Author response:

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

eLife Assessment

This study is useful as it provides further analysis of previously published data to address which specific genes are part of the masculinizing actions of E2 on female zebra finches, and where these key genes are expressed in the brain. However the data supporting the conclusion of masculinizing the song system are incomplete as the current manuscript is a re-analysis of differential gene expression modulated by E2 treatment between male/female zebra finches without manipulation of gene expression. The conclusions (and title) regarding song learning are also incompletely supported with no gene manipulation or song analysis. Importantly, the use of WGCNA for a question of sex-chromosome expression in species without dosage compensation is considered inadequate. As the experimental design did not include groups to directly test for song learning, and there was also no analysis of song performance, these data were also considered inadequate in that regard.

We are sorry the editor felt the manuscript so incomplete and inadequate. Though the tone of this assessment seems more severe than the below reviewer comments, we are also happy to see that the editor has considered our paper further for a revised publication, based on the reviewer’s comments. We address the editor’s comments as follows:

While we agree that manipulation of some of the genes we discovered, whose expression levels are E2-sensitive in the song system, would take the study further in validating some proposed hypothesis in the discussion of the paper, we don’t think the outcome of gene manipulations would change the major conclusions from the results of the paper. In this study we performed estrogen hormone manipulations, with causal consequences on gene expression in song nuclei and associated song behavior. In a way this is analogous to gene manipulations, but manipulating directly the action of estrogen. The categories of genes impacted, and the differences among the sex chromosomes wouldn’t change.

For the comment on WGCNA being inadequate for addressing questions on sex chromosome expression in species without dosage compensation, we think the evidence in our data does not bear that out. One main result of this paper is the separation of Z chromosome transcripts whose expression is most strongly regulated by chromosomal dosage (WGCNA module E) across regions from those subject to additional sources of regulation in song nuclei (other modules). It seems to us that rather than being confounded by the lack of dosage compensation, WGCNA allowed us to better resolve the effects of dosage on different genes within the sex chromosomes. We have added a new figure more directly examining sex chromosome transcript abundance within different modules. Briefly, we found that module E assigned Z chromosome genes exhibited almost exactly the male-biased expression ratio expected from no dosage compensation while the Z chromosome genes in song nuclei assigned to other modules were expressed below the dosage predicted value, consistent with module E containing those genes whose expression are most strongly regulated by dose across all brain regions sampled.

At its core, WGCNA finds sets of correlated genes. The biological reality of the zebra finch transcriptome is that Z chromosome expression is largely anti-correlated with W chromosome due to dosage. However, this dosage effect is not felt equally by all genes and WGCNA provides an unbiased computational framework which can be used to separate dose from other potential sources of gene regulation. This is why roughly ⅓ of Z chromosome genes are not assigned to module E; for example the growth hormone receptor is assigned to module G based on its correlation with genes upregulated within HVC.

“As the experimental design did not include groups to directly test for song learning, and there was also no analysis of song performance, these data were also considered inadequate in that regard.”

Concerning the comment on no analysis on song performance in the paper, all such analyses were conducted on our previous study on the same animals (Choe et al. 2021, Hormones & Behavior). The birds considered here were sacrificed at PHD30, prior to the onset of learned song behavior. However, females treated with E2 the same at the same time and allowed to mature into adulthood, went onto to develop rudimentary song. Further, induction of rudimentary song learning in females following E2 treatment has been well established since the early ‘80s. We have added the following text toward the end of the intro to make this more clear:

“While the birds for this study were sacrificed prior to the developmental presentation of song behavior, we have previously shown that female finches treated in exactly the say way with E2 go on to produce rudimentary imitative songs as adults (Choe et al 2021), consistent with the known induction of vocal learning in females by E2 (REF).”

Reviewer #1 (Recommendations For The Authors):

Overall, this is a wonderfully designed and executed study that takes full advantage of new resources, such as the most complete zebra finch genome assembly yet, as well as the latest methods. I have very few suggestions as to the improvement of the manuscript. They are as follows:

Results Section:

In the paragraph "Identification of gene expression modules in song nuclei":

"The E2-treated females in this study had similarly sized song system nuclei as males, indicating that E2 treatment prevented atrophy."

Clarify if this comparison is to treated and/or untreated males.

We thank the reviewer for their comment. The relative differences in the song nuclei sizes between the E2-treated females and the other groups is more complex that our original sentence implied. We have revised the main the text as follows

“In our previous study, we found that estradiol treatment in PHD30 females caused HVC to enlarge and Area X to appear when it normally does not develop in females, but both at sizes less than in untreated or treated males.The sizes of PHD30 female LMAN RA were already the sizes as seen in males, as the later has not atrophied yet at this age(25).”

In the paragraph "Sex- and micro-chromosome gene expression across the telencephalon": "These animal and chromosome specific shifts in the transcriptomes could represent the systemic effects of allelic chromosomal structural variation..."

The authors should clarify the meaning of a"llelic chromosomal structural variation" in this context, as it is an unusual phrase. Major chromosomal structural variation seems unlikely to produce these effects. Is it also possible that animal-specific modules with brain-wide higher could also result from laboratory contamination between all samples from one animal? This is not too likely but perhaps should be acknowledged or ruled out.

We have removed the word allelic, which was unnecessary. We can’t envision how laboratory contamination could occur such that all of one animal’s samples would be affected to produce the observed result which is module and chromosome specific. An animal wide effect could emerge during sacrifice, but we can think of no reason that would affect these modules and not others. Rather, the most likely explanation is biological natural difference between animals. We have added this consideration of alternative explanations.

In the section "Candidate gene drivers of HVC specialization in E2-treated females":

When discussing GHR's role in cell growth and proliferation, the authors' argument could be expanded by including the documented role of GH signaling in anti-apoptotic protection of neurons from rounds of neural pruning during development as documented in the chicken, e.g. • Harvey S, Baudet M-L, Sanders EJ. 2009. Growth Hormone-induced Neuroprotection in the Neural Retina during Chick Embryogenesis. Annals of the New York Academy of Sciences, 1163: 414-416. https://doi.org/10.1111/j.1749-6632.2008.03641.x

We thank the reviewer for sharing this publication with us.. We have added the following sentence to our discussion with the above citation. “Further, our results are consistent with growth hormone’s known role in avian anti-apoptotic protection, with elevated signaling associated with the survival of chicken neurons during rounds of pruning in the developing

retina.”

The authors' argument of the relevance of the passerine GH duplication would be strengthened by citing:

• Rasband SA, Bolton PE, Fang Q, Johnson PLF, Braun MJ. 2023. Evolution of the Growth Hormone Gene Duplication in Passerine Birds, Genome Biol Evol, 15(3) https://doi.org/10.1093/gbe/evad033. Greatly expands on the Yuri et al. paper cited by characterizing of the molecular evolution of these genes across hundreds of avian species, supporting positive selection on multiple amino acid sites identified in both ancestral and duplicate (passerine) growth hormone.

• Xie F, London SE, Southey BR et al. 2010. The zebra finch neuropeptidome: prediction, detection and expression. BMC Biol 8, 28. https://doi.org/10.1186/1741-7007-8-28 The authors report significantly different expression of the ancestral GH gene in the adult male zebra finch auditory forebrain after different song exposure experiences.

We have amended the results section sentence and added all suggested citations. The sentence now reads: “The gene which encodes growth hormone receptor’s ligand, growth hormone, is interestingly duplicated and undergoing accelerated evolution in the genomes of songbirds (Rasband et al 2023); the GH ligand has been found to be upregulated in the zebra finch auditory forebrain following the presentation of familiar song (Xie et al 2010).”

Figures:

- Figure 1B. "Duration of sex typing" being a shorter bar compared to the others is not fully explained in the experimental design. Presumably at the end of this time period, the sex is non-invasively, phenotypically evident. I suggest an arrow pointing to the PHD/PHD range when sex is apparent in plumage/anatomy.

- Figure 4. Caption appears to be truncated; "across all... genes"?

Fixed

- Figure 5. For 5E, 5F, 5G, 5H, consider enlarging the plots so overlapping gene symbols are readable. Alternately, smaller numbers or symbols could be used with a key in areas where overlapping symbols are hard to prevent.

We agree that these are not the easiest to read; we originally offset the symbols in R to minimize overlaps, but it can only do so much for the more crammed panels. We have now added a supplemental .xlsx file with the underlying data from each of the 4 tests for readers that want to examine the data in more detail.

Reviewer #2 (Recommendations For The Authors):

Since WGCNA methods will inherently draw together sex-chromosome genes into the same module in systems without dosage compensation, I suggest the authors rerun the WGCNA using only female samples and only male samples. Then identify the composition of modules that differ between E2 and vehicle-treated females and compare these genes to males. Then from male WGCNA identify the composition of modules that differ between E2 and vehicle-treated males and compare to female modules.

We thank the reviewer for their suggestions. However, we believe it is not as strong as the approach we used, which is grouping data from both sexes in the WGCNA analyses in a study that is looking for sex differences. The reviewer's proposed approach amounts to computing modules twice (once per sex), determining song system specialized modules and E2 responsive modules in both settings, then intersecting the two sets to find corresponding modules, all done to prevent the non-dose compensated sex chromosome genes from being drawn into the same module.

While WGCNA does group the majority of sex chromosome genes into module E, it does not categorize them all this way (Fig 3). The module classification instead differentiates those sex chromosome genes whose expression are most explained by chromosome dosage / sex across regions (modE) from those whose expression is controlled by other sources of regulation; for an example of the latter, the growth hormone receptor (GHR) is one of several Z chromosome genes classified into modG as its expression better correlates with the genes specialized to HVC than it does with the majority of dosage-dependent Z chromosome genes found in modE. Further, to remove biological sex as a variable in a WGCNA analysis that is focused on sex differences seems counterintuitive.

Instead, to quantitatively address the reviewer’s concern, we conducted additional analyses, that led to an added new figure, associated text, and tables, that better describes sex/chromosome dosage effects on the abundance (FPKM) and expression ratios of sex chromosome transcripts by module irrespective of brain region (Fig. 5). We find that the Z chromosome genes in modE were expressed at the expected chromosome dosage in the non-vocal surrounding regions (65.06% observed vs 66.6% expected) while in other modules, other Z chromosome genes were expressed at intermediate levels between equal expression and the expected chromosomal dosage. For example, the Z chromosome content of modules D and H exhibited near equal expression between sexes. Within the song system, Z chromosome gene content of modG was highly expressed in males beyond what is expected from chromosome dosage, consistent with modG’s male-specific upregulation in song nuclei relative to surrounds in the absence of E2. These results better demonstrate that in our WGCNA on the combined dataset we are able to separate those Z chromosome genes whose expression is predominantly dosage controlled from those subject to additional regulation such as song system specialization.

Fig. S3 Legend: 'Black arrow' -> 'Red arrow'

Change made.

Fig. S5 - What part of the figure shows the 'human convergent signature'? Also, simply listing the number of genes mapped to a chromosome is misleading to readers unfamiliar with the zebra finch genome, you should either provide the number of genes on each chromosome or present as corrected by that number.

Fig. S5 was the same type of analyses in Fig. 3 but with an older zebra finch genome assembly, where we had not included the panel a for enrichments with genes convergent in expression between songbird song regions and humans speech brain regions. However, we see that Fig. S5 was not adding any new important information to the paper, so we removed it.

For the chromosome analyses in Fig. 3b, we provide both the total raw number of module assigned genes broken down by chromosome (The black bar plots on the right) as well as a statistical fold-enrichment value of modules per chromosome. Given the number of genes per chromosome and genes per module in our data, we computed the fold-enrichment for each intersection (observed intersection size / expected intersection size). To test for the significance of these enrichments, we bootstrapped FDR corrected p values for the enrichment of each chromosome-module pairing by randomizing the mapping of genes to modules to construct a null distribution of fold enrichments for each intersection. Our intent was not to describe the size of the chromosomes themselves, information readily available elsewhere, but to show the disproportionate chromosomal origins of the gene sets considered by this study. Performing this enrichment test using all annotated genes per chromosome would artificially increase enrichment values and make the analysis less conservative by confounding the results with the inherent enrichment for “brain function” in the assigned genes relative to all genes.

At several places you say "we correlated expression of each sex chromosome transcript with sexual dimorphism within each region, such that expressed W genes would be positively correlated and depleted Z chromosome genes would be anticorrelated." What was the sexual dimorphism that was being correlated with? Is this the eigengene?

We thank you for this comment. Our language was less clear than it could be. We tested for correlations of both the eigengene and the individual gene expression profiles with the biological sex of the animals. We have changed the text to:

“To do this, we tested for a correlation between the expression of each sex chromosome transcript to the animals’ sex within each brain region. We found that female-enriched transcripts were positively correlated with sex and male-enriched transcripts were anticorrelated (Fig. 4f,g).”

Fig. 4A: The 'true/false' boxes and animal A-L is confusing and unnecessary. I'd suggest just using M and F (or sex symbols) with a horizontal line below each set of 3 for respective E2 and Veh.

Change made.

Reviewer #3 (Recommendations For The Authors):

General comments:

After the initial characterization of the datasets and module identification, it is quite hard to follow the logic of the data presentation in the various other Results sections or to clearly understand how they relate to the main stated goal to identify factors related to sex differences in vocal learning. The most relevant findings relate to the presumed actions of hormone treatment and sex chromosome gene dosage in song nuclei, whereas analyses of other brain areas, other chromosomes, or speech-related genes serve more as controls and/or appear as distractions from the main theme. A suggestion to increase the clarity of the presentation and potential impact of the study is to change the order of the presentation, focusing first on the specific analyses and comparisons that most directly speak to the main goals of the study, and then secondarily and more briefly presenting the controls or less related comparisons.

The reviewer’s suggestion for the results section organization is exactly what we had tried to do. We opened the first paragraph on identification of modules, then presented the song nuclei specific modules, followed by E2-changes to those modules; and the followed by other specific results for the remainder of the paper, including module enrichments to specific chromosomes. The reviewer mentioned our analyses of “other brain areas” (which we assume to mean the non-vocal surround regions), other chromosomes (which we assume means autosomes) and speech-related genes as controls were a distraction in the paper; but within our analysis, these other brain regions are essential controls needed to assess the song-system specificity of any observed sex differences observed from the very first paragraphs of the results; the autosomes were not controls for sex chromosome results, but primary results in of themselves; the overlap with speech-related genes was also not a control, but a novel discovery. We have revised these points in the paper to make them clearer, and revised some of the section titles and transitions between sections to help increase clarity of the main storyline of the paper.

A related comment is that many of the inferences drawn from the WGCNA analysis were quite complex, thus independent verification of some predictions would be quite valuable. For example, consider the passage: "In non-vocal learning juvenile females, interestingly LMAN was specialized relative to the AN by the same gene modules as in males (B, F, and I) as well as an additional module G (Fig. 2b); RA was specialized by module A as in males, but not module L and by additional modules A and G. In contrast, neither juvenile female HVC nor Area X exhibited significant gene module expression specializations relative to their surrounds." Providing in situ hybridization verification of these regional gene expression predictions with a few representative genes seems quite feasible given the group's expertise and would considerably strengthen confidence in the module-based inferences.

We performed in-situ independent validation of 36 candidate genes in our first study with this dataset (Choe et al 2021). We now mention this validation in the revised paper. The reviewer’s selection of one of our sentences though made us realize that our grammar used to explain the results was not as clear as it needs to be. We thus cleaned up the grammar of our module descriptions so that it should be communicated with less complexity, the main issue noted by the reviewer.

Because this is a re-analysis of a previously published dataset, the authors should more explicitly describe somewhere in the Discussion how the present analysis advances the understanding of sex differences in songbird neuroanatomy and behavior beyond the previous analysis.

We have added an additional sentence into the discussion more clearly separating the results of the current study from our previous work.

Specific comments:

Abstract:

There is evidence (from Frank Johnson's lab) that RA does not completely atrophy in female zebra finches, but is still present with more preserved connectivity than previously thought, possibly related to non-singing function(s). A term like 'marked reduction' of female RA may more accurately reflect the current state of knowledge.

We have changed the text to “partial atrophy”.

The term "driver" is undefined and unclear at this point of the paper; a clear definition for "driver" is also lacking in the Intro.

We now define “driver” or “genetic driver” as understood to mean “a genetic locus whose expression and/or inheritance strongly regulates the trait of interest”.

When citing the literature on studies that identified "specific genes with specialized up- or down-regulated expression in song and speech circuits relative to the surrounding motor control circuits", the authors should also cite studies from other labs (e.g. Li et al., PNAS, 2007; Lovell et al, Plos One 2008; Lovell et al, BMC Genomics 2018; Nevue et al, Sci Rep. 2020), to be accurate and fair.

Citations added

For clarity, the authors should explicitly formulate the hypothesis they are proposing at the end of the Summary.

We thank the reviewer for this comment. We have replaced the final sentence of the summary with: “We present a hypothesis where reduced dosage and expression of these Z chromosome genes changes the developmental trajectory of female HVC, partially preventable by estrogen treatment, contributing to the loss of song learning behavior.”

Introduction:

Vocal learning is arguably the ability to imitate 'vocal' sounds, this could be clarified here.

We have amended the sentence to “Vocal learning is the ability to imitate heard sounds using a vocal organ…”

Given they are currently considered sister taxa, can the author briefly explain what is the basis for assuming that songbirds and parrots independently evolved vocal learning?

Although songbirds and parrots belong to a monophyletic clade, they are not sister taxa. There are two clades separating them that are vocal non-learners. We have cited the reference that demonstrated this (e.g. Jarvis et al 2014 Science).

Why use Taeniopygia castanotis rather than the more broadly used Taeniopygia guttata?

Zebra finches were recently reclassified and T.castanotis is now more accurate. The Indonesian Timor zebra finch retained T.guttata while the Australian finch, used here, was classified as T.castanotis.

The authors state: "...vocal learning is strongly sexually dimorphic in zebra finches and many other vocal learning species" and cite Nottebohm and Arnold, Science, 1978. That landmark paper only shows dimorphism in song nuclei (not learning) in two songbird species. The authors should provide citations for other species and behavior, or modify the statement.

We have added an additional citation (Odom et al.) to this sentence which covers the phylogeny more broadly.

The authors refer to the nucleus RA as being located in the lateral intermediate arcopallium (LAI). Other labs have described this domain as the dorsal part of the intermediate arcopallium, thus AId or AID (Mello et al., JCN, 2019; Yuan and Bottjer, J Neurophys 2019; Yuan and Bottjer, eNeuro, 2020; Nevue et al., BCM Genomics, 2020). The authors should acknowledge this discrepancy in nomenclature so that data and conclusions can be more readily compared across studies.

We thank the reviewer and agree that this is helpful. We have added a note at the first mention of LAI.

The authors state that data from the gynandromorph bird described by Agate et al implicates "sex chromosome gene expression within the song system" as involved in the song system sexual dimorphism. That study, however, only rules out circulating gonadal steroids, and while suggesting a cell-autonomous mechanism like sex chromosome genes, it does not necessarily exclude other brain-autonomous factors like sex differences in local production of sex steroids.

We say that this study “implicated” sex chromosome gene expression, which is accurate per the results and discussion of that study. We are unsure what “brain autonomous factors like sex differences in local production of sex steroids” means?. “Brain autonomous” and “local production” in the brain seem contradictory in this context?

Results:

The authors state that "the E2-treated females in this study had similarly sized song system nuclei as males, indicating that E2 treatment prevented atrophy". Can they clarify whether the VEH-treated females actually had smaller RAs than E2-treated females or VEH-treated males at this age? This is still quite early in development and it is unclear to what extent RA's marked sexual dimorphism in adults or later developmental ages has already taken place in untreated (or VEH-treated) birds. A related comment is that the authors state later on: "We interpret these findings to indicate that: LMAN and RA atrophy later in juvenile female development..." Does this mean these nuclei actually did not show the marked decreases predicted earlier in the text? Clarifying this point would be helpful.

We thank the reviewer for pointing out this discrepancy, which reviewer #1 asked for clarification as well. RA size at this age is similar in males and females. However, HVC and Area X is smaller and absent respectively in females and E2 treatment partially prevents this atrophy. The text now reads:

“In our previous study, we found that estradiol treatment in PHD30 females caused HVC to enlarge and Area X to appear when it normally does not develop in females, but both at sizes less than in untreated or treated males.The sizes of PHD30 female LMAN RA were already the sizes as seen in males, as the later has not atrophied yet at this age(25).”

The authors acknowledge that area X is absent in untreated and VEH-treated females. Could they please clarify how area X and the surrounding stratal tissue that excludes area X were identified for laser capture dissections in juvenile females?

We have added the following statement to the main text portion discussing the dissections.

“In the case of vehicle-treated females which lack Area X, a piece of striatum from the same location of where Area X is found in males was taken. “

Some passages in Results discussing the authors' interpretation of the modules seem quite speculative and possibly belong instead in the Discussion. For example: "... that module A and G genes could be associated with the start of this atrophy; HVC and Area X are likely the first to atrophy or not develop; and lack of any gene module specialization in them at this age could mean that they would be more sensitive to estrogen prevention of vocal learning loss."

As suggested, we have removed this text from the results; these ideas were already presented in the Discussion. We have merged the resulting small paragraph with the preceding paragraph.

The authors state: "To assess the effects of chronic exogenous estrogen on the developing song system, we first performed a control analysis of modules in the E2-treated juvenile males." How can an assessment of estrogen effects be a "control" analysis? Does this refer to a contrast with females? Please clarify the language here.

The reviewer is correct, that E2 treatment in males should not be considered a control experiment. We removed the word “control”.

When discussing the GO-enriched terms for module G, it is unclear how the authors reached the conclusion about "proliferative", as the enriched terms do not refer to processes more directly indicative of proliferation like "cell division" or "cell cycle regulation". Rather, these terms seem more related to differentiation and growth, which do not necessarily imply proliferation. The authors also refer to "HVC proliferation" later on in the Discussion. However, there is conclusive evidence from several labs that proliferative events associated with postnatal neuronal addition and/or replacement in song nuclei occur in the subventricular zone, not in song nuclei like HVC itself, and that the growth of song nuclei largely reflects cell survival, as well as growth in size and complexity under the regulation of sex steroids.

We agree that “proliferative” may have been a poor word choice here. We did not mean to indicate that cell division was occuring in HVC itself. Instead we meant to indicate that HVC is able to accommodate the new born neurons from the SVZ. We have replaced the word “proliferative” throughout. In the instance the reviewer mentions specifically we replaced it with,“...potentially act to integrate and differentiate late born neurons.”

With regard to module E, referring to a telencephalon-wide sexually dimorphic gene expression program seems quite a stretch, given that only a few regions were sampled and compared between sexes. These related statements should be toned down.

We have replaced “telencephalon-wide” with “more distributed across the finch telencephalon” and other similar language in each instance.

The following passage is very speculative and should shortened and/or moved to the Discussion: "Based on the findings in these gene sets, we hypothesize that without excess estrogen in females, HVC expansion is prevented by not specializing the growth and neuronal migration promoting genes in module G to the HVC lineage by late development. This is potentially enacted by depleting necessary gene products from the Z sex chromosome, such as GHR, which are already present in only one copy."

We have deleted this portion of the text, as the idea is already present in the discussion.

Figure 5: To this reviewer, the comparisons of sex differences and of female response to E2 are the most relevant and informative ones, whereas the regional differences between song nuclei and surrounds refer to different cell populations and cell types where other processes may be occurring, independently of what occurs in song nuclei. It thus seems like the intersection analysis in panel 5i may be subtracting out important "core genes" in terms of E2 effects and/or sex differences in the most relevant cell populations, i.e. in this case within song nucleus HVC.

Song learning and the vocal learning brain regions are specialized behaviors and associated nuclei which have a set of hundreds of specialized genes compared to the surrounds. Our previous findings shows that E2 drives the appearance of these specializations in female zebra finches. Thus, we considered this the most interesting question to focus on, which we have further highlighted. Nevertheless, in response to the reviewers suggestion, we have added a .xlsx supplemental file containing the results from each of the individual tests so readers may examine any single comparison, or set of comparisons, in more detail.

Discussion:

It is unclear what the term "critical period" refers to in: "during the critical period of atrophy for the female vocal circuit"; please clarify.

We agree that our language was nebulous. We have replaced it with “as several male song control nuclei begin to expand and female nuclei partially atrophy”

In: "HVC appeared unspecialized at the level of gene module expression in control females", does "unspecialized" refer to a lack of difference in gene expression when compared to surroundings? Please clarify. The same comment applies to other uses of "unspecialized" in this paragraph.

Yes, unspecialized means lack of difference in gene expression in the song nucleus. To clarify this point, we have reworked that and the following sentence as follows:

“HVC appeared unspecialized compared to the surrounding nidopallium at the level of gene module expression in control females, with no significantly differentially expressed MEGs . However, in E2-treated females, HVC exhibited a subset of the observed male HVC gene expression specializations. Similarly, the vehicle-treated female striatum located where Area X would be also lacked any specialized gene module expression, but the E2-treated female Area X exhibited a subset of the male Area X specializations, consistent with the known absence of Area X in vehicle-treated females and presence in E2-treated females.”

The authors state: "...we surprisingly found that the most specialized genes were disproportionately from the Z chromosome", when discussing module G in HVC. Why is this so surprising? In a sense, this could be taken as consistent with the findings of Friedrich et al, 2022, where sex differences in the RA transcriptome were predominantly Z related on 20 dph. Arguably 20 dph is still quite close to 30 dph in the present study, when compared to 50 dph in Friedrich et al, when autosomes predominate.

Our bioRxiv was originally posted in July 2021, prior to the publication of Friedrich et al, 2022; however we had previously added to our discussion that several of our results are consistent with the observations of Friedrich et al..

We have a different interpretation of Z chromosome gene results in Friedrich et al.. While the percentage of specialized genes from the Z chromosome decreased, the absolute number of specialized Z chromosome genes actually increased over this interval. In Fig. 3a from Friedrich et al. it appears that ~28% of Z chromosome genes were sexually dimorphic in their expression in RA at PHD20 but that ~39% of Z chromosome genes were similarly dimorphic at PHD50. We interpret this result as the Z chromosome genes being among the earliest genes differentially expressed between the sexes, not that their differential expression or role ever subsequently decreased. We have reworked this portion of the discussion to make our point more clear:

“This model of sex chromosome influenced song system development is consistent with recent observations comparing male and female zebra finch transcriptomes from RA at young juvenile (PHD20) and young adult (PHD50) ages in un-manipulated birds (Friedrich et al. 2022)57. While that study proposes that the role of the sex chromosome in maintaining transcriptomic sex differences diminishes across development, as the proportion of specialized genes that originate on the sex chromosomes diminishes, this effect was driven by large increases in differentially expressed autosomal genes rather than by any reduction in sex chromosome dimorphism; the percentage of differentially expressed Z chromosome genes increased from PHD20 (28%) to PHD50 (39%) (Friedrich et al). This leads us to conclude that sexually dimorphic Z chromosome expression at juvenile ages precedes the sexually dimorphic expression of the autosomes seen in adults. This is consistent with our hypothesis that sufficient expression of select Z chromosome gene products (GHR, etc..) is necessary for subsequent autosomal song system specializations (modG).”

Further, when we write ”When examining the module G HVC specialization induced by E2-treatment in female HVC, we surprisingly found that the most specialized genes were disproportionately from the Z chromosome” we are referring to the upregulation of module G by E2 in female HVC, not the sex difference described in RA by Friedrich et al. which only utilized un-treated RA samples and thus is more likely related to our observations of module E.

The term "sexual dimorphism" has been more traditionally used for sex differences that are very marked, like features that are highly regressed or absent in one sex, most often in females. Quantitative differences in gene expression, including dosage differences like those related to module E, are more appropriately described as sex differences rather than dimorphisms. That usage would be more consistent with most of the literature, and thus preferable.

We did a google search for common definitions, and found more the opposite. Sexual dimorphism being used more often as differences of degree (with the zebra finch example as one of the top hits), and sex differences being used often as more absolute differences (like presence vs absence of the Y chromosome). Further, as in the reviewer’s first sentence, the definition of sexual dimorphism is a sex difference. That is, the two phrases can be interchangeable. Thus, we prefer to keep sexual dimorphism.

Several references are incomplete or seem truncated, like 9 and 10.

Fixed

Table S2: Please examine and take into account the W gene curation presented in Table S3 of Friedrich et al., 2022.

We have added additional supplementals (supplemetal_w_chrom_express.csv and supplemetal_z_chrom_express.csv) of the data provided in new Fig 5 incorporating the curation information from Table S3 from Friedrich et al.

Data availability:

Genes for all the main modules identified should be presented in a Supplemental Table, or through a link to a stable data repository.

We have added an additional Supplemental Table supplemental_gene_module_assignment.csv with this information.

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

Learn more at Review Commons

Reply to the reviewers

Reply to the Reviewers

We are very grateful to the reviewers for their time and care in reviewing our manuscript. We have tried to incorporate all of their feedback to the best of our ability, and we feel that this has greatly improved the manuscript.

Reviewer #1

This study provides a strong support for the relationship between replication starting point competition and initial factor concentration. However, some predictive conclusions, such as "the origin of high efficiency may not be activated earlier", are still preliminary. Can the author further clarify the scope of these predictions and any potential mechanism in the discussion part to improve the rigor of this study?

__Response: __In the discussion, we now emphasize the complexity of predicting origin firing time distributions, which are influenced by multiple interrelated factors beyond efficiency alone.

The resolution and accuracy of the model prediction are obvious to all, but the specific generalization ability is still unknown, which makes the further promotion slightly insufficient. Does the author consider conducting additional experiments? To detect the replication time and efficiency in yeast cells with changed levels of key initiation factors (such as Cdc45 or Dpb11). The empirical data can be compared with the model prediction by editing CRISPR gene or manipulating the initial factor abundance through overexpression vector.

__Response: __We fully agree that this would be a very interesting direction, but as this is a theoretical study focused on mathematical modelling, conducting further wet lab experiments would be beyond the scope of this work.

The model currently uses single values for the initiation factor number and recycling rate, though these parameters may vary across cell cycles or under different growth conditions. It is suggested that sensitivity analysis should be added to supplementary materials to explore how the changes of these parameters affect the model output, such as replication time distribution and origin efficiency.

__Response: __Sensitivity analysis of how the model fit and validation is affected by using different recycling rates and initial firing factor counts will be conducted.

While the authors use mean absolute error (MAE) to assess model fit, it is suggested to add other statistical methods, such as root mean square error or correlation analysis, to further evaluate the model's accuracy and robustness. In addition, this model lacks comparison with other studies on fitting yeast replication time, and it is difficult to evaluate the effect of this model compared with other models from the specific performance.

__Response: __We have now included the root mean squared error (RMSE) alongside the mean absolute error (MAE) and R-squared value to compare the simulated replication timing profiles with the experimental data. We agree that we could have been more detailed in comparing our model to other approaches. We have now added a lengthened discussion of this. In some cases, a direct comparison of performance is difficult due to fundamental differences between the approaches, but we have highlighted why this is the case.

Although the code is open, it is suggested to provide specific instructions or examples of the running code in supplementary materials, so as to facilitate reproduction and application by other researchers.

__Response: __The GitHub repository will be updated to enable the running of the entire pipeline. This update will include code for processing replication timing data from Müller et al. (2014) and extracting origin positions from the OriDB. Code will also be provided for writing Beacon Calculus scripts with different parameters and origin firing rates. Instructions on the recommended sequence in which scripts should be executed will also be provided. To enable users to run the model locally on their own computers, a smaller version focused on chromosome 2 will be included in the supplementary information and GitHub repository, along with example input data and expected outputs.

In Figure 2(a), compared with other chromosomes, the fitting effect of chromosome 1 seems to be not good. Has the author ever thought about the reason? In addition, what is the guiding significance of this model in practical applications, such as online services, forecasting tools, or experiments? Can the author give relevant application examples in this regard?

__Response: __Potential explanations for the poorer fit of the replication timing profile for chromosome 1 are now discussed. The y-axis range has also now been set as the same for all subplots in Figure 2a to make the replication timing profiles for each chromosome more easily comparable. In the discussion, we highlight how the intuitive and flexible nature of the model places it as a valuable tool which could be adapted to predict the effect of different perturbations on DNA replication dynamics.

Reviewer #2

In figure 5, the authors demonstrate that replication dynamics are robust to an increase in the number of available firing factors. However, experimental data from strains in which these limiting factors are overexpressed indicate that replication dynamics are substantially altered (e.g. PMID 22081107 and 23562327) since dNTPs become limiting. So the conclusions of the analysis in figure 5 are at best an oversimplification and at worst rather misleading. If adding dNTPs as a factor that becomes limiting only at higher firing factor concentrations is not technically feasible, the authors should be more circumspect in their description and discussion of the results in figure 5.

__Response: __We now discuss the interpretation of the effect of increasing the number of firing factors, given that factors such as dNTP availability are not included in the model.

The analysis of replication dynamics appears to exclude origins within the rDNA, which in the average strain account for ~20-25% of all replication origins in S. cerevisiae depending on the origin list chosen. Ignoring this large number of origins likely has a substantial impact on the model: if rDNA origins are intentionally ignored due to the difficulty of modeling repetitive regions or of having multiple identical origins in the competition model, this should be explicitly addressed in the text.

__Response: __We now emphasize that our model restricts initiation to specific sites and note that some low-efficiency origins, such as those in rDNA, have not been included.

Reviewer #3

Can the authors provide some insight into the model's dependency on the Müller, 2014 replication data set? They initialize and converge to this dataset so this paper's findings are highly contingent on treating this data set as ground truth.

__Response: __In the discussion, we now highlight that, despite the model's reliance on the Müller, 2014 replication data set for fitting, its ability to reproduce other features of DNA replication demonstrates its ability to reflect DNA replication dynamics more broadly.

The authors describe their model as one that simplifies the origin firing mechanisms compared to more complex models. Is there a direct comparison available that can quantify this advantage? Likewise, how does their model compare to a naive discriminative model, such as one that performs peak finding on the replication timing data. For example, the replication fork directionality can be estimated, naively, using a peak finding algorithm. This type of analysis will provide a stronger argument for the usage of their model.

__Response: __Quantitative comparisons between our model and other published models are challenging due to differences in underlying assumptions and metrics used to assess goodness of fit. However, we have now added a discussion addressing these challenges and highlighting how our model's design contrasts with that of other models.

Currently the code is available as supplemental data. Ideally, the code should be available and provided to run the entire pipeline beginning with the initialization of the origin firing program from the Müller, 2014 data set.

__Response: __The GitHub repository will be updated to enable the running of the entire pipeline. This update will include code for processing replication timing data from Müller et al. (2014) and extracting origin positions from the OriDB.

The authors mention that origin firing factors and their recycling time to be the basis of how this model is constructed. While also describing the recycle time as a general timing delay that is dependent on a number of reasons such as diffusion and replisome complex formation. Can the authors discuss the limitations of their model towards this simplification?

__Response: __Limitations of our model's assumptions of constant recycling rates of firing factors are now discussed, as well as our assumption that the firing rates of origins and the maximum number of available firing factors remain constant between simulations.

Author response:

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

eLife Assessment

(1) This is a valuable manuscript that successfully integrates several data sets to determine genomic interactions with nuclear bodies.

In this paper we both challenge and/or revise multiple long-standing “textbook” models of nuclear genome organization while also revealing new features of nuclear genome organization. Therefore, we argue that the contributions of this paper extend well beyond “valuable”. Specifically, these contributions include:

a. We challenge a several decades focus on the correlation of gene positioning relative to the nuclear lamina. Instead, through comparison of cell lines, we show a strong correlation of di4erences in gene activity with di4erences in relative distance to nuclear speckles in contrast to a very weak correlation with di4erences in relative distance to the nuclear lamina. This inference of little correlation of gene expression with nuclear lamina association was supported by direct experimental manipulation of genome positioning relative to the nuclear lamina. Despite pronounced changes in relative distances to the nuclear lamina there was little change relative to nuclear speckles and little change in gene expression.

b. We similarly challenge the long-standing proposed functional correlation between the radial positioning of genes and gene expression. Here, and in a now published companion paper (doi.org/10.1038/s42003-024-06838-7), we demonstrate how nuclear speckle positioning relative to nucleoli and the nuclear lamina varies among cell types, as does the inverse relationship between genome positioning relative to nuclear speckles and the nuclear lamina. Again, this is consistent with the primary correlation of gene activity being the positioning of genes relative to nuclear speckles and also explains previous observations showing a strong relationship between radial position and gene expression only in some cell types.

c. We identified a new partially repressed, middle to late DNA replicating type of chromosome domain- “p-w-v fILADs”- by their weak interaction with the nuclear lamina, which, based on our LMNA/LBR KO experimental results, compete with LADs for nuclear lamina association. Moreover, we show that when fLADs convert to iLADs, most conversions are to this p-w-v fiLAD state, although ~ one third are to a normal, active, early replicating iLAD state. Thus, fLADs can convert between repressed, partially repressed, and active states, challenging the prevailing assumption of the division of the genome into two states – active, early replicating A compartment/iLAD regions versus inactive, late replicating, B compartment/LAD regions.

d. We identified nuclear speckle associated domains as DNA replication initiation zones, with the domains showing strongest nuclear speckle attachment initiating DNA replication earliest in S-phase.

e. We describe for the first time an overall polarization of nuclear genome organization in adherent cells with the most active, earliest replicating genomic regions located towards the equatorial plane and less expressed genomic regions towards the nuclear top or bottom surfaces. This includes polarization of some LAD regions to the nuclear lamina at the equatorial plane and other LAD regions to the top or bottom nuclear surfaces.

