DOI: 10.1007/s11064-024-04257-y

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DOI: 10.1007/s11064-024-04257-y

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Referee #2

Evidence, reproducibility and clarity

This study by Takenaka et al. explores the role of the abnormal oocyte (ao) gene in Drosophila melanogaster. Historically, ao was thought to regulate histone gene expression, causing maternal-effect lethality when disrupted. However, the lack of critical reagents including some alleles limited further testing of this hypothesis. In this study, the authors created new CRISPR/Cas9-generated ao knockouts and an epitope-tagged allele to clarify its role. Findings confirmed ao's maternal-effect lethality, which can be rescued by reducing histone copy number or increasing Y-chromosome heterochromatin. Contrary to previous studies, they found ao does not repress histone transcript levels, leaving its lethality mechanism unresolved. This work challenges the previously assumed role of ao in histone regulation at the level of transcription.

Major comments: Overall, this manuscript was a joy to read, and the experiments are well controlled and done and presented in a clear and precise manner.

Minor comments: My only two suggestions are if there is a way to include that ao is a conserved gene in the introduction that may broaden the readership of this manuscript. Also, there are many supplementary data panels showing Ao expression that could be condensed.

Referees cross-commenting

All the comments sound good to me.

Significance

The significance of the study lies in its challenge to the previously accepted role of ao gene in regulating histone expression. While earlier research suggested ao acted as a repressor of histone expression, this study, using new and well-controlled tools, did not find evidence supporting that role. The findings not only question the previous assumptions but also emphasize the complexity of ao's function, highlighting the need for further exploration of the mechanisms underlying maternal-effect lethality and its genetic interactions with heterochromatin and histone genes. This will contribute to a better understanding of gene regulation during early development.

a.m.a.s

Author response:

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

Reviewer #1 (Public Review):

The manuscript by Poltavski and colleagues describes the discovery of previously unreported enteric neural crestderived cells (ENCDC) which are marked by Pax2 and originating from the Placodes. By creating multiple conditional mouse mutants, the authors demonstrate these cells are a distinct population from the previously reported ENCDCs which originate from the Vagal neural crest cells and express Wnt1.

These Pax2-positive ENCDCs are affected due to the loss of both Ret and Ednrb highlighting that these cells are also ultimately part of the canonical processes governing ENCDC and enteric nervous system (ENS) development. The authors also make explant cultures from the mouse GI tract to detect how Ednrb signaling is important for Ret signaling pathways in these cells and rediscovers the interactions between these 2 pathways. One important observation the authors make is that CGRP-positive neurons in the adult distal colon seem to be primarily derived from these Pax2-positive ENCDCs, which are significantly reduced in the Ednrb mutants, thus highlighting the role of Ednrb in maintaining this neuronal type.

I appreciate the amount of work the authors have put into generating the mouse models to detect these cells, but there isn't any new insight on either the nature of ENCDC development or the role of Ret and Ednrb. Also, there are sophisticated single-cell genomics methods to detect rare cell type/states these days and the authors should either employ some of those themselves in these mouse models or look at extensively publicly available single-cell datasets of the developing wildtype and mutant mouse and human ENS to map out the global transcriptional profile of these cells. A more detailed analysis of these Pax2-positive cells would be really helpful to both the ENS community as well as researchers studying gut motility disorders.

We would like to point out that the reviewer’s comments in both Public Review and in some cases reiterated in Recommendations for the Authors are rooted in several misunderstandings. The reviewer writes “Pax2-positive ENCDCs”, as if the Pax2 lineage (properly, the Pax2Cre-labeled lineage) of the ENS is a subset of neural crest, and states that “there isn’t any new insight” from our study on ENS development. Our conclusion is quite different, that the Pax2Cre lineage (placode-derived) is distinct from the neural crest-derived cell lineage. The reviewer may not have appreciated that our study establishes a fundamental reinterpretation of the very long-standing dogma that the ENS is derived solely from neural crest. We believe that finding and characterizing the unique contribution of an independent cell lineage to the ENS provides critical new perspectives into ENS development and the etiology of Hirschsprung disease. One feature of the Pax2Cre (placodal) lineage is as the source of CGRP-positive mechanosensory neurons in the colon (as the reviewer mentioned), but this is one feature of the larger conceptual discovery of the existence of a separate lineage contribution to the ENS, not the most important observation in and of itself.

The reviewer continues by saying that we “rediscovered” the interaction between Ednrb and Ret in ENS development. In our study we show that the two lineages (placode-derived and neural crest-derived) employ Ednrb and Ret signaling in distinct ways. This isn’t simply rediscovery, this is new insight. To the extent that both lineages utilize both signaling axes (albeit with mechanistic differences) is a primary reason why the unique placodal lineage contribution to the ENS remained unsuspected until now. We have revised the text to make these points more clear in our revised manuscript.

The reviewer also suggests single cell genomic methods, which is addressed below in our response to the reviewer’s first recommendation.

Reviewer #2 (Public Review):

This manuscript by Poltavski and colleagues explores the relative contributions of Pax2- and Wnt1- lineagederived cells in the enteric nervous system (ENS) and how they are each affected by disruptions in Ret and Endrb signaling. The current understanding of ENS development in mice is that vagal neural crest progenitors derived from a Wnt1+ lineage migrate into and colonize the developing gut. The sacral neural crest was thought to make a small contribution to the hindgut in addition but recent work has questioned that contribution and shown that the ENS is entirely populated by the vagal crest (PMID: 38452824). GDNF-Ret and Endothelin3-Ednrb signaling are both known to be essential for normal ENS development and loss of function mutations are associated with a congenital disorder called Hirschsprung's disease. The transcription factor Pax2 has been studied in CNS and cranial placode development but has not been previously implicated in ENS development. In this work, the authors begin with the unexpected observation that conditional knockout of Ednrb in Pax2-expressing cells causes a similar aganglionosis, growth retardation, and obstructed defecation as conditional knockout of Ednrb in Wnt1-expressing cells. The investigators then use the Pax2 and Wnt1 Cre transgenic lines to lineage-trace ENS derivatives and assess the effects of loss of Ret or Ednrb during embryonic development in these lineages. Finally, they use explants from the corresponding embryos to examine the effects of GDNF on progenitor outgrowth and differentiation.

Strengths:

-  The manuscript is overall very well illustrated with high-resolution images and figures. Extensive data are presented.

