mesh used to calculate deformations within the particle population. Proper generation of the mesh is critical for obtaining useful results and must often be determined empirically. Despite several attempts, we were unable to obtain results from 3DFlex comparable to what we observed with our other methods. Even using the results from our 3DVA as prior input to 3DFlex, the largest conformational change we observed was a slight wiggling at the bottom of the D3a subdomain (Movie S12). The authors of 3DFlex note that 3DFlex struggles to model intricate motions, and the implementation of custom tetrahedral meshes currently requires a non-cyclical fusion strategy between mesh segments. Given these limitations, and the complexity of sACE conformational dynamics, it appears that sACE, as a system, is not well-suited to analysis via 3DFlex in its current implementation.

      (6) Movie Consistency: <br /> The MD simulation movies should use the same color coding as the first four movies for consistency. Similarly, the 3DVar analysis map should be color-coded to enhance interpretability. 

      MD simulation movies are re-colored.

      (7) MD Simulations - Data Extraction and Validation: <br /> The manuscript includes several long-timescale MD simulations, but further analysis is needed to extract meaningful dynamic information. Suggested analyses include: <br /> a. RMSF (Root Mean Square Fluctuation) Analysis: Calculate RMSF from MD trajectories and compare it with local resolution variations in cryo-EM maps. 

      RMSF values were included in the new figure 4 along with structural depictions colored by RMSF value to localize variation to the structure.

      b. Assess whether regions exhibiting lower dynamics correspond to higher resolution in cryo-EM. 

      Information is added to Figure 4, Figure S3, S5, S6.

      c. Compare RMSF between simulations with and without glycans to identify potential effects. 

      This has been done in Figure 4.

      d. Clustering Analysis: Use the four solved structures as reference states to cluster MD simulation trajectories. Determine if the population states observed in MD simulations align with cryo-EM findings. 

      This has been done in supplementary figure S10.

      e. Principal Component Analysis (PCA): Perform PCA on MD trajectories and compare with dynamics inferred from cryo-EM analyses (3DVar, cryoDRGN, and RECOVAR) to ensure consistency. 

      This has been done in supplementary figure S11.

      f. Correction of RMSF Analysis or the y-axis label in Figure S9: The RMSF values cannot be negative by definition. The authors should carefully review the code used for this calculation or explicitly define the metric being measured. 

      The Y-axis label has been corrected to clarify that the plot depicts the change in RMSF values when comparing the glycosylated and non-glycosylated MD simulations.

      (8) Discussion on Coordinated Motion and Allostery: <br /> The discussion of coordinated motion and allosteric regulation between sACE-N domains should be explicitly connected to experimental evidence mentioned in the introduction: <br /> "Enzyme kinetics analysis suggests negative cooperativity between two catalytic domains (31-33). However, ACE also exhibits positive synergy toward Ab cleavage and allostery to enhance the activity of its binding partner, the bradykinin receptor (11, 34)." 

      (9) The authors should elaborate on how their new insights provide a mechanistic explanation for these experimental observations. 

      (10) Connection to Therapeutic Implications: <br /> The discussion section should more explicitly connect the structural findings to potential therapeutic applications, which would significantly enhance the impact of the study. 

      These three points (8-10) were addressed in a significant overhaul to the discussion section.

      In summary, this study makes a valuable contribution to the field of ACE structural biology and dynamics. The combination of cryo-EM and MD simulations is particularly powerful, and with major revisions, this manuscript has the potential to make a strong impact. Addressing the points outlined above will significantly improve clarity, strengthen the scientific claims, and enhance the manuscript's accessibility to a broader audience. I appreciate the authors' rigorous approach to this complex topic and encourage them to refine their work to fully highlight the significance of their findings. 

      Reviewer #3 (Recommendations for the authors): 

      (1) The authors incorrectly refer to their ACE construct as full-length throughout the manuscript. Given that they are purifying the soluble region (aa 1-1231), saying full-length ACE is not the correct nomenclature. I suggest removing full-length and using soluble ACE (sACE) throughout the text. 

      We utilize the term full-length to highlight the fact that our structures contain both the N and C domains for both subunits in the dimer, in contrast to the previously published ACE cryo-EM structure. We have clarified in the text that we refer to the full-length soluble region of ACE (sACE), and sACE is used to specifically refer to our construct throughout the text, except when referring to ACE in a more generalized biological context in the introduction and discussion.

      (2) The authors could show differences between the different structural states by measuring and displaying the alpha carbon distances. For example, in Figures 2A, B, 3A, and 4B and C. 

      Alpha carbon displacements for each residue have been added to the new figure 4.

      (3) Most figures, with a few exceptions (Figures 2 and S11), are of low quality. Perhaps they are not saved in the same format. In addition, the color schemes used throughout the figures and movies are not consistent. For example, in Figure 1 D2 domains are in green, while they appear yellow in Figure 2 and later. Please double-check all coloring schemes and keep them consistent throughout the manuscript. In addition, it would be good to keep the labeling of the domains in the subsequent figures, as it is difficult to remember which domain is which throughout the manuscript. 

      We are unsure of how to address the low quality issue, our files and the online versions appear to be of suitable high quality. We will work with editorial staff to ensure all files are of suitable quality. The color scheme has been revised throughout the manuscript to ensure consistency and better differentiate between domains and chains.

      (4) Figure 1. Indicate exactly where in panel A ACE-N ends and ACE-C starts. Also, the pink and magenta, as well as aqua vs. light blue, are hard to distinguish. 