We have now rewritten the text to make the significance of these new findings clearer.

(2) Strength of evidence: The evidence supporting the central claims is varied in its strength ranging from solid to incomplete. Orthogonal evidence validating the novel methodologies with alternative approaches would better support the central claims.

We argue that our work exploited methods, data, and analyses equal to or more rigorous than the current state-of-the-art. This indeed includes orthogonal evidence using alternative methods which both supported our novel methodologies as well as demonstrating their robustness relative to more conventional approaches. This explains how we were able to challenge/revise long-standing models and discover new features of nuclear genome organization. More specifically:

a. Unlike most previous analyses, we have integrated both genomic and imaging approaches to examine the nuclear genome organization relative to not one, but several di4erent nuclear locales and we have done this across several cell types. To our knowledge, this is the first such integrated approach and has been key to our success in appreciating new features of nuclear genome organization.

b. The 16-fraction DNA replication Repli-seq data we developed and applied to this project represents the highest temporal mapping of DNA replication timing to date.

c. The TSA-seq approach that we used remains the most accurate sequence-based method for estimating microscopic distance of chromosome regions to di4erent nuclear locales. As implemented, this method is unusually robust and direct as it exploits the exponential micron-scale gradient established by the di4usion of the free-radicals generated by peroxidase labeling to measure relative distances of chromosome regions to labeled nuclear locales. We had previously demonstrated that TSA-seq was able to estimate the average distances of genomic regions to nuclear speckles with an accuracy of ~50 nm, as validated by light microscopy. The TSA-seq 2.0 protocol we developed and applied to this project maintained the original resolution of TSA-seq to estimate to an accuracy of ~50 nm the average distances of genomic regions from nuclear speckles, as validated by light microscopy, while achieving more than a 10-fold reduction in the required number of cells.

We have rewritten the text to address the reviewer concerns that led them to their initial characterization of the TSA-seq as novel and not yet validated.

First, we have added a discussion of how the use of nuclear speckle TSA-seq as a “cytological ruler” was based on an extensive initial characterization of TSA-seq as described in previous published literature. In that previous literature we showed how the conventional molecular proximity method, ChIP-seq, instead showed local accumulation of the same marker proteins over short DNA regions unrelated to speckle distances. Second, we reference our companion paper, now published, and describe how the extension of TSA-seq to measure relative distances to nucleoli was further validated and shown to be robust by comparison to NAD-seq and extensive multiplexed immuno-FISH data. We further discuss how in the same companion paper we show how nucleolar DamID instead was inconsistent with both the NAD-seq and multiplexed immuno-FISH data as well as the nucleolar TSA-seq.

Third, we have added scatterplots showing exactly how highly the estimated microscopic distances to all three nuclear locales, measured in IMR90 fibroblasts, correlate with the TSA-seq measurements in HFF fibroblasts. This addresses the concern that we were not using the exact same fibroblast cell line for the TSA-seq versus microscopic measurements. The strong correlation already observed would only be expected to become even stronger with use of the exact same fibroblast cell lines for both measurements.

Fourth, we have addressed the reviewer concern that the nuclear lamin TSA-seq was not properly validated because it did not match nuclear lamin Dam-ID. We have now added to the text a more complete explanation of how microscopic proximity assays such as TSA-seq measure something di4erent from molecular proximity assays such as DamID or NAD-seq. We have added further explanation of how TSA-seq complements molecular proximity assays such as DamID and NAD-seq, allowing us to extract further information than either measurement alone. We also briefly discuss why TSA-seq succeeds for certain nuclear locales using multiple independent markers whereas molecular proximity assays may fail against the same nuclear locales using the same markers. This includes brief discussion from our own experience attempting unsuccessfully to use DamID against nucleoli and nuclear speckles.

Reviewer #1 (Public Review):

(1) The weakness of this study lies in the fact that many of the genomic datasets originated from novel methods that were not validated with orthogonal approaches, such as DNAFISH. Therefore, the detailed correlations described in this work are based on methodologies whose efficacy is not clearly established. Specifically, the authors utilized two modified protocols of TSA-seq for the detection of NADs (MKI67IP TSA-seq) and LADs (LMNB1-TSA-seq).

We disagree with the statement that the TSA-seq approach and data has not been validated by orthogonal approaches. We have now addressed this point in the revised manuscript text:

a) We added text to describe how previously FISH was used to validate speckle TSA-seq by demonstrating a residual of ~50 nm between the TSA-seq predicted distance to speckles and the distance measured by light microscopy using FISH:

"In contrast, TSA-seq measures relative distances to targets on a microscopic scale corresponding to 100s of nm to ~ 1 micron based on the measured diffusion radius of tyramide-biotin free-radicals (Chen et al., 2018). Exploiting the measured exponential decay of the tyramide-biotin free-radical concentration, we showed how the mean distance of chromosomes to nuclear speckles could be estimated from the TSA-seq data to an accuracy of ~50 nm, as validated by FISH (Chen et al., 2018)."

b) We note that we also previously have validated lamina (Chen et al, JCB 2018) and nucleolar (Kumar et al, 2024) TSA-seq and further validated speckle TSA-seq (Zhang et al, Genome Research 2021) by traditional immuno-FISH and/or immunostaining. The overall high correlation between lamina TSA-seq and the orthogonal lamina DamID method was also extensively discussed in the first TSA-seq paper (Chen et al, JCB 2018). Included in this discussion was description of how the di4erences between lamina TSA-seq and DamID were expected, given that DamID produces a signal more proportional to contact frequency, and independent of distance from the nuclear lamina, whereas TSA-seq produces a signal that is a function of microscopic distance from the lamina, as validated by traditional FISH.

c) We added text to describe how the nucleolar TSA-seq previously was validated by two orthogonal methods- NAD-seq and multiplexed DNA immuno-FISH:

"We successfully developed nucleolar TSA-seq, which we extensively validated using comparisons with two different orthogonal genome-wide approaches (Kumar et al., 2024)- NAD-seq, based on the biochemical isolation of nucleoli, and previously published direct microscopic measurements using highly multiplexed immuno-FISH (Su et al., 2020)."

d) We have now added panels A&B to Fig. 7 and a new Supplementary Fig. 7 demonstrating further validation of TSA-seq based on showing the high correlation between the microscopically measured distances of many hundreds of genomic sites across the genome from di4erent nuclear locales and TSA-seq scores. As discussed in response #2 below, we have used comparison of distances measured in IMR90 fibroblasts with TSA-seq scores measured in HFF fibroblasts. We would argue therefore that these correlations are a lower estimate and therefore the correlation between microscopic distances and TSAseq scores would likely have been still higher if we had performed both assays in the exact same cell line.

(2) Although these methods have been described in a bioRxiv manuscript by Kumar et al., they have not yet been published. Moreover, and surprisingly, Kumar et al., work is not cited in the current manuscript, despite its use of all TSA-seq data for NADs and LADs across the four cell lines.

The Kumar et al, Communications Biology, 2024 paper is now published and is cited properly in our revision. We apologize for this oversight and confusion our initial omission of this citation may have created. We had been writing this manuscript and the Kumar et al manuscript in parallel and had intended to co-submit. We planned to cross-reference the two at the time we co-submitted, adding the Kumar et al reference to the first version of this manuscript once we obtained a doi from bioRxiv. But we then submitted the Kumar et al manuscript several months earlier, but meanwhile forgot that we had not added the reference to our first manuscript version.

(3) Moreover, Kumar et al. did not provide any DNA-FISH validation for their methods.

As we described in our response to Reviewer 1's comment #1, we had previously provided traditional FISH validation of lamina TSA-seq in our first TSA- seq paper as well as validation by comparison with lamina DamID (Chen et al, 2018).

We also described how the nucleolar TSA-seq was extensively cross-validated in the Kumar et al, 2024 paper by both NAD-seq and the highly multiplexed immuno-FISH data from Su et al, 2020).

We note additionally that in the Kumar et al, 2024 paper the nucleolar TSA-seq was additionally validated by correlating the predicted variations in centromeric association with nucleoli across the four cell lines predicted by nucleolar TSA-seq with the variations observed by traditional immunofluorescence microscopy.

(4) Therefore, the interesting correlations described in this work are not based on robust technologies.

This comment was made in reference to the Kumar et al paper not having been published, and, as noted in responses to points #2 and #3, the paper is now published.

But we wanted to specifically note, however, that our experience is that TSA-seq has proven remarkably robust in comparison to molecular proximity assays. We've described in our responses to the previous points how TSA-seq has been cross-validated by both microscopy and by comparison with lamina DamID and nucleolar NAD-seq. We note also that in every application of TSA-seq to date, all antibodies that produced good immunostaining showed good TSA-seq results. Moreover, we obtained nearly identical results in every case in which we performed TSA-seq with different antibodies against the same target. Thus anti-SON and antiSC35 staining produced very similar speckle TSA-seq data (Chen et al, 2018), anti-lamin A and anti-lamin B staining produced very similar lamina TSA-seq data (Chen et al, 2018), antinucleolin and anti-POL1RE staining produced very similar DFC/FC nucleolar TSA-seq data (Kumar et al, 2024), and anti-MKI67IP and anti-DDX18 staining produced very similar GC nucleolar TSA-seq data (Kumar et al, 2024).

This independence of results with TSA-seq to the particular antibody chosen to label a target differs from experience with methods such as ChIP, DamID, and Cut and Run/Tag in which results can differ or be skewed based on variable distance and therefore reactivity of target proteins from the DNA or due to other factors such as non-specific binding during pulldown (ChIP) or differential extraction by salt washes (Cut and Tag).

Our experience in every case to date is that antibodies that produce similar immunofluorescence staining produce similar TSA-seq results. We attribute this robustness to the fact that TSA-seq is based only on the original immunostaining specificity provided by the primary and secondary antibodies plus the diffusion properties of the tyramide-free radical.

We've now added the following text to our revised manuscript:

"As previously demonstrated for both SON and lamin TSA-seq (Chen et al., 2018), nucleolar TSA-seq was also robust in the sense that multiple target proteins showing similar nucleolar staining showed similar TSA-seq results (Kumar et al., 2024); this robustness is intrinsic to TSA-seq being a microscopic rather than molecular proximity assay, and therefore not sensitive to the exact molecular binding partners and molecular distance of the target proteins to the DNA."

(5) An attempt to validate the data was made for SON-TSA-seq of human foreskin fibroblasts (HFF) using multiplexed FISH data from IMR90 fibroblasts (from the lung) by the Zhuang lab (Su et al., 2020). However, the comparability of these datasets is questionable. It might have been more reasonable for the authors to conduct their analyses in IMR90 cells, thereby allowing them to utilize MERFISH data for validating the TSA-seq method and also for mapping NADs and LADs.

We disagree with the reviewer's overall assessment that that the use of the IMR90 data to further validate the TSA-seq is questionable because the TSA-seq data from HFF fibroblasts is not necessarily comparable with multiplexed immuno-FISH microscopic distances measured in IMR90 fibroblasts.

In response we have now added panels to Fig. 7 and Supplementary Fig. 7, showing:

a) There is very little di4erence in correlation between speckle TSA-seq and measured distances from speckles in IMR90 cells whether we use IMR90 or HFF cells SON TSA-seq data (R² = 0.81 versus 0.76) (new Fig. 7A).

b) There is also a high correlation between lamina (R² = 0.62) and nucleolar (R² = 0.73) HFF TSA-seq and measured distances in IMR90 cells. Thus, we conclude that this high correlation shows that the multiplexed data from ~1000 genomic locations does validate the TSA-seq. These correlations should be considered lower bounds on what we would have measured using IMR90 TSA-seq data. Thus, the true correlation between distances of loci from nuclear locales and TSA-seq would be expected to be either comparable or even stronger than what we are seeing with the IMR90 versus HFF fibroblast comparisons.

c) This correlation is cell-type specific (Fig. 7B, new SFig. 7). Thus, even for speckle TSAseq, highly conserved between cell types, the highest correlation of IMR90 distances with speckle TSA-seq is with IMR90 and HFF fibroblast data. For lamina and nucleolar TSA-seq, which show much lower conservation between cell types, the correlation of IMR90 distances is high for HFF data but much lower for data from the other cell types. This further justifies the use of IMR90 fibroblast distance measurements as a proxy for HFF fibroblast measurements.

Thus, we have added the following text to the revised manuscript:

"We reasoned that the nuclear genome organization in the two human fibroblast cell lines would be sufficiently similar to justify using IMR90 multiplexed FISH data [43] as a proxy for our analysis of HFF TSA-seq data. Indeed, the high inverse correlation (R= -0.86) of distances to speckles measured by MERFISH in IMR90 cells with HFF SON TSA-seq scores is nearly identical to the inverse correlation (R= -0.89) measured instead using IMR90 SON TSA-seq scores (Fig. 7A). Similarly, distances to the nuclear lamina and nucleoli show high inverse correlations with lamina and nucleolar TSA-seq, respectively (Fig. 7A). These correlations were cell type specific, particularly for the lamina and nucleolar distance correlations, as these correlations were reduced if we used TSA-seq data from other cell types (SFig. 7A). Therefore, the high correlation between IMR90 microscopic distances and HFF TSA-seq scores can be considered a lower bound on the likely true correlation, justifying the use of IMR90 as a proxy for HFF for testing our predictions."

Reviewer #2 (Public Review):

Weaknesses:

(1) The experiments are largely descriptive, and it is difficult to draw many cause-andeffect relationships...The study would benefit from a clear and specific hypothesis.

This study was hypothesis-generating rather than hypothesis-testing in its goal. Our research was funded through the NIH 4D-Nucleome Consortium, which had as its initial goal the development, benchmarking, and validation of new genomic technologies. Our Center focused on the mapping of the genome relative to different nuclear locales and the correlation of this intranuclear positioning of the genome with functions- specifically gene expression and DNA replication timing. By its very nature, this project took a discovery-driven versus hypothesis-driven scientific approach. Our question fundamentally was whether we could gain new insights into nuclear genome organization through the integration of genomic and microscopic measurements of chromosome positioning relative to multiple different nuclear compartments/bodies and their correlation with functional assays such as RNA-seq and Repliseq.

Indeed, this study resulted in multiple new insights into nuclear genome organization as summarized in our last main figure. We believe our work and conclusions will be of general interest to scientists working in the fields of 3D genome organization and nuclear cell biology. We anticipate that each of these new insights will prompt future hypothesis-driven science focused on specific questions and the testing of cause-and-effect relationships.

However, we do want to point out that our comparison of wild-type K562 cells with the LMNA/LBR double knockout was designed to test the long-standing model that nuclear lamina association of genomic loci contributes to gene silencing. This experiment was motivated by our surprising result that gene expression differences between cell lines correlated strongly with differences in positioning relative to nuclear speckles rather than the nuclear lamina. Despite documenting in these double knockout cells a decreased nuclear lamina association of most LADs, and an increased nuclear lamina association of the “p-w-v” fiLADs identified in this manuscript, we saw no significant change in gene expression in any of these regions as compared to wild-type K562 cells. Meanwhile, distances to nuclear speckles as measured by TSA-seq remained nearly constant.

We would argue that this represents a specific example in which new insights generated by our genomics comparison of cell lines led to a clear and specific hypothesis and the experimental testing of this hypothesis.

(2) Similarly, the paper would be very much strengthened if the authors provided additional summary statements and interpretation of their results (especially for those not as familiar with 3D genome organization).

We appreciate this feedback and agree with the reviewer that this would be useful, especially for those not familiar with previous work in the field of 3D genome organization. In an earlier draft, we had included additional summary and interpretation statements in both the Introduction and Results sections. At the start of each Results section, we had also previously included brief discussion of what was known before and the context for the subsequent analysis contained in that section. However, we had thought we might be submitting to a journal with specific word limits and had significantly cut out that text.

We have now restored this text and, in certain cases, added additional explanations and context.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

Figures 1C and D. Please add the units at the values of each y-axis.

We have done that.

The representation of Figure 2C lacks clarity and is diJicult to understand. The x-axis labeling regarding the gene fraction number needs clarification.

We've modified the text to the Fig. 2C legend: "Fraction of genes showing significant di=erence in relative positioning to nuclear speckles (gene fraction, x-axis) versus log2 (HFF FKPM / H1 FKPM) (y-axis);"

"We next used live-cell imaging to corroborate that chromosome regions close to nuclear speckles, primarily Type I peaks, would show the earliest DNA replication timing." This sentence requires modification as Supplementary Figure 3F does not demonstrate that Type I peaks exhibit the earliest DNA replication timing; it only indicates that the first PCNA foci in S-phase are in proximity to nuclear speckles.

We've modified the text to: "We next used live-cell imaging to show that chromosome regions close to nuclear speckles show the earliest DNA replication timing; this is consistent with the earliest firing DNA replication IZs, as determined by Repli-seq, aligning with Type 1 peaks that are closely associated with nuclear speckles."

In Figure 5, the authors employed LaminB1-DamID to quantify LADs in LBR-KO and LMNA/LBR-DKO K562 cells. These are interesting results. However, for these experiments, it is crucial to assess LMNB1 signal at the nuclear periphery via immunofluorescence (IF) to confirm the absence of changes, ensuring that the DamID signal solely reflects contacts with the nuclear lamina. Furthermore, in this instance, their findings should be validated through DNA-FISH.

Immunostaining of LMNB1 was performed and showed a normal staining pattern as a ring adjacent to the nuclear periphery. Images of this staining were included in the metadata tied to the sequencing data sets deposited on the 4D Nucleome Data portal. We thank the reviewer for bringing up this point, and have added a sentence mentioning this result in the Results Section:

"Immunostaining against LMNB1 revealed the normal ring of staining around the nuclear periphery seen in wt cells (images deposited as metadata in the deposited sequencing data sets)."

Because both TSA-seq and DamID have been extensively validated by FISH, as detailed in our previous responses to the public reviewer comments, we feel it is unnecessary to validate these findings by FISH.

p-w-v-fiLADs should be labelled in Figure 5B.

We've added labeling as suggested.

"The consistent trend of slightly later DNA replication timing for regions (primarily p-w-v fiLADs) moving closer to the lamina" is not visible in the representation of the data of Figure 5G.

We did not make a change as we believed this trend was apparent in the Figure.

To reduce the descriptive nature of the data, it would be pertinent to conduct H3K9me3 and H3K27me3 ChIP-seq analyses in both the parental and DKO mutant cells. This would elucidate whether p-w-v-fiLADs and NADs anchoring to the nuclear lamina undergo changes in their histone modification profile.

We believe further analysis of the reasons underlying these shifts in positioning, including such ChIP-seq or equivalent analysis, is of interest but beyond the scope of this publication. We see such measurements as the beginning of a new story but insuJicient alone to determine mechanism. Therefore we believe such experiments should be part of that future study.

The description of Figure 7 lacks clarity. Additionally, it appears that TSA-seq for NADs and LADs may not be universally applicable across all cell types, particularly in flat cells, whereas DamID scores demonstrate less variation across cell lines, as also stated by the authors.

TSA-seq is a complement to rather than a replacement for either DamID or NAD-seq. TSAseq reports on microscopic distances whereas both DamID and NAD-seq instead are more proportional to contact frequency with the nuclear lamina or nucleoli, respectively, and insensitive to distances of loci away from the lamina or nucleoli. Thus, TSA-seq provides additional information based on the intrinsic diJerences in what TSA-seq measures relative to molecular proximity methods such as DamID or NAD-seq. The entire point is that the convolution of the exponential point-spread-function of the TSA-seq with the shape of the nuclear periphery allows us to distinguish genomic regions in the equatorial plane versus the top and bottom of the nuclei. The TSA-seq is therefore highly "applicable" when properly interpreted in discerning new features of genome organization. As we stated in the revised manuscript, the lamina DamID and TSA-seq are complementary and provide more information together then either method along. The same is true for the NAD-seq and nucleolar TSA-seq comparison, as described in more detail in the Kumar, et al, 2024 paper.

Introduction:

The list of methodologies for mapping genomic contacts with nucleoli (NADs) should also include recent technologies, such as Nucleolar-DamID (Bersaglieri et al., PMID: 35304483), which has been validated through DNA-FISH.

We did not include nucleolar DamID in the mention in the Introduction of methods for identifying diJerential lamina versus nucleolar interactions of heterochromatin- either from our own collaborative group or from the cited reference- because we did not have confidence in the accuracy of this method in identifying NADs. In the case of the published nucleolar DamID from our collaborative group, published in Wang et al, 2021, we later discovered that despite apparent agreement of the nucleolar DamID with a small number of published FISH localization the overall correlation of the nucleolar DamID with nucleolar localization was poor. As described in detail in the Kumar et al, 2024 publication, this poor correlation of the nucleolar DamID was established using three orthogonal methods- nucleolar TSA-seq, NAD-seq, and multiplexed immuno-FISH measurements from ~1000 genomic locations. Instead, we found that this nucleolar DamID showed high correlation with lamina DamID. We note that many strong NADs are also LADs, which we think is why validation with only several FISH probes is inadequate to demonstrate overall validation of the approach.

We could not compare our nucleolar-DamID data in human cells with the alternative nucleolar-DamID results cited by the reviewer which were performed in mouse cells. We note that in this paper the nucleolar DamID FISH validation only included several putative NAD chromosome regions and, I believe, one LAD region. However, our initial comparison of the nucleolar DamID cited by the reviewer with unpublished TSA-seq data from mouse ESCs produced by the Belmont laboratory and with NAD-seq data from the Kaufman laboratory shows a similar lack of correlation of the nucleolar DamID signal with nucleolar TSA-seq and NAD-seq, as well as multiplexed immuno-FISH data from the Long Cai laboratory, as we saw in our analysis of own nucleolar DamID data in human cells.

We have added explanation concerning the lack of correlation of our nucleolar DamID with orthogonal measurements of nucleolar proximity in the added text (below) to our revised manuscript:

"Nucleolar DamID instead showed broad positive peaks over large chromatin domains, largely overlapping with LADs mapped by LMNB1 DamID (Wang et al., 2021). However, this nucleolar DamID signal, while strongly correlated with lamin DamID, showed poor correlation with either NAD-seq or nucleolar distances mapped by multiplexed immunoFISH (Kumar et al., 2024). We suspect the problem is that with molecular proximity assays the output signals are disproportionally dominated by the small fraction of target proteins juxtaposed in su=icient proximity to the DNA to produce a signal rather than the amount of protein concentrated in the target nuclear body. "

Our mention of nucleolar TSA-seq was in the context of why we focused on nucleolar TSAseq and excluded our own nucleolar DamID. We chose not to discuss the second nucleolar DamID method cited above 1) because it was not appropriate to our discussion of our own experimental approach and 2) also because we cannot yet make a definitive statement of its accuracy for nucleolar mapping.

Reviewer #2 (Recommendations For The Authors):

(1) The authors start the manuscript by describing the 'radial genome organization' model and contrast it with the 'binary model' of genome organization. It would be helpful for the authors to contextualize their results a bit more with regard to these two diJerent models in the discussion.

We have added several sentences in the first paragraph of the Discussion to accomplish this contextualization. The new paragraph reads:

"Here we integrated imaging with both spatial (DamID, TSA-seq) and functional (Repli-seq, RNA-seq) genomic readouts across four human cell lines. Overall, our results significantly extend previous nuclear genome organization models, while also demonstrating a cell-type dependent complexity of nuclear genome organization. Briefly, in contrast to the previous radial model of genome organization, we reveal a primary correlation of gene expression with relative distances to nuclear speckles rather than radial position. Additionally, beyond a correlation of nuclear genome organization with radial position, in cells with flat nuclei we show a pronounced correlation of nuclear genome organization with distance from the equatorial plane. In contrast to previous binary models of genome organization, we describe how both iLAD / A compartment and LAD / B compartment contain within them smaller chromosome regions with distinct biochemical and/or functional properties that segregate di=erentially with respect to relative distances to nuclear locales and geometry."

(2) Data should be provided demonstrating KO of LBR and LMNA - immunoblotting for both proteins would be one approach. In addition, it would be helpful to provide additional nuclear morphology measurements of the DKO cells (volume, surface area, volume of speckles/nucleoli, number of speckles/nucleoli).

We've added additional description describing the generation and validation of the KO lines:

"To create LMNA and LBR knockout (KO) lines and the LMNA/LBR double knockout (DKO) line, we started with a parental "wt" K562 cell line, clone #17, expressing an inducible form of Cas9 (Brinkman et al., 2018). The single KO and DKO were generated by CRISPR-mediated frameshift mutation according to the procedure described previously (Schep et al., 2021). The "wt" K562 clone #17 was used for comparison with the KO clones.

The LBR KO clone, K562 LBR-KO #19, was generated, using the LBR2 oligonucleotide GCCGATGGTGAAGTGGTAAG to produce the gRNA, and validated previously, using TIDE (Brinkman et al., 2014) to check for frameshifts in all alleles as described elsewhere (Schep et al., 2021). The LMNA/LBR DKO, K562 LBR-LMNA DKO #14, was made similarly, starting with the LBR KO line and using the combination of two oligonucleotides to produce gRNAs:

LMNA-KO1: ACTGAGAGCAGTGCTCAGTG, LMNA-KO2: TCTCAGTGAGAAGCGCACGC.

Additionally, the LMNA KO line, K562 LMNA-KO #14, was made the same way but starting with the "wt" K562 cell line. Validation was as described above; additionally, for the new LMNA KO and LMNA/LBR DKO lines, immunostaining showed the absence of anti-LMNA antibody signal under confocal imaging conditions used to visualize the wt LMNA staining while the RNA-seq from these clones revealed an ~20-fold reduction in LMNA RNA reads relative to the wt K562 clone."

As suggested, we also added morphological data for the DKO line in a modified SFig.5.

(3) The rationale for using LMNB1 TSA-seq and LMNB1 DAMID is not immediately clear. The LMNB1 TSA-seq is more variable across cell types and replicates than the DAMID. Could the authors please compare the datasets a bit more to understand the diJerences? For example, the authors demonstrate that "40-70% of the genome shows statistically significant diJerences in Lamina TSA-seq over regions 100 kb or larger, with most of these regions showing little or no diJerences in speckle TSA-seq scores." If the LMNB1 DAMID data is used for this analysis or Figure 2D, is the same conclusion reached? Also, in Figure 6, the authors conclude that C1 and C3 LAD regions are enriched for constitutive LADs, while C2 and C4 LAD regions are fLADs. This is a bit surprising because the authors and others have previously shown that constitutive LADs have higher LMNB1 contact frequency than facultative LADs (Kind, et al Cell 2015, Figure 3C).

Indeed, in the first TSA-seq paper (Chen et al, 2018) we did observe that cLADs had the highest LMNB TSA-seq scores; this was for K562 cells with round nuclei in which there is therefore no diJerence in lamina TSA-seq scores produced by nuclear shape over the entire nucleus.

However, there are diJerences between TSA-seq and DamID in terms of what they measure and we refer the reviewer to the first TSA-seq paper (Chen et al, 2018) that explains in greater depth these diJerences. This first paper explains how DamID is indeed related to contact frequency but how the TSA-seq instead estimates mean distances from the target, in this case the nuclear lamina. This is because the diJusion of tyramide free radicals from the site of their constant HRP production produces an exponential decay gradient of tyramide free radical concentration at steady state.

We have summarized these diJerences in in text we have added to introduce both DamID and TSA-seq in the second Results section:

"DamID is a well-established molecular proximity assay; DamID applied to the nuclear lamina divides the genome into lamina-associated domains (LADs) versus nonassociated “inter-LADs” or “iLADs” (Guelen et al., 2008; van Steensel and Belmont, 2017). In contrast, TSA-seq measures relative distances to targets on a microscopic scale corresponding to 100s of nm to ~ 1 micron based on the measured diJusion radius of tyramide-biotin free-radicals (Chen et al., 2018)... While LMNB1 DamID segments LADs most accurately, lamin TSA-seq provides distance information not provided by DamID- for example, variations in relative distances to the nuclear lamina of diJerent iLADs and iLAD regions. These diJerences between the LMNB1 DamID and LMNB TSA-seq signals are also crucial to a computational approach, SPIN, that segments the genome into multiple states based on their varying nuclear localization, including biochemically and functionally distinct lamina-associated versus near-lamina states (Consortium et al., 2024; Wang et al., 2021).

Thus, lamin DamID and TSA-seq complement each other, providing more information together than either one separately."

We note that these diJerences in lamina DamID and TSA-seq are crucial to being able to gain additional information by comparing variations in the lamina TSA-seq for LADs in Figs. 6&7. See our response to point (4) below, for further explanation.

(4) In 7B/C, the authors show that the highest LMNB1 regions in HFF are equator of IMR90s. However, in Figure 7G, their cLAD score indicates that constitutive LADs are not at the equator. This is a bit surprising given the point above and raises the possibility that SON signals (as opposed to LMNB1 signals) might be more responsible for correlation to localization relative to the equator. Hence, it might be helpful if the authors repeat the analyses in Figures 7B/C in regions with diJering LMNB1 signals but similar SON signals (and vice versa).

Again, this is based on the apparent assumption by the reviewer that DamID and TSA-seq work the same way and measure the same thing. But as explained above in the previous point, this is not true.

In our first TSA-seq paper (Chen et al, 2018) we showed how we could use the exponential decay point-spread-function produced by TSA, measured directly by light microscopy, to convert sequencing reads from the TSA-seq into a predicted mean distance from nuclear speckles, approximated as point sources. These mean distances predicted from the SON TSA-seq data agreed with measured FISH distances to nuclear speckles to within ~50 nm for a set of DNA probes from diJerent chromosome regions. Moreover, varying TSA staining conditions changed the decay constants of this exponential decay, thus producing diJerences in the SON TSA-seq signals. By using these diJerent exponential decay functions to convert the TSA-seq scores from these independent data sets to estimated distances from nuclear speckles, we again observed a distance residual of ~50 nm; in this case though this distance residual of ~50 nm represented the mean residual observed genome-wide. This gives us great confidence that the TSA-seq is working as we have modeled it.

As we mentioned in our response to point 3 above, we did see the highest LMNB TSA-seq signal for cLADs in K562 cells with round nuclei (Chen et al, 2018).

But as we now show in our simulation performed in this paper for Fig. 7, the observed tyramide free radical exponential decay gradient convolved with the flat nuclear lamina shape produces a higher equatorial LMNB1 TSA-seq signal for LADs at the equatorial plane. We confirmed that LADs with this higher TSA-seq signal were enriched at the equatorial plane by mining the multiplexed IMR90 imaging data. Similar mining of the multiplexed FISH IMR90 data showed localization of cLADs away from the equatorial plane.

We are not clear about the rationale for what the reviewer is suggesting about SON signals "being more responsible for correlation to localization to the equator". We have provided an explanation for the higher lamina TSA-seq scores for LADs near the equator based on the measured spreading of the tyramide free radicals convolved with the eJect of the nuclear shape. This makes a prediction that the observed variation in lamina TSA-seq scores for LADs with similar DamID scores is related to their positioning relative to the equatorial plane as we then validated through our mining of the IMR90 multiplexed FISH data.

(5) FISH of individual LADs, v-fiLADs, and p-w-v-fiLADs relative to the lamina and speckle would be helpful to understand their relative positioning in control and LBR/LMNA double KO cells. This would significantly bolster the claim that "histone mark enrichments..more precisely revealed the diJerential spatial distribution of LAD regions...".

Adequately testing these predictions made from the lamina/SON TSA-seq scatterplots by direct FISH measurements would require measurements from large numbers of diJerent chromosome regions through a highly multiplexed immuno-FISH approach. We are not equipped currently in any of our laboratories to do such measurements and we leave this therefore for future studies.

Rather our statement is based on our use of TSA-seq analyzed through these 2D scatterplots and should be valid to the degree that our TSA-seq measurements do indeed correlate with microscopy derived distances.

However, we do now include demonstration of a high correlation of speckle, lamina, and nucleolar TSA-seq with highly multiplexed immuno-FISH measurement of distances to these locales in a revised Fig. 7. The high correlation shown between the TSA-seq scores and measured distances does therefore add additional support to our claim that the reviewer is discussing, even without our own multiplexed FISH validation.

(6) "In contrast, genes within genomic regions which in pair-wise comparisons of cell lines show a statistically significant diJerence in lamina TSA-seq show no obvious trend in their expression diJerences (Figure 2C).". This appears to be an overstatement based on the left panel of 2D.

We do not follow the reviewer's point. In Fig. 2C we show little bias in the diJerences in gene expression between the two cell types for regions that showed diJerences in lamina TSA-seq. The reviewer is suggesting something otherwise based on their impression, not explicitly stated, of the left panel of Fig. 2D. But we see similar shades of blue extending vertically at low SON values and similar shades of red extending vertically at high SON values, suggesting a correlation of gene expression only with the SON TSA-seq score but not with the LMNB1 TSA-seq score displayed on the y-axis. This is also consistent with the very small and/or insignificant correlation coeJicients measured in our linear model relating diJerences in LMNB1 TSA-seq to diJerences in expression but the large correlation coeJicient observed for SON TSA-seq (Fig. 2E). Thus, we see Fig. 2C-E as self-consistent.

(7) In the section on "Polarity of Nuclear Genome Organization" - "....Using the IMR90 multiplexed FISH data set [43]...." - The references are not numbered.

We thank the reviewer for this correction.

(8) I believe there is an error in the Figure 7B legend. The descriptions of Cluster 1 and 2 do not match those indicated in the figure.

We again thank the reviewer for this correction.

Author response:

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

Reviewer #1 (Public review):

Summary:

In their manuscript, Gomez-Frittelli and colleagues characterize the expression of cadherin6 (and -8) in colonic IPANs of mice. Moreover, they found that these cdh6-expressing IPANs are capable of initiating colonic motor complexes in the distal colon, but not proximal and midcolon. They support their claim by morphological, electrophysiological, optogenetic, and pharmacological experiments.

Strengths:

The work is very impressive and involves several genetic models and state-of-the-art physiological setups including respective controls. It is a very well-written manuscript that truly contributes to our understanding of GI-motility and its anatomical and physiological basis. The authors were able to convincingly answer their research questions with a wide range of methods without overselling their results.

We greatly appreciate the reviewer’s time, careful reading and support of our study.

Weaknesses:

The authors put quite some emphasis on stating that cdh6 is a synaptic protein (in the title and throughout the text), which interacts in a homophilic fashion. They deduct that cdh6 might be involved in IPAN-IPAN synapses (line 247ff.). However, Cdh6 does not only interact in synapses and is expressed by non-neuronal cells as well (see e.g., expression in the proximal tubuli of the kidney). Moreover, cdh6 does not only build homodimers, but also heterodimers with Chd9 as well as Cdh7, -10, and -14 (see e.g., Shimoyama et al. 2000, DOI: 10.1042/02646021:3490159). It would therefore be interesting to assess the expression pattern of cdh6proteins using immunostainings in combination with synaptic markers to substantiate the authors' claim or at least add the possibility of cell-cell-interactions other than synapses to the discussion. Additionally, an immunostaining of cdh6 would confirm if the expression of tdTomato in smooth muscle cells of the cdh6-creERT model is valid or a leaky expression (false positive).

We agree with the reviewer that Cdh6 could be mediating some other cell-cell interaction besides synapses between IPANs, and we noted it in the discussion. Cdh6 primarily forms homodimers but, as the reviewer points out, has been known to also form heterodimers with some other cadherins. We performed RNAscope in the colonic myenteric plexus with Cdh7 and found no expression (data not shown). Cdh10 is suggested to have very low expression (Drokhlyansky et al., 2020), possibly in putative secretomotor vasodilator neurons, and Cdh14 has not been assayed in any RNAseq screens. We attempted to visualize Cdh6 protein via antibody staining (Duan et al., 2018) but our efforts did not result in sufficient signal or resolution to identify synapses in the ENS, which remain broadly challenging to assay. Similarly, immunostaining with Cdh6 antibody was unable to confirm Cdh6 protein in tdT-expressing muscle cells, or by RNAscope. We have addressed these caveats in the discussion section.

(1) E. Drokhlyansky, C. S. Smillie, N. V. Wittenberghe, M. Ericsson, G. K. Griffin, G. Eraslan, D. Dionne, M. S. Cuoco, M. N. Goder-Reiser, T. Sharova, O. Kuksenko, A. J. Aguirre, G. M. Boland, D. Graham, O. Rozenblatt-Rosen, R. J. Xavier, A. Regev, The Human and Mouse Enteric Nervous System at Single-Cell Resolution. Cell 182, 1606-1622.e23 (2020).

(2) X. Duan, A. Krishnaswamy, M. A. Laboulaye, J. Liu, Y.-R. Peng, M. Yamagata, K. Toma, J. R. Sanes, Cadherin Combinations Recruit Dendrites of Distinct Retinal Neurons to a Shared Interneuronal Scaffold. Neuron 99, 1145-1154.e6 (2018).

Reviewer #2 (Public review):

Summary:

Intrinsic primary afferent neurons are an interesting population of enteric neurons that transduce stimuli from the mucosa, initiate reflexive neurocircuitry involved in motor and secretory functions, and modulate gut immune responses. The morphology, neurochemical coding, and electrophysiological properties of these cells have been relatively well described in a long literature dating back to the late 1800's but questions remain regarding their roles in enteric neurocircuitry, potential subsets with unique functions, and contributions to disease. Here, the authors provide RNAscope, immunolabeling, electrophysiological, and organ function data characterizing IPANs in mice and suggest that Cdh6 is an additional marker of these cells.