-  The identification of Pax2 expression as a lineage marker that distinguishes a subset of cells in the ENS that may be distinct from cells derived from Wnt1+ progenitors is an interesting new observation that challenges the current understanding of ENS development.

-  Pax2 has not been previously implicated in ENS development - this manuscript does not directly test that role but hints at the possibility.

-  Interrogation of two distinct signaling pathways involved in ENS development and their relative effects on the two purported lineages.

The reviewer provided a succinct and accurate summary of our analysis. We correct just the one statement that the ENS is entirely populated by vagal crest. The paper cited by the reviewer (PMID: 38452824) used Wnt1DreERT2 to lineage label the NC population, so of course only looked at neural crest (comparing vagal vs. sacral NC). The advance in our study is to newly document the independent contribution of the placodal lineage.

Weaknesses:

-  The major challenge with interpreting this work is the use of two transgenic lines, rather than knock-ins, Wnt1Cre and Pax2-Cre, which are not well characterized in terms of fidelity to native gene expression and recombination efficiency in the ENS. If 100% of cells that express Wnt1 do not express this transgene or if the Pax2 transgene is expressed in cells that do not normally express Pax2, then these observations would have very different interpretations and not support the conclusions made. The two lineages are never compared in the same embryo, which also makes it difficult to assess relative contributions and renders the evidence more circumstantial than definitive.

We do not agree that the Cre lines being transgenics rather than knock-ins changes the utility of these reagents or the interpretation of the results; there are also potential problems with knock-in alleles. Wnt1Cre has been in use for 25 years as a pan-neural crest lineage cell marker with exceptional efficiency and specificity (including numerous studies of the ENS), so we disagree that it is not well characterized. Pax2Cre of course has not previously been studied in the ENS, but it has been broadly used in other contexts (e.g., craniofacial, kidney). That said, and as noted in our original manuscript, we are aware that an issue of this study is the uniqueness of the recombination domains of the two Cre lines.  As we wrote, Wnt1Cre and Pax2Cre cannot be combined into the same embryo because they are both Cre lines, and we do not have a suitable nonCre recombinase line to substitute for either. Instead, we demonstrate that the two lines recombine in distinct territories of the early embryonic ectoderm, and that the two lineages thus labeled are distinct in marker expression at the initial onset of their delamination, utilize Edn3-Ednrb and GDNF-Ret in distinct ways during their migration to the hindgut, and contribute to different terminal cell fates in the colon. We think this evidence of the distinct nature of the two lineages from start to finish is compelling rather than merely circumstantial.

-  Visualization of the Pax2-Cre and Wnt-1Cre induced recombination in cross-sections at postnatal ages would help with data interpretation. If there is recombination induced in the mesenchyme, this would particularly alter the interpretation of Ednrb mutant experiments, since that pathway has been shown to alter gut mesenchyme and ECM, which could indirectly alter ENS colonization.

We have several thoughts about this comment. First, we are uncertain why postnatal analysis would be informative, as ENS colonization occurs (or fails to occur in mutants) during embryogenesis. The reviewer might be thinking of a juvenile stage additional contribution to the ENS, which is addressed below (responses to Recommendations for the Authors) but as we discuss there is not relevant to our analysis. Second, we did examine recombination in the distal hindgut at E12.5 during ENS colonization (Fig. 1f and 1h) and did not see overlap between either Cre recombination domain and Edn3 mRNA expression (which is expressed by the nonENS mesenchyme). Furthermore, Ednrb is not expressed in the gut mesenchyme during ENS colonization (Fig. 7figure supplement 1), thus ectopic mesenchymal Cre expression, if any, by either line would have no impact in Cre/Ednrb mutants. Lastly, the reviewer’s idea could have been a plausible hypothesis at the onset of the project, but here we show positive evidence for a different explanation. We do not rigorously exclude the reviewer’s hypothesis, nor other theoretically possible models, but we think we have provided a strong case to support the direct involvement of Ret and Ednrb in ENS progenitors rather than in surrounding non-neural mesenchyme.

-  No consideration of glia - are these derived from both lineages?

To properly address this question would require new reagents and analyses that we have not yet initiated. While an interesting question from a developmental biology standpoint, we don’t think that this investigation would change any of the interpretations that we make in the manuscript.

-  No discussion of how these observations may fit in with recent work that suggests a mesenchymal contribution of enteric neurons (PMID: 38108810).

The recent paper cited by the reviewer is very explicit in describing this mesenchymal contribution to the ENS as occurring after postnatal day P11. Other than the terminal Hirschsprung phenotype, all of our analysis of cell lineage migration and fate and colonic aganglionosis was conducted at embryonic or early (P9) postnatal stages. We therefore do not see a relation of our work to this study. In light of this paper, however, we do agree that it would be worthwhile in a future study to explore Wnt1Cre and Pax2Cre lineage dynamics in the ENS of older mice.

Reviewer #1 (Recommendations For The Authors):

(1) The authors should reanalyze multiple single-cell RNA-seq datasets available now, to see if these cells are detected in those studies and then look at the global transcriptional profile of these Pax2-positive cells compared to the other vagal neural crest-derived ENCDCs. Some of these datasets can be found here - PMIDs: 33288908, 37585461, and https://www.gutcellatlas.org/.

We disagree that the datasets from previous studies provide additional insights that are relevant to the current study. It must be appreciated that Wnt1Cre and Pax2Cre are genetic lineage tracers and that migratory ENS progenitor cells labeled with these reagents do not maintain expression of Wnt1 and Pax2 mRNA or protein. The Wnt1 and Pax2 genes are only transiently expressed within their distinct regions of the ectoderm, and their expression turns off as cells delaminate and begin migration. Thus, Pax2Cre-labeled ENS progenitor cells are not Pax2-positive thereafter. The single cell RNA-Seq studies suggested by the reviewer were collected from older embryos and postnatal mice, and do not represent the E10.5-E11.5 period that accounts for genesis of Ret-mediated and Ednrb-mediated Hirschsprung disease pathology. Even with the most recent work by Zhou et al (Dev Cell, 2024) that included E10.5 cells, this analysis only evaluated neural crest-derived Sox10Cre lineage cells, which does not include the placode-derived Pax2Cre lineage (as we show explicitly in Fig. 2-figure supplement 2).  Consequently, it would not be possible to find the “Pax2-positive cells” in these datasets. Performing a new transcriptomic analysis by isolating Pax2Cre-lineage and Wnt1Cre-lineage cells at the appropriate developmental time points could be the basis of future studies, but we think these are beyond the scope of the present paper. 