      We have updated coloring scheme.

      (5) Figure 2. In the figure legend, the use of brackets for defining closed, intermediate, and open states is confusing, given that the panels are also described with brackets, and some letters match between them. Using a hyphen or bolding the abbreviations could help. Also, define chains A and B, make the black lines that I assume indicate distances in C bold or thicker as they are very hard to see in the figure, and add to the legend what those lines mean. 

      The abbreviations have been changed from parentheses to quotes, and suggestions have been implemented.

      (6) Figure 4 is confusing as shown. Since the authors mention the general range of motion in sACE-N first in the text, wouldn't it make more sense to show panel B first and then panel A? Also, can you point and label the "tip connecting the two long helices of the D1a subdomain" in the figure? It is not clear to me where this region is in B. In addition, add a description of the arrows in B and C to the figure legend. 

      Most changes incorporated. The order should make more sense now in light of other changes.

      (7) Figure 5. Can the authors add a description to the legend as to what the arrows indicate and their thickness? 

      Done

      (8) Add a scale bar to the micrograph images in the supplementary figures. 

      Figure S2 and S4 need the scale bar.

      (9) Provide a more comprehensive description of buffers used in the DF analysis, as this information could be useful to others. 

      We have included the data in Table S1.<br /> (10) Line 51: Reference format not consistent with other references: (Wu et al., 2023). 

      Fixed

      (11) Line 66: Define "ADAM". 

      The definition has been added.

      (12) Line 90: The authors say: Recent open state structures of sACE-N, sACE monomer, and a sACE-N dimer, along with molecular dynamics (MD) simulations of sACE-C, have begun to reveal the conformational heterogeneity, though it remains under-studied (27-29)." Can the authors clarify what "it" refers to? The full-length ACE, sACE, or its specific domains? 

      The sentence now reads: Recent open state structures of sACE-N, sACE monomer, and a sACE-N dimer, along with molecular dynamics (MD) simulations of sACE-C, have begun to reveal ACE conformational dynamics, though they remain under-studied (29-31).

      (13) Line 204: "The comparison of our dimeric sACE cryoEM structures of reveals the conformational dynamics of sACE catalytic domains." The second "of" should be removed. 

      Fixed<br /> (14) Line 268: "From room mean square fluctuation (RMSF) analysis..." "room" should be replaced with "root."

      Fixed

      AuthorResponse
    Visit annotations in context

    Tags

    Annotators

    URL

    biorxiv.org/content/10.1101/2025.01.09.632263v5
  13. www.nature.com www.nature.com
    Antigen specificity shapes distinct aging trajectories of memory CD8⁺ T cells
    2
    Visit annotations in context

    Tags

    Annotators

    URL

    nature.com/articles/s41467-025-61627-y
  14. www.nature.com www.nature.com
    IFNγ augments TKI efficacy by alleviating protein unfolding stress to promote GSDME-mediated pyroptosis in hepatocellular carcinoma
    31
    Visit annotations in context

    Tags

    Annotators

    URL

    nature.com/articles/s41419-025-07839-y
  15. www.biorxiv.org www.biorxiv.org
    A developmental program of early residency promotes the differentiation of divergent uterine NK cell subsets in humans
    1
    Visit annotations in context

    Tags

    Annotators

    URL

    biorxiv.org/content/10.1101/2025.07.05.663267v1
  16. pmc.ncbi.nlm.nih.gov pmc.ncbi.nlm.nih.gov
    Exploring ID4 as a driver of aggression and a therapeutic target in triple-negative breast cancer
    1
    Visit annotations in context

    Tags

    Annotators

    URL

    pmc.ncbi.nlm.nih.gov/articles/PMC12246117/
  17. www.nature.com www.nature.com
    A divergent astrocytic response to stress alters activity patterns via distinct mechanisms in male and female mice
    1
    Visit annotations in context

    Tags

    Annotators

    URL

    nature.com/articles/s41467-025-61643-y
  18. www.sciencedirect.com www.sciencedirect.com
    Optogenetics-enabled discovery of integrated stress response modulators
    1
    Visit annotations in context

    Tags

    Annotators

    URL

    sciencedirect.com/science/article/pii/S0092867425006907
  19. ucite-h12o.github.io ucite-h12o.github.io
    Pruebas radiologicas bai - UCITE Matriz
    4
    2. ucite_matriz 15 Jul 2025
      *En caso de mujeres embarazadas/edad fértil el manejo diagnóstico es equivalente.
      • Embarazada inestable e inconsciente: igual (TAC). Inestable: TAC, insistiendo a la paciente la importancia de la prueba para mujer y feto.
      • Estable y consciente: se podrá valorar la realización de una ecografía dirigida (+- con contraste) que tenga aceptable definición orgánica, en vez de la TAC.
    3. ucite_matriz 15 Jul 2025
      ecografía
      • Modificación de la imagen para incluir las ventanas del extendido (bases pulmonares y deslizamiento)
      • Generar dos supuestos, el de "ecografía en paciente estable" y el de "FAST en inestable para la toma de decisiones".
      • En qué pacientes repetir la exploración a los pocos minutos (ej pacientes con tiempo prehospitalario corto en el cuál el primero es negativo o con poco líquido para la inestabilidad objetivada)
    4. ucite_matriz 15 Jul 2025
      radiografía de tórax AP

      Plantear el supuesto en el que no se realiza Rx tórax en el BAI (ej paciente estables en los que se va a realizar TAC body/torácico y en los que no se ha realizado ningún procedimiento invasivo torácico).