Strengths:

This paper would likely be of interest to a focused enteric neuroscience audience and increase information regarding the properties of IPANs in mice. These data are useful and suggest that prior data from studies of IPANs in other species are likely translatable to mice.

We appreciate the reviewer’s support of our study and insightful critiques for its improvement.

Weaknesses:

The advance presented here beyond what is already known is minimal. Some of the core conclusions are overstated and there are multiple other major issues that limit enthusiasm. Key control experiments are lacking and data do not specifically address the properties of the proposed Cdh6+ population.

Major weaknesses:

(1) The novelty of this study is relatively low. The main point of novelty suggests an additional marker of IPANs (Cdh6) that would add to the known list of markers for these cells. How useful this would be is unclear. Other main findings basically confirm that IPANs in mice display the same classical characteristics that have been known for many years from studies in guinea pigs, rats, mice and humans.

We appreciate the already existing markers for IPANs in the ENS and the existing literature characterizing these neurons. The primary intent of this study was to use these well-established characteristics of IPANs in both mice and other species to characterize Cdh6-expressing neurons in the mouse myenteric plexus and confirm their classification as IPANs.

(2) Some of the main conclusions of this study are overstated and claims of priority are made that are not true. For example, the authors state in lines 27-28 of the abstract that their findings provide the "first demonstration of selective activation of a single neurochemical and functional class of enteric neurons". This is certainly not true since Gould et al (AJP-GIL 2019) expressed ChR2 in nitrergic enteric neurons and showed that activating those cells disrupted CMC activity. In fact, prior work by the authors themselves (Hibberd et al., Gastro 2018) showed that activating calretinin neurons with ChR2 evoked motor responses. Work by other groups has used chemogenetics and optogenetics to show the effects of activating multiple other classes of neurons in the gut.

We thank the reviewer for bringing up this important point and apologize if our wording was not clear. Whilst single neurochemical classes of enteric neurons have been manipulated to alter gut functions, all such instances to date do not represent manipulation of a single functional class of enteric neurons. In the given examples, multiple functional classes are activated utilizing the same neurotransmitter, as NOS and calretinin are each expressed to varying degrees across putative motor neurons, interneurons and IPANs. In contrast, Chd6 is restricted to IPANs and therefore this study is the first optogenetic investigation of enteric neurons from a single putative functional class. Our abstract and discussion emphasizes this point and differentiates this study from those previous.

(3) Critical controls are needed to support the optogenetic experiments. Control experiments are needed to show that ChR2 expression a) does not change the baseline properties of the neurons, b) that stimulation with the chosen intensity of light elicits physiologically relevant responses in those neurons, and c) that stimulation via ChR2 elicits comparable responses in IPANs in the different gut regions focused on here.

We completely agree controls are essential. However, our paper is not the first to express ChR2 in enteric neurons. Authors of our paper have shown in Hibberd et al. 2018 that expression of ChR2 in a heterogeneous population of myenteric neurons did not change network properties of the myenteric plexus. This was demonstrated in the lack of change in control CMC characteristics in mice expressing ChR2 under basal conditions (without blue light exposure). Regarding question (b), that it should be shown that stimulation with the chosen intensity of light elicits physiologically relevant responses in those neurons. We show the restricted expression of ChR2 in IPANs and that motor responses (to blue light) are blocked by selective nerve conduction blockade.

Regarding question (c), that our study should demonstrate that stimulation via ChR2 elicits comparable responses in IPANs in the different gut regions. We would not expect each region of the gut to behave comparably. This is because the different gut regions (i.e. proximal, mid, distal) are very different anatomically, as is anatomy of the myenteric plexus and myenteric ganglia between each region, including the density of IPANs within each ganglia, in addition to the presence of different patterns of electrical and mechanical activity [Spencer et al., 2020]. Hence, it is difficult to expect that between regions stimulation of ChR2 should induce similar physiological responses. The motor output we record in our study (CMCs) is a unified motor program that involves the temporal coordination of hundreds of thousands of enteric neurons and a complex neural circuit that we have previously characterized [Spencer et al., 2018]. But, never has any study until now been able to selectively stimulate a single functional class of enteric neurons (with light) to avoid indiscriminate activation of other classes of neurons.

(1) T. J. Hibberd, J. Feng, J. Luo, P. Yang, V. K. Samineni, R. W. Gereau, N. Kelley, H. Hu, N. J. Spencer, Optogenetic Induction of Colonic Motility in Mice. Gastroenterology 155, 514-528.e6 (2018).

(2) N. J. Spencer, L. Travis, L. Wiklendt, T. J. Hibberd, M. Costa, P. Dinning, H. Hu, Diversity of neurogenic smooth muscle electrical rhythmicity in mouse proximal colon. American Journal of Physiology-Gastrointestinal and Liver Physiology 318, G244–G253 (2020).

(3) N. J. Spencer, T. J. Hibberd, L. Travis, L. Wiklendt, M. Costa, H. Hu, S. J. Brookes, D. A. Wattchow, P. G. Dinning, D. J. Keating, J. Sorensen, Identification of a Rhythmic Firing Pattern in the Enteric Nervous System That Generates Rhythmic Electrical Activity in Smooth Muscle. The Journal of Neuroscience 38, 5507–5522 (2018).

(4) The electrophysiological characterization of mouse IPANs is useful but this is a basic characterization of any IPAN and really says nothing specifically about Cdh6+ neurons. The electrophysiological characterization was also only done in a small fraction of colonic IPANs, and it is not clear if these represent cell properties in the distal colon or proximal colon, and whether these properties might be extrapolated to IPANs in the different regions. Similarly, blocking IH with ZD7288 affects all IPANs and does not add specific information regarding the role of the proposed Cdh6+ subtype.

Our electrophysiological characterization was guided to be within a subset of Cdh6+ neurons by Hb9:GFP expression. As in the prior comment (1) above, we used these experiments to confirm classification of Cdh6+ (Hb9:GFP+) neurons in the distal colon as IPANs. We have clarified in the results and methods that these experiments were performed in the distal colon and agree that we cannot extrapolate that these properties are also representative of IPANs in the proximal colon. We apologize that this was confusing. Finally, we agree with the reviewer that ZD7288 affects all IPANs in the ENS and have clarified this in the text.

(5) Why SMP IPANs were not included in the analysis of Cdh6 expression is a little puzzling. IPANs are present in the SMP of the small intestine and colon, and it would be useful to know if this proposed marker is also present in these cells.

We agree with the reviewer. In addition to characterizing Cdh6 in the myenteric plexus, it would be interesting to query if sensory neurons located within the SMP also express Cdh6. Our preliminary data (n=2) show ~6-12% tdT/Hu neurons in Cdh6-tdT ileum and colon (data not shown). We have added a sentence to the discussion.

(6) The emphasis on IH being a rhythmicity indicator seems a bit premature. There is no evidence to suggest that IH and IT are rhythm-generating currents in the ENS.

Regarding the statement there is no evidence to suggest that IH and IT are rhythm-generating currents in the ENS. We agree with the reviewer that evidence of rhythm generation by IH and IT in the ENS has not been explicitly confirmed. We are confident the reviewer agrees that an absence of evidence is not evidence of absence, although the presence of IH has been well described in enteric neurons. We have modified the text in the results to indicate more clearly that IH and IT are known to participate in rhythm generation in thalamocortical circuits, though their roles in the ENS remain unknown. Our discussion of the potential role of IH or IT in rhythm generation or oscillatory firing of the ENS is constrained to speculation in the discussion section of the text.

(7) As the authors point out in the introduction and discuss later on, Type II Cadherins such as Cdh6 bind homophillically to the same cadherin at both pre- and post-synapse. The apparent enrichment of Cdh6 in IPANs would suggest extensive expression in synaptic terminals that would also suggest extensive IPAN-IPAN connections unless other subtypes of neurons express this protein. Such synaptic connections are not typical of IPANs and raise the question of whether or not IPANs actually express the functional protein and if so, what might be its role. Not having this information limits the usefulness of this as a proposed marker.

We agree with the reviewer that the proposed IPAN-IPAN connection is novel although it has been proposed before (Kunze et al., 1993). As detailed in our response to Reviewer #1, we attempted to confirm Cdh6 protein expression, but were unsuccessful, due to insufficient signal and resolution. We therefore discuss potential IPAN interconnectivity in the discussion, in the context of contrasting literature.

(1) W. A. A. Kunze, J. B. Furness, J. C. Bornstein, Simultaneous intracellular recordings from enteric neurons reveal that myenteric ah neurons transmit via slow excitatory postsynaptic potentials. Neuroscience 55, 685–694 (1993).

(8) Experiments shown in Figures 6J and K use a tethered pellet to drive motor responses. By definition, these are not CMCs as stated by the authors.

The reviewer makes a valid criticism as to the terminology, since tethered pellet experiments do not record propagation. We believe the periodic bouts of propulsive force on the pellet is triggered by the same activity underlying the CMC. In our experience, these activities have similar periodicity, force and identical pharmacological properties. Consistent with this, we also tested full colons (n = 2) set up for typical CMC recordings by multiple force transducers, finding that CMCs were abolished by ZD7288, similar to fixed pellet recordings (data not shown).

(9) The data from the optogenetic experiments are difficult to understand. How would stimulating IPANs in the distal colon generate retrograde CMCs and stimulating IPANs in the proximal colon do nothing? Additional characterization of the Cdh6+ population of cells is needed to understand the mechanisms underlying these effects.

We agree that the different optogenetic responses in the proximal and distal colon are challenging to interpret, but perhaps not surprising in the wider context. It is not only possible that the different optogenetic responses in this study reflect regional differences in the Chd6+ neuronal populations, but also differences in neural circuits within these gut regions. A study some time ago by the authors showed that electrical stimulation of the proximal mouse colon was unable to evoke a retrograde (aborally) propagating CMC (Spencer, Bywater, 2002), but stimulation of the distal colon was readily able to. We concluded that at the oral lesion site there is a preferential bias of descending inhibitory nerve projections, since the ascending excitatory pathways have been cut off. In contrast, stimulation of the distal colon was readily able to activate an ascending excitatory neural pathway, and hence induce the complex CMC circuits required to generate an orally propagating CMC. Indeed, other recent studies have added to a growing body of evidence for significant differences in the behaviors and neural circuits of the two regions (Li et al., 2019, Costa et al., 2021a, Costa et al., 2021b, Nestor-Kalinoski et al., 2022). We have expanded this discussion.

(1) N. J. Spencer, R. A. Bywater, Enteric nerve stimulation evokes a premature colonic migrating motor complex in mouse. Neurogastroenterology & Motility 14, 657–665 (2002).

(2) Li Z, Hao MM, Van den Haute C, Baekelandt V, Boesmans W, Vanden Berghe P, Regional complexity in enteric neuron wiring reflects diversity of motility patterns in the mouse large intestine. Elife 8:e42914 (2019).

(3) Costa M, Keightley LJ, Hibberd TJ, Wiklendt L, Dinning PG, Brookes SJ, Spencer NJ, Motor patterns in the proximal and distal mouse colon which underlie formation and propulsion of feces. Neurogastroenterology & Motility e14098 (2021a).

(4) Costa M, Keightley LJ, Hibberd TJ, Wiklendt L, Smolilo DJ, Dinning PG, Brookes SJ, Spencer NJ, Characterization of alternating neurogenic motor patterns in mouse colon. Neurogastroenterology & Motility 33:e14047 (2021b).

(5) Nestor-Kalinoski A, Smith-Edwards KM, Meerschaert K, Margiotta JF, Rajwa B, Davis BM, Howard MJ, Unique Neural Circuit Connectivity of Mouse Proximal, Middle, and Distal Colon Defines Regional Colonic Motor Patterns. Cellular and Molecular Gastroenterology and Hepatology 13:309-337.e303 (2022).

Recommendations for the Authors:

Reviewer #1 (Recommendations for the authors):

As mentioned above, immunolocalization of cdh6 would be helpful to substantiate the claims regarding IPAN-IPAN synapses.

As mentioned in our response to both reviewers’ public reviews, we attempted to visualize Cdh6 protein via antibody staining (Duan et al., 2018), but our efforts did not result in sufficient signal or resolution to identify Cdh6+ synapses.

(1) X. Duan, A. Krishnaswamy, M. A. Laboulaye, J. Liu, Y.-R. Peng, M. Yamagata, K. Toma, J. R. Sanes, Cadherin Combinations Recruit Dendrites of Distinct Retinal Neurons to a Shared Interneuronal Scaffold. Neuron 99, 1145-1154.e6 (2018).

Reviewer #2 (Recommendations for the authors):

(1) The authors repeatedly refer to IPANs as "sensory" neurons (e.g. in title, abstract, and introduction) but there is some debate regarding whether these cells are truly "sensory" because the information they convey never reaches sensory perception. This is why they have classically been referred to as intrinsic primary afferent (IPAN) neurons. It would be more appropriate to stick with this terminology unless the authors have compelling data showing that information detected by IPANs reaches the sensory cortex.

We thank the reviewer for their comment, but respectfully disagree. The term “sensory neuron” is well established in the ENS. The first definitive proof that “sensory neurons” exist in the ENS was published in Kunze et al., 1995. We note that this paper did not use the word “IPAN” but used the term “sensory neuron”. Furthermore, mechanosensory neurons were published in Spencer and Smith (2004).

Regarding the reviewer’s comment that the authors would need compelling data showing that information detected by IPANs reaches the sensory cortex before the term “sensory neuron” should be valid, it is important to note that many sensory neurons do not provide direct information to the cortex.

(1) W. A. A. Kunze, J. C. Bornstein, J. B. Furness, Identification of sensory nerve cells in a peripheral organ (the intestine) of a mammal. Neuroscience 66, 1–4 (1995).

(2) N. J. Spencer, T. K. Smith, Mechanosensory S-neurons rather than AH-neurons appear to generate a rhythmic motor pattern in guinea-pig distal colon. The Journal of Physiology 558, 577–596 (2004).

(2) Important information regarding the gut region shown and other details are absent from many figure legends.

We apologize for this omission. We have updated the figure legends to include information on gut regions.

Briefing Document: Autonomie des établissements et réussite scolaire : le paradoxe français Source: Excerpts from "Autonomie des établissements et réussite scolaire : le paradoxe français" (Podcast interview with Éric Charbonnier, analyst at the OECD and member of the Conseil d'évaluation de l'école).

Thèmes principaux:

L'autonomie des établissements scolaires en France: Un sujet sensible et souvent mal compris, source de peurs et de résistances.

Le lien entre autonomie et performance des élèves: Les études internationales (PISA) montrent une corrélation positive entre autonomie des établissements et réussite des élèves, particulièrement lorsque l'autonomie est accordée aux acteurs de terrain (chefs d'établissement, enseignants).

L'importance d'une culture d'établissement: La création d'un esprit d'équipe, de coopération, et d'échange de bonnes pratiques est essentielle pour la progression des élèves.

Les freins à l'autonomie en France: Centralisation excessive, rôle limité du chef d'établissement, programmes scolaires rigides, manque de flexibilité dans la gestion des ressources humaines.

La nécessité d'une évaluation cohérente des politiques éducatives: Manque de constance et de cohérence dans les réformes, absence d'évaluation rigoureuse de leur impact, conduisant à un sentiment de découragement chez les acteurs de terrain.

Le rôle crucial du bien-être: Le bien-être des personnels et des élèves est un facteur déterminant de la réussite scolaire, souvent négligé au profit d'une focalisation excessive sur les fondamentaux. Idées et faits importants:

La peur de l'autonomie est infondée: "Je crois que c'est important de partir du en terme de début sur le fait qu'il y a des mots qui font peur en France quand on parle d'autonomie [...] il y a tout de suite une peur qui qui se crée dans la communauté éducative [...] Je crois qu'il faut qu'on sorte déjà de de de cette idée que dès qu'on parle d'autonomie ça va être forcément au détriment du système éducatif." L'autonomie ne doit pas être perçue comme une menace mais comme un levier d'amélioration.

L'autonomie locale est bénéfique, la centralisation est contre-productive: "Ce qui est assez fascinant c'est qu'on voit quand l'autonomie est donnée aux établissements et à leurs acteurs [...] les résultat des élèves s'en trouvent systématiquement améliorés.

À contrario quand l'autonomie est donné aux autorités nationales dans des pays centralisés comme le nôtre on voit qu'il y a un effet négatif sur la performance." Les décisions doivent être prises au plus près du terrain pour être efficaces.

Les exemples internationaux montrent la voie: Des pays comme le Portugal, le Royaume-Uni, le Canada, l'Australie, le Japon et la Corée ont réussi à améliorer la performance de leurs élèves grâce à une forte culture de l'autonomie des établissements, à la formation des chefs d'établissement et des enseignants, et à la coopération.

La France souffre d'une culture individualiste: "On voit qu'on a encore une culture très individualiste dans nos collèges, l'enseignant a la liberté pédagogique mais finalement il est seul face à ces problèmes et ça l'autonomie ça peut apporter ça aussi créer de la coopération [...]"

L'autonomie doit favoriser le travail d'équipe et l'échange de bonnes pratiques.

L'autonomie RH, un tabou français: Dans un tiers des pays de l'OCDE, le chef d'établissement est responsable du recrutement d'une partie des enseignants. Cette flexibilité permet de souder les équipes pédagogiques et de mieux adapter les compétences aux besoins de l'établissement. "Est-ce qu'on imagine ça en France avoir un chef d'établissement au collège et lycée qui peut choisir ces enseignants [...] c'est monnaie courante dans beaucoup de pays et ça permet de la flexibilité."

L'autonomie budgétaire est un levier de performance:

L'autonomie permet aux établissements d'utiliser leur budget de manière plus efficace en fonction de leurs priorités et de leurs besoins spécifiques. "L'autonomie ça offre au pays la possibilité d'utiliser leur budget comme ils le souhaitent de décider du contenu de la formation continue."

L'effet chef d'établissement est mesurable: Des études montrent que les chefs d'établissement performants ont un impact significatif sur la réussite des élèves, notamment dans les établissements les plus difficiles. "Il y a beaucoup d'études qui ont été faites notamment si on prend aux États-Unis ou dans les établissements les plus difficiles on on affectait les meilleurs chefs d'établissement [...] et ça conduisaient à l'amélioration des résultats des élèves."

Le bien-être est une condition essentielle de la réussite: "Quel que soit le métier euh s'il y a pas un environnement où les gens puissent s'épanouir être heureux [...] on tombe facilement dans dans l'ennui si c'est pas le cas et le bien-être est ultra important." Le bien-être des personnels et des élèves doit être au cœur des politiques éducatives.

La cohérence et la constance des réformes sont indispensables: "Il faudrait avoir une obligation de garder un cap au moins une dizaine d'années pour éviter que on on détricote en permanence qu'on ce qu'on a pu faire avant et que finalement on puisse jamais juger le bénéfice de ces réformes." Les changements de cap fréquents et l'absence d'évaluation rigoureuse des réformes nuisent à leur efficacité.

Les programmes scolaires doivent être allégés et recentrés sur l'essentiel: "Il faut équiper les jeunes avec les compétences du 21e siècle [...] et finalement la solution qui a été choisi dans beaucoup de pays c'est d'alourdir les programmes on rajoute et on rajoute [...] et du coup ça crée du stress pour tout le monde."

L'alourdissement des programmes crée du stress et entrave l'acquisition des compétences essentielles. La qualité des enseignants est le facteur clé de la réussite: "La qualité d'un système éducatif n'excède jamais la qualité de ces enseignants et de son chef d'établissement voilà c'est le levier premier de la réussite." La formation, le bien-être et la valorisation des enseignants sont des priorités absolues.

Il faut mieux utiliser les données disponibles: La France dispose de données de qualité, tant au niveau national qu'international, mais il faut les utiliser de manière plus systématique pour identifier les bonnes pratiques et adapter les politiques éducatives aux besoins spécifiques des établissements.

Conclusion:

L'interview met en lumière le paradoxe français : un système éducatif centralisé et rigide, malgré une volonté affichée d'autonomie.

Les études internationales montrent que l'autonomie des établissements, lorsqu'elle est associée à une culture de coopération, à la formation des personnels, et à une évaluation rigoureuse des politiques, est un facteur clé de la réussite des élèves.

Il est impératif de lever les freins à l'autonomie en France, de valoriser le rôle des chefs d'établissement et des enseignants, et de se concentrer sur le bien-être de tous les acteurs de l'éducation.

La valorisation et la diffusion des études de l'OCDE sont un pas important vers cette direction.

Liens utiles mentionnés:

PISA website: Recherche PISA Site internet Publication "Regards sur l'éducation": Recherche OCDE Regards sur l'éducation TALIS website: Recherche TALIS site internet

What kinds of connections are there?

la question du témoignage et du traumatisme.

La première partie explore le recueil de la parole des enfants victimes de violence dans des "salles Mélanie," espaces conçus pour faciliter leur expression, soulignant l'importance d'une écoute adaptée et spécialisée.

On y entend des extraits d'auditions poignantes, révélant les défis et les émotions auxquels sont confrontés les enquêteurs de la brigade des mineurs

Thèmes Principaux et Idées Clés: vers 0h40min

Salles Mélanie et Recueil de la Parole des Enfants Victimes de Violence :

L'émission met en lumière les "salles Mélanie", des espaces spécialement conçus pour recueillir la parole des enfants victimes de violence. Ces salles visent à créer un environnement plus confortable et sécurisant pour faciliter le témoignage.

L'extrait souligne l'importance de l'audition de l'enfant comme un moment clé de l'enquête : "L'audition de l'enfant mineur victime, c'est vraiment un moment clé de l'enquête puisque c'est là que l'enfant va révéler le traumatisme qu'il a pu vivre."

L'aménagement des salles est crucial : "Ce sont des pièces spécialement aménagées avec du mobilier, des jeux pour enfants, des couleurs chaudes et qui permettent un petit peu comme un petit coconir la parole de ses enfants de manière plus efficace et plus délicate."

Témoignages d'Enfants et Difficultés du Recueil de la Parole :

L'émission présente des extraits poignants de témoignages d'enfants victimes de violence sexuelle, soulignant la difficulté pour les enquêteurs d'aborder ces sujets délicats et d'obtenir des informations précises.

Les enfants utilisent souvent leur propre vocabulaire pour décrire les événements, ce qui nécessite une interprétation attentive de la part des enquêteurs : "Ils vont me dire je sais rien le sexe parce qu'elle a un âge où elle peut très bien dire le sexe comme elle peut dire je sais pas la choupette la pépette j'en sais rien et chacun a son terme."

Le langage non verbal est essentiel : "Tout le gestuel est retranscrit, tout ce qu'elle fait en fait. Donc là pareil he elle recommence à dire à faire un signe de la tête.

En fait c'est sa manière de nous répondre et c'est une réponse."

Les enquêteurs utilisent des outils comme des dessins ou des poupées pour aider les enfants à s'exprimer : "Ce qui fait que ça permet à l'enfant de matérialiser s'il souhaite pas parler. Mais finalement, il peut nous expliquer à travers les les poupées."

Impact Émotionnel sur les Enquêteurs et Stratégies d'Adaptation :

Les enquêteurs de la brigade des mineurs sont confrontés à des situations difficiles qui peuvent les affecter émotionnellement.

Ils développent des mécanismes de défense et partagent leurs expériences avec leurs collègues pour gérer le stress et l'impact émotionnel : "On discute d'autres choses après aussi hein. On parle de nos dossiers. On est change beaucoup. Donc c'est vrai que le fait d'en parler aussi, c'est aussi une façon de de se libérer quoi, de passer à autre chose, de pas garder tout pour nous."

La dérision est parfois utilisée comme une stratégie pour faire face à l'horreur : "Certaines situations des fois peuvent nous faire rire aussi donc on tourne ça aussi des fois la dérision c'est une façon de détourner en fait."

Procédure d'Enquête et Difficultés de Preuve : L'émission montre les différentes étapes d'une enquête pour viol sur mineur, depuis le recueil du témoignage de l'enfant jusqu'à l'interrogatoire du suspect et la décision du magistrat.

L'importance d'étayer les déclarations de l'enfant avec des preuves matérielles ou des témoignages est soulignée : "Il faut recueillir le maximum d'éléments pour que quand on aura en face de nous le agresseur pourrait lui dire écoutez monsieur ce que dit l'enfant c'est la vérité c'est pas quelque chose qu'il a inventé"

Les enquêteurs sont confrontés à la difficulté de prouver les faits lorsque l'agresseur nie et qu'il n'y a pas de preuves matérielles : "On est toujours contre une parole, contre une autre. On a rien de plus."

Le principe du doute qui profite à l'accusé est mis en avant, ce qui peut entraîner le classement sans suite de l'affaire malgré les soupçons : "Le doute profite à l'accusé. Donc ouais, il faut faire avec."

• Purpose & Motivation

  We show that for a broad class of CRUD (Create, Read, Update, Delete) applications, this gap can be bridged.

  • This work addresses the difficulty many web authors face in creating interactive, data-driven applications due to limited programming and data-schema knowledge.

• Core Contribution

  Mavo extends the declarative syntax of HTML to describe Web applications that manage, store and transform data.

  • The system introduces a novel approach where authors define schemas implicitly by marking up HTML elements, effectively eliminating the need for complex server-side CMS solutions or JavaScript frameworks.

• Related Work Context

  These three systems introduced powerful ideas: extending HTML to mark editable data in arbitrary web pages, spreadsheet-like light computation, a hierarchical data model, and independence from back-end functionality.

  • Mavo differentiates itself from prior efforts (e.g., Dido, Quilt, Gneiss) by combining lightweight computation, purely client-side data management, and seamless integration with arbitrary HTML layouts.

• HTML-Based Declarative Syntax

  We chose to use HTML elements, attributes, and classes instead of new syntax for Mavo functionality because our target authors are already familiar with HTML syntax.

  • Mavo leverages standard HTML attributes—like property, data-multiple, and data-store—to mark elements for data binding, persistence, and editing.

• Editing & Storage

  Mavo can store data locally in the page (data-store=#elementid\), in the browser’s local storage (data-store=\local\), in an uploaded / downloaded file (data-store=\file\) or on one of the supported persistent storage services.

  • Once marked, any element or text becomes directly editable on the rendered page; changes are saved back to the chosen store, enabling WYSIWYG content management within the browser.

• Objects & Collections

  Properties that contain other properties become grouping elements (objects in programming terminology); this permits a user to define multi-level schemas.

  • By adding data-multiple, users enable collection-like behavior, allowing repeated sections or items to be dynamically added or removed.

• Lightweight Computation & Expressions

  Expressions are delimited by square brackets ([]) by default and can be placed anywhere inside the Mavo instance, including in HTML attributes.

  • This spreadsheet-like capability supports referencing other properties, performing aggregations (e.g., sum(), average(), count()), and conditional logic (iff()).

• Implementation Details

  Mavo is implemented as a JavaScript library that integrates into a web page to simulate native support for our syntax.

  • On page load, Mavo constructs an internal tree representation of the HTML and expressions, updating all references reactively whenever data changes.

• User Studies: Structured Tasks

  We found that the majority of users were easily able to mark up the editable portions of their mockups to create applications with complex hierarchical schemas.

  • Participants successfully transformed static HTML pages (for a ‘Decisions’ app and a ‘Foodie log’) into dynamic CRUD applications, achieving a 100% success rate on the basic CRUD tasks.

• Common Difficulties

  Some participants frequently copied and pasted expressions when they needed the same calculation in different places.

  • Most challenges arose around conditionals (iff()) and more advanced expressions (e.g., filtered counts), underscoring that more complex logic remains harder for novices.

• User Studies: Freestyle Tasks

  We asked them to use Mavo to make their mockup fully functional in any way they chose.

  • Users brought their own HTML for an ‘Address Book’ concept. All were able to implement multi-level data (e.g., multiple phone numbers) with minimal changes.

• Direct Manipulation Design

  Instead of crafting a data model and then deciding how to template and edit it, a Mavo author’s manipulation of the visual layout of an application automatically implies the data model.

  • This makes building and editing data-backed pages more natural for users who think first in terms of presentation.

• Intended Audience & Scalability

  Mavo is aimed at a broad population of users. There is no hard limit to what it can do, since its expressions also accept arbitrary JavaScript.

  • The system is best suited to ‘small data’ applications such as personal information management or single-author web publishing, but can also facilitate multi-user scenarios with suitable back-end access controls.

• Semantic Web Alignment

  Mavo syntax for naming elements is based on a simplified version of RDFa… as a result, at runtime any Mavo instance becomes valid RDFa that can be consumed by any program that needs it.

  • Authors who add property attributes gain structured, machine-readable data without explicitly learning RDFa or JSON.

• Planned Improvements

  A more direct way to declaratively express these operations [sorting, searching, filtering] is needed… sorting, searching and filtering were recurring themes.

  • Future work will focus on refining conditional syntax, improving data migrations when HTML structures change, and incorporating advanced multi-user access models.

• Conclusion

  We show that HTML authors can quickly learn use Mavo attributes to transform static mockups to CRUD applications, and, to a large extent, use Mavo expressions to perform dynamic calculations and data transformations.

  • The researchers envision a future where editing and storing structured data via standard HTML becomes ubiquitous, lowering barriers for both novice and skilled web authors.

Voici un résumé minuté de la vidéo de Sarah Hill sur le cerveau et la pilule contraceptive :

• 0:07 Introduction à l'épisode sur l'impact de la pilule contraceptive sur le cerveau.
• 0:20 Risques connus de la pilule (AVC, embolie pulmonaire, prise de poids) et impact sur la vie des femmes.
• 0:53 La pilule contraceptive empêche la grossesse, mais quel est son impact sur le cerveau ?
• 1:19 Il est difficile de critiquer la pilule contraceptive car elle a été bénéfique pour les femmes.
• 1:46 La pilule a permis aux femmes de devenir plus indépendantes politiquement et financièrement des hommes.
• 3:07 Il ne faut pas hésiter à parler des hormones sexuelles féminines et de leur rôle dans le cerveau.
• 4:13 Les hormones féminines ne sont pas imprévisibles contrairement aux idées reçues.
• 5:38 Les niveaux de testostérone chez les hommes sont plus variables que les niveaux d'hormones chez les femmes.
• 6:18 Les hormones influencent le cerveau et le comportement des femmes et des hommes.
• 6:36 Il est important de parler de l'impact de la pilule contraceptive sur le cerveau des femmes.
• 6:48 La pilule contraceptive modifie les niveaux d'hormones sexuelles des femmes, ce qui change leur cerveau et qui elles sont.
• 7:40 La pilule contraceptive peut diminuer le désir et la fonction sexuelle chez les femmes.
• 8:36 Le profil hormonal créé par la pilule est caractérisé par des niveaux élevés de progestérone et de faibles niveaux d'œstrogènes et de testostérone.
• 9:05 La pilule contraceptive peut changer l'attirance des femmes envers les hommes en diminuant leur préférence pour les traits masculins.
• 10:57 Les femmes sous pilule choisissent des partenaires avec des visages moins masculins.
• 11:36 Les hormones sexuelles influencent de nombreux systèmes dans le corps, y compris la réponse au stress.
• 12:01 Le cortisol aide à gérer et à apprendre du stress.
• 13:20 Les femmes sous pilule contraceptive ont une réponse au cortisol atténuée ou absente face au stress, ce qui peut entraîner des problèmes d'adaptation, de régulation émotionnelle et de mémoire.
• 14:26 La prise de pilule contraceptive pendant l'adolescence peut augmenter le risque de dépression majeure plus tard dans la vie.
• 14:39 Toutes les formes de contraception hormonale peuvent avoir ces effets en inhibant l'ovulation avec de la progestérone synthétique.
• 15:56 Il est plus difficile de vendre la contraception hormonale aux hommes car ils sont moins motivés à éviter une grossesse.
• 16:28 Les changements hormonaux chez les femmes ont des répercussions sur leur entourage et sur le monde.
• 17:04 Il faut plus de conversations sur le cerveau des femmes et la pilule contraceptive, et il faut plus d'options non hormonales.
• 17:42 Les femmes devraient être informées de l'impact de la pilule sur leur corps.
• 17:53 Pour en savoir plus, lire le livre "This is Your Brain on Birth Control".

Cette source est la transcription d'une vidéo vulgarisant des concepts biologiques fondamentaux liés à la reproduction et à la biodiversité.

L'objectif principal est de démystifier des idées fausses sur la reproduction, comme l'idée du père plantant une graine, en expliquant le rôle réel des gènes et l'investissement maternel.

La vidéo aborde ensuite la nature dynamique de la biodiversité, soulignant qu'elle n'est pas un équilibre statique mais un processus constant d'apparition et de disparition d'espèces.

Un thème central est l'effondrement actuel des effectifs des populations, qui menace cette dynamique et pourrait mener à une crise de la biodiversité, avec une forte insistance sur le fait que la perte de diversité en elle-même n'est pas un problème si elle est compensée par la dynamique globale de la nature, ce qui n'est plus le cas aujourd'hui.

• Reproduction et hérédité : La reproduction est une caractéristique majeure des êtres vivants, permettant de produire des descendants ressemblant à leurs parents tout en étant différents.

Cette variation illimitée et héritable est essentielle pour l'évolution et l'adaptation des organismes. La reproduction n'est pas une simple reproduction à l'identique des individus, mais plutôt une reproduction des gènes.

• Reproduction sexuée vs asexuée : La reproduction sexuée implique la combinaison de matériel génétique de deux parents, tandis que la reproduction asexuée produit des descendants génétiquement identiques à un seul parent.

La reproduction asexuée est plus efficace à court terme, mais la reproduction sexuée favorise la diversité génétique nécessaire à l'adaptation à long terme.

• Rôles des parents dans la reproduction : Chez de nombreuses espèces, la femelle contribue davantage à la reproduction en fournissant l'ovule et les ressources nécessaires au développement de l'embryon.

Chez les mammifères, la mère transmet la moitié des gènes du noyau et l'intégralité de l'ADN mitochondrial. Le rôle du mâle peut être perçu comme celui d'un "parasite" qui injecte ses gènes dans la femelle.

• Différences entre mâles et femelles : La sélection naturelle favorise les gènes qui permettent aux individus de se reproduire avec succès.

La sélection sexuelle peut entraîner des différences morphologiques entre mâles et femelles, comme des ornements chez les mâles pour attirer les femelles ou des adaptations pour les combats entre mâles.

Les préférences esthétiques des femelles peuvent influencer l'expression de certains gènes chez les mâles.

• Consanguinité et diversité génétique : La consanguinité peut entraîner des problèmes de santé chez les descendants en augmentant la probabilité d'hériter de gènes récessifs défavorables.

Cependant, la consanguinité n'est pas problématique en soi si elle est pratiquée de manière constante, car les gènes défavorables sont progressivement éliminés.

• Diversité des sexes : Il existe une binarité mâle/femelle basée sur la taille des gamètes, mais il existe de nombreuses variations dans les systèmes de reproduction.

Chez certaines espèces, il peut y avoir plusieurs "sexes" définis comme des groupes d'individus qui ne peuvent se reproduire qu'avec d'autres groupes spécifiques.

Certaines espèces peuvent alterner entre reproduction sexuée et asexuée en fonction des conditions environnementales.

• Biodiversité et son évolution : La biodiversité est en constant mouvement, avec des espèces qui apparaissent et disparaissent.

L'équilibre de la biodiversité est dynamique et dépend du mouvement évolutif.

L'extinction d'espèces n'est pas un problème en soi tant que la dynamique de divergence et d'apparition de nouvelles espèces est maintenue.

• Effondrement de la biodiversité : La diminution des effectifs de nombreuses espèces est un signe d'effondrement de la biodiversité.

La perte de biomasse d'insectes volants est un exemple de ce phénomène.

L'effondrement des effectifs menace la dynamique de la biodiversité et peut entraîner une augmentation des extinctions.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

This manuscript investigates a mechanism between the histone reader protein YEATS2 and the metabolic enzyme GCDH, particularly in regulating epithelial-to-mesenchymal transition (EMT) in head and neck cancer (HNC).

Strengths:

Great detailing of the mechanistic aspect of the above axis is the primary strength of the manuscript.

Weaknesses:

Several critical points require clarification, including the rationale behind EMT marker selection, the inclusion of metastasis data, the role of key metabolic enzymes like ECHS1, and the molecular mechanisms governing p300 and YEATS2 interactions.

We would like to sincerely thank the reviewer for the detailed, in-depth, and positive response. We are committed to implementing constructive revisions to the manuscript to address the reviewer’s concerns effectively.

Major Comments:

(1) The title, "Interplay of YEATS2 and GCDH mediates histone crotonylation and drives EMT in head and neck cancer," appears somewhat misleading, as it implies that YEATS2 directly drives histone crotonylation. However, YEATS2 functions as a reader of histone crotonylation rather than a writer or mediator of this modification. It cannot itself mediate the addition of crotonyl groups onto histones. Instead, the enzyme GCDH is the one responsible for generating crotonyl-CoA, which enables histone crotonylation. Therefore, while YEATS2 plays a role in recognizing crotonylation marks and may regulate gene expression through this mechanism, it does not directly catalyse or promote the crotonylation process.

We thank the reviewer for raising this concern. As stated by the reviewer, YEATS2 functions as a reader protein, capable of recognizing histone crotonylation marks and assisting in the addition of this mark to nearby histone residues, possibly by assisting the recruitment of the writer protein for crotonylation. Our data indicates the involvement of YEATS2 in the recruitment of writer protein p300 on the promoter of the SPARC gene, making YEATS2 a regulatory factor responsible for the addition of crotonyl marks in an indirect manner. Thus, we have decided to make changes in the title by replacing the word “mediates” with “regulates”. Therefore, the updated title can be read as: “Interplay of YEATS2 and GCDH regulates histone crotonylation and drives EMT in head and neck cancer”.