(2) Even in their current quantification method of using immunofluorescent cells in a microscopic field, the authors count very few cells. The quantification in Figures 2v-2z is only from 4 embryos and is in the hundreds. This leads to misrepresentation of cell numbers and is best reflected in Figure 2x, where Wnt1Cre/Ret GI tracts have 0 Ret +ve cells, which we now know is not true even in ubiquitous Ret null embryos, where Ret null cells are detected as late as E14.5 (PMID 37585461)

Because of the reviewer’s comment, we recognize that the specific detail about cell numbers wasn’t properly written. We didn’t count a few hundred cells total, it was a few hundred cells per embryo. Exact numbers are provided in the revised figure legend where “cells/embryo” is now explicitly stated. Multiplied by the number of embryos, this means that we evaluated approx. 1000 total cells per genotype and time point in cases where Ret+ and/or GFP+ (lineage+) cells were found. The total absence of such cells in Wnt1Cre/Ret mutants is a rigorous conclusion. Our results do not misrepresent nor contradict the study by Vincent et al (PMID 37585461). Our analyses were performed on gut tissue isolated at E10.5 and E11.5 stages, which is long before Schwann cell precursors (SCPs, the primary focus of the Vincent et al study) colonize the gut (E14.5; Uesaka et al, 2015. PMID: 26156989). Indeed, as the reviewer notes, SCPs migrate into the gut in a Retindependent manner. For being at a much earlier time point, our focus is on the cranial ectoderm sources of ENS progenitors. We have adjusted the text associated with Fig. 2 to make this more clear.

(3) There are multiple sections in the manuscript that rehash already known facts, like the whole section about Wnt1 conditional Ret null mice which show failure of migration of ENCDCs. This has been shown multiple times and doesn't add anything to the author's story.

We think this comment stems from the reviewer’s perception that the Pax2Cre lineage is a subset of neural crest. The Wnt1Cre data (including Ret-deficient and Ednrb-deficient embryos) presented in the manuscript are not intended to rehash what is already known but to establish important similarities and differences between the newly identified placode-derived and the well-established neural crest-derived ENS progenitor cells. In light of the reviewer’s suggestion #8 below, to move the Wnt1Cre lineage analysis to a supplement, this information remains in the main text to provide proper comparison to the Pax2Cre-lineage profile. We think we were fair in the text to the legacy of work on neural crest and ENS development and were explicit in using our Wnt1Cre analysis to compare to the Pax2Cre lineage. Finally, we point out that our analysis was conducted on a different genetic background (outbred ICR) compared to previous studies, and there are strain-specific differences in Hirschsprung-associated lethality between our background and previous studies, so it was not impossible that the behavior of the neural crest cell lineage in the ICR background could be different from past observations on different backgrounds. Although we did not identify any major differences, it is important that the information on NC behavior in this background be presented. 

(4) Also, the conclusion drawn for Figure 5C "this indicates that the Wnt1Cre-derived cells do not harbor a cellautonomous response to GDNF" seems to suggest the authors are not very well versed with the ENS literature. GDNF as well as EDN3 are expressed from surrounding mesenchyme and are cell non-autonomous.

The reviewer seems to have misread or misunderstood the specific statement as well as the more important broader conclusion of the experiment. First, of course the source of GDNF ligand in vivo is the mesenchyme. The explant assay was designed to eliminate this and then to substitute GDNF as provided experimentally. The focus of the experiment was to address the response to GDNF, not the source of GDNF. But more importantly, the experiment revealed a surprising outcome that the reviewer did not appreciate. In Pax2Cre/Ret mutants, the Wnt1Cre lineage still expresses Ret, yet does not grow out from the gut explant when provided with GDNF. This shows that the neural crest lineage requires Ret function in placode-derived cells in order to respond to GDNF. In other words, despite expressing Ret, the NC lineage does not harbor a cellautonomous response to GDNF, as we wrote. Because this might be confusing to some readers, we have revised the description of this analysis to hopefully be more clear.

(5) The fact that Ret and Ednrb signaling pathways interact is not a novel finding and has been reported multiple times in Ret and Ednrb mutant mice and cell lines (PMID: 12355085, 12574515 , 27693352, 31818953), potentially through shared transcription factors (PMID:31313802).It would have been more relevant if the authors could show how the specific tyrosine residue (Y 1015) in Ret is phosphorylated in the presence of Ednrb.

The observation that human mutations in RET and EDNRB both cause Hirschsprung disease is decades old, and of course numerous studies in human, mouse, and cells have addressed the relation between the two signaling pathways. We did not mean to imply that we were the first to discover that Ret and Ednrb signaling pathways interact. The reviewer cites a number of papers all from the Chakravarti lab that address this phenomenon; while these are a valuable contribution to the field, there is still more to be learned. The model elaborated in PMID: 31313802, in which Ret and Ednrb are both enmeshed in a common gene regulatory network, does not readily explain why each has a different phenotypic manifestation and doesn’t take into account the importance of the placodal lineage. The main new contributions of our paper are the existence of a new cell lineage that contributes to the ENS, and that the placodal and neural crest lineages utilize Ret and Ednrb signaling differently. The clarification of how these elements are differentially used by the two lineages explains long-segment and short-segment Hirschsprung disease (Ret and Ednrb mutants, respectively) far better than in past studies. The reviewer unfortunately dismisses these insights and seems to feel that a biochemical exploration of one specific component of the signaling interaction (Y1015 phosphorylation) would be more relevant. This should be the basis of future studies and are beyond the scope of the new findings reported in the present paper. 

(6) What is the mechanism of the presence of Y1015 phosphorylation in 33% of Ednrb deficient Pax2Cre cells? It appears to me what the authors report as absent phosphorylation in the 67% of cells could be just weak staining or cells missing in prep.

The reviewer, referring to Fig. 7q, presumably meant to say Wnt1Cre rather than Pax2Cre. The reviewer overlooked that we provided an explanation for this observation in our original manuscript. This sentence reads “Because Ednrb is expressed only in a subset of Wnt1Cre-derived enteric progenitor cells (Figure 7 – figure supplement 1), the residual Y1015 phosphorylation observed in Wnt1Cre/Ednrb mutant cells is likely to occur in the Ednrb-negative Wnt1Cre-derived cell population”. The sentence is retained unchanged in the revised manuscript. The explanation is not because of weak staining or problems with tissue preparation.