    Visit annotations in context

    Annotators

    URL

    ucite-h12o.github.io/matriz/pruebas_radiologicas_bai/
  20. accessmedicina.mhmedical.com accessmedicina.mhmedical.com
    Dermatosis bacterianas
    1
    Visit annotations in context

    Annotators

    URL

    accessmedicina.mhmedical.com/content.aspx
  21. www.youtube.com www.youtube.com
    Video: Approches expérimentales en éducation (4) - Agir pour l'éducation (2023-2024) (DocDrop)
    1
    1. tanemika 14 Jul 2025

      Document de Synthèse : L'Importance des Compétences Sociales et Comportementales en Éducation

      Source : Extraits de "Approches expérimentales en éducation (4) - Agir pour l'éducation (2023-2024)" - Conférence d'Élise Huillery.

      Introduction : Un Déficit Français en Compétences Clés

      La conférence d'Élise Huillery, chercheuse affiliée à J-PAL et professeure à Dauphine, met en lumière l'importance croissante des compétences sociales et comportementales dans la réussite scolaire et professionnelle.

      Ces compétences, souvent désignées sous d'autres termes tels que "soft skills", "compétences non cognitives" (bien que ce terme soit considéré comme impropre) ou "compétences socio-émotionnelles", sont cruciales non seulement pour l'acquisition de connaissances, mais aussi pour le bien-être général des individus et leur insertion dans la société.

      Un constat alarmant est dressé concernant la France : les élèves français présentent un déficit "très singulier" de ces compétences par rapport aux autres pays de l'OCDE.

      Ce déficit se manifeste à travers plusieurs indicateurs psychologiques et comportementaux, qui ont des répercussions significatives sur leur parcours.

      I. Définition et Mesure des Compétences Sociales et Comportementales

      Élise Willer distingue deux grandes catégories de compétences :

      Compétences Comportementales (rapport à soi) : Confiance en soi / Auto-efficacité : Le sentiment d'être capable de réussir des tâches.

      Optimisme : La capacité à voir le positif et à croire en ses propres capacités.

      État d'esprit de développement (Growth Mindset) : La conviction que l'intelligence et les capacités peuvent progresser avec l'effort et l'entraînement, par opposition à un état d'esprit fixiste où l'intelligence est perçue comme innée et immuable.

      "L'intelligence c'est comme un muscle ça progresse en s'entraînant on va connecter des neurones en s'entraînant et donc on va développer son intelligence".

      Locus de contrôle (interne/externe) : La perception de la source de contrôle de sa propre vie.

      Un locus de contrôle interne signifie que l'individu pense que ce qui lui arrive dépend de ses propres actions, tandis qu'un locus externe attribue la réussite ou l'échec à des facteurs extérieurs incontrôlables (chance, destin).

      Contrôle de soi / Autodiscipline : La capacité à différer une gratification immédiate pour un bénéfice futur plus important, à s'imposer des efforts.

      Persévérance : La capacité à maintenir ses efforts face aux difficultés, étroitement liée à l'état d'esprit de développement.

      Compétences Sociales (rapport aux autres) :

      • Coopération : La capacité à travailler ensemble efficacement pour atteindre un objectif commun.

      • Empathie : La capacité à prendre en considération le point de vue d'autrui et l'impact de son propre comportement sur les autres.

      Respect et Tolérance.

      Sentiment d'appartenance : Le sentiment de faire partie d'un groupe, d'être soutenu et de contribuer à la réussite collective.

      Ces compétences sont mesurées principalement via des questionnaires (avec de multiples items pour construire des indices synthétiques) et parfois par des tâches comportementales (jeux de confiance ou de coopération) considérées comme plus fiables car mettant les individus en situation réelle.

      II. Le Constat d'un Déficit Français Particulier

      Les données des enquêtes internationales comme PISA (Programme international pour le suivi des acquis des élèves) de 2012 et 2018 révèlent un déficit marqué des élèves français :

      État d'esprit de développement : La France est "plutôt dans le bas" par rapport à la moyenne des pays de l'OCDE.

      Anxiété : Les élèves français sont "au-dessus de la moyenne des pays de l'OCDE" en termes d'anxiété.

      Perception de ses compétences : Alors que le niveau en mathématiques des élèves français est proche de la moyenne de l'OCDE, ils se sentent "moins compétents en moyenne que la moyenne", indiquant un "problème de perception de ses compétences".

      Persévérance : Les élèves français "lâchent davantage l'affaire quand ils y arrivent pas", un déficit "très net".

      Ouverture à la résolution de problème : Ils ont "moins plaisir qu'ailleurs à découvrir des problèmes et à essayer de les résoudre".

      Locus de contrôle : Il est "beaucoup plus externe que la moyenne", signifiant une perception que leur réussite dépend de facteurs extérieurs qu'ils ne contrôlent pas. Coopération :

      La France affiche le "niveau de coopération le plus faible de tous les pays de l'OCDE".

      Sentiment d'appartenance : Il est "plutôt négatif" à l'école.

      Résolution collaborative de problèmes : Le score est inférieur à la moyenne.

      Ce tableau psychologique des élèves français est jugé "pas très réjouissant".

      Inégalités Sociales : Ce déficit global est exacerbé par les inégalités sociales.