(2) The study suggests a link between YEATS2 and metastasis due to its role in EMT, but the lack of clinical or pre-clinical evidence of metastasis is concerning. Only primary tumor (PT) data is shown, but if the hypothesis is that YEATS2 promotes metastasis via EMT, then evidence from metastatic samples or in vivo models should be included to solidify this claim.

We appreciate the reviewer’s suggestion. Here, we would like to state that the primary aim of this study was to delineate the molecular mechanisms behind the role of YEATS2 in maintaining histone crotonylation at the promoter of genes that favour EMT in head and neck cancer. We have dissected the importance of histone crotonylation in the regulation of gene expression in head and neck cancer in great detail, having investigated the upstream and downstream molecular players involved in this process that promote EMT. Moreover, with the help of multiple phenotypic assays, such as Matrigel invasion, wound healing, and 3D invasion assays, we have shown the functional importance of YEATS2 in promoting EMT in head and neck cancer cells. Since EMT is known to be a prerequisite process for cancer cells undergoing metastasis(1), the evidence of YEATS2 being associated with EMT demonstrates a potential correlation of YEATS2 with metastasis. However, as part of the revision, we will use publicly available patient data to investigate the direct association of YEATS2 with metastasis by checking the expression of YEATS2 between different grades of head and neck cancer, as an increase in tumor grade is often correlated with the incidence of metastasis(2).

(3) There seems to be some discrepancy in the invasion data with BICR10 control cells (Figure 2C). BICR10 control cells with mock plasmids, specifically shControl and pEGFP-C3 show an unclear distinction between invasion capacities. Normally, we would expect the control cells to invade somewhat similarly, in terms of area covered, within the same time interval (24 hours here). But we clearly see more control cells invading when the invasion is done with KD and fewer control cells invading when the invasion is done with OE. Are these just plasmid-specific significant effects on normal cell invasion? This needs to be addressed.

We appreciate the reviewer for the thorough evaluation of the manuscript. The figure panels in question, Figure 2B and 2C, represent two different experiments performed independently, the invasion assay performed after knockdown and overexpression of YEATS2, respectively. We would like to clarify that both panels represent results that are distinct and independent of each other and that the method used to knockdown or overexpress YEATS2 is also different. As stated in the Materials and Methods section, the knockdown is performed using lentivirus-mediated transfection (transduction) of cells, on the other hand, the overexpression is done using standard method of transfection by directly mixing transfection reagent and the respective plasmids, prior to the addition of this mix to the cells. The difference in the experimental conditions in these two experiments might have attributed to the differences seen in the controls as observed previously(3). Hence, we would like to state that the results of figure panels Figure 2B and Figure 2C should be evaluated independently of each other.

(4) In Figure 3G, the Western blot shows an unclear band for YEATS2 in shSP1 cells with YEATS2 overexpression condition. The authors need to clearly identify which band corresponds to YEATS2 in this case.

The two bands seen in the shSP1+pEGFP-C3-YEATS2 condition correspond to the endogenous YEATS2 band (lower band, indicated by * in the shControl lane) and YEATS2-GFP band (upper band, corresponding to overexpressed YEATS2-GFP fusion protein, which has a higher molecular weight). To avoid confusion, the endogenous band will be highlighted (marked by *) in the lane representing the shSP1+pEGFP-C3-YEATS2 condition in the revised version of the manuscript.

(5) In ChIP assays with SP1, YEATS2 and p300 which promoter regions were selected for the respective genes? Please provide data for all the different promoter regions that must have been analysed, highlighting the region where enrichment/depletion was observed. Including data from negative control regions would improve the validity of the results.

Throughout our study, we have performed ChIP-qPCR assays to check the binding of SP1 on YEATS2 and GCDH promoter, and to check YEATS2 and p300 binding on SPARC promoter. Using transcription factor binding prediction tools and luciferase assays, we selected multiple sites on the YEATS2 and GCDH promoter to check for SP1 binding. The results corresponding to the site that showed significant enrichment were provided in the manuscript. The region of SPARC promoter in YEATS2 and p300 ChIP assay was selected on the basis of YEATS2 enrichment found in the YEATS2 ChIP-seq data. We will provide data for all the promoter regions investigated (including negative controls) in the revised version of the manuscript.

(6) The authors establish a link between H3K27Cr marks and GCDH expression, and this is an already well-known pathway. A critical missing piece is the level of ECSH1 in patient samples. This will clearly delineate if the balance shifted towards crotonylation.

We thank the reviewer for their valuable suggestion. To support our claim, we had checked the expression of GCDH and ECHS1 in TCGA HNC RNA-seq data (provided in Figure 4—figure supplement 1A and B) and found that GCDH showed increase while ECHS1 showed decrease in tumor as compared to normal samples. We hypothesized that higher GCDH expression and decreased ECHS1 expression might lead to an increase in the levels of crotonylation in HNC. To further substantiate our claim, we will check the abundance of ECHS1 in HNC patient samples as part of the revision.

(7) The p300 ChIP data on the SPARC promoter is confusing. The authors report reduced p300 occupancy in YEATS2-silenced cells, on SPARC promoter. However, this is paradoxical, as p300 is a writer, a histone acetyltransferase (HAT). The absence of a reader (YEATS2) shouldn't affect the writer (p300) unless a complex relationship between p300 and YEATS2 is present. The role of p300 should be further clarified in this case. Additionally, transcriptional regulation of SPARC expression in YEATS2 silenced cells could be analysed via downstream events, like Pol-II recruitment. Assays such as Pol-II ChIP-qPCR could help explain this.

Using RNA-seq and ChIP-seq analyses, we have shown that YEATS2 affects the expression of several genes by regulating the level of histone crotonylation at gene promoters globally. The histone writer p300 is a promiscuous acyltransferase protein that has been shown to be involved in the addition of several non-acetyl marks on histone residues, including crotonylation(4). Our data provides evidence for the dependency of the writer p300 on YEATS2 in mediating histone crotonylation, as YEATS2 downregulation led to decreased occupancy of p300 on the SPARC promoter (Figure 5F). However, the exact mechanism of cooperativity between YEATS2 and p300 in maintaining histone crotonylation remains to be investigated. To address the reviewer’s concern, we will perform various experiments to delineate the molecular mechanism pertaining to the association of YEATS2 with p300 in regulating histone crotonylation. Following are the experiments that will be performed:

(a) Co-immunoprecipitation experiments to check the physical interaction between YEATS2 and p300.

(b) We will check H3K27cr levels on the SPARC promoter and SPARC expression in p300-depleted HNC cells.

(c) Rescue experiments to check if the decrease in p300 occupancy on the SPARC promoter can be compensated by overexpressing YEATS2.

(d) As suggested by the reviewer, Pol-II ChIP-qPCR at the promoter of SPARC will be performed in YEATS2-silenced cells to explain the mode of transcriptional regulation of SPARC expression by YEATS2.

(8) The role of GCDH in producing crotonyl-CoA is already well-established in the literature. The authors' hypothesis that GCDH is essential for crotonyl-CoA production has been proven, and it's unclear why this is presented as a novel finding. It has been shown that YEATS2 KD leads to reduced H3K27cr, however, it remains unclear how the reader is affecting crotonylation levels. Are GCDH levels also reduced in the YEATS2 KD condition? Are YEATS2 levels regulating GCDH expression? One possible mechanism is YEATS2 occupancy on GCDH promoter and therefore reduced GCDH levels upon YEATS2 KD. This aspect is crucial to the study's proposed mechanism but is not addressed thoroughly.

The source for histone crotonylation, crotonyl-CoA, can be produced by several enzymes in the cell, such as ACSS2, GCDH, ACOX3, etc(5). Since metabolic intermediates produced during several cellular pathways in the cell can act as substrates for epigenetic factors, we wanted to investigate if such an epigenetic-metabolism crosstalk existed in the context of YEATS2. As described in the manuscript, we performed GSEA using publicly available TCGA RNA-seq data and found that patients with higher YEATS2 expression also showed a high correlation with expression levels of genes involved in the lysine degradation pathway, including GCDH. Since the preferential binding of YEATS2 with H3K27cr and the role of GCDH in producing crotonyl-CoA was known(6,7), we hypothesized that higher H3K27cr in HNC could be a result of both YEATS2 and GCDH. We found that the presence of GCDH in the nucleus of HNC cells is correlated to higher H3K27cr abundance, which could be a result of excess levels of crotonyl-CoA produced via GCDH. We also found a correlation between H3K27cr levels and YEATS2 expression, which could arise due to YEATS2-mediated preferential maintenance of crotonylation. This states that although being a reader protein, YEATS2 is affecting the promoter H3K27cr levels, possibly by helping in the recruitment of p300 (as shown in Figure 5F). Thus, YEATS2 and GCDH are both responsible for the regulation of histone crotonylation-mediated gene expression in HNC.

We did not find any evidence of YEATS2 regulating the expression of GCDH in HNC cells. However, we found that YEATS2 downregulation reduced the nuclear pool of GCDH in head and neck cancer cells (Figure 7F). This suggests that YEATS2 not only regulates histone crotonylation by affecting promoter H3K27cr levels (with p300), but also by affecting the nuclear localization of crotonyl-CoA producing GCDH. Also, we observed that the expression of YEATS2 and GCDH are regulated by the same transcription factor SP1 in HNC. We found that the transcription factor SP1 binds to the promoter of both genes, and its downregulation led to a decrease in their expression (Figure 3 and Figure 7).

We would like to state that the relationship between YEATS2 and the nuclear localization of GCDH, as well as the underlying molecular mechanism, remains unexplored and presents an open question for future investigation.

(9) The authors should provide IHC analysis of YEATS2, SPARC alongside H3K27cr and GCDH staining in normal vs. tumor tissues from HNC patients.

We thank the reviewer for their suggestion. We are consulting our clinical collaborators to assess the feasibility of including this IHC analysis in our revision and will make every effort to incorporate it.

Reviewer #2 (Public review):

Summary:

The manuscript emphasises the increased invasive potential of histone reader YEATS2 in an SP1-dependent manner. They report that YEATS2 maintains high H3K27cr levels at the promoter of EMT-promoting gene SPARC. These findings assigned a novel functional implication of histone acylation, crotonylation.

We thank the reviewer for the constructive comments. We are committed to making beneficial changes to the manuscript in order to alleviate the reviewer’s concerns.

Concerns:

(1) The patient cohort is very small with just 10 patients. To establish a significant result the cohort size should be increased.

We thank the reviewer for this suggestion. We will increase the number of patient samples to assess the levels of YEATS2 and H3K27cr in normal vs. tumor samples.

(2) Figure 4D compares H3K27Cr levels in tumor and normal tissue samples. Figure 1G shows overexpression of YEATS2 in a tumor as compared to normal samples. The loading control is missing in both. Loading control is essential to eliminate any disparity in protein concentration that is loaded.

In Figures 1G and 4D, we have used Ponceau S staining as a control for equal loading. Ponceau S staining is frequently used as an alternative for housekeeping genes like GAPDH as a control for protein loading(8). It avoids the potential for variability in housekeeping gene expression. However, it may be less quantitative than using housekeeping proteins. To address the reviewer’s concern, we will probe with an antibody against a house keeping gene as a loading control in the revised figures, provided its expression remains stable across the conditions tested.

(3) Figure 4D only mentions 5 patient samples checked for the increased levels of crotonylation and hence forms the basis of their hypothesis (increased crotonylation in a tumor as compared to normal). The sample size should be more and patient details should be mentioned.

A total of 9 samples were checked for H3K27cr levels (5 of them are included in Figure 4D and rest included in Figure 4—figure supplement 1D). However, as a part of the revision, we will check the H3K27cr levels in more patient samples.

(4) YEATS2 maintains H3K27Cr levels at the SPARC promoter. The p300 is reported to be hyper-activated (hyperautoacetylated) in oral cancer. Probably, the activated p300 causes hyper-crotonylation, and other protein factors cause the functional translation of this modification. The authors need to clarify this with a suitable experiment.

In our study, we have shown that p300 is dependent on YEATS2 for its recruitment on the SPARC promoter. As a part of the revision, we propose the following experiments to further substantiate the role of p300 in YEATS2-mediated gene regulation:

(a) Co-immunoprecipitation experiments to check the physical interaction between YEATS2 and p300.

(b) We will check H3K27cr levels on the SPARC promoter and SPARC expression in p300-depleted HNC cells.

(c) Rescue experiments to check if the decrease in p300 occupancy on the SPARC promoter can be compensated by overexpressing YEATS2.

(d) Pol-II ChIP-qPCR at the promoter of SPARC will be performed in YEATS2-silenced cells to explain the mode of transcriptional regulation of SPARC expression by YEATS2.

(5) I do not entirely agree with using GAPDH as a control in the western blot experiment since GAPDH has been reported to be overexpressed in oral cancer.

We would like to clarify that GAPDH was not used as a loading control for protein expression comparisons between normal and tumor samples. GAPDH was used as a loading control only in experiments using head and neck cancer cell lines where shRNA-mediated knockdown or overexpression was employed. These manipulations specifically target the genes of interest and are not expected to alter GAPDH expression, making it a suitable loading control in these instances.

(6) The expression of EMT markers has been checked in shControl and shYEATS2 transfected cell lines (Figure 2A). However, their expression should first be checked directly in the patients' normal vs. tumor samples.

We thank the reviewer for the suggestion. To address this, we will check the expression of EMT markers alongside YEATS2 expression in normal vs. tumor samples.

(7) In Figure 3G, knockdown of SP1 led to the reduced expression of YEATS2 controlled gene Twist1. Ectopic expression of YEATS2 was able to rescue Twist1 partially. In order to establish that SP1 directly regulates YEATS2, SP1 should also be re-introduced upon the knockdown background along with YEATS2 for complete rescue of Twist1 expression.

To address the reviewer’s concern regarding the partial rescue of Twist1 in SP1 depleted-YEATS2 overexpressed cells, we will perform the experiment as suggested by the reviewer. In brief, we will overexpress both SP1 and YEATS2 in SP1-depleted cells and then assess the expression of Twist1.

(8) In Figure 7G, the expression of EMT genes should also be checked upon rescue of SPARC expression.

We thank the reviewer for the suggestion. We will check the expression of EMT markers on YEATS2/ GCDH rescue and update Figure 7G in the revised version of the manuscript.

References

(1) T. Brabletz, R. Kalluri, M. A. Nieto and R. A. Weinberg, Nat Rev Cancer, 2018, 18, 128–134.

(2) P. Pisani, M. Airoldi, A. Allais, P. Aluffi Valletti, M. Battista, M. Benazzo, R. Briatore, S. Cacciola, S. Cocuzza, A. Colombo, B. Conti, A. Costanzo, L. Della Vecchia, N. Denaro, C. Fantozzi, D. Galizia, M. Garzaro, I. Genta, G. A. Iasi, M. Krengli, V. Landolfo, G. V. Lanza, M. Magnano, M. Mancuso, R. Maroldi, L. Masini, M. C. Merlano, M. Piemonte, S. Pisani, A. Prina-Mello, L. Prioglio, M. G. Rugiu, F. Scasso, A. Serra, G. Valente, M. Zannetti and A. Zigliani, Acta Otorhinolaryngol Ital, 2020, 40, S1–S86.

(3) J. Lin, P. Zhang, W. Liu, G. Liu, J. Zhang, M. Yan, Y. Duan and N. Yang, Elife, 2023, 12, RP87510.

(4) X. Liu, W. Wei, Y. Liu, X. Yang, J. Wu, Y. Zhang, Q. Zhang, T. Shi, J. X. Du, Y. Zhao, M. Lei, J.-Q. Zhou, J. Li and J. Wong, Cell Discov, 2017, 3, 17016.

(5) G. Jiang, C. Li, M. Lu, K. Lu and H. Li, Cell Death Dis, 2021, 12, 703.

(6) D. Zhao, H. Guan, S. Zhao, W. Mi, H. Wen, Y. Li, Y. Zhao, C. D. Allis, X. Shi and H. Li, Cell Res, 2016, 26, 629–632.

(7) H. Yuan, X. Wu, Q. Wu, A. Chatoff, E. Megill, J. Gao, T. Huang, T. Duan, K. Yang, C. Jin, F. Yuan, S. Wang, L. Zhao, P. O. Zinn, K. G. Abdullah, Y. Zhao, N. W. Snyder and J. N. Rich, Nature, 2023, 617, 818–826.

(8) I. Romero-Calvo, B. Ocón, P. Martínez-Moya, M. D. Suárez, A. Zarzuelo, O. Martínez-Augustin and F. S. de Medina, Anal Biochem, 2010, 401, 318–320.

DOI: 10.1186/s12885-025-13714-y

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Voici un sommaire minuté de l'affaire Kevin, avec les idées fortes en gras, basé sur les sources fournies:

• 0:02-0:20 Introduction au podcast "Délits Mineurs" d'ARTE Radio, qui présente des affaires jugées au tribunal pour enfants de Bobigny. Le podcast offre une perspective subjective sur le système judiciaire pour mineurs, à travers les yeux d'un assesseur bénévole.

• 0:28-1:20 Présentation de l'assesseur et du contexte de l'audience. Kevin, 16 ans, est jugé pour violences aggravées. La victime est Ryan, 18 ans, représenté par son avocate.

• 1:22-2:24 Rappel des faits : une altercation entre les familles de Kevin et Ryan sur le parking d'un hypermarché dégénère en violence, Kevin frappant Ryan. Kevin assume les coups, mais minimise leur importance par rapport à ce que Ryan lui a fait subir dans le passé.

• 2:24-3:16 Révélation du contexte : Il y a 5 ans, Ryan a agressé sexuellement Kevin lors d'une colonie de vacances. Ryan a été reconnu coupable d'agression sexuelle et condamné à un an de prison avec sursis, une décision mal acceptée par Kevin et sa famille.

• 3:23-4:23 Témoignage du père de Kevin : Il décrit l'impact dévastateur de l'agression sur son fils et toute la famille. La famille se sent lésée par la justice. Déménager est impossible à cause de leur salon de tatouage.

• 4:30-5:24 L'éducatrice de Kevin témoigne de son repli sur lui-même. L'avocate de Ryan rappelle la violence des coups et le traumatisme subi par son client. Elle accuse Kevin de s'être rendu justice lui-même et critique l'irresponsabilité de ses parents.

• 5:30-6:46 Réquisitions du procureur : Il rappelle que le tribunal ne re juge pas le viol, mais les coups portés. Il souligne que la société ne tolère pas la violence, mais juge les mineurs différemment en raison de leur manque de maturité. Il estime que la peine de Ryan était déjà lourde compte tenu de son âge.

• 6:52-7:43 Le procureur se tourne vers les parents de Kevin, les assure de sa compréhension et reconnaît qu'ils ont subi des provocations. Kevin remercie le procureur. Kevin est déclaré coupable de violence et un suivi éducatif est ordonné.

• 7:43-8:07 Première audience : Kevin et sa famille sont soulagés. Le père remercie l'assesseur.

• 8:08-9:30 Six mois plus tard, lors de l'audience de sanction, l'assesseur retrouve une famille toujours rongée par la douleur et le sentiment d'injustice. La peine de prison avec sursis pour Ryan est toujours inacceptable pour eux.

• 9:30-9:51 Décision : Un avertissement judiciaire est prononcé, une mesure symbolique pour inciter Kevin à trouver une autre issue à sa souffrance que la violence.

Voici une brève synthèse des informations clés concernant la demande d'asile en France, basée sur les sources fournies:

• Procédure de demande d'asile [54:30]: La procédure implique d'abord de se rendre à la SPADA (structure de premier accueil des demandeurs d'asile), puis au GUDA (guichet unique pour demandeur d'asile) [54:30]. La loi d'armanin de 2024 prévoit de transformer les GUDA en guichets franceasile, intégrant ainsi l'OFPRA [55:52].

• Conditions d'éligibilité [18:39, 19:54]: Pour obtenir une protection internationale (statut de réfugié, protection subsidiaire ou apatridie), il faut remplir des conditions communes, notamment prouver le rattachement au pays d'origine, la réalité, l'actualité et l'individualisation des craintes, et l'absence de protection dans l'État de rattachement [18:39, 19:54].

• Statut de réfugié vs. protection subsidiaire [33:14]: Les conditions d'accès diffèrent entre le statut de réfugié (basé sur cinq critères non cumulatifs) et la protection subsidiaire (risque réel de subir des atteintes graves) [33:14, 34:03, 42:17].

• Procédure Dublin [1:05:16]: La procédure Dublin désigne un seul État européen responsable de la demande d'asile [1:05:16]. Les pays d'entrée principaux sont l'Espagne, l'Italie et la Grèce [1:05:56]. Cette procédure peut être longue et complexe [1:06:36].

• Procédure accélérée [1:08:35, 1:09:24]: Elle offre moins de garanties juridictionnelles mais vise un traitement plus rapide des demandes [1:08:35, 1:09:24]. Les motifs de placement incluent les pays d'origine sûrs, la demande tardive, etc. [1:09:56, 1:13:33].

• OFPRA et CNDA [56:45, 57:08]: L'OFPRA (Office français de protection des réfugiés et des apatrides) examine les demandes d'asile [56:45]. En cas de rejet, un recours est possible à la CNDA (Cour nationale du droit d'asile) [57:08].

• Droits sociaux [1:38:17, 1:49:11]: Les demandeurs d'asile ont accès aux soins après 3 mois de résidence [1:38:17]. Les bénéficiaires de la protection internationale (BPI) ont le droit de travailler [1:49:11].

• Fin de la demande d'asile [1:41:11]: En cas de rejet définitif, il y a fin de l'hébergement et de l'ADA (allocation pour demandeur d'asile), avec possibilité de régularisation ou situation irrégulière [1:41:30]. En cas d'octroi d'une protection internationale, il y a fin des CMA (conditions matérielles d'accueil) au bout de 6 mois, mais accès à divers droits et facilités (emploi, réunification familiale, naturalisation) [1:42:54].

Voici un résumé structuré de la vidéo "Le sol, royaume du vivant | ARTE", mettant en évidence les idées clés :

• Introduction (0:00-1:00) : Le sol est un écosystème vivant essentiel à la vie sur Terre. Les humains ont un rôle à jouer dans sa préservation et sa restauration.
• Restauration du sol (1:00-2:30) : Techniques de restauration des sols :
  • Recouvrir le sol de carton et de compost pour nourrir les organismes vivants.
  • Le rôle des vers de terre est crucial pour aérer le sol et enrichir le sol.
  • L'agriculture régénératrice avec la culture en bande améliore la qualité des sols et stocke le CO2.
  • L'utilisation des vaches pour revitaliser le sol et produire du fumier.
  • L'importance d'avoir des plantes saines grâce à un sol sain pour une alimentation de qualité.
• Compostage et micro-organismes (2:30-4:00) :
  • Techniques de compostage des déchets organiques pour enrichir le sol.
  • Les micro-organismes renforcent la santé des plantes.
  • La structure du sol est améliorée par les micro-organismes qui créent des cavités pour l'air et l'eau.
  • Les excréments de lapins nourrissent les organismes du sol.
• Réseaux fongiques (4:00-6:00) :
  • Les champignons sont des organismes diversifiés qui forment des réseaux souterrains.
  • Les systèmes symbiotiques et les échanges de nutriments entre les racines et les champignons sont étudiés avec des technologies de pointe.
  • Les champignons stockent le carbone et influent sur le marché des nutriments.
  • Une grande quantité de carbone terrestre est stockée grâce aux champignons.
• Récolte et biodiversité (6:00-7:30) :
  • La récolte et l'importance de laisser les racines travailler dans le sol.
  • Les plantes aux racines profondes ameublissent le sol et favorisent la transformation de l'azote.
  • Le processus de décomposition des plantes en nutriments par les micro-organismes.
  • Les arbres et les plantes sont liés aux micro-champignons, essentiels à leur survie.
• Impact de l'azote et adaptation urbaine (7:30-9:00) :
  • L'excès d'azote dans le sol entraîne la disparition des champignons mycorhiziens et des plantes sauvages.
  • Les champignons mycorhiziens aident les plantes à se développer en milieu urbain.
  • Étude de la biodiversité des champignons sur les toits végétalisés des arrêts de bus en ville.
• Cartographie des sols et importance des sons (9:00-10:30) :
  • La nécessité de cartographier les sols face aux bouleversements mondiaux.
  • L'importance d'utiliser tous les sens pour comprendre le sol, y compris l'écoute des sons qu'il produit.
  • Les protozoaires et les bactéries maintiennent l'équilibre de l'écosystème du sol.
• Culture en bande et agriculture régénératrice (10:30-12:00) :
  • La culture en bande augmente la quantité de matières organiques et capture le CO2.
  • Techniques de labour non invasives pour préserver la structure du sol.
  • Les micro-organismes du sol sont similaires à ceux de l'intestin humain.
• Conclusion (12:00-12:30) : Apprendre à faire confiance au sol vivant pour une collaboration réussie.

Feed Back es ecencial, y es tambien una opción altamente recomendable para proporcionar retorno.

Feedback esta presente en todo el proceso de aprendizaje y no solo es hacer correciones, o palabras de aliento, sino retroalimentaciones

Evaluar no es sólo calificar, aunque sí es una de sus dimenciones no es la única, pese a estar en la Licenciatura en Pedagogía hay colegas que continúan replicando la medición del aprendizaje de las y los estudiantes cuando podemos proponer y aplicar otras manera de evaluar, incluso que las y los mismos estudiantes evalúen el curso o el taller en pro de retroalimentar y mejorar el quehacer pedagógico en los procesos de enseñanza y aprendizaje.

Cuántas y cuántos de nosotros hemos escuchado o hablado, a lo largo de nuestra formación educativa, la preocupación sobre la acreditación u obtención de notas altas en vez de centrarnos en aprender. Diferenciando a la memorización, apelando a una educación bancaria en vez de formativa que propicie el reconocimiento de los saberes propios y adquiridos, además de la transformación del entorno de las y los educandos.

Hola:)

En ese sentido, el diálogo con las comunidades que trabajan metaherramientas e investigación y publicación reproducibles (como la de Grafoscopio, de la cual participo), podría arrojar interesantes polinizaciones cruzadas que permitan abordar los temas de difusión y alfabetismos críticos alrededor de este tipo de publicaciones híbridas y orientadas a la web, en lugar de al impreso.

Creo que la producción de comunes digitales con licenciamientos que expliciten posturas al respecto de los mismos y cómo se piensan las sinergias ayudaría a evidenciar la postura crítica (o no) de tales comunidades informales.

Vale la pena ahondar sobre la sinergia entre lo forma y lo informal para la sostenibilidad de las comunidades a la luz de los hallazgos de la tesis. Creo que allí hay una inquietud amplia y potente sobre la cual la tesis podría brindar más luces, pues la necesidad de esa sinergía y su enunciación breve parece más un punto de partida que una conclusión. La tesis, con sus amplias entrevistas y trabajo podría apoyar más especificidad sobre tales sinergias y, por ejemplo, el puente que pueden establecer los bienes comunes al respecto.

Por el contrario, lo que creo es que hay que visibilizar el conflicto de estas distintas formas de relacionamiento, para poder navegarlo mejor.

Esta idea de múltiples escalas también debería visibilizar los datos pequeños que esta misma tesis usa y, creo, evidenciar de modos más explícitos la crítica a lo que he llamado en mis comentarios hipertextuales al texto la "oda al gigantismo e datos" en el que han caído también las humanidades digitales y sus representantes más visibles (ejp: Manovich).

Esto es visible a lo largo de todo el texto. Creo que hay una aproximación genuina por materialidades argumentativas distintas y en diálogo con la idea del impreso como formato derivado/subordinado y una tesis nacida en digital, conjugando las infraestructuras, el código y los interactivos como elementos importantes de la argumentación misma y otra forma de experimentar la lectura en varios niveles más allá del PDF estático.

Queda la pregunta por el licenciamiento y la posibilidad de que la marginalia interactiva, que yo mismo habilité para la evaluación y lectura estén disponibles a futuros lectores del texto en digital y cómo alentar así comunidades de lectura alrededor de la tesis misma.

Sin embargo es preocupante la postura extrativista y colonial que GitHub cuando entrena grandes modelos de lenguaje a partir del código ajeno, no pago, ni reconocido de quienes alojan el código fuente allí (incluidas estas páginas).

Sería chévere que la interfaz fuera bi/multilingüe, como en Scratch, pero el código fuente usara el inglés como lengua franca pero también en resistencia y, como diría Paulo Freire, para decir en la voz del colono las preocupaciones del colonizado. Esa ha sido nuestra postura en la comunidad de Grafoscopio. El código se escribe en inglés, pero las narrativas de datos y sus preocupaciones son en español e incluso, con proyectos recientes ha dado cuenta de lenguas amazónicas en La Chorrera.

Valdria la pena abrir la conversación sobre la diglosia y cómo ha sido abordada en proyectos con tensiones similares (como expliqué para el caso de Grafoscopio).

Dicha generosidad debe ser promovida no solo de manera factual, brindando las infraestructuras mismas, sino nominal indicando las licencias explícitas que configuran de modo de aplicar claramente esa generosidad.

También, las exploraciones en anchura del comentario previo ayudarían a proveer una mirada panorámica para quienes quieran recorrer caminos paralelos.

Ahora entiendo mejor la elección de Magicbook sobre otros sistemas más ampliamente usados como Pandoc o Quarto que también están enfocados en publicación con salida multiformato a partir de un código fuente único. Se trata más bien de un "accidente histórico" en el sentido que se siguió el camino de un autor conocido en lugar de explorar alternativas que dicho autor no había tomado (algo totalmente válido e incluso habitual).

En mis intentos de lectura hipermedial/infraestructural de esta tesis, intenté tomar el código fuente en Markdown y pasarle Pandoc para la producción de sitios estáticos, pero rápidamente me encontré con problemas de replicabilidad, imagino asociados a la forma particular en que MagicBook construye sus piezas interactivas, como el primer interactivo del mapa de búsqueda de los términos asociados a humanidades digitales en español y portugues (no recuerdo si inglés también).

Sin embargo, el uso de otras infraestructuras para procurar replicabilidad, me permitió ver los supuestos de las acá usadas y me pregunté si , por ejemplo el mapa no podría hacer más portable con snippets autocontenidos de código y datos que produzcan los interactivos exportados desde otros formatos.

Una inquietud para pensar a futuro y que tendría que ver con la exploración en anchura (mas que en profundidad) de alternativas e infraestructuras generosas, mostrando maneras quizás más sencillas de lograr replicabilidad

Me parece súper interesante la opción de construir un sistema de diseño propio para la publicación de la tesis y sin lugar a dudas una búsqueda valiosa. Algo similar hice en la publicación híbrida de la mía, yendo por sistemas LaTeX para preprensa digital, en lugar del formato clásico de tesis.El desarrollo de las secciones posteriores y la manera en que da cuenta de ese sistema es muy rico.

Sin embargo, me queda la inquietud de por qué no adaptar un sistema de diseño preexistente, como la plantila Editorial de HTML5 Up o cualquier otra de GitBook, Read The Docs, etc, dado que ambas podían dar cuenta de los tipos de capítulos enunciados a continuación.

Creo que vale la pena agregar las fechas de tales creaciones y si son obras vivas, las de su primer commit, de ser posible, para ubicar efectivamente estos antecedentes en perspectiva histórica más fácilmente.

Precisamente porque se trata de una obra híbrida, vale la pena que estas fechas estén explícitas y cortas, así el enlace hipertextual contenga los datos completos y extensos, incluida la fecha. Cuando haga falta, colocaré la etiqueta ¿fecha? para sugerir la presencia explícita de este dato.

Muy interesante y bella compilación. Gracias por ella, como por muchas de las que aparecen a lo largo de este escrito.

Nuestra manera de lidiar con la diglosia para el caso de Grafoscopio, fue produciendo el código y las interfaces de usuario en inglés, con mucho de la documentación en español (salvo importantes excepciones, como el Manual de usuario en inglés y la página web bilingue es/en)

Así como aquellas que tienen que ver con epistemologías diseñisticas, por razones como las expuestas en este párrafo.

La idea de humanizar infraestructuras y datos tuvo que ver, en nuestro caso, con prototipos que habitaran comunidades de práctica (Grafoscopio) y espacios físicos (HackBo, el eje cafetero, varios eventos nacionales e internacionales). Son caminos que vale la pena poner en diálogo.

Algo similar intenté en mi propia tesis, procurando además una comunidad de práctica y un artefacto digital Grafoscopio, que exploran de manera innovadora la relación de transformación entre metaherramientas digitales y comunidades de base.

Es reconfortante encontrar que esta tesis resuena ahora en la PUJ con estas investigaciones emprendidas hace casi década y media (2010) en la Universidad de Caldas. Creo que da cuenta de motivaciones y hallazgos resonantes, propios del espíritu de una época, que se expresan más allá de los circuitos académicos tradicionales y aunque conexos de modos incluso incidentales, reflejan una intensión de exederlos.

La web en cambio me parece incidental para los ensayos interactivos y muchos de ellos existieron antes de la web, desde los tiempos del Dynabook de Kay, Ingalls y Golberg hasta el Hypercard o el Dynamicland en dicha tradición.

A contracorriente, suelo pensar el diseño web y la web en general no cómo una plataforma de desarrollo sino como un "exportation target", que ahora se vuelve más multilingüe, más allá de Javascript y todas sus falencias gracias a posibilidades como las abiertas con los Sistemas Hipermediales.

¿Cómo podemos dar cuenta del impacto o el relacionamiento con esas formas de divulgación mas amplias? Por ejemplo, a través de la lectura anotada hipertextual yo mostré mi relacionamiento con el texto. Las bifurcaciones al repositorio fuente podrían hacer otro tanto (aunque no está clara la licencia del mismo).

Dar cuenta de esas otras formas de circulación y vinculación con otros públicos para estas otras publicaciones, incluso durante los tiempos de escritura de la tesis, es clave a la hora de evidenciar esas otras formas de relacionamiento e impacto que investigaciones+creaciones alternas pueden tener debido a las propiedades diferenciales de sus materialidades y formas de circulación.