(7) The references the authors cite regarding the previous discovery of Ret expression in the nucleus are incorrect. The review articles the authors cite do not mention anything about Ret expression in the nucleus. The evidence of nuclear localization of Ret previously comes from overexpression studies in HEK293 cells (PMID: 25795775). Such overexpression studies are fraught with generating noisy data for well-documented reasons. But if this observation is correct, the authors miss a great opportunity to identify what the Ret protein is doing in the nucleus. Is it in direct contact with its known transcription factors like Sox10 and Rarb? This would shed a lot of light on the possible mechanism of Ret LoF observed in Ret mutant mice

The reviewer overlooked that the one of the review articles that we cited (Chen, Hsu, & Hung, 2020) has a dedicated paragraph for RET (section 3.14), which summarizes the work by Barheri-Yarmand et al (PMID: 25795775) which is the very paper noted by the reviewer in the comment above. The reviewer also somewhat misstated the results of the Barheri-Yarmand et al study. By immunostaining, this paper showed nuclear localization of endogenous Ret, albeit a version of Ret with a disease-associated mutation that makes it constitutively active by constitutive autophosphorylation. Nonetheless, this was endogenous Ret. The paper also used overexpression of GFP-tagged RET in HEK293 cells to show that wildtype RET can behave in a similar manner, at least under these circumstances. Our point is simply that Ret (and other receptor tyrosine kinases) can be found in the nucleus in certain biological contexts, and our observations are consistent with this precedent.

The reviewer also suggests a biochemical follow-up analysis related to this observation, which we agree would be of interest. Such an investigation however is beyond the scope of the present study.

(8) The manuscript could benefit from a major rewrite by reorganizing sections to make it easy for the readers to follow the narrative.

Many sections about the role of Ret and Ednrb in Wnt1cre-derived ENCDCs can be moved to a supplement. These facts are well-documented and have been proven before.

This was addressed in our response to comment #3 of this reviewer. The figures have been kept as main figures in the revised manuscript to allow side-by-side comparison to parallel analysis of the Pax2Cre lineage.

- The observation that only a handful of Pax2Cre cells at E10.5 express Ret and the observation that conditional Ret null abrogates these cells at E11.5, are not presented together and makes connecting these two facts difficult.

Ret expression at E10.5 and E11.5 are both shown in the same figure (Fig. 2). In the presentation of these results, we first describe in normal development that Ret is expressed differently in E10.5 ENS progenitors between the Pax2Cre and Wnt1Cre lineages. This is additional support for the argument that the two lineages are molecularly distinct. Then comes evaluation of postnatal fates with different markers before we return to embryonic Ret expression. We acknowledge that this can make it difficult to connect these observations. We decided to retain the original organization in order to not lose this important conclusion. However, we have revised the text to hopefully make this connection between the sections more congruent.

Reviewer #2 (Recommendations For The Authors):

- The labeling of some as "figure supplements" is really hard to follow in the text and confusing to interpret when a main figure or supplemental figure is being referenced, and which one.

We understand this comment, but this is journal style and outside of our control. We have kept the journal format in the revised manuscript.

- The data in Figures 3b-c is well established in the field and somewhat misinterpreted. NOS1 neurons in the mouse ENS and their projections have been well described (Sang and Young, 1996, and other studies). CGRP immunoreactivity would reflect both ENS CGRP-expressing neurons and visceral afferents from DRG.

There of course is a history of analysis of NOS1, CGRP, and other markers in the ENS. The focus of the analysis in Fig. 3 is to demonstrate how the cells that express these markers are impacted by gene manipulation in the Wnt1Cre and Pax2Cre lineages. For the giant migrating contractions that are associated with defecation, ample past electrophysiological studies have established that mechanosensory CGRP+ neurons trigger NOS+ inhibitory neurons (and ACh+ excitatory neurons) of the myenteric plexus to propel colonic contents. Thus, these are the relevant markers to explain the lack of colonic peristalsis in Ednrb-deficient mice. To our awareness, our results with NOS1 do not contradict any past study, including the Sang and Young 1996 description. Regarding CGRP, indeed the reviewer is correct that this marker is expressed by both neuronal subtypes. Two arguments support the specific derivation of ENS mechanosensory neurons from the Pax2 lineage. First, the ENS and DRG neurons can be distinguished by the location of their cell bodies and their axon extensions in the gut wall; only the ENS neurons are deficient in Pax2Cre/Ednrb mutants (as documented in Fig. 3). Second, the DRG population is derived from neural crest and is not labeled by Pax2Cre. If this population of CGRP+ neurons had functional relevance to colonic peristalsis, this would not be altered in Pax2Cre/Ednrb mutants. Indeed, the CGRP+ afferent nerve endings of DRG origin in the distal colon are mechanical distension sensors but do not modulate either ENS or autonomic nervous system activity (PMID: 37541195). We believe that our interpretation is correct.

- The evidence in Figure 3 supporting the claim that NOS1 and CGRP-expressing enteric neurons come from distinct lineages is weak. IHC for CGRP is notoriously poor at labeling soma in the ENS. IHC for tdTomato to ensure the detection of low levels of Tomato expression and quantification of observations would strengthen this claim.

CGRP is a vesicular peptide which is stored and transported in vesicles, therefore the antibody against CGRP labels vesicular particles of soma and synaptic vesicles along the axons of those CGRP-producing neurons.

It is not expected to label the entire cytoplasm (or the range of subcellular organelles) as NOS antibody does. We did included quantification of data in Figure 3-figure supplement 1 in the manuscript to support the claim of lineage derivation. As described in the Methods section of the manuscript, we used binary threshold selection for Tomato+ cell count using Fiji-Image J, which detects both TomatoHigh and TomatoLow cells as Tomato+; we feel this is equal to or even superior to IHC for this analysis. 

- IHC panels in Figures 3h-o are largely uninterpretable. Most of the signal seems to be non-specific background staining in the mucosa and quantification of mucosal signal in this context does not seem meaningful.  