      À niveau scolaire égal, les élèves d'origine sociale défavorisée se perçoivent comme "moins bons que les élèves favorisés".

      Le même phénomène est observé chez les filles par rapport aux garçons.

      III. Les Effets Multiples de ces Compétences : Le "Triple Dividende"

      La recherche montre que ces compétences ne sont pas de simples "traits culturels" sans impact, mais qu'elles jouent un rôle "important" sur :

      • La Réussite Scolaire :
      • État d'esprit de développement : Des études en Norvège (2019), aux États-Unis (Yeager, 2019) et une étude menée en France par Élise Willer (Chouet, Allan) montrent qu'une intervention ciblée sur la plasticité cérébrale et l'état d'esprit de développement peut augmenter les notes scolaires et réduire le redoublement.

      Le programme "Énergie Jeune" (France) :

      Mis en place dans des collèges REP et REP+, ce programme de 12 séances sur 4 ans (de la 6ème à la 3ème), animé par des bénévoles, vise à déconstruire l'idée fixiste du niveau scolaire et à renforcer le locus de contrôle interne.

      • Résultats : Une augmentation des notes de 7% d'un écart-type, un effet "très important relativement à l'investissement que ça demande".

      Cet effet est comparable à des dispositifs beaucoup plus coûteux comme le dédoublement des classes de CP.

      Le programme change les perceptions des chances de réussite, améliore les comportements en classe (diminution des retards, absences, sanctions en 3ème) et a un "petit effet sur également donc les ambitions scolaires à plus long terme".

      • Limites : L'effet est plus fort chez les élèves ayant des comportements disciplinés à l'entrée en 6ème, et moins chez les élèves les plus difficiles, qui sont pourtant ceux "à risque de décrochage".

      L'Insertion Professionnelle :

      • Programme longitudinal de Montréal (démarré dans les années 80) : Visant des garçons défavorisés avec des comportements disruptifs pour renforcer le contrôle de soi et les habiletés sociales.

      • Effets : Amélioration de l'autonomie et de la confiance entre pairs.

      À très long terme (jusqu'à 37 ans), ce programme a montré des effets "très forts" sur : * Augmentation des revenus du travail (+20%). * Baisse du taux d'inactivité (-10 points). * Baisse de la criminalité (-33% d'arrestations). * Baisse de la dépendance aux aides sociales.

      Perry Preschool Project :

      Un autre programme axé sur les interactions de groupe et l'autodiscipline, montrant également des hausses de salaire et des études plus longues à l'âge adulte.

      Autres Bénéfices Publics :

      La réduction des coûts liés * aux redoublements, * aux programmes d'éducation spéciale, * à la criminalité et * aux aides sociales.

      • Analyse coût-bénéfice : Le programme de Montréal a rapporté "11 dollars" à la société et à l'individu pour "1 euro investi" à l'âge de 7-9 ans, démontrant que ces programmes doivent être perçus comme des "investissements sociaux".

      IV. Recommandations pour l'École et Perspectives

      La bonne nouvelle est que ces compétences "ne sont pas fixes" et qu'il est "facile à faire évoluer" ces traits.

      Pour rendre l'école plus performante, il est recommandé d'agir sur trois piliers, de manière plus systématique :

      • L'apprentissage coopératif : Enseigner aux élèves à apprendre ensemble. Le coût est faible (formation des enseignants) pour un impact potentiellement "substantiel" (près de 40% d'un écart-type selon l'Education Endowment Foundation).

      • Le développement de l'état d'esprit de développement :

      Continuer les efforts pour faire comprendre aux élèves que leurs capacités ne sont pas figées.

      • Les méthodes d'évaluation : Les méthodes actuelles françaises sont jugées comme accentuant un "état d'esprit fixiste" et un "sentiment d'incompétence".

      Il est nécessaire de "retravailler nos modes d'évaluation" pour des approches qui "expliquent ce qu'il a bien fait, ce qu'il a mal fait et donne des conseils", sans stigmatiser par des "points rouges".

      • Ces changements passent "forcément par la formation des enseignants", à la fois initiale et continue.

      Un nouveau programme expérimental, Motiveaction, est en cours de développement avec les collègues d'Élise Willer.

      Il vise à former 750 enseignants sur ces trois modules (état d'esprit de développement, évaluation pour favoriser les progrès, apprentissage coopératif) afin de "généraliser motivation énergie jeune grâce à une auprès des enseignants" et d'amplifier les gains en compétences et en apprentissages scolaires des élèves. Les résultats de cette expérimentation sont attendus pour 2025.

      En synthèse, la conférence insiste sur un paradoxe français : un fort déficit en compétences sociales et comportementales, mais une grande facilité à les faire évoluer avec des investissements raisonnables, générant des bénéfices considérables à long terme.

      L'école est identifiée comme un levier essentiel pour cette transformation.

      Collège de France 2024 CPS webinaire conférence vidéo compétences psychosociales
    Visit annotations in context

    Tags

    Annotators

    URL

    youtube.com/watch
  22. www.youtube.com www.youtube.com
    Video: Approches expérimentales en éducation (5) - Agir pour l'éducation (2023-2024) (DocDrop)
    1
    1. tanemika 14 Jul 2025

      Ce document de synthèse analyse en profondeur l'importance des interventions de soutien à la parentalité pour le développement cognitif et socio-émotionnel des enfants, ainsi que pour la réduction des inégalités sociales.