约翰·麦克塔格特·埃利斯·麦克塔格特 (John McTaggart Ellis McTaggart, 1866-1925) 是一位重要的英国唯心主义哲学家，尽管他通常被归类为黑格尔主义者，但他实际上对黑格尔的哲学进行了深刻而严厉的批判，尤其是在逻辑和形而上学领域。 他的著作，特别是《黑格尔辩证法研究》（Studies in the Hegelian Dialectic, 1896）和《黑格尔逻辑注释》（A Commentary on Hegel's Logic, 1910），是研究黑格尔哲学的经典文献，同时也是对黑格尔体系最具挑战性的批评之一。

要详细解释麦克塔格特对黑格尔哲学的批判，我们需要从以下几个方面展开：

一、 麦克塔格特对黑格尔哲学的总体态度：批判性的继承

尽管麦克塔格特对黑格尔提出了许多重要的批评，但他并非完全否定黑格尔的哲学。 更准确地说，他的态度是 批判性的继承 或者说是 “友善的敌人”。

• 早期的黑格尔主义倾向： 麦克塔格特早年深受黑格尔哲学的影响，并将其视为当时最重要的哲学体系。 他在《黑格尔辩证法研究》中，最初的目标并非完全批判，而是 澄清和解释 黑格尔辩证法的运作方式，并试图 捍卫 黑格尔哲学的某些核心思想。
• 逐渐深入的批判： 随着研究的深入，麦克塔格特逐渐发现了黑格尔哲学体系中他认为无法克服的 内在矛盾和逻辑缺陷。 特别是在逻辑和形而上学领域，他开始对黑格尔的辩证法、绝对唯心主义的形而上学基础提出尖锐的批评。
• 对黑格尔“绝对唯心主义” 的接受与改造： 麦克塔格特基本上认同黑格尔的 唯心主义立场， 认为宇宙的本质是 精神性的 (Spiritual)。 但他 拒绝 黑格尔的 “绝对精神” (Absolute Spirit) 概念， 以及黑格尔将 “绝对” 理解为 逻辑终点和完美统一体 的方式。 麦克塔格特的唯心主义更倾向于 多元主义 和 人格主义， 他认为宇宙是由 无数个独立的、永恒的 “自我” (Selves) 或 “人格中心” (Personal Centres) 构成的， 而非统一于一个 “绝对” 之中。 可以说，麦克塔格特试图 在唯心主义框架内，构建一个与黑格尔不同的、更符合逻辑和经验的形而上学体系。

二、 麦克塔格特批判的核心领域：逻辑与形而上学

麦克塔格特的批判主要集中在黑格尔哲学的 逻辑体系 (辩证法) 和 形而上学基础 (绝对唯心主义) 两个核心领域。

• 逻辑体系的批判： 辩证法的逻辑有效性质疑

  • 关注焦点：辩证法的 “必然性” (Necessity) 和 “逻辑有效性” (Logical Validity)： 麦克塔格特并非简单地否定辩证法，而是深入分析辩证法的 逻辑结构和论证过程， 并质疑其 “从一个概念必然地推导出另一个概念” 的主张。 他认为黑格尔的辩证法 缺乏严格的逻辑证明， 其 “必然性” 更多是 修辞上的或描述性的， 而非 逻辑上的必然性。
  • 正题、反题、合题 (Thesis, Antithesis, Synthesis) 模式的质疑： 麦克塔格特仔细考察了黑格尔辩证法中 “正题-反题-合题” 的三段式结构， 并指出 “反题” 的出现并非总是从 “正题” 逻辑必然地推导出来的， 很多时候只是 人为地 “强加” 或 “解释” 出来的， 缺乏真正的逻辑必然性。 他认为黑格尔经常 过度强调概念之间的 “矛盾” 和 “对立”， 并 预设了 “合题” 必然优于 “正题” 和 “反题” 的结论， 这都是 有偏颇和缺乏论证 的。
  • 具体概念辩证发展过程的分析与批判： 麦克塔格特在《黑格尔辩证法研究》和《黑格尔逻辑注释》中， 详细分析了黑格尔《逻辑学》中 “存在论” (Doctrine of Being)、 “本质论” (Doctrine of Essence)、 “概念论” (Doctrine of Concept) 等各个阶段的具体概念辩证发展过程， 逐个概念地 检验其逻辑推演的有效性。 他指出， 在很多情况下， 黑格尔的 概念过渡和推演 都 缺乏逻辑上的必然性， 甚至是 含糊不清、模棱两可 的。 他认为黑格尔的辩证法并非真正的 “逻辑方法”， 而更像是一种 “修辞手法” 或 “描述框架”。

  [Image of Hegel's Dialectical Triad Diagram]

• 形而上学基础的批判： 绝对唯心主义的内在矛盾与困境

  • 对 “绝对精神” 概念的质疑： 麦克塔格特 拒绝接受黑格尔的 “绝对精神” (Absolute Spirit) 概念， 认为这是一个 模糊不清、难以理解 的概念。 他质疑 “绝对精神” 如何能够既是 “精神” (具有意识和自我) 又是 “绝对” (无限和完美)？ 他认为将 “绝对” 与 “精神” 结合起来会产生内在矛盾。 如果 “绝对精神” 是一个 “人格化的上帝”， 那么它就 限制了个人的自由和独立性； 如果 “绝对精神” 只是一个 “抽象的理性原则”， 那么它就 缺乏 “精神” 的特性， 无法解释意识和人格的起源。
  • 对 “绝对” 概念的逻辑分析与批判： 麦克塔格特深入分析了黑格尔 “绝对” (Absolute) 概念的 逻辑含义， 并指出 “绝对” 概念本身就存在逻辑上的困难和矛盾。 他认为， 如果 “绝对” 是 “完全的、包含一切的”， 那么它就 无法进行任何 “否定” 和 “辩证运动”， 因为 “否定” 和 “运动” 都意味着 “不完整” 和 “差异”， 而 “绝对” 则应该是 “完整” 和 “统一” 的。 因此， 黑格尔的 “绝对” 概念与辩证法自身就存在内在冲突。
  • 主张多元主义的形而上学： “自我” (Selves) 的实在性： 麦克塔格特 反对黑格尔的 “一元论” (Monism) 的绝对唯心主义， 转而 主张多元主义的形而上学。 他认为 “自我” (Selves) 或 “人格中心” (Personal Centres) 才是最基本的实在， 宇宙是由 无数个独立的、永恒的 “自我” 构成的。 他认为 “自我” 的意识、经验、关系 才是 真正可以理解的精神实体， 而 抽象的 “绝对精神” 概念是多余的、甚至是误导性的。 他的代表作《存在的本质》（The Nature of Existence）详细阐述了他的多元唯心主义形而上学体系。

三、 麦克塔格特的主要批评观点：

总结麦克塔格特对黑格尔哲学的批判，可以归纳为以下几个主要观点：

1. 辩证法缺乏逻辑必然性： 麦克塔格特认为黑格尔的辩证法 并非真正的逻辑方法， 其概念推演过程 缺乏严格的逻辑证明， “正题-反题-合题” 模式更多是一种 修辞框架， 而非 逻辑必然性。 他质疑辩证法 “从一个概念必然地推导出另一个概念” 的主张。
2. “绝对精神” 概念的内在矛盾： 麦克塔格特认为 “绝对精神” 概念本身就存在内在矛盾， 难以理解和自洽。 他质疑 “绝对” 与 “精神” 的结合 是否可能， 以及 “绝对精神” 如何能够既是 “绝对” 又是 “动态发展” 的？
3. “绝对” 概念的逻辑困境： 麦克塔格特分析认为 “绝对” (Absolute) 概念本身就存在逻辑上的困难， 例如 “绝对” 如何进行 “否定” 和 “辩证运动”？ “绝对” 概念与辩证法自身存在内在冲突。
4. 黑格尔体系的武断和教条： 麦克塔格特认为黑格尔哲学体系 过于庞大和系统化， 试图将一切都纳入其辩证法的框架， 这使得其体系带有一定的 武断和教条主义 色彩。 他认为黑格尔 预设了辩证法可以解决一切哲学问题， 并 强行将各种概念和现象纳入辩证法的框架， 缺乏足够的 灵活性和开放性。
5. 偏离经验和直觉： 麦克塔格特认为黑格尔哲学 过于抽象和思辨， 脱离了经验和直觉。 他认为黑格尔 过分强调理性思维的力量， 忽视了 感性经验、个体意识和常识判断 的重要性。 他主张哲学应该 更加贴近经验和直觉， 从 更具体、更直接的经验出发 构建形而上学体系。

四、 麦克塔格特的修正与替代：多元唯心主义

麦克塔格特在批判黑格尔哲学的过程中， 也逐渐发展出自己的哲学体系， 即 多元唯心主义 (Pluralistic Idealism)。 他的多元唯心主义可以被视为对黑格尔 一元论的绝对唯心主义 的一种 修正和替代。

• 多元 “自我” (Selves) 的实在性： 麦克塔格特认为 “自我” (Selves) 或 “人格中心” (Personal Centres) 才是宇宙最基本的实在。 宇宙是由 无数个独立的、永恒的 “自我” 构成的， 每个 “自我” 都具有 意识、经验、意志。 他将 “自我” 的经验和关系 视为理解 实在本质的关键。
• 拒绝 “绝对精神” 和 “一元论”： 麦克塔格特 彻底拒绝了黑格尔的 “绝对精神” 概念， 认为宇宙 并非统一于一个 “绝对” 之中， 而是 多元的、分散的。 他认为 “一元论” 的形而上学无法解释个体的独特性和自由意志， 而 “多元主义” 的形而上学更符合经验和理性。
• 关系 (Relations) 的重要性： “爱的关系” (Relation of Love) 的中心地位： 麦克塔格特强调 “关系” (Relations) 在形而上学中的重要性。 他认为 “自我” 之间通过 “关系” 相互联系和相互作用， 构成一个复杂的、动态的宇宙。 在所有关系中， 麦克塔格特特别强调 “爱的关系” (Relation of Love) 的中心地位， 认为 “爱” 是最高形式的关系， 也是最能体现 “自我” 价值和意义的关系。 他甚至认为， 整个宇宙的最终目的， 就是实现 “自我” 之间更丰富、更完善的 “爱的关系”。

• 时间非实在 (Time is Unreal)： “B系列” 时间的批判： 麦克塔格特提出了著名的 “时间非实在论” (The Unreality of Time) 观点。 他在其重要论文《时间的非实在性》（The Unreality of Time, 1908）中， 论证了 我们通常理解的时间 (A系列和B系列时间) 是 逻辑上自相矛盾的、最终是幻象 (illusion)。 他区分了两种时间序列：

  • A系列 (A-series): 基于 “过去-现在-未来” 的时间序列， 强调时间的 “流逝” (passage) 和 “变迁” (becoming)。 A系列时间是 主观的、动态的、与意识体验密切相关 的时间。
  • B系列 (B-series): 基于 “先后顺序” (earlier-later) 的时间序列， 强调事件之间的 时间关系 (temporal relations)， 例如 “X事件早于Y事件” 或 “X事件晚于Y事件”。 B系列时间是 客观的、静态的、与物理时间测量密切相关 的时间。

  麦克塔格特认为 A系列时间是逻辑上自相矛盾的， 因为 “现在” 的性质是不断变化的， 一个事件在某一时刻是 “未来”， 在另一时刻变成 “现在”， 又在另一时刻变成 “过去”， 这种 性质的不断变化 会导致 逻辑上的无限倒退 (infinite regress)。 而 B系列时间虽然没有逻辑矛盾， 但它只是事件之间静态的 “先后顺序关系”， 无法真正捕捉到时间 “流逝” 和 “变迁” 的动态特性。 因此， 麦克塔格特结论认为， 我们通常理解的时间 (A系列和B系列时间) 最终都是非实在的， 是幻象。 真正的实在应该 超越时间， 是 永恒的、不变的。

五、 麦克塔格特批判的影响与评价

麦克塔格特的著作，特别是《黑格尔辩证法研究》和《黑格尔逻辑注释》， 对黑格尔哲学研究产生了 深远的影响。

• 对黑格尔辩证法的有力批判： 麦克塔格特的批判被认为是 对黑格尔辩证法最具逻辑性和挑战性的批评之一。 他的著作迫使黑格尔主义者 认真反思辩证法的逻辑有效性， 并 重新审视黑格尔体系的形而上学基础。
• 推动了分析哲学对黑格尔哲学的研究： 麦克塔格特的批判 使用了分析哲学的逻辑分析方法， 这为后来的分析哲学家研究黑格尔哲学 开辟了道路。 许多分析哲学家受到麦克塔格特的影响， 开始从 逻辑分析的角度 来解读和评价黑格尔哲学， 而不再仅仅停留在 历史和文化背景的解读。
• 促进了唯心主义哲学的多元发展： 麦克塔格特的多元唯心主义 挑战了黑格尔的一元论绝对唯心主义， 为唯心主义哲学 提供了另一种可能的发展方向， 即 多元主义的、人格主义的唯心主义。 他的 “自我” 中心 和 “爱的关系” 优先 的思想， 对后来的 个人主义和社群主义 思潮也产生了一定的影响。
• 引发了关于时间本质的哲学辩论： 麦克塔格特的 “时间非实在论” 观点， 引发了哲学界关于 时间本质 的 广泛而持久的辩论， 至今仍然是 时空哲学领域的重要议题。 他的 A系列和B系列时间的区分 成为了分析时间哲学的 经典框架。

评价与争议：

麦克塔格特的批判虽然有力， 但也并非没有争议。 一些黑格尔主义者 反驳 麦克塔格特对辩证法的理解， 认为他 过于字面化和形式化地解读辩证法， 忽视了辩证法的 “精神” 和 “动态性”。 他们认为辩证法并非旨在提供 形式逻辑意义上的 “证明”， 而是一种 “理解模式” 或 “思维方式”， 其价值在于 揭示概念之间的内在联系和矛盾， 推动思想的深入和发展。 此外， 麦克塔格特的 多元唯心主义 和 时间非实在论 也面临着来自其他哲学流派的 质疑和挑战。

总结：

约翰·麦克塔格特对黑格尔哲学的批判是 深刻而富有洞见的。 他以 严谨的逻辑分析和敏锐的哲学洞察力， 深入考察了黑格尔哲学的 逻辑体系和形而上学基础， 提出了 许多重要的批评， 并 发展出自己的多元唯心主义哲学。 他的著作不仅 推动了黑格尔哲学研究的深入， 也 促进了唯心主义哲学的多元发展， 并引发了关于时间本质的持久哲学辩论。 麦克塔格特是 20世纪初期英国唯心主义的重要代表人物， 他的批判性工作对于理解黑格尔哲学以及西方哲学从唯心主义向其他流派转变的历史进程都具有重要的意义。 研究麦克塔格特对黑格尔的批判， 有助于我们 更深入地理解黑格尔哲学的复杂性和局限性， 并拓展我们对逻辑、形而上学和时间本质的哲学视野。

O quizás unas nuevas post-humanidades digitales que se cuestionen tanto el gigantismo de los datos y las tecnlogías computacionales, como el de las grandes instituciones humanísticas, revelando el valor de lo pequeño, lo conexo, lo convivencial y comunitario.

Otra alternativa, que seguimos en Grafoscopio para lograr ese enraízamiento recontextualizado alimentado por las particularidades, fue optar por la creación de metaherramientas digitales y sistemas maleables, así como proveer y configurar alfabetismos críticos al respecto, en lugar de sólo herramientas.

Esto nos da una especie de "escalabilidad en horizontal" y en pequeño, no universalizante: más comunidades en condiciones de adaptar metaherramientas digitales, en lugar de una herramienta digital única que sirva a muchas comunidades.

Sin embargo, incluso en los Critical SCS y las HD hay una preocupación por las herramientas y poca explicitación de las metaherramientas y su relación con las infraestructuras.

Colocaría dentro de esas hegemonías digitales, desafortunadamente invisibles incluso para personas cercanas a las HD y los Critical STS lugares como GitHub, con evidentes prácticas extractivistas, como las de Copilot y el código de generación probabilística.

Muchos practicantes de las DH usan y promueven estas infraestructuras maximalistas, extrativistas y panópticas sin ningún inconveniente o búsqueda de alternativas.

Sería valioso politizar de manera evidente esta postura. ¿Cómo ocurre la reciprocidad desde la generación de bienes comunes y cómo ellos a través de cosas como el licenciamiento garantizan aperturas comunicantes y agrietamientos institucionales que permitan sostenibilidad genuina más allá de los modelos extractivistas neoliberales a los cuales la academia está también sometida.

Reflexiones como las de Dmitry Kleiner con sus espectros de licenciamiento, Michael Bauwens con las reflexiones de producción entre pares y Nadia Eghbal sobre la sostenibilidad del software libre y sus crisis, pueden ilustrar este caracter más concreto de la reflexión a la que invitaría.

Licencias explícita ayudan a percibir una reflexión más detallada y granular sobre el sostenimiento de esa deriva estructural y por ello sería bueno colocarlas a esta tesis.

Esta definición pareciera aplicarse a distintos mundos, más allá de los del arte. Es decir esa lectura ecológica, cibernética y autopoiética, es propia de lo social, en la cual caen las HD, pero no logro apreciar acá las particularidades que atañe a las HD, referidas, por ejemplo a una forma particular de conjunción de lo formal y lo informal.

Valdría la pena en esta sección anunciar, así sea brevemente parte de las reflexiones más puntuales del capítulo al respecto. por ejemplo, las referidas a la deriva estructural o maneras de reciprocidad específicas entre lo formal e informal que ayuden a la sostenbilidad en perspectiva cibernética/autopoiética.

Creo que resonamos en este párrafo y en el comentario anterior.

Sin embargo, creo que lo propio y la interoperabilidad no están en riña, sino que tienen que ver con cómo hay acceso diferenciado, pero interoperable a infraestructuras digitales dispuestas por y para las comunidades y alejadas de grandes oligopolios.

Por ejemplo, nuestro reciente proyecto de Cartofonías para la revitalización lingüística en La Chorrera, Amazonas usa tecnologías interoperables, pero se distancia de tecnologías hegemónicas y sus centros, de este modo no hospedamos el código en el privativo GitHub) ni usamos el motor wiki libre y complicado de la Wikimedia/Wikipedia, ni hospedamos las memorias en Meta/Instagram. Nuestra curaduría e interconexión de alternativas (Fossil, TiddlyWiki, Internet Archive) es interoperable pero alejadas de las hegemonías tecnoeconómicas libres o privativas. Las comunidades además son las que deciden sobre los accesos a los datos y una posible intranet, podría extender el computador en territorio dónde ahora están hospedados localmente, para que evolucione y crezca con condiciones de acceso distintas y más allá de lo que ahora están en línea.

Tal vez incluso para superar el proyecto de lo humano o enmarcarlo como menos preponderante, en medio de esas otredades de las Humanidades eurocéntricas y con "H" mayúscula han omitido histórcamente.

En ese sentido valdría la pena explicitar esos vínculos que este y el siguiente párrafo anuncian y cómo las HD, en particular las Latinoamericanas se conectan con esas miradas críticas.

Al menos pareciera que la mirada lejana de los movivimento hacktivistas y de tecnologías cívicas de las HD ha sido más bien "apolitico" y academicista, aunque quizás, salvo excepciones crítica y políticamente informadas, como las ejemplificadas en este párrafo.

Así como criticar la visibilidad y el papel de las llamadas "grandes instituciones humanistas" en funciones que no sólo las exceden, sino que han estado largamente por fuera a pesar de que esto se reconoce en miradas, precisamente menos institucionalizadas.

Me recuerda la postura de Gabriela Coleman en su tesis doctoral respecto a no definir lo hacker, acotándolo y por el contrario mantener las tensiones vivas y creativas.

En el caso de Grafoscopio, que ha sido y sigue sosteniéndose principalmente gracias a las economías de los afectos y cuidados, la sostenibilida económica y de código ha ocurrido en tres frentes:

• Lo uso para consultorías adaptándolo a necesidades de nuestros clientes. El código resultado queda libre y las narrativas de datos son del cliente.
• Lo uso para proyectos educativos e investigativos, tanto en la universidad, como en el hackerspace y otras comunidades de base.
• Aplico a becas que implican la mejora de Grafoscopio, su documentación y adaptación de funcionalidades.

Las dos primeras han sido fuentes más constantes de mejoras y este año el software cumple su primera década desde el primer commit, transformándose junto con la infraestructura subyacente en la que está basado.

Desde la comunidad de Grafoscopio, hemos encontrado en la metaherramienta TidddlyWiki esta adaptabilidad extrema y facilidad de uso multiplataforma y multilingüe, incluso para procesos de catalogación y memoria viva.

También hemos acuñado lo que he llamado "programación intersticial" que permite extender las infraestructuras y, en general los sistemas sociotécnicos pequeños, no desde sus entrañas, sino desde los intersticios con otras infraestructuras, comunidades y sistemas. Así, cuando TiddlyWiki no tiene una funcionalidad particular, la conectamos con Grafoscopio/Pharo o con Fossil para tener las ventajas de todas sin las desventajas particulares de sólo una de ellas.

La posibilidad de adaptación se facilita con metaherramientas. Sin embargo, la computación de hoy en día y el paradigma ganador de sistemas tipo Unix, las invisibiliza y dificulta constantemente, a diferencia de los paradigmas Lisp/Symbolics y Smalltak/Dynabook, que viven entre nosotros, desconocidos ampliamente incluso en el campo de las HD, a pesar de preocupaciones por la adaptación y los alfabetismos críticos, como los establecidos acá.

Precisamente, en aras del establecimiento claro de reglas, es que me parece que el repositorio de esta tesis debería tener una postura explícita frente al licenciamiento que favorezca los comunes.

También estaría el grupo de los Scanponies que trabajan escaners desde el hazlo tú mismo, hazlo con otros y tienen miembros de la colectiva de edición independiente con software libre, Miau y una presencia latinoamericana importante y activa en la red de Telegram.

Es raro. Pandoc me ha servido para propósitos similares y también hemos tenidos que adaptarlo con hacks, pero estos son soportados por el sistema para que ocurran de modo relativamente sofisticado, vía filtros en Lua. Me pregunto qué hace de Magicbook un sistema tan inflexible y no logré ubicarlo en el capítulo 10, donde hice comentarios más extensos entre dichas alternativas de publicación reproducible y abierta.

La definición de infraestructuras de bolsillo precisamente quería resaltar otro tipo de metáforas contrapuestas a "la nube" que dieran cuenta de los contextos del Sur Global.

La versión beta es posterior a la alfa, que es más inmadura. Acá lo propio sería hablar de release candidate como siguiente versión hasta llegar a software en producción. Véase Software release life cycle.

Acá también con Grafoscopio y sus elecciones no habituales estamos en beta permanente, pero el software al ser un sistema maleable y una metaherramienta, introduce nuevas caracterísitcas para adaptarse a sus usuarios y contextos en lugar de al contrario.

Es interesante como Grafoscopio ha evitado varias de estas fallas al hacer elecciones extrañas como ser desarrollado en Pharo (que de entrada le da interfaz gráfica y modelos de persistencia de datos ad-hoc), organizando talleres informales como las Data Week y las Data Rodas que crean conocimiento localizado y hacen una diglosia puente en lugar de abismo y basarse en las economías del cuidado y los afectos, reconociéndolas para no requerir tanto dinero inicial. Si bien se comparten las fragilidades de los proyectos de pequeña y mediana, por ejemplo respecto a el número pequeño de desarrolladores, vale la pena visibilizar también estas estrategias diferenciadas para lidiar con estos problemas comunes.

Aunque también muchas son construídas desde economías de los afectos y cuidados y con bajo presupuesto.

Por ejemplo vía metaherramientas, de modo que las infraestructuras ofrecidas contienen dentro de sí las formas de cambiarlas.

interesante además que sólo funcionen con conectividad o se vuelvan "tiestos". Soluciones de conectividad nula, baja o intermitente, como las de las infraestructuras de bolsillo, se colocan criticamente en este lugar.

También valdría la pena preguntarse por formatos mixtos, como los de las narrativas de datos antes mencionadas, que van más allá de sólo el código, combinándolo con prosa, datos y salidas enriquecidas (visualizaiones, interactivos, etc) y en alguna medida explorando de antemano en qué consisten esas condiciones localistas y los cotidianos de quienes escriban tales narrativas.

No sé hasta qué punto se pueda afirmar eso. Depende de lo que si se programa en inglés tiene que ver con la vida cotidiana o no. En el caso de las tecnologías cívicas el código que escribimos en inglés, permanentemente tiene que ver con lo local y el cotidiano. Por eso son los usos significativos, de los que habla este párrafo al final, donde estaría el centro del posible desarraigo (o su ausencia), sin desconocer que hay usos totalmente enagenados de muchos lenguajes de programación.

Nuesta aproximación es construir narrativas de datos profusamente comentadas en español, con código totalmente en inglés, tanto en los talleres de la comunidad de Grafoscopio, como en los pregrados de Ciencia de la Información. Si bien esto enfrente a læs aprendices a las tensiones de la diglosia, también instaura un puente frente a ella.

No sé si se pueda marcar una relación tan directa entre la diglosia y el desarraigo. Puede ocurrir como diría Freira, que se aprenda el lenguaje colono para decir la voz de colonizando y hablar precisamente de preocupaciones locales. Comentarios de los esfuerzos en ese sentido de reflejar lo local en código, vía narrativas de datos, metaherramientas y alfabetismos críticos, han sido hechos a lo largo del texto.

Contra esta tendencia, las infraestructuras de bolsillo, las metaherramientas y desarrollos posteriores como los de An app can be a home-cooked meal y Home-Cooked Software and Barefoot Developers parecen indicar una contratendencia a software altamente específico desarrollado por y para contextos locales y pequeños.

Desafortunadamente, experiencias políticamente informadas como las de Grafoscopio respecto a producir código recontextualizable y en pequeño, están lejos de las humanidades digitales tando académicas como informales y también de lo que las investigaciones al respecto cartografían.

La alternativa es intentar deescalabilidad para lograr recontextualización, precisamente para reflejar las relaciones con los contextos locales donde dicha recontextualización ocurre. Las metaherramientas facilitan estos procesos.

Otra manera es no pensar en escalabilidad vertical, sino horizontal y recontextualización, como hemos hecho con Grafoscopio (las etiquetas de esta anotación pueden ayudar a encontrar notas similares a lo largo del texto donde comento el fenómeno en mayor detalle).

Escalar hacia abajo y en horizontal, vía infraestructuras de bolsillo, por ejemplo fue nuestra manera de responder a los desafíos como los planteados en esta sección.

Otra forma es lidiar con la diglosia de maneras similares a como lo hicimos con Grafoscopio, con software, interfaces gráficas y código en inglés, que tiene una amplia documentación y narrativas de datos en español o incluso haciendo puentes con procesos de revitalización lingüística en Lenguas del Amazonas Colombiano.

Una de las cosas interesante de Pandoc y otros los lenguajes de etiquetado ligero, es la insistencia de su autor, John MacFarlane, por no usar palabras angloparlantes en las marcas, como se ve, no sólo en la especificación de Pandoc, sino en algunos comentarios sobre otros lenguajes diseñados por él como Djot.

Podrían otro tipo de redes sociales, como NOSTR, donde el algoritmo puede personalizarse, generar rotaciones de atención sin tantos centralismos,

Colocaría acá las economías de los afectos y cuidados (familia, amigos), que aunque también invisibles, hacen posible el desarollo de infraestructuras propias y apropiadas desde estos contextos y constituyen ese empujón de largo aliento necesario, como evidencié y agradecí en primera persona con el desarrollo de Grafoscopio.

Otro tanto tiene que ver con encontrar las grietas para ubicarse de manera que no se compita directamente con lo pre-existente, sino que se complemente lo que este no ofrece, como ocurrió con Grafoscopio también, que se ubicó en la intersección de campos como metaherramientas, sistemas malleables investigación/publicación reproducible, tecnologías cívicas, de una forma en que otras herramientas con mayor visibilidad (Jupyter, por ejemplo) no habían hecho y con ventajas y recorridos particulares no suplidos por las alternativas que emergieron luego.

Allí se ubican investigaciones y materialidades argumentativas por fuera de ese circuito, que, desde mi historia personal, iniciaron en 2011, con mi doctorado, dando cuenta de inquietudes resonantes con este punto y con este escrito.

El problema del estandar y las métricas son su condición de externos, más que la universalización sobre la base de de interoperabilidad. APIs extensibles e interoperables que dan cuenta de unos mínimos a la vez que recontextualizan estándares han sido definidas en caso como los de NOSTR

La forma de superar estos binarismos y navegar estas tensiones, al menos desde la comunidad de Grafoscopio es desarrollar tecnologías propias que reorganizan stacks tecnológicos alternos y agregan metaherramientas a la mezcla, de manera que podamos disfrutar las ventajas de la interoperabilidad y la alta localización y recontextualización.

Agregaría los trabajos de Silvia Buitrago sobre redes inalámbricas de Fusa Libre, los procesos de tecnificación con Arduino del tostado de café y de la comunidad de Grafoscopio; así como el trabajo del profesor Carlos Barreneche sobre la red ciudadana de calidad del aire y sus sensores de hardware abierto, iniciados en HackBo o mi trabajo con Grafoscopio, metaherramientas y las tecnologías cívicas

De ahí lo importante de señalar críticamente la fascinación de las HD y muchos de sus representantes con el gigantismo de datos en detrimento de estas miradas locales y en pequeño.

Otro interactivo que muestra sesgos de género, como he referido en otras anotaciones

En la comunidad de Grafoscopio hemos curado, usado y desarrollado stacks alternos de infraestructura para recorrer futuros alternos, con inspiraciones frecuentes en cosas como el Solarpunk.

Salvo en perpectivas críticas como las feministas tipo Data Feminism o Feminist AI que precisamente visibilizan lo que ya mayoría de enfoques invisibiliza.

Así como invisibilizar lo interesante local y que ocurre a pequeñas escalas de datos y computación.

Mucho de eso ocurre con lo que llamo Inteligencias Aparentes que no sólo invisibilizan sino que principalmente expropian el trabajo humano.

Star también fue clave en mi tesis doctoral. Mis reflexiones también han agregado algunas propiedades a las infraestructuras.

En este sentido me refiero a la importancia de una interoperabilidad recontextualizable en el comentario del siguiente capítulo.

Dado que la lectura ocurre principalmente en digital,agregaría un botón de pausa a la simulación, pues intentar concentrarse en la lectura del texto circundante con la visión periférica distraída por el movimiento de la partículas resulta muy difícil.

Algo similar experimentamos con las Data Weeks y Data Rodas en Grafoscopio, lo que nos llevó a establecer una serie de principios que incluían cosas como las prácticas de cuidado mútuo y reconocemos el carácter flotante de la mayoría de læs participantes y el duradero de muy pocos (por ello y otras cosas es clave la creación permanente de memoria viva hipertextual en nuestras infraestructuras de bolsillo).

En resonancia con esto, debemos implementar métricas abiertas y no uniformes para visibilizar lo que hacemos en los márgenes, como lo propuesto en la publicación futura de Plurimétrica.

Otra posibilidad es la construcción de memoria viva durante y entre los talleres que les de un sentido de continuidad y progreso y que permita valorar la lógica de los talleres para construir tecnologías propias en lugar de pensarla sólo para la apropiación de tecnologías externas, muy en resonancia con lo dicho en este comentario.

Si bien tenemos aún problemas, en Grafoscopio, para el aprendizaje entre pares gradual, la memoria viva y los problemas altamente contextuales hacen de ellos problemas encarnados que asumimos en talleres futuros y vínculos entre comunidades de práctica y espacios institucionalizados

O también les subsume dentro de las lógicas burocráticas, heterónomas y no convivenciales señaladas por Illich en textos como La Sociedad Desescolarizada (1972).

En general el tono de institución y academicismo como estable y deseable y comunidad de práctica como pasajera y no tan deseada se percibe a lo largo de este capítulo, entre más avanzan las secciones, informado por los testimonios recolectados y la experiencia misma que ha estado marcada por esas comunidades de práctica pasajeras e identidades institucionalizadas en lugar de robustas e identitarias al margen de las instuciones.

Por supuesto, es una apuesta del escrito consecuente, pero permanentemente me lleva a preguntas sobre cómo podría esto enriquecerse con experiencias y testimonios fuera de la red más académicista e institucionalizada de las HD.

En el caso de HackBo, lo que hemos hecho es tener "instituciones amigas" de miembros del espacio o cercanas al mismo, que pueden captar recursos que terminan ayudando al sostenimiento del espacio. Esto nos permite usar infraestructuras legales y jurídicas ya establecidas sin cambiar la vocación ni el estatus no legal del espacio.

Si bien esta por fuera de los alcances, vale la pena también pensar en cómo trabajos como este conectan memoria, particularmente en la revisión de antecedentes no sólo sobre las temáticas, sino sobre las materialidades que soportan estas publicaciones híbridas y lo que se ha hecho con tesis de doctorado como la mía (2010 - 2019) o el trabajo de publicación reproducible realizado en pregrado por Felipe Vera y publicado este año en el repositorio institucional.

Agregaría yo, vínculos más visibles con las instituciones sin estar subsumidas en ellas o sus lógicas. Esto pasa por métricas y formatos distintos de investigación y publicación y evaluación, como aquellos a los que esta tesis se inscribe y contribuye, siendo parte de un pequeño preo creciente acero de tesis en postgrado y pregrado a los que se suma ahora nuestra facultad.

Vale la pena construir también repositorios alternos, resilientes y distribuidos que muestren estas crecientes alternativas y mediaciones. Yo en particular he estado pensando en usar Brea, durante 2025 y su caracter de "CMS desacoplado" para evidenciar trabajos como este y los mencionados, que pueden y deben leerse de modos distintos y a los cuales las plataformas maximalistas e institucionales no les harían tanta justicia.

Otro tanto ocurrirá con Plurimétrica, pero ese es un proyecto que espero anunciar como parte de una conversación más larga a la que esta evaluación invita.

Pareciera ser que la la RCHD es una comunidad de práctica particularmente jóven (en rasgos, más que en tiempo) y poco resiliente respecto a sus procesos de memoria, paradógicamente siendo este uno de los temas de las HD. Esto contrasta con otras comunidades como las de software libre, como se ha indicado en otros comentarios, donde la resiliencia de la memoria, en principio en los repositorios de código, pero también en cosas como narrativas de datos y wikis, es parte constitutiva de las mismas. Quizás esa experiencia particular de la RCHD hace que se solape la informalidad con lo efímero, en contraste con las comunidades de software libre.

Un contraste interesante sería por qué las redes hacktivistas con vínculos eventuales e itinerantes pero frágiles con las universidades, logran mantener el ímpetu y una rotación de protagonismos, a pesar de lo demandante de esfuerzos de largo aliento como el FLISoL, que completa ya sus 20 años y se ha expandido ampliamente por Latinoamérica o HackBo con 14 años en Bogotá.

Este tipo de contrastes creo que pueden alimentar la reflexión sobre las funciones de las HD que están ocurriendo fuera de la autorreconocída red de HD e inspiraciones para lograr cruces y abordar tensiones y problemáticas comunes a partir de las diferencias.

Este sesgo tiene que ver con que se confunde lo informal con lo efímero, cuando son ejes ortogonales. Pueden haber vínculos informales de largo aliento, como se explica en el comentario anterior.

Aunque también existen comunidades de práctica cuyo carácter informal no es equivalente a lo efímero o a un envolvente corto, como ocurre con varias comunidades de software libre, y espacios maker/hacker. Por el contrario, son procesos indentitarios de largo aliento que persisten incluso cuando sus miembros cambian de trabajo o filiaciones institucionales.

Por ejemplos como estos es que la noción de institución me cuesta a menos que se refiera en el plano general de aquello instituido, en lugar de una acepción de organización más bien constreñida y contra la que se lucha, por ejemplo en la propuesta de Illich de pasar de la vida institucionalidada (como en el caso académico y estatal) a las organizaciones conviviales (como el hackerspace). Matizar la noción de institución ayuda a diferenciar mejor y a dialogar con los afuera, en lugar de la acepción en la cual todo es institución.

Creo que habría que distinguir lo legítimo de lo estatal/legal y de lo institucional. Desde muchas comunidades hacker/maker y hacktivistas sostenemos prácticas legitimas no institucionalizadas y dentro de un marco legal general, pero sin una constitución legal específica pues no fuimos nunca a una notaría pública a registrar el acta de constitución del hackerspace, ni contamos con gerente o con cargos formales a pesar de que sí reconocemos estructuras de poder relativamente planas y transitorias, configuradas por experticias diferenciales.

Algo muy similar ocurre con los hacktivistas.

Interesante. Siguiendo a Raw Data is an oxymoron, la eleccón del lugar de muestreo deja por fuera los lugares poco activos en Twitter/X, por decisión política, como varios hackerspaces. En estudios futuros valdría la pena revisar qué nos pueden decir esas redes periféricas que precisamente no usan Humanidades Digitales como su punto de enunciación o anclaje a pesar de compartir muchas de las 3 preocupaciones fundantes que se han dado a las humanidades.

Pasó literalmente así en el caso de la comunidad y la metaherramienta de Grafoscopio, repitiendo la historia de otras herramientas y comunidades que, en la medida en que se definen mutuamente, comparte incluso el mismo nombre, como Debian, Python, Arch Linux, Pharo, etc.

O, como digo en el comentario anterior, dadas las materialidades argumentativas mixtas, ocupar roles simultáneamente, como hace Victor, en la medida en que, a través de una materialidad discursiva específica, en un espacio concreto que cuida da cuenta de unas preocupaciones intelectuales que son enunciadas por esa materialidad en ese espacio y en conexión con otros.

Dado el carácter mixto de los campos y prácticas afines, uno podría pensar que esos roles no están tan diferenciados en las HD y de hecho es provechoso. Bret Victor, por ejemplo con el Dynamicland cumple las tres funciones de manera concomitante, vela por un espacio en la medida en que estudia dominios de la cultura humana construyendo artefactos para ello. Y se podría decir otro tanto de muchos de sus colaboradores en ese espacio o de quienes cumplen funciones igualmente mezcladas y múltiples en varios lugares de Sur Global aunque con mayor invisibilidad.

Veo que la difrenciación entre roles y personas se establece más adelante.

No queda claro qué diferencia a ese centro en la participación y colaboración cuando ocurre en otros lugares o en un mundo del arte. De nuevo, me parece que esas características son aplicables a tantos lugares que no logro acotar las particularidades de la definición de mundos del arte.

No queda claro acá que diferencia a los mundos del arte de otros mundos o si todos los mundos serían mundos del arte, dado lo amplia de esa definición aplicable prácticamente para cualquier agrupación humana regalada.

• Denotational Design as a Real Process

Denotational design is the process that has been elaborately developed by Conal Elliott.

• Core Principle of Stepping Back from Implementation

We don't want to jump in and say, ‘An image is an array of pixels.’ That’s too soon yet that’s where most of us start.

• Abstract Definition of an Image

An image is just a function from a pixel location, so an X, Y coordinate to color, where X, Y are in the real number space.

• Emphasis on Algebraic Properties and Category Theory

He uses algebraic properties and category theory. I think algebraic properties are a very good indicator that you are, ‘on to something’ in the design.

• Incremental, Iterative Refinement

You have to go back and revise and you make an attempt in a certain direction, and you learn something, and you bring that back to the beginning.

• Four Steps of the Denotational Design Process

These are the four steps that I see... This first one is to...like a Zenning out and forgetting all implementation assumptions...Then you explore...Then you align with category theory concepts...Then the final thing is actually implementing it.

• Challenges with Haskell’s Type System

Haskell has no type for real numbers. Most languages don’t...Another thing is, when you’re talking about say, the Monad laws or the Functor laws...there’s no way to do that equality comparison.

• Similar Difficulties in Clojure

I do think it's a little harder than in Haskell, but I also think that most of the design part is happening in your head.

• The Essence of Denotational Design

It’s about going back to first principles, building things up, understanding how things compose, and following a different gradient from what most people use when they design.

Reviewer #1 (Public review):

Summary:

This research focuses on C. elegans klinotaxis, a chemotactic behavior characterized by gradual turning, aiming to uncover the neural circuit mechanism responsible for the context-dependent reversal of salt concentration preference. The phenomenon observed is that the preferred salt concentration depends on the difference between the pre-assay cultivation conditions and the current environmental salt levels.

The authors propose that a synaptic-reversal plasticity mechanism at the primary sensory neuron, ASER, is critical for this memory- and context-dependent switching of preference. They build on prior findings regarding synaptic reversal between ASER and AIB, as well as the receptor composition of AIY neurons, to hypothesize that similar "plasticity" between ASER and AIY underpins salt preference behavior in klinotaxis. This plasticity differs conceptually from the classical one as it does not rely on any structural changes but rather synaptic transmission is modulated by the basal level of glutamate, and can switch from inhibitory to excitatory.