We disagree with the reviewer’s comment. As described in the response above, CGRP+ mechanosensory neurons send their peripheral axon projections to innervate mucosa (sensory epithelial cells), and NOS+ inhibitory motor axons innervate the circular muscle. Thus, panels h-o of Fig. 3 focus on the axonal profile and are not intended to visualize soma, which is why sagittal views are presented instead of flatmount views. All of the controls were performed side-by-side to confirm that the signal is real and interpretable.

Note also that the colon does not have villi so this annotation should be revised.

We appreciate that the reviewer brought this misstatement to our attention. We corrected this error in the revised manuscript.

- Phospho-RET staining in Figure 7 is difficult to discern and interpret with high background. Positive and negative controls would strengthen these data.

Fig. 7 shows phospho Ret-Y1015 staining in lineage-labeled Wnt1Cre/Ednrb/R26nTnG mutants. The strength of the signal to noise in the figure is a matter of Ret expression level and the quality of the anti-pY1015 antibody. We are not aware of a meaningful positive control that has been validated in the literature that we could use for comparison. The ideal negative control would be to perform the same analysis in Wnt1Cre/Ret/R26nTnG mutants, but because this manipulation eliminates the entire NC cell lineage from the colon, there would be no NC cells in which to visualize background staining in this lineage with this antibody when Ret protein is not present. We note that anti-pY1096 did not show a difference in staining between control and mutant, which supports the interpretation of a specific impact on pY1015. We also point out here, as in the text, that we do not yet have any validation that phosphorylation of Y1015 is functionally important in NC migration to the distal colon. Clearly, more work to address this role and to demonstrate the mechanism of phosphorylation of this specific residue in response to Edn3-Ednrb signaling will be needed.

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What is this?

performer must provide himself an out that is other than the actual reason

letting people keep their secrets

example two people having a convo that's private with others around other random unrelated people offer the courtesy or tact of not paying attention two people won't completely cease having conversation or make dramatic show of secrecy but will refrain from discussing all things out right maintain some level of secrecy while respecting or acknowledging other's "tact"

THIISSSSS

If I say that I know that you know I know you know- the whole performance reality breaks - shame or laughter

audience allows nurse to fulfill role by participating in something they didn't want to do

outsiders make performers aware of their own prescence

outsiders themselves work so that the performance can maintain its own impression

dramaturgical discipline- disassociation from own role as a performance- real or fake intellectual and emotional involvement in activity at stake

example- dramturgical loyalty threatened when clerks say candidly what products are actually worth buying

sometimes performer pleas to audience for their mercy because of a failure or inability to continue performing.

sometimes this threat of politene appearances is for a certain purpose

facts about performers (of past and present relevance) can pose as threat to illusion

staging talk- talk regarding performance itself

unofficial communication- communication that always has potential to be denied, works to communicate and test relations between two teams communicate roles, how the intimacy of relationships should be- communicate boundaries, etc.

sometimes teammates communicate things as a part of performance but only that those "in the know" pick up on

derisive collusion- done by performer and hidden from higher-ups , for the performers own purpose

some of these aren't for bonding or fun- for the sake of performance itself aka stage cues

derogation of audience actually serves a purpose- keeps morale of the team and bonds them in a way that allows them to maintain face during performance

include the rapid swtich in beahviors?

other type of communication referred for backstage- using cynical or strictly technical language to describe performance behaviors

first type- trash talking audience

4 types of communication not for the front stage?

some colleague groups collective identity more tied than others could racial groups be considered a colleague group?

colleagues let colleagues down when don't retain secretive info

confidants- people who performers relay full feelings to- people whom they tell that the performance was a performance and what aspects felt like one.

sometimes acquisition of specialist themself is apart of the performance

why emphasis on discretion so important- that trust is hard to build when there's little incentive for it to be mutual.

service specialists attend to front stage but often, must obtain backstage view and destructive information to do their job

givens servants functions as almost invisible but visible when necessary, confusion around what behavior acceptable before them

shill, spotters, shoppers, and go-betweens perform role but don't adopt it?

audience knows what theyve been allowed to percieved

inside secrets- objective intellectual content

often time intruding audience ignored or asked to leave - no use acting normal

one may feel more out of character (or still our of character) backstage in performance to other team members

downfall of upward mobility- forget the backstage world that all performances (such as our own) had

"If you like a church don't join it"

Three nuances of backstage informality: 1. team still wants to seem trustworthy like they can be trusted with secrets of the team 2. teammate must perform for sake of other teammates moral 3. divides on other factors amongst team members

no pure types

performance often relies on the backrooms and the privacy of them

break room at work

audience holds director to performance more than anyone else responding with higher demands on other performers, sets them apart

performers must form unanimous agreements while concealing the fact that such agreements had to be made amongst members

Lol this is so ironic- each member must perform as though they embody a role so wholly that they didn't even need to decide on something

performer rarely performs part in team performance for the sake of his other teammates- define the other as someone " in the know"

The audience doesn't even need to be other people- it can be an imagined outward judgement or assessment of action that directs a performance when no one is looking

that one Margaret Atwood quote

like teachers switching up and calling each other "Ms. -----" in front of students

the expression of one role reliant on the proper expression of the other roles in performance

oftentimes- the performance requires participants who perform roles which require adopting demeanor's

can't think of presentation as just an extension of the performer

because even the liars and honest want to be presumes as honest, looking at how the nonhonest perform and work to legitimate their authority shows us how the already legitimate do it?

One can maintain a status quo without rigid adherence to all demands of performance in the relationship

larger number of matters and parts of relationship = more points of secrecy

A lot of ways to lie without directly lying, yet, "bare-faced" lies are the ones with all the consequences

"really" as a flexible term of legitimating one's performance - they're not "really" something can be something objective or subjective

have empathy for those with one hidden thing vs those who conceal everything

When we find out someone is a fraud, what we discover is that they did not have authority to play the part they played- they don't have the means of status by the right materials or legitimating factors

in our heads- a true or honest performer- would not mess up. Assumption that there IS an honest performer or someone who embodies something without a need for front. This view isn't analytically very applicable

as members, we assess validity or phoniness of performance

we do not let or are expected not to allow momentary emotions to impact performance- should be homogenous

we usually designate strict social expectations of presentation to other cultures but exist well within our own.

takes very little to signal this meaning- susceptible to misinterpretation

conceal motives that break idea of what a "good" clergymen should do

Can you be a good clergymen and still wish to progress socailly?

training extensive to five image that they are set apart

fulfill idealized ideas of routines and roled

never a performance about bettering or reflecting what is really there, people embody identities for a certain result

performance is more focused on living up to standard societal expectations than an individuals actual behabior

people often choose a certain routine to invest own ego into

merchants overcharge for things that look expensive (are in which to role of money is dramatically fulfilled) so that they can pay for what costs but is not dramatic

issue- exists a nonbelief in other people that are doing fulfilling their role unless they see a dramatized representation of the task

issues of taks that are "between ranks" arise- in the instance of nurse and doctor it relates to the capability of the professional but also what is "right" for the position.