      Il se base principalement sur les recherches et les méta-analyses présentées par Carlo Barone, sociologue et professeur à Sciences Po.

      Thèmes Principaux et Idées Clés

      1. L'impact Précoce et Cumulatif des Inégalités de Développement

      Manifestation Précoce des Inégalités: Les inégalités de développement se manifestent dès les premières années, voire les premiers mois de vie des enfants, et ont des répercussions à long terme sur leur réussite scolaire.

      Par exemple, le vocabulaire réceptif des enfants de 4 ans en France est fortement lié au niveau d'éducation des parents.

      Ce constat est "préoccupant parce que nous savons que le vocabulaire réceptif avec la conscience phonologique est un des deux prédicteurs les plus importants des apprentissages en lecture et en écriture à l'école primaire".

      Apprentissage Cumulatif et Plasticité Cérébrale: Tout apprentissage est cumulatif, et la plasticité cérébrale des enfants est maximale pendant les premières années de vie, soulignant "l'intérêt d'intervenir dès le plus jeune âge pour favoriser l'égalité des chances dans l'éducation"**.

      2. Les Limites des Interventions Éducatives Scolaires Seules

      • Concentration Exclusive sur l'Environnement Scolaire: Les politiques éducatives en France (dédoublement des classes, dispositifs d'aide aux devoirs) bien que produisant des effets, sont parfois "moins efficaces que l'on espérait notamment par rapport aux moyens financiers humains importants qui sont alloués".

      Une limite majeure est qu'elles "se concentrent uniquement sur l'environnement scolaire et n'interviennent pas sur les inégalités flagrantes entre les environnements familiaux".

      • Complémentarité des Approches: L'implicite est souvent qu'on ne peut ou ne devrait pas intervenir sur ce qui se passe à la maison, alors que "les deux stratégies d'intervention peuvent être complémentaires".

      3. Le Rôle Crucial et Souvent Sous-Estimé des Parents

      • Temps Passé avec les Enfants: Les parents passent un temps considérable avec leurs enfants, un temps qui a "augmenté au fil du temps", y compris le "temps de qualité" (activités de jeu, lecture).

      Cette augmentation est plus marquée pour les parents des catégories socio-professionnelles favorisées.

      • Influence Permanente: Contrairement aux enseignants et camarades de classe qui changent, les parents "constituent une influence permanente et à long terme".

      • Aspirations Éducatives Élevées: Les parents, y compris ceux issus de milieux défavorisés ou immigrés, ont des aspirations éducatives élevées pour leurs enfants.

      Une implication apparemment moindre de la part de familles socialement défavorisées "reflète probablement surtout un ensemble de barrières sociales auquel ces familles sont confrontées plutôt que un manque d'intérêt des parents pour le développement et la réussite scolaire de leurs enfants".

      • Manque de Connaissance et Barrières Informationnelles: Beaucoup de parents "ne réalisent pas à quel point ce qui se passe à la maison a des conséquences importantes pour ce qui se passe à l'école".

      Des activités comme la lecture, les jeux de société, la cuisine partagée sont des opportunités d'apprentissage informel, mais "tous les parents ne sont pas conscients du potentiel de ces apprentissages informels".

      Les parents des milieux populaires ont moins accès aux informations d'experts et à leur circulation dans leurs cercles sociaux.

      4. Typologie des Interventions de Soutien à la Parentalité

      Une revue de 109 études randomisées identifie quatre stratégies principales :

      • Compétences Langagières et Cognitives (36%): Encourager des activités stimulant les apprentissages informels (lecture, puzzles, jeux de société).

      • Développement Socio-émotionnel (25%): Stimuler la réceptivité et la réactivité des parents aux besoins développementaux des enfants.

      • Règles à la Maison (Moins Développé): Pratiques et routines familiales (usage des écrans, sommeil, devoirs) influençant les capacités d'autorégulation des enfants.

      • Approches Hybrides/Holistiques (20%): Combinaison des stratégies précédentes.

      5. Exemples d'Interventions et Leurs Leçons

      • Ready4K (États-Unis): Intervention par SMS ciblant les parents d'enfants de 2 à 4 ans.

      Envoi de trois SMS par semaine (information, suggestion, encouragement).

      "Les enfants avec des compétences langagières moins développées ont connu une amélioration importante de leur vocabulaire et leurs compétences langagère". Points forts :

      • Stratégie de Communication Innovante: Utilisation des SMS pour toucher un maximum de parents, y compris ceux qui ne peuvent pas se rendre aux réunions scolaires.

      • Adaptation aux Contraintes Parentales: Messages simples, en plusieurs langues, ne demandant pas un investissement de temps important, mais intégrant des activités ludiques dans les routines quotidiennes.

      "Concevoir des interventions qui prennent en compte l'ensemble des contraintes parentales et propose des conseils réalisables pour tous les parents".

      • Coût-Bénéfice Favorable: Environ 5 € par enfant par an.

      Intervention sur la Lecture Partagée (France):

      Étude menée par Carlo Barone et ses collègues.

      Distribution de flyers informatifs et prêt gratuit de livres adaptés à l'âge, avec des conseils pour rendre l'activité agréable et l'intégrer au rituel du coucher. Suivi par SMS et appels téléphoniques.

      • Résultats: Augmentation significative de la fréquence de lecture parentale, particulièrement chez les parents moins éduqués, et amélioration du vocabulaire des enfants issus de familles moins éduquées et bilingues.

      Ces effets ont persisté 6 mois après l'intervention.