To test this hypothesis, the study employs a previously established neuroanatomically grounded model [4] and demonstrates that reversing the ASER-AIY synapse sign in the model agent reproduces the observed reversal in salt preference. The model is parameterized using a computational search technique (evolutionary algorithm) to optimize unknown electrophysiological parameters for chemotaxis performance. Experimental validity is ensured by incorporating constraints derived from published findings, confirming the plausibility of the proposed mechanism.

Finally. the circuit mechanism allowing C. elegans to switch behaviour to an exploration run when starved is also investigated. This extension highlights how internal states, such as hunger, can dynamically reshape sensory-motor programs to drive context-appropriate behaviors.

Strengths and weaknesses:

The authors' approach of integrating prior knowledge of receptor composition and synaptic reversal with the repurposing of a published neuroanatomical model [4] is a significant strength. This methodology not only ensures biological plausibility but also leverages a solid, reproducible modeling foundation to explore and test novel hypotheses effectively.

The evidence produced that the original model has been successfully reproduced is convincing.

The writing of the manuscript needs revision as it makes comprehension difficult.

One major weakness is that the model does not incorporate key findings that have emerged since the original model's publication in 2013, limiting the support for the proposed mechanism. In particular, ablation studies indicate that AIY is not critical for chemotaxis, and other interneurons may play partially overlapping roles in positive versus negative chemotaxis. These findings challenge the centrality of AIY and suggest the model oversimplifies the circuit involved in klinotaxis.

Reference [1] also shows that ASER neurons exhibit complex, memory- and context-dependent responses, which are not accounted for in the model and may have a significant impact on chemotactic model behaviour.

The hypothesis of synaptic reversal between ASER and AIY is not explicitly modeled in terms of receptor-specific dynamics or glutamate basal levels. Instead, the ASER-to-AIY connection is predefined as inhibitory or excitatory in separate models. This approach limits the model's ability to test the full range of mechanisms hypothesized to drive behavioral switching.

While the main results - such as response dependence on step inputs at different phases of the oscillator - are consistent with those observed in chemotaxis models with explicit neural dynamics (e.g., Reference [2]), the lack of richer neural dynamics could overlook critical effects. For example, the authors highlight the influence of gap junctions on turning sensitivity but do not sufficiently analyze the underlying mechanisms driving these effects. The role of gap junctions in the model may be oversimplified because, as in the original model [4], the oscillator dynamics are not intrinsically generated by an oscillator circuit but are instead externally imposed via $z_\text{osc}$. This simplification should be carefully considered when interpreting the contributions of specific connections to network dynamics. Lastly, the complex and context-dependent responses of ASER [1] might interact with circuit dynamics in ways that are not captured by the current simplified implementation. These simplifications could limit the model's ability to account for the interplay between sensory encoding and motor responses in C. elegans chemotaxis.

Appraisal:

The authors show that their model can reproduce memory-dependent reversal of preference in klinotaxis, demonstrating that the ASER-to-AIY synapse plays a key role in switching chemotactic preferences. By switching the ASER-AIY connection from excitatory to inhibitory they indeed show that salt preference reverses. They also show that the curving/turn rate underlying the preference change is gradual and depends on the weight between ASER-AIY. They further support their claim by showing that curving rates also depend on cultivated (set-point).

Thus within the constraints of the hypothesis and the framework, the model operates as expected and aligns with some experimental findings. However, significant omissions of key experimental evidence raise questions on whether the proposed neural mechanisms are sufficient for reversal in salt-preference chemotaxis.

Previous work [1] has shown that individually ablating the AIZ or AIY interneurons has essentially no effect on the Chemotactic Index (CI) toward the set point ([1] Figure 6). Furthermore, in [1] the authors report that different postsynaptic neurons are required for movement above or below the set point. The manuscript should address how this evidence fits with their model by attempting similar ablations. It is possible that the CI is rescued by klinokinesis but this needs to be tested on an extension of this model to provide a more compelling argument.

The investigation of dispersal behaviour in starved individuals is rather limited to testing by imposing inhibition of the SMB neurons. Although a circuit is proposed for how hunger states modulate taxis in the absence of food, this circuit hypothesis is not explicitly modelled to test the theory or provide novel insights.

Impact :

This research underscores the value of an embodied approach to understanding chemotaxis, addressing an important memory mechanism that enables adaptive behavior in the sensorimotor circuits supporting C. elegans chemotaxis. The principle of operation - the dependence of motor responses to sensory inputs on the phase of oscillation - appears to be a convergent solution to taxis. Similar mechanisms have been proposed in Drosophila larvae chemotaxis [2], zebrafish phototaxis [3], and other systems. Consequently, the proposed mechanism has broader implications for understanding how adaptive behaviors are embedded within sensorimotor systems and how experience shapes these circuits across species.

Although the reported reversal of synaptic connection from excitatory to inhibitory is an exciting phenomenon of broad interest, it is not entirely new, as the authors acknowledge similar reversals have been reported in ASER-to-AIB signaling for klinokinesis ( Hiroki et al., 2022). The proposed reversal of the ASER-to-AIY synaptic connection from inhibitory to excitatory is a novel contribution in the specific context of klinotaxis. While the ASER's role in gradient sensing and memory encoding has been previously identified, the current paper mechanistically models these processes, introducing a hypothesis for synaptic plasticity as the basis for bidirectional salt preference in klinotaxis.

The research also highlights how internal states, such as hunger, can dynamically reshape sensory-motor programs to drive context-appropriate behaviors.

The methodology of parameter search on a neural model of a connectome used here yielded the valuable insight that connectome information alone does not provide enough constraints to reproduce the neural circuits for behaviour. It demonstrates that additional neurophysiological constraints are required.

Additional Context

Oscillators with stimulus-driven perturbations appear to be a convergent solution for taxis and navigation across species. Similar mechanisms have been studied in zebrafish phototaxis [3], Drosophila larvae chemotaxis [2], and have even been proposed to underlie search runs in ants. The modulation of taxis by context and memory is a ubiquitous requirement, with parallels across species. For example, Drosophila larvae modulate taxis based on current food availability and predicted rewards associated with odors, though the underlying mechanism remains elusive. The synaptic reversal mechanism highlighted in this study offers a compelling framework for understanding how taxis circuits integrate context-related memory retrieval more broadly.

As a side note, an interesting difference emerges when comparing C. elegans and Drosophila larvae chemotaxis. In Drosophila larvae, oscillatory mechanisms are hypothesized to underlie all chemotactic reorientations, ranging from large turns to smaller directional biases (weathervaning). By contrast, in C. elegans, weathervaning and pirouettes are treated as distinct strategies, often attributed to separate neural mechanisms. This raises the possibility that their motor execution could share a common oscillator-based framework. Re-examining their overlap might reveal deeper insights into the neural principles underlying these maneuvers.

(1) Luo, L., Wen, Q., Ren, J., Hendricks, M., Gershow, M., Qin, Y., Greenwood, J., Soucy, E.R., Klein, M., Smith-Parker, H.K., & Calvo, A.C. (2014). Dynamic encoding of perception, memory, and movement in a C. elegans chemotaxis circuit. Neuron, 82(5), 1115-1128.

(2) Antoine Wystrach, Konstantinos Lagogiannis, Barbara Webb (2016) Continuous lateral oscillations as a core mechanism for taxis in Drosophila larvae eLife 5:e15504.

(3) Wolf, S., Dubreuil, A.M., Bertoni, T. et al. Sensorimotor computation underlying phototaxis in zebrafish. Nat Commun 8, 651 (2017).

(4) Izquierdo, E.J. and Beer, R.D., 2013. Connecting a connectome to behavior: an ensemble of neuroanatomical models of C. elegans klinotaxis. PLoS computational biology, 9(2), p.e1002890.

Author response:

eLife Assessment 

The authors utilize a valuable computational approach to exploring the mechanisms of memorydependent klinotaxis, with a hypothesis that is both plausible and testable. Although they provide a solid hypothesis of circuit function based on an established model, the model's lack of integration of newer experimental findings, its reliance on predefined synaptic states, and oversimplified sensory dynamics, make the investigation incomplete for both memory and internal-state modulation of taxis.  

We would like to express our gratitude to the editor for the assessment of our work. However, we respectfully disagree with the assessment that our investigation is incomplete, if the negative assessment is primarily due to the impact of AIY interneuron ablation on the chemotaxis index (CI) which was reported in Reference [1]. It is crucial to acknowledge that the CI determined through experimental means incorporates contributions from both klinokinesis and klinotaxis [1]. It is plausible that the impact of AIY ablation was not adequately reflected in the CI value. Consequently, the experimental observation does not necessarily diminish the role of AIY in klinotaxis. Anatomical evidence provided by the database (http://ims.dse.ibaraki.ac.jp/ccep-tool/) substantiates that ASE sensory neurons and AIZ interneurons, which have been demonstrated to play a crucial role in klinotaxis [Matsumoto et al., PNAS 121 (5) e2310735121], have the highest number of synaptic connections with AIY interneurons. These findings provide substantial evidence supporting the validity of the presented minimal neural network responsible for salt klinotaxis.

Public Reviews: 

Reviewer #1 (Public review): 

Summary: 

This research focuses on C. elegans klinotaxis, a chemotactic behavior characterized by gradual turning, aiming to uncover the neural circuit mechanism responsible for the context-dependent reversal of salt concentration preference. The phenomenon observed is that the preferred salt concentration depends on the difference between the pre-assay cultivation conditions and the current environmental salt levels. 

We would like to express our gratitude for the time and consideration you have dedicated to reviewing our manuscript.

The authors propose that a synaptic-reversal plasticity mechanism at the primary sensory neuron, ASER, is critical for this memory- and context-dependent switching of preference. They build on prior findings regarding synaptic reversal between ASER and AIB, as well as the receptor composition of AIY neurons, to hypothesize that similar "plasticity" between ASER and AIY underpins salt preference behavior in klinotaxis. This plasticity differs conceptually from the classical one as it does not rely on any structural changes but rather synaptic transmission is modulated by the basal level of glutamate, and can switch from inhibitory to excitatory. 

To test this hypothesis, the study employs a previously established neuroanatomically grounded model [4] and demonstrates that reversing the ASER-AIY synapse sign in the model agent reproduces the observed reversal in salt preference. The model is parameterized using a computational search technique (evolutionary algorithm) to optimize unknown electrophysiological parameters for chemotaxis performance. Experimental validity is ensured by incorporating constraints derived from published findings, confirming the plausibility of the proposed mechanism. 

Finally. the circuit mechanism allowing C. elegans to switch behaviour to an exploration run when starved is also investigated. This extension highlights how internal states, such as hunger, can dynamically reshape sensory-motor programs to drive context-appropriate behaviors.  

We would like to thank the reviewer for the appropriate summary of our work. 

Strengths and weaknesses: 

The authors' approach of integrating prior knowledge of receptor composition and synaptic reversal with the repurposing of a published neuroanatomical model [4] is a significant strength.

This methodology not only ensures biological plausibility but also leverages a solid, reproducible modeling foundation to explore and test novel hypotheses effectively.

The evidence produced that the original model has been successfully reproduced is convincing.

The writing of the manuscript needs revision as it makes comprehension difficult.  

We would like to thank the reviewer for recognizing the usefulness of our approach. In the revised version, we will improve the explanation.  

One major weakness is that the model does not incorporate key findings that have emerged since the original model's publication in 2013, limiting the support for the proposed mechanism. In particular, ablation studies indicate that AIY is not critical for chemotaxis, and other interneurons may play partially overlapping roles in positive versus negative chemotaxis. These findings challenge the centrality of AIY and suggest the model oversimplifies the circuit involved in klinotaxis.

We would like to express our gratitude for the constructive feedback we have received. We concur with some of your assertions. In fact, our model is the minimal network for salt klinotaxis, which includes solely the interneurons that are connected to each other via the highest number of synaptic connections. It is important to note that our model does not consider redundant interneurons that exhibit overlapping roles. Consequently, the model is not applicable to the study of the impact of interneuron ablation. In the reference [1], the influence of interneuron ablations on the chemotaxis index (CI) has been investigated. The experimentally determined CI value incorporates the contributions from both klinokinesis and klinotaxis. Consequently, it is plausible that the impact of AIY ablation was not significantly reflected in the CI value. The experimental observation does not necessarily diminish the role of AIY in klinotaxis. 

Reference [1] also shows that ASER neurons exhibit complex, memory- and context-dependent responses, which are not accounted for in the model and may have a significant impact on chemotactic model behaviour. 

As pointed out by the reviewer, our model does not incorporate the context-dependent response of the ASER. Instead, the salt concentration-dependent glutamate release from the ASRE [S. Hiroki et al. Nat Commun 13, 2928 (2022)] as the result of the ASER responses is considered in the present study.

The hypothesis of synaptic reversal between ASER and AIY is not explicitly modeled in terms of receptor-specific dynamics or glutamate basal levels. Instead, the ASER-to-AIY connection is predefined as inhibitory or excitatory in separate models. This approach limits the model's ability to test the full range of mechanisms hypothesized to drive behavioral switching.  

We would like to thank the reviewer for the helpful comments. In the revised version, we will mention the limitation.

While the main results - such as response dependence on step inputs at different phases of the oscillator - are consistent with those observed in chemotaxis models with explicit neural dynamics (e.g., Reference [2]), the lack of richer neural dynamics could overlook critical effects. For example, the authors highlight the influence of gap junctions on turning sensitivity but do not sufficiently analyze the underlying mechanisms driving these effects. The role of gap junctions in the model may be oversimplified because, as in the original model [4], the oscillator dynamics are not intrinsically generated by an oscillator circuit but are instead externally imposed via $z_¥text{osc}$. This simplification should be carefully considered when interpreting the contributions of specific connections to network dynamics. Lastly, the complex and contextdependent responses of ASER [1] might interact with circuit dynamics in ways that are not captured by the current simplified implementation. These simplifications could limit the model's ability to account for the interplay between sensory encoding and motor responses in C. elegans chemotaxis. 

We might not understand the substance of your assertions. However, we understand that the oscillator dynamics were not generated by an oscillator neural circuit in our modeling. On the other hand, the present study focuses on how the sensory input and resulting interneuron dynamics regulate the oscillatory activity of SMB motor neurons to generate klinotaxis. 

Appraisal: 

The authors show that their model can reproduce memory-dependent reversal of preference in klinotaxis, demonstrating that the ASER-to-AIY synapse plays a key role in switching chemotactic preferences. By switching the ASER-AIY connection from excitatory to inhibitory they indeed show that salt preference reverses. They also show that the curving/turn rate underlying the preference change is gradual and depends on the weight between ASER-AIY. They further support their claim by showing that curving rates also depend on cultivated (set-point).  

We would like to thank the reviewer for assessing our work.

Thus within the constraints of the hypothesis and the framework, the model operates as expected and aligns with some experimental findings. However, significant omissions of key experimental evidence raise questions on whether the proposed neural mechanisms are sufficient for reversal in salt-preference chemotaxis.  

We agree with your opinion. The present hypothesis should be verified by experiments.

Previous work [1] has shown that individually ablating the AIZ or AIY interneurons has essentially no effect on the Chemotactic Index (CI) toward the set point ([1] Figure 6). Furthermore, in [1] the authors report that different postsynaptic neurons are required for movement above or below the set point. The manuscript should address how this evidence fits with their model by attempting similar ablations. It is possible that the CI is rescued by klinokinesis but this needs to be tested on an extension of this model to provide a more compelling argument.  

We would like to express our gratitude for the constructive feedback we have received. In the reference [1], the influence of interneuron ablations on the chemotaxis index (CI) has been investigated. It is important to acknowledge that the experimentally determined CI value encompasses the contributions of both klinokinesis and klinotaxis. It is plausible that the impact of AIY ablation was not reflected in the CI value. Consequently, these experimental observations do not necessarily diminish the role of AIY in klinotaxis. The neural circuit model employed in the present study constitutes a minimal network for salt klinotaxis, encompassing solely interneurons that are connected to each other via the highest number of synaptic connections. Anatomical evidence provided by the database (http://ims.dse.ibaraki.ac.jp/cceptool/) substantiates that ASE sensory neurons and AIZ interneurons, which have been demonstrated to play a crucial role in klinotaxis [Matsumoto et al., PNAS 121 (5) e2310735121], have the highest number of synaptic connections with AIY interneurons. Our model does not take into account redundant interneurons with overlapping roles, thus rendering it not applicable to the study of the effects of interneuron ablation.

The investigation of dispersal behaviour in starved individuals is rather limited to testing by imposing inhibition of the SMB neurons. Although a circuit is proposed for how hunger states modulate taxis in the absence of food, this circuit hypothesis is not explicitly modelled to test the theory or provide novel insights.  

As pointed out by the reviewer, the neural circuit that inhibits the SMB motor neurons was not explicitly incorporated in our model. We then examined whether our minimal network model could reproduce dispersal behavior under starvation conditions solely due to the experimentally identified inhibitory effect of SMB motor neurons.

Impact : 

This research underscores the value of an embodied approach to understanding chemotaxis, addressing an important memory mechanism that enables adaptive behavior in the sensorimotor circuits supporting C. elegans chemotaxis. The principle of operation - the dependence of motor responses to sensory inputs on the phase of oscillation - appears to be a convergent solution to taxis. Similar mechanisms have been proposed in Drosophila larvae chemotaxis [2], zebrafish phototaxis [3], and other systems. Consequently, the proposed mechanism has broader implications for understanding how adaptive behaviors are embedded within sensorimotor systems and how experience shapes these circuits across species.

We would like to express our gratitude for useful suggestion. We will add the argument that the reviewer mentioned in the revised version.  

Although the reported reversal of synaptic connection from excitatory to inhibitory is an exciting phenomenon of broad interest, it is not entirely new, as the authors acknowledge similar reversals have been reported in ASER-to-AIB signaling for klinokinesis ( Hiroki et al., 2022). The proposed reversal of the ASER-to-AIY synaptic connection from inhibitory to excitatory is a novel contribution in the specific context of klinotaxis. While the ASER's role in gradient sensing and memory encoding has been previously identified, the current paper mechanistically models these processes, introducing a hypothesis for synaptic plasticity as the basis for bidirectional salt preference in klinotaxis.  

The research also highlights how internal states, such as hunger, can dynamically reshape sensory-motor programs to drive context-appropriate behaviors.  

The methodology of parameter search on a neural model of a connectome used here yielded the valuable insight that connectome information alone does not provide enough constraints to reproduce the neural circuits for behaviour. It demonstrates that additional neurophysiological constraints are required.  

We would like to acknowledge the appropriate recognition of our work.

Additional Context 

Oscillators with stimulus-driven perturbations appear to be a convergent solution for taxis and navigation across species. Similar mechanisms have been studied in zebrafish phototaxis [3],

Drosophila larvae chemotaxis [2], and have even been proposed to underlie search runs in ants.

The modulation of taxis by context and memory is a ubiquitous requirement, with parallels across species. For example, Drosophila larvae modulate taxis based on current food availability and predicted rewards associated with odors, though the underlying mechanism remains elusive. The synaptic reversal mechanism highlighted in this study offers a compelling framework for understanding how taxis circuits integrate context-related memory retrieval more broadly.  

We would like to express our gratitude for the insightful commentary. In the revised version, we will incorporate the discussion that the similar oscillator mechanism with stimulus-driven perturbations has been observed for zebrafish phototaxis [3] and Drosophila larvae chemotaxis [2].

As a side note, an interesting difference emerges when comparing C. elegans and Drosophila larvae chemotaxis. In Drosophila larvae, oscillatory mechanisms are hypothesized to underlie all chemotactic reorientations, ranging from large turns to smaller directional biases (weathervaning). By contrast, in C. elegans, weathervaning and pirouettes are treated as distinct strategies, often attributed to separate neural mechanisms. This raises the possibility that their motor execution could share a common oscillator-based framework. Re-examining their overlap might reveal deeper insights into the neural principles underlying these maneuvers. 

We would like to acknowledge your thoughtfully articulated comment. As pointed out by the reviewer, from the anatomical database (http://ims.dse.ibaraki.ac.jp/ccep-tool/), we found that the neural circuits underlying weathervaning and pirouettes in C. elegans are predominantly distinct but exhibit partial overlap. When we restrict our search to the neurons that are connected to each other with the highest number of synaptic connections, we identify the projections from the neural circuit of weathervaning to the circuit of pirouettes; however we observed no reversal projections. This finding suggests that the neural circuit of weathervaning, namely, our minimal neural network, is not likely to be affected by that of pirouettes, which consists of AIB interneurons and interneurons and motor neurons the downstream. 

(1) Luo, L., Wen, Q., Ren, J., Hendricks, M., Gershow, M., Qin, Y., Greenwood, J., Soucy, E.R., Klein, M., Smith-Parker, H.K., & Calvo, A.C. (2014). Dynamic encoding of perception, memory, and movement in a C. elegans chemotaxis circuit. Neuron, 82(5), 1115-1128. 

(2) Antoine Wystrach, Konstantinos Lagogiannis, Barbara Webb (2016) Continuous lateral oscillations as a core mechanism for taxis in Drosophila larvae eLife 5:e15504. 

(3) Wolf, S., Dubreuil, A.M., Bertoni, T. et al. Sensorimotor computation underlying phototaxis in zebrafish. Nat Commun 8, 651 (2017). 

(4) Izquierdo, E.J. and Beer, R.D., 2013. Connecting a connectome to behavior: an ensemble of neuroanatomical models of C. elegans klinotaxis. PLoS computational biology, 9(2), p.e1002890. 

Reviewer #2 (Public review): 

Summary: 

This study explores how a simple sensorimotor circuit in the nematode C. elegans enables it to navigate salt gradients based on past experiences. Using computational simulations and previously described neural connections, the study demonstrates how a single neuron, ASER, can change its signaling behavior in response to different salt conditions, with which the worm is able to "remember" prior environments and adjust its navigation toward "preferred" salinity accordingly.  

We would like to express our gratitude for the time and consideration the reviewer has dedicated to reviewing our manuscript.

Strengths: 

The key novelty and strength of this paper is the explicit demonstration of computational neurobehavioral modeling and evolutionary algorithms to elucidate the synaptic plasticity in a minimal neural circuit that is sufficient to replicate memory-based chemotaxis. In particular, with changes in ASER's glutamate release and sensitivity of downstream neurons, the ASER neuron adjusts its output to be either excitatory or inhibitory depending on ambient salt concentration, enabling the worm to navigate toward or away from salt gradients based on prior exposure to salt concentration.

We would like to thank the reviewer for appreciating our research. 

Weaknesses: 

While the model successfully replicates some behaviors observed in previous experiments, many key assumptions lack direct biological validation. As to the model output readouts, the model considers only endpoint behaviors (chemotaxis index) rather than the full dynamics of navigation, which limits its predictive power. Moreover, some results presented in the paper lack interpretation, and many descriptions in the main text are overly technical and require clearer definitions.  

We would like to thank the reviewer for the constructive feedback. As the reviewer noted, the fundamental assumptions posited in the study have yet to be substantiated by biological validation. Consequently, these assumptions must be directly assessed by biological experimentation. The model performance for salt klinotaxis is evaluated by multiple factors, including not only a chemotaxis index but also the curving rate vs. bearing (Fig. 4a, the bearing is defined in Fig. A3) and the curving rate vs. normal gradient (Fig. 4c). The subsequent two parameters work to characterize the trajectory during salt klinotaxis. In the revised version, we will meticulously revise the manuscript according to the suggestions by the reviewer. We would like to express our sincere gratitude for your insightful review of our work.

Reviewer #3 (Public review):

Summary:

This study examines prediction errors, information gain (Kullback-Leibler [KL] divergence), and uncertainty (entropy) from an information-theory perspective using two experimental tasks and pupillometry. The authors aim to test a theoretical proposal by Zénon (2019) that the pupil response reflects information gain (KL divergence). In particular, the study defines the prediction error in terms of KL divergence and speculates that changes in pupil size associated with KL divergence depend on entropy. Moreover, the authors examine the temporal characteristics of pupil correlates of prediction errors, which differed considerably across previous studies that employed different experimental paradigms. In my opinion, the study does not achieve these aims due to several methodological and theoretical issues.

Strengths:

(1) Use of an established Bayesian model to compute KL divergence and entropy.

(2) Pupillometry data preprocessing, including deconvolution.

Weaknesses:

(1) Definition of the prediction error in terms of KL divergence:

I'm concerned about the authors' theoretical assumption that the prediction error is defined in terms of KL divergence. The authors primarily refer to a review article by Zénon (2019): "Eye pupil signals information gain". It is my understanding that Zénon argues that KL divergence quantifies the update of a belief, not the prediction error: "In short, updates of the brain's internal model, quantified formally as the Kullback-Leibler (KL) divergence between prior and posterior beliefs, would be the common denominator to all these instances of pupillary dilation to cognition." (Zénon, 2019).

From my perspective, the update differs from the prediction error. Prediction error refers to the difference between outcome and expectation, while update refers to the difference between the prior and the posterior. The prediction error can drive the update, but the update is typically smaller, for example, because the prediction error is weighted by the learning rate to compute the update. My interpretation of Zénon (2019) is that they explicitly argue that KL divergence defines the update in terms of the described difference between prior and posterior, not the prediction error.

The authors also cite a few other papers, including Friston (2010), where I also could not find a definition of the prediction error in terms of KL divergence. For example [KL divergence:] "A non-commutative measure of the non-negative difference between two probability distributions." Similarly, Friston (2010) states: Bayesian Surprise - "A measure of salience based on the Kullback-Leibler divergence between the recognition density (which encodes posterior beliefs) and the prior density. It measures the information that can be recognized in the data." Finally, also in O'Reilly (2013), KL divergence is used to define the update of the internal model, not the prediction error.

The authors seem to mix up this common definition of the model update in terms of KL divergence and their definition of prediction error along the same lines. For example, on page 4: "KL divergence is a measure of the difference between two probability distributions. In the context of predictive processing, KL divergence can be used to quantify the mismatch between the probability distributions corresponding to the brain's expectations about incoming sensory input and the actual sensory input received, in other words, the prediction error (Friston, 2010; Spratling, 2017)."

Similarly (page 23): "In the current study, we investigated whether the pupil's response to decision outcome (i.e., feedback) in the context of associative learning reflects a prediction error as defined by KL divergence."

This is problematic because the results might actually have limited implications for the authors' main perspective (i.e., that the pupil encodes prediction errors) and could be better interpreted in terms of model updating. In my opinion, there are two potential ways to deal with this issue:

a) Cite work that unambiguously supports the perspective that it is reasonable to define the prediction error in terms of KL divergence and that this has a link to pupillometry. In this case, it would be necessary to clearly explain the definition of the prediction error in terms of KL divergence and dissociate it from the definition in terms of model updating.

b) If there is no prior work supporting the authors' current perspective on the prediction error, it might be necessary to revise the entire paper substantially and focus on the definition in terms of model updating.

(2) Operationalization of prediction errors based on frequency, accuracy, and their interaction:

The authors also rely on a more model-agnostic definition of the prediction error in terms of stimulus frequency ("unsigned prediction error"), accuracy, and their interaction ("signed prediction error"). While I see the point here, I would argue that this approach offers a simple approximation to the prediction error, but it is possible that factors like difficulty and effort can influence the pupil signal at the same time, which the current approach does not take into account. I recommend computing prediction errors (defined in terms of the difference between outcome and expectation) based on a simple reinforcement-learning model and analyzing the data using a pupillometry regression model in which nuisance regressors are controlled, and results are corrected for multiple comparisons.

(3) The link between model-based (KL divergence) and model-agnostic (frequency- and accuracy-based) prediction errors:

I was expecting a validation analysis showing that KL divergence and model-agnostic prediction errors are correlated (in the behavioral data). This would be useful to validate the theoretical assumptions empirically.

(4) Model-based analyses of pupil data:

I'm concerned about the authors' model-based analyses of the pupil data. The current approach is to simply compute a correlation for each model term separately (i.e., KL divergence, surprise, entropy). While the authors do show low correlations between these terms, single correlational analyses do not allow them to control for additional variables like outcome valence, prediction error (defined in terms of the difference between outcome and expectation), and additional nuisance variables like reaction time, as well as x and y coordinates of gaze.

Moreover, including entropy and KL divergence in the same regression model could, at least within each task, provide some insights into whether the pupil response to KL divergence depends on entropy. This could be achieved by including an interaction term between KL divergence and entropy in the model.

(5) Major differences between experimental tasks:

More generally, I'm not convinced that the authors' conclusion that the pupil response to KL divergence depends on entropy is sufficiently supported by the current design. The two tasks differ on different levels (stimuli, contingencies, when learning takes place), not just in terms of entropy. In my opinion, it would be necessary to rely on a common task with two conditions that differ primarily in terms of entropy while controlling for other potentially confounding factors. I'm afraid that seemingly minor task details can dramatically change pupil responses. The positive/negative difference in the correlation with KL divergence that the authors interpret to be driven by entropy may depend on another potentially confounding factor currently not controlled.

(6) Model validation:

My impression is that the ideal learner model should work well in this case. However, the authors don't directly compare model behavior to participant behavior ("posterior predictive checks") to validate the model. Therefore, it is currently unclear if the model-derived terms like KL divergence and entropy provide reasonable estimates for the participant data.

(7) Discussion:

The authors interpret the directional effect of the pupil response w.r.t. KL divergence in terms of differences in entropy. However, I did not find a normative/computational explanation supporting this interpretation. Why should the pupil (or the central arousal system) respond differently to KL divergence depending on differences in entropy?

The current suggestion (page 24) that might go in this direction is that pupil responses are driven by uncertainty (entropy) rather than learning (quoting O'Reilly et al. (2013)). However, this might be inconsistent with the authors' overarching perspective based on Zénon (2019) stating that pupil responses reflect updating, which seems to imply learning, in my opinion. To go beyond the suggestion that the relationship between KL divergence and pupil size "needs more context" than previously assumed, I would recommend a deeper discussion of the computational underpinnings of the result.

Diferencia con la demencia es que la demencia es un trastorno crónico y progresivo que implica deterioro gradual de funciones cognitivas como la memoria y el pensamiento

Corregir.

Dividr en dos o más oraciones

Voici un sommaire minuté de la présentation, mettant en évidence les idées fortes :

• 0:00-0:01:50: Introduction de Thomas Andrillon et de son équipe de recherche, la "dream team", qui étudie le sommeil et les rêves à l'Institut du cerveau. Le séminaire se concentrera sur ce que le cerveau fait pendant le sommeil : se coupe-t-il du monde extérieur ou continue-t-il à traiter des informations ?.

• 0:01:50-0:03:00: Le paradoxe du sommeil : comment définir la limite entre l'éveil et le sommeil ? Le sommeil est un état de non-réponse réversible.

• 0:03:00-0:04:20: Étude du réveil et de l'intensité sonore nécessaire pour réveiller quelqu'un. Il est plus difficile de se réveiller en début de nuit qu'en fin de nuit. La saillance du stimulus est importante.

• 0:04:20-0:05:00: La physiologie du sommeil : ralentissement cérébral et relaxation musculaire. Le thalamus joue un rôle clé dans la perception sensorielle. L'endormissement a été longtemps considéré comme un état de déconnexion sensorielle à cause du filtrage thalamique.

• 0:05:00-0:06:00: Disséquer les différentes étapes du traitement du son, de l'entrée vers la sortie, et voir comment le sommeil impacte ces étapes. La notion de filtrage thalamique n'est pas complète.

• 0:06:00-0:07:00: Le cerveau continue d'encoder et de recevoir les informations du monde extérieur pendant le sommeil. Le cortex auditif réagit à l'information auditive même pendant le sommeil.

• 0:07:00-0:09:00: Utilisation de l'électroencéphalographie (EEG) pour inférer les traitements cognitifs pendant le sommeil. Présentation de stimulis audio et alignement de l'activité cérébrale. Les stimulis familiers continuent d'être traités même pendant le sommeil.

• 0:09:00-0:11:00: Étude des différents niveaux de sommeil et de leur impact sur le traitement sensoriel. Le cerveau humain a une transition éveil/sommeil non instantanée. Le thalamus est la première région à s'endormir, suivi par différentes régions du cortex.

• 0:11:00-0:12:00: L'endormissement est un processus qui prend du temps, avec des variations dans les capacités de traitement cognitif.

• 0:12:00-0:14:00: Étude de la prise de décision pendant le sommeil. Les individus en sommeil peuvent continuer à sélectionner la bonne réponse à des tâches de décision lexicale et sémantique.

• 0:14:00-0:15:00: Toutes les aires du cerveau ne dorment pas de la même manière au même moment. Des aires cérébrales peuvent se réveiller dans un contexte de cerveau globalement endormi.

• 0:15:00-0:16:00: Le dormeur se protège des perturbations en surexprimant des rythmes de sommeil. Modulation fine du sommeil et concept de sommeil local.

• 0:16:00-0:17:00: En sommeil profond, plus on est sensible à l'information auditive, moins on est capable de la traiter. Le cortex en sommeil profond est bistable, avec des phénomènes d'auto-inhibition.

• 0:17:00-0:18:00: Absence de préparation motrice en sommeil paradoxal. L'hypothèse : on est distrait par le rêve. La complexité cérébrale corrèle avec la capacité à répondre à l'éveil et en sommeil lent léger, mais inversement en sommeil paradoxal.

• 0:18:00-0:19:00: Les mouvements oculaires pendant le sommeil paradoxal sont associés au contenu visuel du rêve. Les cortex sensoriels répondent à des activations endogènes.

• 0:19:00-0:21:00: Différents types de filtres empêchent de traiter les informations pendant le sommeil : thalamique, cortical et attentionnel. Le paradigme de cocktail party montre qu'on peut sélectionner ce qu'on veut traiter pendant le sommeil. Les ondes lentes ont un double rôle : couper du monde extérieur et créer des fenêtres d'éveil.

• 0:21:00-0:23:00: On peut répondre pendant le sommeil. Le somnambulisme est interprété comme un réveil brutal du sommeil profond. Les rêveurs lucides peuvent communiquer depuis leur rêve en utilisant des codes.

• 0:23:00-0:24:00: Fluctuations de la vigilance pendant le sommeil et impact sur la perméabilité aux informations extérieures. Difficulté d'étudier l'incorporation des stimuli dans les rêves.

• 0:24:00-0:25:00: L'apprentissage pendant le sommeil : un vieux rêve. Les mots entendus pendant le sommeil sont traités comme des mots nouveaux au niveau de la mémoire explicite, mais il y a une trace implicite.

• 0:25:00-0:27:00: Étude de l'apprentissage de bruit blanc pendant le sommeil. Les sons entendus pendant le sommeil lent profond donnent des performances encore moindres que des sons nouveaux.

• 0:27:00-0:29:00: Le sommeil pourrait avoir une influence sur la plasticité synaptique. L'acétylcholine joue un rôle clé. Le sommeil favorise à la fois la consolidation des souvenirs et l'oubli.

• 0:29:00-0:30:00: Conditionnement pendant le sommeil : association d'odeurs chez les fumeurs. L'apprentissage pendant le sommeil est limité, de bas niveau, implicite et fragile.

• 0:30:00-0:33:00: Le sommeil n'est pas uniforme. On peut être dans des états hybrides. Le principe de localité du sommeil s'applique aussi à l'éveil. Étude de l'impact des ondes lentes sur l'attention pendant l'éveil.

• 0:33:00-0:34:00: Conclusion : le sommeil et l'éveil sont sur un continuum. Importance d'étudier le sommeil pour comprendre le fonctionnement du cerveau.

• 0:34:00-0:36:30: Questions et discussion sur le statut de la conscience durant le sommeil.

Are you seeing the axis labels and legend? I thought they were there the first time I looked, but now I do not see them. Odd.

How does one interpret a relevance score? is it relevant to the search or standardized somehow?

Voici un résumé des points clés concernant la fréquentation des sites adultes par les mineurs, selon l'étude de l'Arcom de mai 2023:

• 2,3 millions de mineurs visitent des sites adultes chaque mois en 2022, ce qui représente 30% des internautes mineurs.
• Ce nombre a augmenté de 36% en 5 ans.
• Les mineurs passent en moyenne 50 minutes par mois sur ces sites.
• Plus de la moitié des garçons de 12 à 17 ans fréquentent ces sites. Ils y passent environ une heure par mois.
• Pornhub est le site le plus visité par les mineurs, avec 1,4 million de visiteurs mineurs en décembre 2022, soit 18% de leur audience. Ce chiffre a augmenté de 0,9 million en 5 ans.
• Les mineurs représentent une part significative de l'audience de plusieurs sites, dépassant 10%. Par exemple, ils constituent 17% de l'audience de Pornhub.
• L'utilisation de ces sites par les mineurs se fait principalement via mobile. 75% des mineurs utilisent exclusivement leur mobile pour accéder à ces sites.
• Il existe un écart important entre les genres : les hommes sont 2,5 fois plus nombreux à visiter ces sites et y passent trois fois plus de temps que les femmes. Dès 12 ans, plus de la moitié des garçons visitent des sites adultes chaque mois.
• L'audience des mineurs est concentrée sur un petit nombre de sites. Les cinq premiers sites captent 59% du temps passé sur les sites adultes par les mineurs, comparativement à 43% pour les adultes.

Voici un bref compte rendu de la vidéo, qui traite du sommeil, du replay et de l'apprentissage :

• Introduction Le séminaire porte sur le sommeil, le replay et l'éducation, en particulier sur la façon dont le cerveau se modifie par l'apprentissage. Le sommeil pourrait avoir pour fonction de modifier nos circuits cérébraux et d'approfondir les connaissances acquises pendant la journée. Le replay est un phénomène où le cerveau rejoue ce qu'il a appris.