Feels a but more about organization that social expectation tbh

often the front or selection of t- or title- isn't suitable for all the tasks at hand

appearance are status and other identifications (can show things like what you do for work) Manner tells us of oncoming action within a persons role (what the person expects to happen or how others are to respond)

so little distinction between performing and becoming

there is often a demand to be insincere that the cynical performers, in their hyperawareness of the gap between reality and the performers, more willingly offer up this insincerity.

not every cynical performer evil- some work to do good with such performance

MMEEEE

other extreme- individual isn't at all convinced of own performance, may not actually care if its real me asf

except this time, the audience is the performers

A mi juicio, nadie tiene más derecho de matar a nadie especialmente, incluso el estado.

https://docdrop.org/pdf/hcfea_rapport_dehors_25_10_2024-zblo8.pdf/

La ville à hauteur d'enfants : Pour un accès serein à la nature, à l'extérieur et aux espaces publics Introduction

Ce rapport explore les multiples facettes de l'accès des enfants à la nature, à l'extérieur et aux espaces publics.

Il examine les obstacles, les opportunités et les initiatives qui façonnent l'expérience des enfants dans la ville.

I. Un environnement urbain contraignant pour l'enfant

Un périmètre aux règlementations parfois trop contraignantes

Cette section analyse le cadre institutionnel et règlementaire qui régit l'aménagement urbain et met en lumière les contraintes et les limites qu'il impose à la création d'environnements adaptés aux enfants.

Des règlementations spécifiques : contraintes, limites et points de vigilance

Ce chapitre explore les réglementations spécifiques qui impactent l'aménagement urbain et leur application, souvent en contradiction avec l'objectif de créer des espaces adaptés aux enfants.

Quels soutiens pour des projets tournés vers les enfants, la nature et les espaces publics ?

Ce point examine les dispositifs de soutien et les financements disponibles pour la réalisation de projets qui favorisent l'accès des enfants à la nature et aux espaces publics.

Les quartiers prioritaires de la politique de la ville particulièrement visés par les financements « verts »

Ce passage se concentre sur les quartiers prioritaires de la politique de la ville et l'importance des financements "verts" pour améliorer leur environnement.

L'enjeu du logement dégradé

Cette partie met en lumière le problème du logement dégradé et son impact sur le bien-être et le développement des enfants.

L'éducation par la ville

Ce chapitre explore la notion de la ville comme un lieu d'apprentissage et d'éducation pour les enfants.

Une prise de conscience institutionnelle et dans les collectivités

Ce point analyse la prise de conscience croissante des institutions et des collectivités locales quant à l'importance de créer des villes plus adaptées aux enfants.

II. Repenser la ville à hauteur d'enfant : des initiatives et des solutions

La ville à hauteur d’enfants : c’est possible

Cette section met en avant des initiatives concrètes et des solutions innovantes pour repenser la ville en fonction des besoins des enfants.

Spécificité de l’enfance – Connaître et reconnaître la spécificité de l’enfance et son besoin d’être dehors

Ce chapitre souligne l'importance de comprendre les besoins spécifiques des enfants et leur besoin vital d'accès à l'extérieur et à la nature.

Se déplacer : sortir, s’aventurer, grandir

Ce passage met l'accent sur l'importance de la mobilité et de l'exploration pour le développement des enfants, en proposant des solutions pour des déplacements plus sécurisés et adaptés.

Rencontrer : grandir avec les autres, apprendre la citoyenneté

Ce point explore le rôle des interactions sociales et de l'apprentissage de la citoyenneté dans les espaces publics pour le développement des enfants.

Jouer : un droit fondamental pour apprendre et grandir

Ce chapitre rappelle l'importance du jeu comme un droit fondamental pour l'apprentissage et le développement des enfants, en plaidant pour des espaces de jeux plus libres et créatifs.

Apprendre : explorer, expérimenter, connaître, découvrir

Ce passage met en avant l'importance de l'apprentissage par l'exploration, l'expérimentation et la découverte dans la ville et la nature.

Imaginer : laisser place à la créativité, à la rêverie

Ce point souligne l'importance de stimuler l'imagination et la créativité des enfants en leur offrant des espaces propices à la rêverie et à l'expression artistique.

Grandir : de l'enfance à l'adolescence, vers l'autonomie

Ce chapitre analyse les besoins spécifiques des adolescents et les aménagements urbains qui peuvent les accompagner vers l'autonomie.

Se protéger : de la vigilance aux dangers invisibles

Ce passage met en lumière les dangers auxquels les enfants sont exposés dans la ville et propose des solutions pour les protéger, en abordant des thèmes tels que la violence, le harcèlement et la pollution.

Découvertes – Faire l’expérience de l’ailleurs

Ce point explore l'importance des séjours scolaires et des voyages pour élargir les horizons des enfants et favoriser leur ouverture au monde.

Conclusion

Ce rapport appelle à une transformation profonde de la ville pour la rendre plus accueillante et stimulante pour les enfants, en intégrant leurs besoins spécifiques dans les politiques d'aménagement urbain et en encourageant des initiatives qui leur permettent de s'épanouir pleinement.

Il met en évidence l'importance d'une collaboration entre les institutions, les collectivités locales, les associations et les familles pour créer un environnement urbain où les enfants peuvent grandir sereinement et se développer pleinement.

Annexes

La Loi Notre et les directions centrales de l'aménagement du territoire

Glossaire

Source : hcfea_rapport_dehors_25_10_2024.pdf

Responsabilité des collectivités territoriales pour la sécurité des enfants aux abords des locaux scolaires

1. Introduction

Ce rapport analyse la responsabilité des collectivités territoriales pour la sécurité des enfants aux abords des locaux scolaires.