      • Limites: Pas d'impact sur les familles complètement allophones.

      Les effets à long terme n'ont pas pu être observés.

      Méta-analyse sur la Lecture Parentale:

      Sur 30 études randomisées (0-6 ans), les interventions de "lecture dialogique" ont des impacts importants sur le développement langagier (25-26% d'écart type), tandis que les autres interventions ont des impacts très faibles.

      Cependant, les impacts positifs des interventions en lecture dialogique se concentrent principalement sur les familles socialement favorisées, suggérant un risque d'augmentation des inégalités.

      Les effets ont tendance à diminuer rapidement après l'intervention.

      6. Leçons des Méta-Analyses Générales

      Méta-analyse de Jeong et al. (2021) - 102 études (0-3 ans):

      • Deux Catégories d'Intervention: Messages de stimulation d'apprentissages informels et promotion de styles parentaux autoritatifs (chaleur et soutien élevés, attentes claires et cohérentes).

      • Impacts Multiples:

      • Effets positifs importants sur le développement cognitif (32% d'écart type) et langagier,

      • encourageants sur le développement socio-émotionnel et moteur, et sur la qualité de la relation parents-enfants.

      • Moins d'effet sur la santé mentale des parents.

      • Variabilité des Impacts: Grande variabilité dans l'efficacité des interventions, certains étant très efficaces, d'autres non.

      • Contextes Nationaux: Impacts moyens plus importants dans les pays en développement, mais restent significatifs dans les pays riches. Une autre méta-analyse (Francis Steves et al.) suggère une "transportabilité élevée" des interventions entre pays riches.

      • Combinaison des Approches: Les interventions les plus efficaces combinent la stimulation cognitive et la réceptivité parentale.

      • Format: Pas de différences systématiques d'efficacité entre les visites à domicile et les espaces communautaires, ni entre les programmes individuels et collectifs.

      C'est "une excellente nouvelle parce qu'évidemment les les coûts de ces formats sont très différents".

      • Méta-analyse de Prime et al. (0-6 ans): Confirme les conclusions de Jeong et al.

      • Hétérogénéité Sociale: Ces interventions se révèlent "plus efficaces sur les familles socialement favorisées".

      Cela pose un "dilemme potentiel entre l'objectif d'améliorer le niveau moyen de développement [...] et la réduction des inégalités". Une solution est de cibler les interventions sur les familles défavorisées, mais des interventions universalistes qui réduisent les inégalités seraient préférables.

      • Importance des Pères: Les interventions ciblant spécifiquement les pères sont encore rares et leurs résultats peu concluants, mais c'est une piste de recherche prometteuse.

      • Effets à Long Terme: La principale limite de ces méta-analyses est qu'elles ne considèrent que les effets à court terme.

      La durabilité des effets reste une question ouverte. "Nous ne sommes pas encore en mesure d'indiquer quels interventions produisent les effets les plus durables".

      7. Barrières Comportementales et Stratégies pour les Surmonter

      • Défis de l'Implication Parentale: Les parents peuvent être fatigués, stressés, manquer de temps, et nos messages "rivalisent avec toutes ces préoccupations pour attirer leur attention". Le passage de l'intention à l'action est difficile.

      • Nécessité de Plus que de Simples Informations: Fournir des informations et des conseils est utile mais souvent "insuffisant".

      Il faut "réussir à attirer l'attention des parents, rendre les contenus des interventions accessibles et pertinents par rapport au contexte de vie des parents, faire des demandes de temps raisonnables qui s'inscrivent dans les routines parentales, faire face au phénomène d'autocensure des parents notamment les parents plus socialement défavorisés et réussir à maintenir les parents impliqués dans la durée".

      • Incredible Years: Programme de groupe (10-14 parents, 3 mois) testé dans 8 pays européens (pas la France).

      Encourage des relations chaleureuses, le jeu interactif et une discipline constructive. Utilise des méthodes basées sur la pratique (résolution de problèmes, discussion de vidéos, jeux de rôles).

      L'accès aux réunions est facilité (garde d'enfants, transport). Résultats robustes et similaires pour les familles favorisées et défavorisées (30-38% d'écart type sur le développement cognitif et socio-émotionnel).

      8. Faciliter l'Accès aux Services de la Petite Enfance

      • Effets Positifs des Services de Qualité: L'accès à des services de petite enfance de bonne qualité a des effets positifs, surtout pour les enfants défavorisés.

      • Inégalités d'Accès en France: L'accès aux crèches et assistantes maternelles est "très inégalitaire en France", avec une sous-représentation des enfants de familles défavorisées et immigrées.

      Barrières à l'Accès:

      • Accessibilité: Critères d'éligibilité, distribution territoriale de l'offre.
      • Économiques: Coûts directs et coûts d'opportunité.
      • Informationnelles: Connaissance des coûts, critères, modalités de candidature.
      • Administratives: Difficultés à interagir avec la bureaucratie, remplir des formulaires.
      • Interventions Possibles: Atténuer les barrières informationnelles et administratives par des interventions qui "apportent des informations aux familles sur le fonctionnement de ces services [...] et qui les accompagnent dans le processus de candidature". Une étude en Allemagne a montré l'efficacité de ces dispositifs à réduire les inégalités d'accès.

      Conclusion Générale

      • Les interventions de soutien à la parentalité représentent un levier important et "trop souvent négligé par les décideurs politiques" pour favoriser le développement des enfants et réduire les inégalités.