• Gilles Laurent Gilles Laurent étudie le sommeil dans l'évolution, en particulier la place de l'évolution dans le sommeil. Il aborde l'évolution du sommeil chez les animaux et certains travaux sur le sommeil chez les reptiles.

• Le mystère du sommeil Une des principales raisons pour lesquelles le sommeil est intéressant, c'est qu'on ne sait pas très bien pourquoi il existe et que ses fonctions sont encore un mystère. La question abordée est de savoir si les humains sont les seuls à dormir.

• Évolution de la vie et du cerveau La vie a commencé il y a environ 4 milliards d'années, et le cerveau est apparu il y a 700 millions d'années. Il existe de nombreuses façons de construire un cerveau, avec une histoire commune et une histoire spécifique à chaque lignée.

• Convergence et divergence La convergence fonctionnelle est une obligation de l'évolution. Les cerveaux ont commencé avec l'invention de la motricité et des récepteurs sensoriels, nécessitant une interface entre les deux pour la prise de décision. La convergence fonctionnelle fait que l'on observe des phénomènes similaires dans différentes espèces, qui ne sont pas nécessairement le résultat d'un ancêtre commun.

• Sommeil universel? Si le sommeil est universel, cela peut être dû à des origines communes ou à une convergence fonctionnelle.

• Rythmes biologiques Le cerveau est adapté à la physique du monde, et les rythmes biologiques répondent à la rotation de la Terre. Les rythmes circadiens ont une période de 24 heures, et les rythmes saisonniers sont liés à la rotation autour du soleil. Les rythmes circadiens ont été découverts par Jean-Jacques d'Ortous de Mairan. Les mécanismes des rythmes circadiens sont maintenant bien compris grâce aux travaux sur la mouche.

• Histoire de l'étude du sommeil L'étude scientifique du sommeil a commencé il y a environ 100 ans avec les travaux de Nathaniel Kleitman. Le sommeil est soumis à une pression circadienne et une pression homéostatique.

• Définition du sommeil Le sommeil requiert un système nerveux. Il existe trois types de définitions du sommeil : comportementale, électrophysiologique et fonctionnelle.

• Définitions comportementales Les définitions comportementales du sommeil comprennent l'immobilité, le changement de posture, la réversibilité, un seuil d'éveil élevé et la régulation homéostatique.

• Définitions électrophysiologiques Les définitions électrophysiologiques utilisent l'EEG, l'EOG et l'EMG pour classifier les états du cerveau : l'état éveillé, le sommeil lent et le sommeil paradoxal (REM).

• Définitions fonctionnelles Les définitions fonctionnelles du sommeil incluent l'homéostasie métabolique, le développement, l'apprentissage, la mémoire et l'immunité.

• Difficultés de comparaison Il est difficile de comparer l'électrophysiologie du sommeil entre les espèces en raison des différences cérébrales et des méthodes d'enregistrement.

• Exemples de sommeil chez les animaux Il existe un accord général sur la présence de sommeil chez les vertébrés, les insectes et les céphalopodes. Des exemples incluent le sommeil chez le lézard et le poulpe. La mouche est un modèle important pour l'étude du sommeil.

• L'hydre L'hydre, un animal primitif sans système nerveux central, montre des signes de sommeil.

• Problèmes et variabilité Il existe des problèmes liés aux définitions du sommeil et à la généralisation des résultats obtenus chez les espèces de laboratoire. Les phénotypes du sommeil sont extrêmement variables.

• Évolution du sommeil Au début de l'évolution biologique, il y a eu l'invention des rythmes circadiens intracellulaires. Chez les eucaryotes pluricellulaires, il est nécessaire de synchroniser toutes les cellules. Le sommeil commence avec l'invention des systèmes nerveux chez les métazoaires.

• Fonctions primitives Les fonctions primitives du sommeil pourraient être liées à la régulation du métabolisme et de l'immunité. Plus tard, il y a eu une complexification de l'activité du cerveau pendant le sommeil, avec l'apparition du sommeil lent, puis du sommeil paradoxal.

• Pression du sommeil La mouche est utilisée comme modèle pour étudier la pression du sommeil et le rôle potentiel de l'adénosine. Des neurones dans le "fan-shaped body" du cerveau de la mouche sont impliqués dans la régulation du sommeil. Une hypothèse est que les protéines modifiées par l'oxydation et les canaux potassiques jouent un rôle dans l'excitabilité des neurones et le déclenchement du sommeil.

• Questions sur les fonctions du sommeil Quelles sont les fonctions respectives du sommeil lent et du sommeil paradoxal? Le sommeil lent est lié à l'apprentissage, et le sommeil paradoxal pourrait être lié aux aspects émotionnels de la mémoire. Il existe une diversité des périodes de sommeil entre les espèces, liée à leur adaptation aux niches écologiques.

• Sommeil et développement Les jeunes ont besoin de plus de sommeil pour la construction de représentations internes. Le cerveau est peu développé à la naissance et a beaucoup de choses à construire.

Voici un sommaire minuté des idées principales de la vidéo :

• 0:08-4:00 Introduction au séminaire sur le sommeil, le "replay" et l'apprentissage, soulignant l'importance du sommeil dans la modification des circuits cérébraux et l'approfondissement des connaissances acquises. Présentation de Gilles Laurent, neuroscientifique spécialiste du sommeil chez différentes espèces animales.

• 4:00-6:00 Le sommeil est un mystère, on ne sait pas pourquoi il existe. La question centrale est de savoir si les humains sont les seuls à dormir. Aperçu de la présence du sommeil chez divers animaux, des mammifères aux invertébrés. Questionnement sur la définition du sommeil et son évolution.

• 6:00-8:00 L'évolution de la vie et du cerveau : la vie a commencé il y a environ 4 milliards d'années, mais le cerveau est apparu plus tard, il y a 700 millions d'années. Diversification des plans d'organisation des cerveaux pendant le Cambrien. Importance de la convergence fonctionnelle due à la pression sélective de l'évolution.

• 8:00-12:00 Le cerveau est adapté à la physique du monde, y compris aux rythmes de rotation de la Terre. Discussion des rythmes circadiens et saisonniers, et de leur découverte par Jean-Jacques d'Ortous de Mairan. Explication des mécanismes des rythmes circadiens, avec l'exemple de la mouche et des travaux de Ron Konopka et Seymour Benzer.

• 12:00-15:00 Le sommeil est-il un héritage d'une origine commune ou d'une convergence fonctionnelle? Les biorythmes sont le résultat de l'adaptation à la planète. L'étude scientifique du sommeil a commencé il y a environ 100 ans avec Nathaniel Kleitman. Le sommeil est influencé par une pression circadienne et une pression homéostatique. Définition du sommeil basée sur des observations chez l'humain et d'autres mammifères, nécessitant un système nerveux.

• 15:00-18:00 Définitions comportementales du sommeil (immobilité, changement de posture, réversibilité, seuil d'éveil élevé, régulation homéostatique) proposées par Henri Piéron. Définitions électrophysiologiques (EEG, EOG, EMG) et identification des phases du sommeil : sommeil lent et sommeil paradoxal (REM). Définitions fonctionnelles du sommeil : homéostasie métabolique, développement, apprentissage, mémoire et immunité.

• 18:00-22:00 Difficultés de comparer l'électrophysiologie du sommeil entre espèces différentes en raison des différences cérébrales et des méthodes d'enregistrement. Accord général sur la présence du sommeil chez les vertébrés, les insectes et les céphalopodes. Exemples de sommeil chez le lézard (reptile) et le poulpe (invertébré), illustrant différentes manifestations du sommeil. La mouche est un modèle important pour l'étude du sommeil, avec des critères spécifiques et des actogrammes pour mesurer l'activité. L'hydre, un animal primitif sans système nerveux central, montre également des signes de sommeil.

• 22:00-25:00 Problèmes liés aux définitions du sommeil et à la généralisation des résultats obtenus chez des espèces de laboratoire. Variabilité des phénotypes du sommeil (durée, rythme, proportion de sommeil lent et paradoxal). Résumé conceptuel de l'évolution du sommeil : rythmes circadiens intracellulaires, synchronisation cellulaire chez les eucaryotes pluricellulaires, et apparition du sommeil avec les systèmes nerveux chez les métazoaires. Fonctions primitives du sommeil liées à la régulation métabolique et de l'immunité. Complexification du sommeil avec l'évolution des cordés et l'apparition du sommeil paradoxal.

• 25:00-30:00 Exemples illustrant l'évolution : le sommeil paradoxal et la pression du sommeil. La mouche comme modèle pour étudier la pression du sommeil et le rôle potentiel de l'adénosine. Identification de neurones dans le "fan-shaped body" du cerveau de la mouche, impliqués dans la régulation du sommeil. Hypothèse sur le rôle des protéines modifiées par l'oxydation et des canaux potassiques dans l'excitabilité des neurones et le déclenchement du sommeil.

• 30:00-34:00 Questions sur les fonctions respectives du sommeil lent et du sommeil paradoxal, et sur leur lien avec l'apprentissage et les rêves. Discussion sur la diversité des périodes de sommeil entre espèces et leur adaptation aux niches écologiques. Déficits cognitifs observés chez les mouches privées de sommeil.

• 34:00-38:00 Sommeil et développement : les jeunes ont besoin de plus de sommeil pour la construction de représentations internes. Comparaison avec le sommeil chez les larves de nématodes.

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Reply to the reviewers

Response to Reviewers

We thank the reviewers for their comments and suggestions, which we think are helpful and will improve the manuscript, and intend to address with the changes and planned revisions below.

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

Bello et al look at the SNP rs28834970 associated with Alzheimer's disease (AD), with C being the risk allele, on chromatin accessibility and expression of a nearby gene, PTK2B, in microglia. Their contention is that the single SNP affects chromatin accessibility and binding of the transcription factor CEBP[beta] in an intronic region of PTK2B and thereby affects PTKB expression. I had a few questions that I think are critical to be addressed. Please note that my numbering of panels is based on the figures, not the legends, which do not seem to quite agree with each other. There are also some figure legends that say "IFNg" while the figures say "LPS", which should be fixed.

We apologise for the mistake in the figure legend that made this confusing, which we have now revised.

The abstract says that editing a line that is homozygous for protective alleles to homozygous for risk results in "subtle downregulation of PTK2B expression". It isn't clear to me that the presented data fully supports this contention, which is central to the argument of the paper. In figure 2e, the authors show in both RNAseq and ddPCR that there is numerically lower PTK2B expression but this is not indicated to be statistically significant by one-way paired ANOVA. If there is no nominally significant difference in the edited lines, compared to the proposed significant differences in lines carrying the full risk haplotype (figure 1), then it would not seem sensible to ascribe the effects to the single edited base pair.

We agree with the reviewer that given the effect of the SNP on PTK2B expression in the edited lines is small and only significant in macrophages, we should not interpret the effects to be mediated solely through PTK2B expression, and have substantially reworded the manuscript accordingly.

Whilst the effects in the eQTL analysis are significant, it is worth noting that this is likely due to the much larger number of donors (133-217) giving greater power to detect the subtle changes in expression (~1.1 to 2 fold in eQTL). This change is of a similar magnitude in our SNP edited lines (~1.2 fold in SNP edited lines) as would be expected of most common regulatory variants so we believe that it could be the primary causal variant. However, we cannot exclude that other variants in the haplotype could contribute to the effect, so have also reworded accordingly to make this clear.

Given this uncertainty about the overall strength of effect of the single base pair change it would seem important to evaluate the proposed mechanism of CEBPb binding. It wasn't clear whether the ATAC-seq data summarized in the volcano plot in 2C is proposed to be a cause or a consequence of the CEBPb binding change. I am assuming that the 'fold change' estimate here is CC compared to TT, which would be consistent with direction of effect in figure 1, but please clarify.

We apologise for the mistake in the figure legend that made this confusing, which we have now revised along with clarification in the revised text. It is difficult to be sure whether changes in chromatin accessibility are a cause or consequence of CEBPb binding, but the fact that the binding of CEBPb is increased in the CC allele (Fig 2a, Fig 2c), that the C allele better matches the consensus sequence (Fig 2b) and there is increased chromatin accessibility (Fig 2a, Supp Fig 3b) suggests that CEBPb binding is causing the formation of the region of chromatin accessibility.

In contrast to the subtle effects at PTK2B, the global transcriptional effects in figure 3 look quite strong. Are any of these changes dependent on PTK2B, that is to say, are they mimicked by partial suppression of PTK2B expression or activity?

We agree that the downstream effects of the SNP are much stronger than the effects on PTK2B expression, and we have substantially reworded the manuscript to make it clear that we are unsure that the effects of the SNP are all mediated via PTK2B.

However, we note that there is evidence in the literature of a loss in CCL4 and CCL5 expression upon PTK2B knockout in macrophages (https://www.nature.com/articles/s41467-021-27038-5) and inhibition of PTK2B in monocytes results in a reduction in CCL5 and CXCL1 (https://www.nature.com/articles/s41598-019-44098-2) consistent with our observations.

Experiments to manipulate PTK2B expression in microglia and readout changes at the RNA level would take a few months to complete, but we would be willing to do this if the reviewer felt this was necessary.

Finally, in figure 4, it should be clarified as to why lower expression of PTK2B would be expected to have a detrimental effect on Alzheimer's risk. If understood correctly, and again fixing the figure legends would be helpful, the CC edited lines (risk) have lower chemokine induction than the unedited TT lines.

We apologise for the error in this figure which we have corrected in the revised version. You are correct that the CC lines have a lower chemokine level in both unstimulated and stimulated cells, and we have now discussed further how this may be linked to increased disease risk.

"Even though overexpression of these chemokines is characteristic of neuroinflammation, correlated with disease progression and found in late stages of AD, knockout of chemokines, such as CCL2, and chemokine receptors, such as CCR2 and CCR5, in mice is associated with increased Aβ deposition and accumulation [47,50-52,107]. It has also been found that patients carrying CCR5Δ32 mutation, which prevents CCR5 surface expression, develop AD at a younger age[108]. Therefore, we hypothesize that in individuals carrying the C/C allele of rs28834970 downregulation of these chemokines in macrophages and microglia harbouring the C/C allele of rs28834970 affects Aβ-induced microglia chemotaxis, leukocytes recruitment and clearance of Aβ, and may increase the risk of developing symptomatic AD"

Reviewer #1 (Significance (Required)):

Going from GWAS hits, which represent blocks of high LD inherited variants, to single functional variants is a difficult problem in human genetics. The current paper attempts to isolate the effect of a single variant within an LD block on IPSC derived macrophages and microglia. This idea might be useful in nominating PTK2B as a therapeutic target for AD, although there is some question in my mind as to direction of effect.

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

SUMMARY: In this manuscript the authors explore the biological effects of an intronic SNP in the PTK2B gene, previously shown to be associated with late onset Alzheimer's disease (AD) risk. Based on the likely effect of the SNP locus on PTK2B expression in the macrophage lineage, the authors explore the consequences of introducing with the Crispr/Cas9 technique the biallelic SNP base change (C/C vs T/T) in a human IPSC line that is then differentiated into macrophages or microglia. They observe that C/C increases chromatin accessibility and CEBPb binding in comparison to T/T, with a slight decrease in PTK2B expression, significant in macrophages but not in microglia. The authors then investigate the transcriptome changes induced by the C/C mutation and find alteration in many genes, including a decreased expression of a number of cytokine or receptor proteins involved in inflammatory responses. The authors also mention a decreased effect on IFNg-induced reduced mobility but the data are missing (see Figure errors below). Overall the authors propose that the risk SNP is associated with a decreased PTK2B expression and hypothesize a link between this change and a decreased function of macrophages/microglia that may contribute to AD pathology.

MAJOR COMMENTS

1- The authors claim that their results show that the investigated SNP has a causal effects in "microglial function" (Title) and in Alzheimer's disease (AD) (Abstract 2nd sentence "Here we validate a causal single nucleotide polymorphism (SNP) associated with an increased risk of Alzheimer's disease". The word "causal" is repeated many times. However the authors should qualify their claim with respect to AD. Their results do show that the SNP has an effect on chromatin accessibility, CEBP binding, PTK2B expression and transcriptome, but the link between these changes is not formally demonstrated and their potential role in AD-like phenotype is not explored. The "causal" role is not formally and logically demonstrated. It remains an interesting, plausible hypothesis and the results provide strong arguments in support of that hypothesis but do not prove it, yet.

Concerning the title, "causal effects on microglial function" is awkward, anything that has effects is logically "causal" in these effects. The title should be "... has effects on microglial functions" or "... alters microglial function".

We agree with the reviewer that given the effect of the SNP on PTK2B expression in the edited lines is small and only significant in macrophages, we should not interpret the effects to be mediated solely through PTK2B expression, or that they cause AD. We have substantially reworded the manuscript throughout to account for this.

2- One major difficulty in the results is to link the slight decrease in PTK2B transcript, which is only significant in macrophages, with the rest of the phenotype. Because what matters to make this link is not the mRNA but the protein, and because mRNA levels are often not strictly correlated with the protein levels, the authors should measure the PTK2B/PYK2 protein levels in their differentiated cell lines in basal conditions and following activation (as they do for other readouts) using immunoblotting. A robust and significant diminution in PYK2 protein would strongly support its role in linking PTK2B expression and transcriptome change.

We have performed preliminary analyses of PTK2B expression by Western blot in these cell lines after differentiation, but were unable to observe a significant change in abundance in the edited cell lines. This is not unexpected given the results at the RNA level, since the effect size of this common regulatory variant is likely very small (estimated to be ~1.2 fold from the eQTL analysis), and likely within the variability of this assay.

As mentioned above, we have reworded the manuscript to avoid interpreting that the effects of rs28834970 are mediated solely through effects on PTK2B expression. We think that an experiment to manipulate PTK2B levels (see next point) may be a better way to demonstrate whether these effects are mediated through PTK2B expression.

An optional additional key experiment would be to reverse the transcriptome phenotype by increasing the expression of PTK2B (e.g. by cDNA transfection). Note that these points are important because an alternative hypothesis to explain the effects of C/C mutation on macrophage function would be that the C/C mutation has a long distance effect on other chromatin regions with key role in regulating these cells.

We agree that this would be a valuable experiment, and are planning additional experiments to investigate the effect of manipulating PTK2B levels (through knockout) on microglia.

3- The manuscript contains several errors in the figures and figure legends. In Fig. 2 the legends for the figure items are shuffled. Figure 4 and Supplementary Figure 5 are duplicates of the same one. Consequently important data are not presented.

We apologise for the errors in these figures that were due to a mistake during uploading where the incorrect versions were used. The legends for figure 2 and panels in figure 4 have now been corrected, and show the effects of rs28834970 on microglial migration and chemokine release in the presence or absence of IFNg.

4- When the number of replicates is small (e.g. n = 3) it is preferable to use non parametric tests (rank analysis, e.g. Mann Whitney's test) rather than t test. This applies to Figures 2D (current legend 2A), 2E (current legend 2B), Figure 4A-C, Supplementary Figures 2A, 2B. In Supplementary Fig 4E (MARCO) the number of replicates (presumably 3 because based on RNAseq) and the used test are not indicated. Is it the RNAseq statistical analysis?

We thank the reviewer for this comment. We acknowledge that the t-test may lead to inflated false discovery rates. However, it has been shown that for small sample sizes parametric tests have a power advantage compared to non-parametric ones that may outweigh the possibly exaggerated false positives. See https://genomebiology.biomedcentral.com/articles/10.1186/s13059-022-02648-4#Sec3 which states:

"In conclusion, when the per-condition sample size is less than 8, parametric methods may be used because their power advantage may outweigh their possibly exaggerated false positives."

We have also modified the legend of supplementary figure 4E to clarify the number of replicates used.

5- In addition to the above comment on tests, when the number of replicates is small it is not appropriate (and misleading) to show box plots or bars with SEM. In the indicated figures the individual data points should be shown.

We now show individual replicates on box plots (Figure 2D, 2E and supp figure 4E).

MINOR COMMENTS:

a- Macrophages and microglia are very similar cell types. Could the authors comment more on the differences they observe and how they are related to those previously described?

We have now referenced the original papers and commented on the markers that we see differentially expressed, notably P2RY12 which is a key homeostatic microglia marker that distinguishes these cells from macrophages.

b- In Fig. 2A CEBPb cut and run plot, the differences are not limited to the SNP immediate vicinity, there are also visible differences between T/T and C/C plots in at least a 40-kb range. Is it due to multiple interactions of CEBPb? How can the point difference have broad consequences? Please explain this potentially interesting and relevant finding.

Whilst there may be small changes in CEBPb binding at the second intronic PTK2B chromatin peak, this is not statistically significant given the variability between repeats. In fact, the only significant change we see in CEBPb binding genome-wide is at the locus overlapping the SNP (Fig 2c).

c- Potentially cis-altered genes near the SNP include CHRNA2 and EPHX2 (see Sup. Fig. 3a). Their expression may not be detected in macrophage lineage. If this is the case please indicate in the text, otherwise please include the corresponding data in Sup. Fig. 3b to show the presence or absence of SNP-induced change.

You are correct that CHRNA2 and EPHX2 are not expressed in our macrophages or microglia, and we have now explicitly stated this in the revised text.

d- In general the Figures are not of very high quality and are difficult to read or understand without constantly going back and forth to the legends (which are mislabeled in some instances). To improve:

. Please increase font size whenever possible.

. Please improve Fig. 1d by indicating the position of the SNP, numbering the exons (an intermediate scale plot may be necessary and lines on bottom trace are hardly visible).

. Please indicate the correct color code for T/T and C/C in Fig 3a and b, left panels, which currently doesn't match.

. Please label the Venn's diagrams comparisons in Sup. Fig. 4b.

. In the text and legends the Figure items are identified with letters in upper case, in the figures they are in lower case. Please be consistent.

We have improved the resolution of the images in the pdf and Fig 1d has been revised to include the position of the SNP. The colour code for T/T and C/C is correct in fig 3a and 3b, but since the PCA plots are independently created, we would not always expect the position of the T/T and C/C alleles to be the same. The Venn diagrams in Sup Fig 4b have been updated, and the letters for the figure panels made consistently upper case throughout.

e- In Fig. 2D and 2E, the Y axes should start at zero to avoid artificially increasing the visual differences. If there is a strong reason not to do so (I don't see any here), the Y axis should be clearly interrupted to avoid confusion.

We have altered this accordingly.

f- In the introduction the authors provide some background about previous work about the potential role of PTK2B/PYK2 in AD pathophysiology. The cited preclinical results suggest that PTK2B activity could have a deleterious effect (references in the manuscript). In contrast, some other reports (PMID: 29803828, 33718872) suggest a protective effect of PTK2B/PYK2. Because the evidence in the current manuscript suggests that the risk-associated SNP results in a decreased function of PTK2B/PYK2 (through decreased levels), at least in cells of the macrophage lineage, the authors could broaden their discussion to include these results.

We have now discussed the conflicting evidence in the revised manuscript.

Reviewer #2 (Significance (Required)):

ADVANCE: Late onset Alzheimer's disease is a major medical issue. It has a complex genetic risk component with many associated loci identified in GWAS. Most of these have only a small individual impact on the risk. One of the SNPs associated with increased risk (rs28834970) is located in an intron of the PTK2B gene. Although various reports have investigated the role of the PTK2B gene product, the tyrosine kinase PYK2, in several AD models, the possible link with rs28834970, is unclear.

An important point is to determine whether TàC SNP corresponding to rs28834970 alters PTK2B expression and how it does so. An alternative hypothesis could be that the SNP has a strong linkage disequilibrium with an unidentified allele in human populations that could be responsible for AD risk. The current manuscript is a significant step forward in addressing that question. By generating a biallelic C/C SNP mutation in a human IPSC line the current study allows to eliminate such linked contribution.

The strength of the manuscript is to show an effect on chromatin accessibility, CEBP binding and possibly PTK2B transcripts. It also provides interesting evidence of a broad effect of the C/C mutation on the transcriptome of macrophage lineage cells. In its current form the manuscript presents weaknesses that could be improved. These flaws include issues with the presentation discussed above and the uncomplete demonstration that it is the decrease in PTK2B expression that causes the macrophage/microglia phenotype. If these flaws were overcome the paper would represent a significant advance.

AUDIENCE: The expected audience is specialized in AD with a possible broader range if all weaknesses are addressed.

REVIEWER EXPERTISE: Basic science close to the field.

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

Learn more at Review Commons

Referee #2

Evidence, reproducibility and clarity

Summary: In this manuscript the authors explore the biological effects of an intronic SNP in the PTK2B gene, previously shown to be associated with late onset Alzheimer's disease (AD) risk. Based on the likely effect of the SNP locus on PTK2B expression in the macrophage lineage, the authors explore the consequences of introducing with the Crispr/CAS9 technique the biallelic SNP base change (C/C vs T/T) in a human IPSC line that is then differentiated into macrophages or microglia. They observe that C/C increases chromatin accessibility and CEBPb binding in comparison to T/T, with a slight decrease in PTK2B expression, significant in macrophages but not in microglia. The authors then investigate the transcriptome changes induced by the C/C mutation and find alteration in many genes, including a decreased expression of a number of cytokine or receptor proteins involved in inflammatory responses. The authors also mention a decreased effect on IFNg-induced reduced mobility but the data are missing (see Figure errors below). Overall the authors propose that the risk SNP is associated with a decreased PTK2B expression and hypothesize a link between this change and a decreased function of macrophages/microglia that may contribute to AD pathology.

Major comments:

1. The authors claim that their results show that the investigated SNP has a causal effects in "microglial function" (Title) and in Alzheimer's disease (AD) (Abstract 2nd sentence "Here we validate a causal single nucleotide polymorphism (SNP) associated with an increased risk of Alzheimer's disease". The word "causal" is repeated many times. However the authors should qualify their claim with respect to AD. Their results do show that the SNP has an effect on chromatin accessibility, CEBP binding, PTK2B expression and transcriptome, but the link between these changes is not formally demonstrated and their potential role in AD-like phenotype is not explored. The "causal" role is not formally and logically demonstrated. It remains an interesting, plausible hypothesis and the results provide strong arguments in support of that hypothesis but do not prove it, yet. Concerning the title, "causal effects on microglial function" is awkward, anything that has effects is logically "causal" in these effects. The title should be "... has effects on microglial functions" or "... alters microglial function".
2. One major difficulty in the results is to link the slight decrease in PTK2B transcript, which is only significant in macrophages, with the rest of the phenotype. Because what matters to make this link is not the mRNA but the protein, and because mRNA levels are often not strictly correlated with the protein levels, the authors should measure the PTK2B/PYK2 protein levels in their differentiated cell lines in basal conditions and following activation (as they do for other readouts) using immunoblotting. A robust and significant diminution in PYK2 protein would strongly support its role in linking PTK2B expression and transcriptome change. An optional additional key experiment would be to reverse the transcriptome phenotype by increasing the expression of PTK2B (e.g. by cDNA transfection). Note that these points are important because an alternative hypothesis to explain the effects of C/C mutation on macrophage function would be that the C/C mutation has a long distance effect on other chromatin regions with key role in regulating these cells.
3. The manuscript contains several errors in the figures and figure legends. In Fig. 2 the legends for the figure items are shuffled. Figure 4 and Supplementary Figure 5 are duplicates of the same one. Consequently important data are not presented.
4. When the number of replicates is small (e.g. n = 3) it is preferable to use non parametric tests (rank analysis, e.g. Mann Whitney's test) rather than t test. This applies to Figures 2D (current legend 2A), 2E (current legend 2B), Figure 4A-C, Supplementary Figures 2A, 2B. In Supplementary Fig 4E (MARCO) the number of replicates (presumably 3 because based on RNAseq) and the used test are not indicated. Is it the RNAseq statistical analysis?
5. In addition to the above comment on tests, when the number of replicates is small it is not appropriate (and misleading) to show box plots or bars with SEM. In the indicated figures the individual data points should be shown.

Minor comments:

• a. Macrophages and microglia are very similar cell types. Could the authors comment more on the differences they observe and how they are related to those previously described?
• b. In Fig. 2A CEBPb cut and run plot, the differences are not limited to the SNP immediate vicinity, there are also visible differences between T/T and C/C plots in at least a 40-kb range. Is it due to multiple interactions of CEBPb? How can the point difference have broad consequences? Please explain this potentially interesting and relevant finding.
• c. Potentially cis-altered genes near the SNP include CHRNA2 and EPHX2 (see Sup. Fig. 3a). Their expression may not be detected in macrophage lineage. If this is the case please indicate in the text, otherwise please include the corresponding data in Sup. Fig. 3b to show the presence or absence of SNP-induced change.
• d. In general the Figures are not of very high quality and are difficult to read or understand without constantly going back and forth to the legends (which are mislabeled in some instances). To improve:
  • Please increase font size whenever possible.
  • Please improve Fig. 1d by indicating the position of the SNP, numbering the exons (an intermediate scale plot may be necessary and lines on bottom trace are hardly visible).
  • Please indicate the correct color code for T/T and C/C in Fig 3a and b, left panels, which currently doesn't match.
  • Please label the Venn's diagrams comparisons in Sup. Fig. 4b.
  • In the text and legends the Figure items are identified with letters in upper case, in the figures they are in lower case. Please be consistent.
• e. In Fig. 2D and 2E, the Y axes should start at zero to avoid artificially increasing the visual differences. If there is a strong reason not to do so (I don't see any here), the Y axis should be clearly interrupted to avoid confusion.
• f. In the introduction the authors provide some background about previous work about the potential role of PTK2B/PYK2 in AD pathophysiology. The cited preclinical results suggest that PTK2B activity could have a deleterious effect (references in the manuscript). In contrast, some other reports (PMID: 29803828, 33718872) suggest a protective effect of PTK2B/PYK2. Because the evidence in the current manuscript suggests that the risk-associated SNP results in a decreased function of PTK2B/PYK2 (through decreased levels), at least in cells of the macrophage lineage, the authors could broaden their discussion to include these results.

Significance

Advance: Late onset Alzheimer's disease is a major medical issue. It has a complex genetic risk component with many associated loci identified in GWAS. Most of these have only a small individual impact on the risk. One of the SNPs associated with increased risk (rs28834970) is located in an intron of the PTK2B gene. Although various reports have investigated the role of the PTK2B gene product, the tyrosine kinase PYK2, in several AD models, the possible link with rs28834970, is unclear.

An important point is to determine whether TC SNP corresponding to rs28834970 alters PTK2B expression and how it does so. An alternative hypothesis could be that the SNP has a strong linkage disequilibrium with an unidentified allele in human populations that could be responsible for AD risk. The current manuscript is a significant step forward in addressing that question. By generating a biallelic C/C SNP mutation in a human IPSC line the current study allows to eliminate such linked contribution.

The strength of the manuscript is to show an effect on chromatin accessibility, CEBP binding and possibly PTK2B transcripts. It also provides interesting evidence of a broad effect of the C/C mutation on the transcriptome of macrophage lineage cells. In its current form the manuscript presents weaknesses that could be improved. These flaws include issues with the presentation discussed above and the uncomplete demonstration that it is the decrease in PTK2B expression that causes the macrophage/microglia phenotype. If these flaws were overcome the paper would represent a significant advance.

Audience: The expected audience is specialized in AD with a possible broader range if all weaknesses are addressed.

Reviewer Expertise: Basic science close to the field.

Voici un résumé des idées principales du documentaire, en mettant en évidence les points clés en gras :

• Les aliments ultratransformés (AUT) sont conçus par les géants de l'alimentation pour maximiser l'attrait et la consommation, souvent au détriment de la santé. Ces produits subissent des transformations industrielles poussées et contiennent des ingrédients rarement utilisés dans la cuisine traditionnelle, avec des ajouts importants de sel, de sucre et de matières grasses.
• Carlos Montero a créé la classification NOVA, qui divise les aliments en quatre groupes selon leur degré de transformation, mettant en évidence les AUT comme étant distincts des aliments frais ou peu transformés.
• Les aliments ultratransformés sont liés à divers problèmes de santé, tels que l'obésité, le diabète de type 2, les maladies cardiovasculaires, le cancer, et les troubles mentaux. Des études montrent que les régimes riches en AUT entraînent une consommation excessive de calories et une prise de poids.
• L'industrie agroalimentaire utilise des techniques de marketing sophistiquées, y compris la recherche du "point de félicité" pour optimiser le goût et rendre les produits irrésistibles. La neuro-imagerie est utilisée pour comprendre et manipuler les circuits de récompense du cerveau.
• La texture des aliments ultratransformés est conçue pour encourager une consommation rapide et excessive, en exploitant le concept de « densité calorique évanescente ».
• L'industrie agroalimentaire s'inspire des stratégies de marketing de l'industrie du tabac pour fidéliser les consommateurs dès le plus jeune âge, en créant des extensions de gamme et en ciblant différents segments de marché.
• Les aliments ultratransformés contiennent de nombreux additifs, tels que des émulsifiants, des édulcorants et des colorants, dont les effets à long terme sur la santé sont préoccupants. Des études suggèrent que certains émulsifiants peuvent perturber le microbiote intestinal et favoriser l'inflammation.
• Les entreprises agroalimentaires déploient des stratégies pour minimiser les risques perçus de leurs produits, en finançant des études, en manipulant l'étiquetage et en semant le doute sur les preuves scientifiques.
• Face à ces enjeux, certains pays mettent en place des réglementations, comme l'étiquetage d'avertissement en Colombie, pour informer les consommateurs et encourager une alimentation plus saine. Des initiatives visent également à inciter les entreprises à reformuler leurs produits et à promouvoir des choix alimentaires plus équilibrés.
• Une alimentation saine devrait être basée sur des aliments frais et peu transformés, préparés à la maison, plutôt que sur des produits ultratransformés.

Author response:

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

We thank Reviewers for highlighting the strengths of our work along with suggestions for future directions.

We agree with the Reviewers that RPS26 depletion may impact not only RAN translation initiation and codon selection (as showed in the experiments in Figure 4G), but also other mechanisms, such as speed of PIC scanning, as we stated in the discussion. Although, we did provide the data showing that mRNA of exogenous FMR1-GFP does not change upon RPS26 depletion (Figure 3B&C), hence observed effect most likely stems from translation regulation. In addition, an experiment with ASO-ACG treatment (Figure 4G) suggests that near cognate start codon selection or speed of PIC scanning may be a part of the regulation of RAN translation sensitive to RPS26 depletion. In addition, our latest unpublished results (Niewiadomska D. et al., in revision), indicate that FMRpolyG in fusion with GFP is fairly stable, in particular, while derived from long repeats (>90xCGG), suggesting that the protein stability is not at play in RPS26-dependent regulation.

We would like to stress that in order to avoid bias in result interpretation and to mimic the natural situation, the majority of experiments concerning levels of FMRpolyG were performed in cell models with stable expression of ACG-initiated FMRpolyG. Currently, we do not possess a cell model with stable expression of AUG-initiated FMRpolyG, and the experiments based on transient transfection system would not necessarily be comparable to the results obtained in stable expression system. However, we believe that the experiment presented in Figure 2B serves as a good control for overall translation level upon RPS26 depletion indicating that RPS26 insufficiency does not affect global translation and the observed regulation is specific to some mRNAs including the one encoding FMRpolyG frame. We also show that the level of ca. 80% of identified canonical proteins, including FMRP, did not change upon RPS26 silencing (SILAC-MS, Figure 4A). Indeed, we did not explore the ribosome composition upon RPS26 and TSR2 depletion, although, most likely the pool of functional ribosomes in the cell is sufficient enough to support the basal translation level (SUnSET assays, Figure 2B & 5C). However, we cannot exclude possibility that for some mRNAs, including one encoding for FMRpolyG, the observed effect can be partially caused by lowering the number of fully active ribosomes, especially in experiments with transient transfection experiments where transgene expression is hundreds times higher than for average native mRNA.

Finally, we agree with the Reviewer that in vitro translation assay would provide the evidence of direct effect of RPS26 on FMRpolyG level, however, we did not manage to overcome technical difficulties in obtaining cellular lysate devoid of RPS26 from vendor companies.

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

General Comments

We thank Reviewers for the critical comments and experimental suggestions. We considered most of the advices in the revised version of the manuscript, which allowed for a more balanced interpretation of the results presented, and further supported major statement of the manuscript that insufficiency of the RPS26 and RPS25 plays a role in modulating the efficiency of noncanonical RAN translation from FMR1 mRNA, which results in the production of toxic polyglycine protein (FMRpolyG). Firstly, performing new experiments, we showed that silencing of the RPS26 and its chaperone protein TSR2, which regulates loading/exchange of RPS26 in maturing small ribosome subunit, did not elicit global translation inhibition. Secondly, we demonstrated that in contrary to RPS26 and RPS25 depletion, silencing the RPS6 protein, a core component of 40S subunit, did not affect FMRpolyG production, further supporting the specific effect of RPS26 and RPS25 on RAN translation regulation of mutant FMR1 mRNA. We also observed that depletion of RPS26, RPS25 and RPS6 had significant negative effect on cells proliferation which is in line with previously published results indicating that insufficiencies of ribosomal proteins negatively affect cell growth. Moreover, we showed that FMRpolyG production is significantly affected by RPS26 depletion while initiated at ACG, but not other near cognate start codons. Importantly, translation of FMRP initiated at canonical AUG codon of the same mRNA upstream the CGGexp was not affected by RPS26 silencing, similarly to vast majority of the human proteome. This implies that RAN translation of FMR1 mRNA mediated by RPS26 insufficiency is likely to be dependent on start codon selection/fidelity. In essence, we provide a series of evidences indicating that cellular amount of 40S ribosomal proteins RPS26 and RPS25 is important factor of CGGrelated RAN translation regulation. Finally, we also decided to tone down our claims. Now, we state that the RPS26/25/TSR2 insufficiency or depletion, affects RAN translation, rather than composition of 40S ribosomal subunit per se influences RAN translation. We have addressed all specific concerns below and made changes to the new version of manuscript.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

In this manuscript, Tutak et al use a combination of pulldowns, analyzed by mass spectrometry, reporter assays, and fluorescence experiments to decipher the mechanism of protein translation in fragile X-related diseases. The topic is interesting and important.