2. Responsabilité civile des collectivités locales

Responsabilité pour défaut d’entretien des installations ouvertes au public

Ce point examine la responsabilité des collectivités locales pour les accidents survenus suite à un défaut d'entretien des installations ouvertes au public, y compris les abords des écoles.

Idem pour défaut d’entretien du bâti scolaire

Ce passage se concentre sur la responsabilité des collectivités locales en cas d'accident lié à un défaut d'entretien du bâti scolaire.

Que fait la police : la position ministérielle il y a vingt ans. A-t-elle changé ?

Ce point analyse la position du ministère de l'Intérieur concernant la sécurité aux abords des écoles et l'évolution de cette position au cours des 20 dernières années.

La responsabilité scolaire en droit administratif

Ce chapitre se focalise sur la responsabilité des collectivités locales en matière de sécurité scolaire en droit administratif.

3. Responsabilité pénale des agents et des élus locaux

Ce chapitre explore la responsabilité pénale des agents et des élus locaux en cas d'accident impliquant des enfants aux abords des écoles.

4. Proposition d'un projet de lettre au maire pour obtenir l'autorisation d'implanter un terrain d'aventure

Cette section propose un modèle de lettre à adresser au maire pour solliciter l'autorisation d'implanter un terrain d'aventure en invoquant la Convention internationale des droits de l'enfant (CIDE).

45 yaşına gelindiğinde, bu bireylerin yalnızca birkaç dişi (genellikle premolarlar) diş taşı birikintisi olmayan dişlerdi.

La fonction de chef d'établissement dans l'enseignement public du second degré aujourd'hui

Ce document synthétise les idées principales du rapport "chef-etablissement-rapport-cafe-pedagogique-IGESR.pdf". Il met en lumière les réalités, les défis et les perspectives de la fonction de chef d'établissement dans le second degré, en se basant sur une analyse approfondie du contexte actuel.

1. Profil des chefs d'établissement:

Le rapport dresse un portrait détaillé des chefs d'établissement :

Rémunération:

La rémunération, bien que complexe, est jugée globalement satisfaisante en comparaison avec les cadres de la fonction publique. Toutefois, des disparités existent en fonction de la catégorie d'établissement dirigée.

Le rapport préconise une meilleure reconnaissance de la charge et des responsabilités des chefs d'établissement, notamment par l'instauration d'un échelon terminal spécifique à leur grille indiciaire. "La mission recommande cependant de mieux reconnaître la charge et les responsabilités managériales et de pilotage qui incombent aux chefs d’établissement, en proposant d’instaurer la hors échelle lettre Bbis comme échelon terminal de la grille indiciaire du corps des personnels de direction, qui ne serait accessible qu’à des agents exerçant les fonctions de chefs d’établissement."

Formation:

La formation actuelle des chefs d'établissement est jugée "hybride", mêlant formation initiale et continue. Un besoin d'adaptation et d'approfondissement des compétences est mis en avant, notamment face aux nouveaux enjeux du métier.

"La formation dont bénéficient actuellement les personnels de direction présente de nombreuses porosités entre les dispositifs destinés à répondre aux gestes professionnels à l’entrée dans le corps en formation initiale statutaire d’une part, et ceux consacrés à continuer d’acquérir des compétences spécifiques au regard des emplois occupés en formation continue d’autre part."

Gestion des carrières:

La gestion des carrières est partagée entre l'administration centrale et les académies, ce qui peut engendrer un manque de transparence et de fluidité. Le rapport recommande une plus grande implication des académies dans le mouvement des chefs d'établissement, tout en favorisant la mobilité nationale.

"Dans une approche d’autonomie territoriale accrue, certains recteurs revendiquent une déconcentration du mouvement plus poussée, en particulier sur la question des entrants au mouvement inter-académique et sur les retours des personnels détachés à l’étranger ou mis à disposition des collectivités d’outre-mer."

2. Réalités de la mission :

Cadre réglementaire flou: Le cadre réglementaire définissant les missions des chefs d'établissement est jugé peu spécifique, ancien et manque de clarté, notamment concernant la distinction entre les rôles du chef et de son adjoint.

"Finalement, ce sont les textes à très faible autorité normative (un protocole d’accord et son annexe) qui encadrent le plus finement la mission d’un chef d’établissement. Ils datent de l’année 2000 et mériteraient sans doute une actualisation afin de mieux prendre en compte les nouvelles missions attendues."

Multiplication des tâches:

La mission du chef d'établissement est marquée par une multiplication des tâches et une diversification croissante des responsabilités. La gestion des réformes successives, des applications informatiques complexes et des relations avec les usagers (enseignants, familles, partenaires) contribuent à une charge de travail importante et à un sentiment d'isolement.

"L’image est celle d’un déferlement de nouveaux enjeux qui passent systématiquement par l’École donc, dans le second degré, par les collèges et lycées. Le chef d’établissement y représente l’État et deviendrait le relais, le passage obligé pour résoudre toutes préoccupations éducatives."

Mission pédagogique sous-investie:

Bien que la mission pédagogique reste au cœur du métier, elle est souvent reléguée au second plan face aux impératifs administratifs et relationnels.

Les chefs d'établissement expriment le besoin de se recentrer sur la dimension pédagogique et d'être davantage en lien avec les équipes enseignantes.

"Systématiquement interrogés par la mission sur ce sujet, les chefs d’établissement rencontrés affirment n’avoir pas délaissé la dimension pédagogique du métier, mais elle deviendrait sous calibrée dans leur agenda pour laisser la place aux autres missions dont celles qui se surajoutent d’année en année."

3. Ressenti des chefs d'établissement :

Perte de sens: La multiplication des réformes, le manque de confiance de l'institution et la complexité croissante du métier contribuent à un sentiment de perte de sens chez certains chefs d'établissement.

"Mais c’est la perte de sens du métier (notamment par la multiplication des réformes à mettre en place sans prendre le temps d’évaluer la précédente) et le manque de confiance inhérent à leur statut de cadre, responsable d’un établissement autonome qui prévalent dans les ressentis négatifs exprimés par les chefs d’établissement rencontrés."

Recherche d'autonomie et de reconnaissance:

Les chefs d'établissement aspirent à une plus grande autonomie dans leur prise de décision et à une meilleure reconnaissance de leur statut de cadre de terrain.

"La reconnaissance de leur statut de cadre de terrain est un sujet récurrent, dans lequel la rémunération n’est qu’une partie de la problématique."