      Elles sont "peu coûteuses" et offrent une grande flexibilité. L'accessibilité effective est primordiale pour leur efficacité et la durabilité de la réduction des inégalités.

      Cependant, ces interventions doivent être "complétées par d'autres types d'action de nature plus structurelle" (critères d'éligibilité, répartition de l'offre, levée des barrières linguistiques) et prendre en compte les inégalités socio-économiques plus larges (pauvreté, chômage, insécurité économique) qui peuvent entraver la réceptivité des parents.

      En somme, il est essentiel de comprendre et de cibler les défis spécifiques auxquels les familles sont confrontées pour concevoir des interventions pertinentes et efficaces.

      Collège de France parentalité parents famille politique publique REAAP CAF réussite scolaire 2024 sociologie
    Visit annotations in context

    Tags

    Annotators

    URL

    youtube.com/watch
  23. www.biorxiv.org www.biorxiv.org
    Hippocampal single-cell RNA Atlas of chronic methamphetamine abuse-induced cognitive decline in mice
    1
    1. Public_Reviews 14 Jul 2025

      Reviewer #3 (Public review):

      Summary:

      This study aimed to elucidate the intricate mechanisms underlying cognitive decline induced by chronic METH abuse, focusing on the hippocampus at a single-cell resolution. The authors established a robust mouse model of chronic METH exposure. They observed significant impairments in working memory, spatial cognition, learning, and cognitive memory through Y-maze and novel object recognition tests. To gain deeper insights into the cellular and molecular changes, they utilized single-cell RNA sequencing to profile hippocampal cells. They performed extensive bioinformatics analyses, including cell clustering, differential gene expression, cellular communication, pseudotemporal trajectory, and transcription factor regulation.

      Strengths:

      (1) The authors performed a comprehensive suite of bioinformatics analyses, including differential gene expression, cellular cross-talk, pseudotime trajectory, and SCENIC analysis, which enable a multifaceted exploration of METH-induced changes at both the cellular and molecular levels.

      (2) The study demonstrates an awareness of the potential influence of circadian rhythms, dedicating a specific section in the discussion to the disruption of circadian rhythms, which has rarely been mentioned in previous studies on METH. They highlight the frequent occurrence of circadian regulation in their analysis across several cell types.

      (3) The pseudotime analysis provides valuable insights into hindered neurogenesis, showing a shift in NSC differentiation toward astrocytes rather than neuroblasts in METH-treated mice. The detailed analysis of BBB components (endothelial cells, mural cells, SMCs) and their heterogeneous responses to METH is also a significant contribution.

      Weaknesses:

      (1) While the bioinformatics analyses are extensive, the study is primarily descriptive at the molecular level. The absence of experimental validation, such as targeted mRNA/protein quantification and gene knockdown/overexpression to confirm the causal relationship between these identified genes and METH-induced cognitive deficits, is a notable limitation.

      (2) While the discussion extensively covers the functional implications of specific molecular pathways and cell types, it would greatly benefit from a comparison of these findings with existing RNA sequencing data from other METH models in hippocampal tissue.

      (3) The conclusion that "prolonged METH use may progressively impair cognitive function" may not be uniformly supported by the behavioral data: Figures 1C and F (discrimination and preference indexes) exhibited that the 4-week test further declined in the METH group compared to the 2-week. In contrast, Figure 1E and H present a contradictory pattern.

      Review 3
    Visit annotations in context

    Tags

    Annotators

    URL

    biorxiv.org/content/10.1101/2025.05.02.651823v1
  24. zettelkasten.de zettelkasten.de
    Introducing the Antinet Zettelkasten • Zettelkasten Method
    5
    1. offray 13 Jul 2025
      The Antinet’s structure enables you to retain more knowledge and connections than you ever thought possible. You’ll begin to notice yourself reading differently. Certain keyterms you’ve stamped onto your mind by way of the deliberate act of writing them onto your index will start to arise while reading; all you have to do oftentimes is simply write down the keyterm in the book’s margin. Or, if you do not wish to write in a book’s margins, you can write the keyterm down on a notecard you keep with you while reading, which is what Luhmann did. This notecard acts as a staging area for your thoughts before you transform them into full reflections on individual cards.

      Algo similar me pasó con Hypothesis durante la escritura doctoral. Sin embargo, fui grandemente ayudado por los sistemas de autocompleción de etiquetas en Hypothesis, de modo que, cuando estaba etiquetando una lectura, encontraba otras etiquetas que ya había usado y un orden emergente iba ocurriendo en mi forma de leer, que luego empleaba para mi forma de escribir.

      Hypothesis marginalia
    2. offray 13 Jul 2025
      Also stamped in time is that of your own mind and its own context, with its own links that it thinks of at the time of writing and developing thought. In brief, there’s temporal context that is stamped and installed into your Antinet.

      Esto me recuerda a WonderOS y OLLOS de Alexander Obenauer con sus items organizados en cronologías.

      cronologias
    3. offray 13 Jul 2025
      It’s about exploration from one card to the next, and jumping to cards linked to remote branches. The tree structure of the Zettelkasten enables meaningful exploration. One knowledge scientist goes on to say, “secondary memories themselves have an inner order that allows for exploration.” (21)21 Such things are enhanced by the tree structure of the Zettelkasten.