Although a role for Rps26-deficient ribosomes in toxic protein translation is plausible based on already available data, the authors' data are not carefully controlled and thus do not support the conclusions of the paper.

We sincerely appreciate your rigorous, insightful, and constructive feedback throughout the revision process. We believe your guidance has been instrumental in significantly enhancing the quality of our research. Below, we have addressed your comments pointby-point.

Strengths:

The topic is interesting and important.

Weaknesses:

In particular, there is very little data to support the notion that Rps26-deficient ribosomes are even produced under the circumstances. And no data that indicate that they are involved in the RAN translation. Essential controls (for ribosome numbers) are lacking, no information is presented on the viability of the cells (Rps26 is an essential protein), and the differences in protein levels could well arise from block in protein synthesis, and cell division coupled to differential stability of the proteins.

We agree that data presented in the first version of the manuscript did not directly address the following processes: ribosome content, global translation rate and cell viability upon RPS26 depletion. Therefore we addressed some of the issues in the revised version of the manuscript. In particular, we showed that RPS26 and TSR2 knock down did not inhibit global translation (new Figure 2B & 4C), hence we concluded that the changes of FMRpolyG level did not arise from general translational shut down. On the other hand, RPS26, RPS25 and RPS6 depletion negatively affected cells proliferation (new Figure 2A,5D,6C), which is in line with a number of previously published researches (e.g. Cheng et al, 2019; Havkin-Solomon et al, 2023). However, the rate of proliferation abnormalities is limited. We agree that observed effects on RAN translation from mutant FMR1 mRNA may stem from the combination of altered protein synthesis, conditions of the cells but also cis-acting factors of mRNA sequence/structure. In new experiments we showed that single nucleotide substitution of ACG by other near cognate start codons change sensitivity of RAN translation to insufficiency of RPS26 (new Figure 4F). Also the inhibitory effect of antisense oligonucleotide binding to the region of 5’UTR containing ACG initiation codon (ASO_ACG) is different in cells differing in amount of RPS26 (new Figure 4G).

We also agree that our data only partially supports the role of RPS26-defficient ribosomes in RAN translation. Therefore, we have toned down our claims. Now, we state that the RPS26/25/TSR2 insufficiency or depletion affects RAN translation. We also changed the title of the manuscript to: “Insufficiency of 40S ribosomal proteins, RPS26 and RPS25, negatively affects biosynthesis of polyglycine-containing proteins in fragile-X associated conditions” (Previously it was: “Ribosomal composition affects the noncanonical translation and toxicity of polyglycine-containing proteins in fragile X-associated conditions”.

Specific points:

(1) Analysis of the mass spec data in Supplemental Table S3 indicates that for many of the proteins that are differentially enriched in one sample, a single peptide is identified. So the difference is between 1 peptide and 0. I don't understand how one can do a statistical analysis on that, or how it would give out anything of significance. I certainly do not think it is significant. This is exacerbated by the fact that the contaminants in the assay (keratins) are many, many-fold more abundant, and so are proteins that are known to be mitochondrial or nuclear, and therefore likely not actual targets (e.g. MCCC1, PC, NPM1; this includes many proteins "of significance" in Table S1, including Rrp1B, NAF1, Top1, TCEPB, DHX16, etc...).

The data in Table S6/Figure 3A suffer from the same problem.

I am not convinced that the mass spec data is reliable.

We thank Reviewer for the comment concerning MS data; however, we believe that it may stem from misunderstanding of the data presented in Table S3 and S6. Both tables represent the output from MaxQuant analysis (so-called ProteinGroup) of MS .raw files, without any filtering. As stated in the Material&Methods, we applied default parameters suggested by MaxQuant developers to analyze MS data, these include identification of proteins based on at least 1 unique peptide, and thus some of the proteins with only 1 unique peptide are shown in Tables S1 and S3. Reviewer is also right that in this output table common contaminants, such as keratins are included. However, these identifications are denoted as “CON_”, and are further filtered out during statistical analysis in Perseus software. During the statistical analysis we first filtered out irrelevant protein groups identifications, such as contaminants, or only identified by site modifications.

We have changed the names of Supplementary Table files, giving more detailed description. We hope this will help to avoid misunderstanding for broader public. Secondly, when comparing the data presented in Table S3 and volcano plot presented in Figure 1B, one can notice that indeed the majority of identified proteins are not statistically significant (grey points), thus not selected for further stratification. Lack of significance of these proteins may be partially due to poor MS identification, however, they are not included in the following parts of the manuscript. Further, we selected only eight proteins (out of over 150) for stratification by orthogonal techniques, thus we argue that this step validates the biological relevance of chosen candidate RAN-translation modifiers. One should also keep in mind that pull down samples analyzed by MS often yield lower intensity and identification rates, when comparing to whole cell analysis, as a result of lower protein input or stringent washes used during sample preparation.

Regarding the data presented in Table S6 (SILAC data), we argue that these data are of very good quality. More than 2,000 proteins were identified in a 125min gradient, with over 80% of proteins that were identified with at least 2 unique peptides. Each of three biological replicates was analyzed three times (technical replicates), giving total of 9 high resolution MS runs. Together, we strongly believe that this data is of high confidence.

(2) The mass-spec data however claims to identify Rps26 as a factor binding the toxic RNA specifically. The rest of the paper seeks to develop a story of how Rps26-deficient ribosomes play a role in the translation of this RNA. I do not consider that this makes sense.

Indeed, we identified RPS26 as a protein that co-precipitated with FMR1 containing expanded CGG repeats (Supplementary Figure 1G) and found that depletion of RPS26 hindered RAN translation of FMRpolyG, suggesting that RPS26 positively affects RAN translation. However, we did not state that RPS26 directly interacts with toxic RNA. In order to confirm the specificity of RAN translation regulation by RPS26 insufficiency, we tested whether depletion of other 40S ribosomal protein, RPS6, affects FMRpolyG synthesis. Our experiments showed that there was no any significant effect on RAN translation efficiency post RPS6 silencing (new Figure 5C). Importantly, we showed that RPS26 depletion did not inhibit global translation (new Figure 2B). In addition, mutagenesis of near-cognate start codon (new Figure 4F) and ASO_ACG treatment (new Figure 4G) provided the evidences that modulation of FMRpolyG biosynthesis by RPS26 level may depend on start codon selection. In essence, our data suggest that RPS26 depletion specifically affects synthesis of FMRpolyG, but not FMRP derived from the same FMR1 mRNA with CGGexp. However, we do not claim that the observed effect is the consequence of a direct interaction between RPS26 and 5’UTR of FMR1 mRNA. Downregulation of FMRpolyG biosynthesis could be an outcome of the alteration of ribosomal assembly, decrease of efficiency and fidelity of PIC scanning/initiation or impeded elongation or a combination of all these processes. In the manuscript we presented the results of experiments which tested many of these possibilities.

(3) Rps26 is an essential gene, I am sure the same is true for DHX15. What happens to cell viability? Protein synthesis? The yeast experiments were carefully carried out under experiments where Rps26 was reduced, not fully depleted to give small growth defects.

We agree with the Reviewer that RPS26 and DHX15 are essential proteins, similarly to all RNA binding proteins, and caution should be taken during experimental design. To address this, we titrated different concentrations of siRPS26, and found that administration of 5 nM siRPS26, which just partially silenced RPS26, decreased FMRpolyG by around 50% (new Figure 1D). This impact was even greater with 15 nM siRPS26, as we observed around 80% decrease of FMRpolyG.

Havkin-Solomon et al. (2023), showed that proliferation rate is decreased in cells with mutated C-terminus of RPS26, which is required for contacting mRNA. In accordance with this study, we showed that cells with knocked down RPS26 proliferate less efficiently (new Figure 2A), but depletion of RPS26 did not impact the global translation (new Figure 2B). In addition, our SILAC-MS data indicates that ~80% of proteins with determined expression level were not affected by RPS26 insufficiency, and ~20% of the proteins turned out to be sensitive to RPS26 decrease. Although, these data do not take into account the protein stability.

(4) Knockdown efficiency for all tested genes must be shown to evaluate knockdown efficiency.

The current version of the manuscript contains representative western blots with validation of knock-down efficiency (for example in Figure 3B, C, E, Figure 6A) and we included knock-down validations where applicable (Figures 1D, 2B, 4G and 5C).

(5) The data in Figure 1E have just one mock control, but two cell types (control si and Rps26 depletion).

Mock control corresponds to the cells treated with lipofectamine reagent and was included in the study to determine the “background” signal from cells treated with delivery agent and reagents used to measure the apoptosis process. These cells were neither expressing FMRpolyG, nor siRNAs. Luminescence signals were normalized to the values obtained from mock control. We added more details describing this assay in the Figure 1 legend.

(6) The authors' data indicate that the effects are not specific to Rps26 but indeed also observed upon Rps25 knockdown. This suggests strongly that the effects are from reduced ribosome content or blocked protein synthesis. Additional controls should deplete a core RP to ascertain this conclusion.

We agree that observed effects may stem from reduced ribosome content, however, we argue that this is the only possibility and explanation. Previously, it was shown that RPS25 regulates G4C2-related RAN translation, but knock out of RPS25 does not affect global translation (Yamada S, 2019, Nat. Neuroscience). Similarly, we showed that KD of RPS26 or TSR2 did not reduce significantly global translation rate (SUnSET assay; new Figure 2B and 5C, respectively).

Moreover, in a new version of manuscript we included a control experiment, where we silenced core ribosomal protein (RPS6) and found that RPS6 depletion did not affect RAN translation from mutant FMR1 mRNA (new Figure 5C), thus strengthening our conclusion about specific RAN translation regulation by the level of RPS26 and RPS25.

Finally, our observation aligns well with current knowledge about how deficiency of different ribosomal proteins alters translation of some classes of mRNAs (Luan Y, 2022, Nucleic Acids Res; Cheng Z, 2019, Mol Cell). It was shown that depletion of RPS26 affects translation rate of different mRNAs compared to depletion of other proteins of small ribosomal subunit.

(7) Supplemental Figure S3 demonstrates that the depletion of S26 does not affect the selection of the start codon context. Any other claim must be deleted. All the 5'-UTR logos are essentially identical, indicating that "picking" happens by abundance (background).

Supplementary Figure 3D represents results indicating that the mutation in -4 position (from G to A) did not affect the RAN translation regardless of RPS26 presence or depletion. However, this result does not imply that RPS26 does not affect the selection of start codon of sequence- or RNA structure-context. We verified this particular -4 position, as it was suggested previously as important RPS26-sensitive site in yeasts (Ferretti M, 2017, Nat Struct Mol Biol). We agree with Reviewer that all 5’UTR logos presented in our paper did not show statistical significance for neither tested position for human mRNAs. On the contrary, we observed that regulation sensitive to RPS26 level depends on the selection of start codon of RAN translation, in particular ACG initiation (new Figure 4F&G). RPS26 depletion affected ACG-initiated but not GTG- or CTG-initiated RAN translation.

In the previous version of the manuscript, we wrote that we did not identify any specific motifs or enrichment within analyzed transcripts in comparison to the background. On the other hand, we found that the GC-content among analyzed transcripts is higher within 5’UTRs and in close proximity to ATG in coding sequences (Figure 4D), what suggests the importance of RNA stable structures in this region. In addition, we showed that mRNAs encoding proteins responding to RPS26 depletion have shorter than average 5’UTRs (new Figure 4E).

(8) Mechanism is lacking entirely. There are many ways in which ribosomes could have mRNA-specific effects. The authors tried to find an effect from the Kozak sequence, unsuccessfully (however, they also did not do the experiment correctly, as they failed to recognize that the Kozak sequence differs between yeast, where it is A-rich, and mammalian cells, where it is GGCGCC). Collisions could be another mechanism.

Indeed, collisions as well as other mechanisms such as skewed start codon fidelity may have an effect on efficiency of FMRpolyG biosynthesis. In the current version of the manuscript, we show that RPS26 amount-sensitive regulation seems to be start codonselection dependent (new Figure 4F&G).

Reviewer #2 (Public Review):

Summary:

Translation of CGG repeats leads to the accumulation of poly G, which is associated with neurological disorders. This is a valuable paper in which the authors sought out proteins that modulate RAN translation. They determined which proteins in Hela cells bound to CGG repeats and affected levels of polyG encoded in the 5'UTR of the FMR1 mRNA. They then showed that siRNA depletion of ribosomal protein RPS26 results in less production of FMR1polyG than in control. There are data supporting the claim that RPS26 depletion modulates RAN translation in this RNA, although for some results, the Western results are not strong. The data to support increased aggregation by polyG expression upon S26 KD are incomplete.

We thank the Reviewer for critical comments and suggestions. We sincerely appreciate your rigorous, insightful, and constructive feedback throughout the revision process.

Below each specific point, we addressed the mentioned issues.

Strengths:

The authors have proteomics data that show the enrichment of a set of proteins on FMR1 RNA but not a related RNA.

We thank Reviewer for appreciation of provided MS-screening results, which identified proteins enriched on FMR1 RNA with expanded CGG repeats.

Weaknesses:

- It is insinuated that RPS26 binds the RNA to enhance CGG-containing protein expression. However, RPS26 reduction was also shown previously to affect ribosome levels, and reduced ribosome levels can result in ribosomes translating very different RNA pools.

In previous version of the manuscript we did not state that RPS26 binds directly to RNA with expanded CGG repeats and we did not show the experiment indicating direct interaction between studied RNA and RPS26. What we showed is that RPS26 was enriched on FMR1 RNA MS samples, however, we did not verify whether it is direct or indirect interaction. We also tried to test hypothesis that lack of RPS26 in PIC complex may affect efficiency of RAN translation initiation via specific, previously described in yeast Kozak context (Ferretti M, 2017, Nat Struct Mol Biol). As we described this hypothesis was negatively validated. However, we showed that other features of 5’UTR sequences (e.g. higher GC-content or shorter leader sequence) are potentially important for translation efficiency in cells with depleted RPS26.

Indeed, RPS26 is involved in 40S maturation steps (Plassart L, 2021, eLife) and its insufficiency or mutations or blocking its inclusion to 40S ribosome may result in incomplete 40S maturation, which subsequently might negatively affect translation per se. However, we did not observe global translation inhibition after RPS26 depletion or depletion of TSR2, the chaperon involved in incorporation/exchange RPS26 to small ribosomal subunit (new Figure 2B and 5C). In addition, our SILAC-MS data indicates that majority of studied proteins (including FMRP, the main product of FMR1 gene) were not affected by RPS26 depletion which can be carefully extrapolated to global translation. In revised manuscript we also showed that relatively low silencing of RPS26 also decreased FMRpolyG production in model cells (new Figure 1D).

We agree that reduced ribosome levels can result in different efficiency of translation of different RNA pools. We enhance this statement in revised manuscript. However, we also showed that the same mRNA containing different near cognate start codons (single/two nucleotide substitution) specific to RAN translation, or targeting this codon with antisense oligonucleotides resulted in altered sensitivity of FMR1 mRNA translation to RPS26 depletion (new Figure 4F).

- A significant claim is that RPS26 KD alleviates the effects of FMRpolyG expression, but those data aren't presented well.

We thank the Reviewer for this comment. In the new version of the manuscript, we have added new microscopic images and improved the explanation of Figure 1E. We have also completed the interpretation of Figure 1F in the main text, figure image as well as figure legend, and we hope that these changes will ameliorate understanding of our data.

Recommendations For The Authors:

- A significant claim is that RPS26 KD alleviates the effects of FMR polyG expression, but those data aren't presented well:

Figure 1D (supporting data in S2) and 2D - the authors need to show representative images of a control that has aggregation and indicate aggregates being counted on an image. The legend states that there are no aggregates, but the quantification of aggregates/nucleus is ~1, suggesting there are at least 1 per cell. It is preferred to show at least a representative of what is quantified in the main figure instead of a bar graph.

The representative images of control and siRPS26-treated cells are now shown in revised version of Figure 1E. Additionally, we completed the Figure legend concerning this part, as well as extended description of the experiment in Materials&Methods section.

Figure 1E - it is unclear what luminescence signal is being measured. Is this a dye for an apoptotic marker? More information is needed in the legend.

This information was added to the legend of modified Figure 1F (previously 1E) as suggested.

- Some of the Western blots are not very convincing. Better evidence for the changes in bar graphs would improve how convincing the data are:

Fig 2B. The western for FMR95G in the first model is not very convincing. The difference by eye for the second siRNA seems to give a larger effect than the first for 95G construct but they appear almost the same on the graph. More supporting information for the quantification is needed.

We provided better explanation for WB quantification in M&M section in the manuscript. Alos, we provided additional blot demonstrating independent biological replicate of the mentioned experiment in supplementary materials (Supplementary Figure S2E).

Figure 4A, the blots for RPS26 and FMR95G are not convincing. They are quite smeary compared to all of the others shown for these proteins in other figures. Could a different replicate be shown?

We provided additional blot demonstrating the effect on transiently expressed FMRpolyG affected by depletion of TSR2 in COS7 cell line (Supplementary Figure S4A).

Figure 5A and 5B blots are not ideal. Could a different replicate be shown? Or show multiple replicates in the supplemental figure?

We provided additional blots from the same experiment, although data is not statistically significant, most likely due to low quality of normalization factor, which is Vinculin (Supplementary Figure S5A). Nevertheless, the level of FMRpolyG is decreased by ~70% after RPS25 silencing in SH-SY5Y cells.

Figure 2C. Please use the same y axes for all four Westerns in B and C. One would like to compare 95 and 15 repeats, but it is difficult when the y axes are different.

Thank you for this comment. The y axis was adjusted as suggested by the Reviewer.

Figure 3D-The text suggests a significant difference between positive and negative responders that is not clear in the figure.

In the main body of the manuscript we state that: “We did not observe any significant differences in the frequency of individual nucleotide positions in the 20-nucleotide vicinity of the start codon relative to the expected distribution in the BG”, which is in line with the graph showed in Figure 4D (previously 3D).

Reviewer #3 (Public Review):

Tutak et al provide interesting data showing that RPS26 and relevant proteins such as TSR2 and RPS25 affect RAN translation from CGG repeat RNA in fragile X-associated conditions. They identified RPS26 as a potential regulator of RAN translation by RNAtagging system and mass spectrometry-based screening for proteins binding to CGG repeat RNA and confirmed its regulatory effects on RAN translation by siRNA-based knockdown experiments in multiple cellular disease models and patient-derived fibroblasts. Quantitative mass spectrometry analysis found that the expressions of some ribosomal proteins are sensitive to RPS26 depletion while approximately 80% of proteins including FMRP were not influenced. Since the roles of ribosomal proteins in RAN translation regulation have not been fully examined, this study provides novel insights into this research field. However, some data presented in this manuscript are limited and preliminary, and their conclusions are not fully supported.

(1) While the authors emphasized the importance of ribosomal composition for RAN translation regulation in the title and the article body, the association between RAN translation and ribosomal composition is apparently not evaluated in this work. They found that specific ribosomal proteins (RPS26 and RPS25) can have regulatory effects on RAN translation (Figures 1C, 2B, 2C, 2E, 4A, 5A, and 5B), and that the expression levels of some ribosomal proteins can be changed by RPS26 knockdown (Figure 3B, however, the change of the ribosome compositions involved in the actual translation has not been elucidated). Therefore, their conclusive statement, that is, "ribosome composition affects RAN translation" is not fully supported by the presented data and is misleading.

We thank the Reviewer for critical comments and suggestions. We agree that the initial title and some statements in the text were misleading and the presented data did not fully support the aforementioned statement regarding ribosomal composition affecting FMRpolyG synthesis. Therefore, in the revised version of the manuscript we included a control experiment indicating that depletion of another core 40S ribosomal protein (RPS6) did not impact the FMRpolyG synthesis (new Figure 5C), which supports our hypothesis that RPS26 and RPS25 are specific CGG-related RAN translation modifiers. To precisely deliver a main message of our work, we changed the title that will indicate the specific effect of RPS26 and RPS25 insufficiency on RAN translation of FMRpolyG. Proposed title: “Insufficiency of 40S ribosomal proteins, RPS26 and RPS25 negatively affects biosynthesis of polyglycine-containing proteins in fragile-X associated conditions”. We also changed all statements regarding “ribosomal composition” in main text of the new version of manuscript.

(2) The study provides insufficient data on the mechanisms of how RPS26 regulates RAN translation. Although authors speculate that RPS26 may affect initiation codon fidelity and regulate RAN translation in a CGG repeat sequence-independent manner (Page 9 and Page 11), what they really have shown is just identification of this protein by the screening for proteins binding to CGG repeat RNA (Figure 1A, 1B), and effects of this protein on CGG repeat-RAN translation. It is essential to clarify whether the regulatory effect of RPS26 on RAN translation is dependent on CGG repeat sequence or near-cognate initiation codons like ACG and GUG in the 5' upstream sequence of the repeat. It would be better to validate the effects of RPS26 on translation from control constructs, such as one composed of the 5' upstream sequence of FMR1 with no CGG repeat, and one with an ATG substitution in the 5' upstream sequence of FMR1 instead of near-cognate initiation codons.

We agree that the data presented in the manuscript implies that insufficiency of RPS26 plays a pivotal role in the regulation of CGG-related RAN translation and in the revised version of the manuscript we included a series of experiments indicating that ACG codon selection seems to be an important part of RPS26 level-dependent regulation of polyglycine production (new Figure 4F&G; see point 3 below for more details). Importantly, in the luciferase assay showed on Figure 4F we used the AUG-initiated firefly luciferase reporter as normalization control.

Moreover, to verify if FMRpolyG response to RPS26 deficiency depends on the type of reporter used, we repeated many experiments using FMRpolyG fused with different tags. The luciferase-based assays were in line with experiments conducted on constructs with GFP tag (new Figure 1D), thus strengthening our previous data. Moreover, in the series of experiments, we show that FMRP synthesis which is initiated from ATG codon located in FMR1 exon 1, was not affected by RPS26 depletion (Figure 3E & 4C), even though its translation occurs on the same mRNA as FMRpolyG. This indicates a specific RPS26 regulation of polyglycine frame initiated from ACG near cognate codon.

(3) The regulatory effects of RPS26 and other molecules on RAN translation have all been investigated as effects on the expression levels of FMRpolyG-GFP proteins in cellular models expressing CGG repeat sequences Figures 1C, 2B, 2C, 2E, 4A, 5A, and 5B). In these cellular experiments, there are multiple confounding factors affecting the expression levels of FMRpolyG-GFP proteins other than RAN translation, including template RNA expression, template RNA distribution, and FMRpolyG-GFP protein degradation. Although authors evaluated the effect on the expression levels of template CGG repeat RNA, it would be better to confirm the effect of these regulators on RAN translation by other experiments such as in vitro translation assay that can directly evaluate RAN translation.

We agree that there are multiple factors affecting final levels of FMRpolyG-GFP proteins including aforementioned processes. We evaluated the level of FMR1 mRNA, which turned out not to be decreased upon RPS26 depletion (Figure 3B&C), therefore, we assumed that what we observed, was the regulation on translation level, especially that RPS26 is a ribosomal protein contacting mRNA in E-site. We believe that direct assays such as in vitro translation may be beneficial, however, depletion of RPS26 from cellular lysate provided by the vendor seems technically challenging, if not completely impossible. Instead, we focused on sequence/structure specific regulation of RAN translation with the emphasis on start-codon initiation selection. It resulted in generating the valuable results pointing out the RPS26 role in start codon fidelity (Figure 4F&G). These new results showed that translation from mRNAs differing just in single or two nucleotide substitution in near cognate start codon (ACG to GUG or ACG to CUG), although results in exactly the same protein, is differently sensitive to RPS26 silencing (new Figure 4F). Similar differences were observed for translation efficiency from the same mRNA targeted or not with antisense oligonucleotide complementary to the region of RAN translation initiation codon (new Figure 4G). These results also suggest that stability of FMRpolyG is not affected in cells with decreased level of RPS26.

(4) While the authors state that RPS26 modulated the FMRpolyG-mediated toxicity, they presented limited data on apoptotic markers, not cellular viability (Figure 1E), not fully supporting this conclusion. Since previous work showed that FMRpolyG protein reduces cellular viability (Hoem G, 2019,Front Genet), additional evaluations for cellular viability would strengthen this conclusion.

We thank the Reviewer for this suggestion. We addressed the apoptotic process in order to determine the effect of RPS26 depletion on RAN translation related toxicity (Figure 1F). In revised version of the manuscript, we also added the evaluation on how cells proliferation was affected by RPS26, RPS25, RPS6 and TSR2 depletion. Our data indicate that TSR2 silencing slightly impacted the cellular fitness (new Figure 5D), whereas insufficiencies of RPS26, RPS25 and RPS6 had a much stronger negative effect on proliferation (new Figure 2A, 5D, 6C), which is in line with previous data (Cheng Z 2019, Mol Cell; Luan Y, 2022, Nucleic Acids Res). The difference in proliferation rate after treatment with siRPS26 makes proper interpretation of cellular viability assessment very difficult.

Recommendations For The Authors:

(1) It would be nice to validate the effects of overexpression of RPS26 and other regulators on RAN translation, not limited to knockdown experiments, to support the conclusion.

We did not performed such experiments because we believed that RPS26 overexpression may have no or marginal effect on translation or RAN translation. It is likely impossible to efficiently incorporate overexpressed RPS26 into 40S subunits, because the concentration of all ribosomal proteins in the cells is very high.

(2) It would be better to explain how authors selected 8 proteins for siRNA-based validation (Figure 1C, 1D, S1D) from 32 proteins enriched in CGG repeat RNA in the first screening.

We selected those candidates based on their functions connected to translation, structured RNA unwinding or mRNA processing. For example, we tested few RNA helicases because of their known function in RAN translation regulation described by other researchers. This explanation was added to the revised version of the manuscript.

(3) Original image data showing nuclear FMRpolyG-GFP aggregates should be presented in Figure 1D.

The representative images of control and siRPS26-treated cells are now shown in modified version of Figure 1E and described with more details in the legend.

(4) Image data in Figure 2A and 2D have poor signal/noise ratio and the resolution should be improved. In addition, aggregates should be clearly indicated in Figure 2D in an appropriate manner.

The stable S-FMR95xG cellular model is characterized by very low expression of RANtranslated FMR95xG, therefore, it is challenging to obtain microscopic images of better quality with higher GFP signal. In the L-99xCGG model expression of transgene is higher. Therefore, we provided new image in the new version of Figure 3D (former 2D). Moreover, we showed aggregates on the image obtained using confocal microscopy (new Supplementary Figure 2D).

(5) The detailed information on patient-derived fibroblast (age and sex of the patient, the number of CGG repeats, etc.) in Figure 2F needed to be presented.

This information was added to the figure legend (Figure 3F; previously 2F) and in the Material and Methods section as suggested.

(6) It would be better to normalize RNA expression levels of FMR1 and FMR1-GFP by the housekeeping gene in Figure S2C, like other RT-qPCR experimental data such as Figure 2B.

Normalization of FMR1-GFP to GAPDH is now shown in modified version of Figure S2C (right graph) as requested by the Reviewer.

(7) It would be better to add information on molecular weight on all Western blotting data.

(8) Marks corresponding to molecular weight ladder were added to all images.

Full blots, including protein ladders were deposited in Zenodo repository, under doi: 10.5281/zenodo.13860370

References

Cheng Z, Mugler CF, Keskin A, Hodapp S, Chan LYL, Weis K, Mertins P, Regev A, Jovanovic M & Brar GA (2019) Small and Large Ribosomal Subunit Deficiencies Lead to Distinct Gene Expression Signatures that Reflect Cellular Growth Rate. Mol Cell 73: 36-47.e10

Havkin-Solomon T, Fraticelli D, Bahat A, Hayat D, Reuven N, Shaul Y & Dikstein R (2023) Translation regulation of specific mRNAs by RPS26 C-terminal RNA-binding tail integrates energy metabolism and AMPK-mTOR signaling. Nucleic Acids Res 51: 4415–4428

Hoem,G., Larsen,K.B., Øvervatn,A., Brech,A., Lamark,T., Sjøttem,E. and Johansen,T. (2019) The FMRpolyGlycine protein mediates aggregate formation and toxicity independent of the CGG mRNA hairpin in a cellular model for FXTAS. Front. Genet., 10, 1–18.

Luan Y, Tang N, Yang J, Liu S, Cheng C, Wang Y, Chen C, Guo YN, Wang H, Zhao W, et al (2022) Deficiency of ribosomal proteins reshapes the transcriptional and translational landscape in human cells. Nucleic Acids Res 50: 6601–6617

Plassart L, Shayan R, Montellese C, Rinaldi D, Larburu N, Pichereaux C, Froment C, Lebaron S, O’donohue MF, Kutay U, et al (2021) The final step of 40s ribosomal subunit maturation is controlled by a dual key lock. Elife 10

DOI: 10.1038/s41467-025-56626-y

Resource: RRID:Addgene_48007

Curator: @scibot

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DOI: 10.1038/s41467-025-56626-y

Resource: None

Curator: @scibot

SciCrunch record: RRID:Addgene_50310

What is this?

DOI: 10.1038/s41467-025-56626-y

Resource: RRID:Addgene_48015

Curator: @scibot

SciCrunch record: RRID:Addgene_48015

What is this?

DOI: 10.1038/s41467-025-56626-y

Resource: RRID:Addgene_48008

Curator: @scibot

SciCrunch record: RRID:Addgene_48008

What is this?

Voici un résumé de l'interview avec le Dr. Jimmy Mohamed, structuré avec un sommaire minuté et les idées fortes en gras :

• Introduction

  • Présentation du Dr. Jimmy Mohamed, médecin spécialisé en nutrition, et de son dernier ouvrage "Je mange bien, je vais bien".
  • L'objectif de l'interview est de comprendre comment l'alimentation influence notre santé et de déconstruire certaines idées reçues.

• Importance de la nutrition

  • La nutrition est centrale pour la santé, avec environ 90 000 repas au cours d'une vie.
  • "Nous sommes ce que nous mangeons" : bien manger maximise les chances d'être en bonne santé.
  • La société actuelle normalise de mauvaises habitudes alimentaires (petit-déjeuner sucré, repas rapides le midi, dîner devant la télé).

• Le petit-déjeuner

  • Le petit-déjeuner n'est pas forcément le repas le plus important pour tout le monde. Il est essentiel pour les enfants et les adolescents.
  • Inutilité de se forcer à manger le matin si on n'a pas faim.
  • L'importance d'écouter ses sensations alimentaires, surtout pendant les vacances.
  • Le corps a des ressources incroyables et on sous-estime la puissance du jeûne.
  • Un lobby industriel américain a influencé l'idée que le petit-déjeuner est indispensable.
  • Les premiers jours sans petit-déjeuner peuvent être difficiles à cause de l'horloge biologique.

• Composition du petit-déjeuner

  • Le sucre est l'ennemi : il crée de l'inflammation, perturbe le microbiote et provoque des fringales.
  • Miser sur les protéines, comme les œufs : un œuf contient une vingtaine de protéines, du bon gras, des vitamines, des minéraux et de la choline (bonne pour la mémoire et le foie).
  • Éviter les céréales et les produits ultra-transformés.
  • Décryptage d'une boîte de céréales : le sucre est le deuxième ingrédient, suivi du sirop de glucose.
  • Les industriels cherchent à faire du profit, sans se soucier des conséquences sur la santé.
  • Privilégier les fruits entiers plutôt que les jus.

• Nutri-score

  • Le Nutri-score est un bon indicateur, mais il n'est pas obligatoire et les industriels peuvent tricher.
  • Il faut être acteur et ne plus acheter les produits mauvais pour la santé afin de faire pression sur les industriels.
  • Applications comme Yuka pour scanner les produits et obtenir des informations nutritionnelles.

• Alternatives au lait

  • Les laits végétaux (soja, avoine, amande) ne sont pas de vrais laits : ils contiennent peu de protéines et de calcium.
  • Le lait d'amande est surtout pour le plaisir, privilégier la consommation d'amandes entières.

• Addiction au sucre

  • Nous sommes programmés pour être accros au sucre, mais il faut éviter les sirops de glucose-fructose présents dans les produits industriels.
  • Ces sirops perturbent le microbiote, le pancréas et les messages de satiété, rendant les produits addictifs.
  • Le sucre des fruits est moins dangereux car il est emprisonné dans les fibres.

• Produits transformés et habitudes alimentaires

  • Les produits ultra-transformés sont dénaturés et nous rendent malades.
  • Un jeune sur trois ne cuisine pas de produits frais tous les jours.
  • L'alimentation est trop souvent associée à la récompense.
  • Il faut une éducation nutritionnelle pour apprendre aux enfants à reconnaître les bons aliments.
  • Les repas sont souvent pris rapidement et mal, avec des conséquences sur la santé.
  • Un jeune Français sur cinq ne sait pas reconnaître une courgette, signe d'une déconnexion avec l'alimentation.

• Protéines et alternatives végétariennes

  • La sardine en boîte est un excellent produit, riche en protéines et en oméga-3.
  • Les lentilles sont une bonne source de protéines végétales, riches en fibres.
  • Les protéines animales sont mieux assimilées, mais il faut en manger moins et privilégier une alimentation plus végétale.
  • Associer différentes légumineuses pour obtenir tous les acides aminés essentiels.

• Hydratation

  • L'eau doit être la boisson principale.
  • Les sodas contiennent du sucre, des additifs et des édulcorants nocifs.
  • Les boissons "zéro sucre" contiennent des édulcorants qui augmentent l'appétit et perturbent le pancréas.
  • Il vaut mieux prendre un vrai soda sucré de temps en temps que de s'habituer aux boissons "zéro".
  • Les boissons fraîches anesthésient les papilles et masquent le goût du sucre.

• Impact de l'alimentation sur la santé mentale

  • L'alimentation influence nos décisions et notre humeur.
  • 40% des personnes dépressives ont de l'inflammation dans le corps.
  • Améliorer son alimentation peut améliorer les symptômes de la dépression.
  • Le café est une bonne boisson, riche en antioxydants, mais il faut faire attention à la quantité et à la sensibilité individuelle.
  • Du thé matcha, un thé vert, est un bon choix pour un effet stimulant plus stable que le café.

• Le goûter

  • Un goûter équilibré se compose d'un produit laitier, d'un produit céréalier et d'un fruit.
  • Éviter les produits ultra-transformés.
  • Les amandes sont une bonne option pour un goûter sain.

• Manger par terre et avec les mains

  • Manger par terre favorise une meilleure digestion et une diminution des apports caloriques.
  • Manger avec les mains permet de mieux respecter les signaux de satiété.

• Quantité et satiété

  • Il faut manger à 80% de sa satiété et ne pas terminer son assiette systématiquement.
  • Réduire ses apports caloriques de 10 à 15% en écoutant son corps.
  • La pomme de terre est un aliment sous-côté, moins calorique que le riz et les pâtes.
  • L'obésité est en augmentation, y compris dans les pays développés, à cause de la qualité et de la quantité des aliments.
  • Bien manger coûte cher, alors que mal manger est souvent moins cher.

• Polluants éternels (PFAS)

  • Les PFAS sont des perturbateurs endocriniens qui perturbent le système hormonal et augmentent le risque de nombreuses maladies.
  • L'ennemi principal est le plastique.
  • Il faut bannir le plastique de sa vie : éviter de réchauffer les plats dans des boîtes en plastique, utiliser des biberons en verre, jeter les poêles antiadhésives abîmées, utiliser des spatules en bois, boire l'eau du robinet plutôt que de l'eau en bouteille.

• Conclusion

  • Il est essentiel de sensibiliser et de s'informer sur les enjeux de l'alimentation.
  • En étant conscient de l'impact de l'alimentation sur la santé, on devient acteur de sa propre santé.
  • Il faut avancer étape par étape et manger des pommes de terre.

Voici un bref compte rendu de la vidéo "Chez les jeunes, une fracture entre les sexes ? | 28 minutes | ARTE", basé sur le sommaire fourni :

• Polarisation Croissante :

Le rapport du Haut Conseil à l'égalité entre les femmes et les hommes révèle une polarisation croissante entre les sexes chez les jeunes en France.

Les jeunes femmes estiment qu'il est difficile d'être une femme, tandis qu'une partie des jeunes hommes pense que la vie est dure pour eux aussi.

• Montée du Masculinisme :

Les jeunes hommes sont exposés à des contenus masculinistes sur les réseaux sociaux, qui prônent la haine des femmes et des minorités sexuelles.

Ces contenus sont souvent accessibles en cherchant des conseils sur la séduction ou la popularité.

• Radicalisation du Féminisme :

Certains discours politiques et courants de pensée antagonisent les groupes sociaux. Le féminisme est une cause universelle, mais certains groupes féministes ont parfois exclu les hommes.

• Rôle des Hommes dans le Féminisme :

Il est important de reconnaître le rôle des hommes dans le combat féministe, tout en soulignant que ce sont principalement les femmes qui défendent ces idées.