      Al comienzo pensaba algo así de cosas como Leo editor, donde organicé mucho de mi conocimiento en árboles con clones que podían aparecer en varias hojas. Sin embargo, dicha estructura podía tonarse complicada de acceder y preordenar (en esa época no tenía la estrategía de ordenar por fecha) y la interfaz no tenía muchas de las conveniencias de la interfaz web.

      Leo editor árboles de conocimiento
    4. offray 13 Jul 2025
      Simply stated, Luhmann’s Zettelkasten structure was not dynamic or fluid in nature. Yet, it was not rigid, either. Examples of a rigid structure are classification systems like the Dewey Decimal Classification System or Paul Otlet’s massive notecard world museum known as, The Mundaneum. These types of systems are helpful for interpersonal knowledge systems; however, they’re not illustrative of what Niklas Luhmann’s system was: an intrapersonal communication system. Luhmann’s notebox system was not logically and neatly organized to allow for the convenience of the public to access. Nor was it meant to be. It seemed chaotic to those who perused its contents other than its creator, Niklas Luhmann.

      Nuestros wikis interpersonales son pensados para ser utilizados por otras personas. Sin embargo, los identificadores únicos vía NanoID, podrían tener también una jerarquía de contadores arborecentes, similares a los de Luhmann.

      Había pensado en esos contadores únicos en cada wiki para TiddlyWiki por si queríamos algún tipo de enlace corto local de ese wiki y el NanoID para los indicadores únicos entre wikis, dadas las bajas probabilidades de colisión. Son ideas que se pueden combinar con otras como el control de versiones.

      El carácter dinámico del wiki no tiene por qué sacrificar la trazabilidad histórica y una cierta memoria visual de los enlaces y sus conexiones. Lo clave es que el wiki sea programable con curvas progresivas entre creación de contenido y de funcionalidad, como lo hace TiddlyWiki y lo propone Cardumem

      wikis interpersonales Cardumem identificadores únicos contadores arborecentes TiddlyWiki
    5. offray 12 Jul 2025
      This is important because it allows one to communicate with the Antinet, transforming it into a communication experience with a second mind, a doppelgänger, or a ghost in a box, as Luhmann called it. (5)5 This is the entity Luhmann referred to when he titled his paper, Communicating with Noteboxes.

      El comentario a una anotación continente habla de Monos de Markov, en el sentido de atribuirle personalidad y vida a algo inanimado, al hablar con él (tanto como se habla con un Tarot u otro juego interpretativo/narrativo). Estos serían los límites del "ghost in a box", que valdría la pena reconocer a pesar de lo útil de la interpretación animista para el archivo.

      ghost in a box Tarot game Markov monkey animismo
    Visit annotations in context

    Tags

    Annotators

    URL

    zettelkasten.de/posts/introduction-antinet-zettelkasten/
  25. pub-979cff47d4d84105ade2d75c354ef020.r2.dev pub-979cff47d4d84105ade2d75c354ef020.r2.dev
    Unit Testing: Principles, Practices, and Patterns
    1
    1. vicentematus 13 Jul 2025
      igure 1.4 The branch metric is calculated as the ratio of thenumber of code branches exercised by the test suite and thetotal number of branches in the production code base

      Prompt: Que es el branch coverage y cómo se calcula?

    Visit annotations in context

    Annotators

    URL

    pub-979cff47d4d84105ade2d75c354ef020.r2.dev/Unit Testing Principles, Practices, and Patterns_ Effective -- Vladimir Khorikov; Safari, an O'Reilly Media Company -- 1st, First Edition, PS, 2019 -- 9781617296277 -- 25329bce8f94e3bc57b1f07cb65734d3 -- Anna’s Archive.pdf
  26. discoversouthcarolina.com discoversouthcarolina.com
    Hike to Twin Falls
    1
    Visit annotations in context

    Annotators

    URL

    discoversouthcarolina.com/articles/hike-to-twin-falls
  27. accessmedicina.mhmedical.com accessmedicina.mhmedical.com
    Células, órganos y microambientes del sistema inmune | KUBY. Inmunología, 8e | AccessMedicina | McGraw-Hill Medical
    1
    Visit annotations in context

    Annotators

    URL

    accessmedicina.mhmedical.com/content.aspx
  28. github.com github.com
    GitHub - c2d7fa/zt: Simple but opinionated plain text-compatible hypertext minor mode for implementing Zettelkasten in Emacs
    1
    1. offray 12 Jul 2025
      I like to think of structure notes as a kind of wormhole from one "side" of the Zettelkasten to the other. If you're using the follower note approach, then your notes are already chained together in some way via the two-way links that are added to each note. So the primary function of a structure note is to let you jump directly from one chain of notes to another one that is very far away – there is little point in adding notes that are already close together in the sequence. Creating structure notes should feel like a creative exercise in compiling seemingly disparate ideas that actually have some hidden connection. Write each structure note like a table of contents for a small book about some very specific topic; if it's a book you actually want to read from one end to the other, then the structure note is successful. If writing the structure note feels feels like chore, then you're doing something wrong.

      Nunca lo había pensado de esta manera. Mis notas de estructura suelen ser mini tablas de contenido de cosas relacionadas en lugar de disintas.

      Me pregunto si algún enfoque algorítmico podría permitir visualizar esas conexiones extrañas entre nodos. He pensado en algo así para las etiquetas y otros elementos de organización de mi información.

      Grafoscopio Zettelkasten visualización de datos serendípia
    Visit annotations in context

    Tags

    Annotators

    URL

    github.com/c2d7fa/zt