Besoin d'accompagnement:

Un besoin d'accompagnement accru est exprimé, tant dans l'exercice quotidien du métier que dans les perspectives de carrière.

"La mission recommande de construire un plan d’accompagnement des chefs d’établissement dans la suite de leur carrière, avec un cadre national pour les orientations stratégiques et une déclinaison académique pour le déploiement auprès de chaque personnel."

4. Conclusion et perspectives :

Le rapport souligne la complexité et l'évolution constante de la fonction de chef d'établissement.

Il appelle à une réflexion approfondie sur le sens du métier, les conditions d'exercice et les perspectives d'avenir de ces cadres de terrain.

La recherche d'un meilleur équilibre entre autonomie et obéissance, la clarification des missions et la mise en place d'un accompagnement individualisé renforcé sont des enjeux majeurs pour garantir l'attractivité et l'efficacité de la fonction de chef d'établissement.

Reviewer #1 (Public review):

Summary:

The authors examine CD8 T cell selective pressure in early HCV infection using. They propose that after initial CD8-T mediated loss of virus fitness, in some participants around 3 months after infection, HCV acquires compensatory mutations and improved fitness leading to virus progression.

Strengths:

Throughout the paper, the authors apply well-established approaches in studies of acute to chronic HIV infection for studies of HCV infection. This lends rigor the to the authors' work.

Weaknesses:

(1) The Discussion could be strengthened by a direct discussion of the parallels/differences in results between HIV and HCV infections in terms of T cell selection, entropy, and fitness.

(2) In the Results, please describe the Barton model functionality and why the fitness landscape model was most applicable for studies of HCV viral diversity.

(3) Recognize the caveats of the HCV mapping data presented.

(4) The authors should provide more data or cite publications to support the authors' statement that HCV-specific CD8 T cell responses decline following infection.

(5) Similarly, as the authors' measurements of HCV T and humoral responses were not exhaustive, the text describing the decline of T cells with the onset of humoral immunity needs caveats or more rigorous discussion with citations (Discussion lines 319-321).

(6) What role does antigen drive play in these data -for both T can and antibody induction?

(7) Figure 3 - are the X and Y axes wrongly labelled? The Divergent ranges of population fitness do not make sense.

(8) Figure S3 - is the green line, average virus fitness?

(9) Use the term antibody epitopes, not B cell epitopes.

period should go inside quotes (all the time!)

Estoy de acuerdo con tu análisis sobre cómo la autora subraya la importancia de la subjetividad para entender la acción social desde la perspectiva de los actores. Efectivamente, como menciona, Schutz defiende esta postura mientras que Parsons prioriza una visión más estructural y objetiva. Quizás podríamos agregar que el enfoque de Schutz permite captar los significados individuales y la riqueza del contexto social, mientras que el de Parsons busca patrones generales, lo cual abre el debate sobre el equilibrio entre ambos enfoques para lograr una comprensión.

lizeth Duarte ID 684503

Según el análisis e identificación de la autora ella no considera suficiente mente completa la explicación de los fenómenos sociales que no tienen en cuenta la perspectiva subjetiva de los actores. Esta Hipótesis se enmarca en la tradición fenomenológica y sociológica que plantea que las ciencias sociales deben diferenciarse de las ciencias naturales al estudiar y analizar al ser humano, que no solo actúa si o que también interpreta y da sentido a sus acciones. La autora presenta y resalta diferentes argumentos como la experiencia subjetiva en el que el punto de partida de acción social , ya que los individuos actúan basándose en su percepción y significados de la realidad el análisis sociológico debe incorporar el contexto de interpretación individual para captar las motivaciones detrás de las acciones. La suposición planteada parece adecuada en la medida en que reconoce la diferencia entre ciencias sociales y ciencias naturales. La subjetividad es en efecto un componente esencial en el análisis de los fenómenos sociales. No obstante, se debe reflexionar sobre la necesidad de un equilibrio: mientras que la inclusión de la subjetividad es valiosa, la investigación también debe buscar cierta objetividad y consistencia para generar teorías aplicables en diferentes contextos.

Schutz critica a Parsons por tratar las razones para actuar de la sociedad de forma superficial y sostiene que es muy importante adoptar una manera pensar subjetiva. Para él, los motivos deben entenderse como significados de las personas que el autor le transmite sus propias acciones, lo cual permite una comprensión más fácil y profunda de las acciones de la sociedad.

La autora indica que las personas en la mayoría de veces actúan basadas en sus experiencia cotidianas, sin cuestionar se. Solo cuando enfrentan situaciones que no son esperadas, buscando afirmar si sus métodos son los mejores para alcanzar sus objetivos.

El texto señala que la acción de las personas no es solo una reacción a estímulos, sino que busca ajustarse a modelos de comportamiento que la sociedad. Esto implica que las decisiones de las personas están influenciadas por reglas sociales, lo que refleja una manera de ver en donde la acción está guiada tanto por objetivos personales como por lo que quiere la sociedad.

Bryan Segura - ID 977365

El ensayo de López se evidencia los diversos argumentos de ambos autores (Alfred Schutz y Talcott Parsons), que muestran puntos a favor y en contra de ambos. Por ejemplo, la importancia de la subjetivada que Schutz sostiene la importancia de un enfoque subjetivo para comprender las acciones sociales, su complejidad y las interacciones en la vida cotidiana.

Pero ahora frente a la hipótesis se podría decir que es adecuada ya que en primera instancia analizar la subjetividad de manera sistemática cuando esta parte del propio comportamiento del individuo, puede llevar a generar sesgos de interpretación y así poner en duda los resultados obtenidos.

De la misma forma, la falta de profundización y la confusión entre los enfoques, generaría una abertura para crear una creencia que considere que todas las interpretaciones son validad, nublando los diferentes patrones en el comportamiento social.

Bryan Segura - ID 977365

Aquí radica el argumento principal sobre el que se plantea la hipótesis. Y se adjunta la distinción entre el conocimiento científico y del sentido común como parte importante de la acción social. El debate llega a un punto en común sobre la teoría de la acción y su carente sentido si no se aplica a un punto de vista subjetivo, aún así va más allá y afirma que el conocimiento de sentido común es importante para entender cómo es que los actores ejecutan sus actividades cotidianas.

Bryan Segura - ID 977365

El sentido de las experiencias cotidianas son mucho mejor comprendidas en este fragmento del texto.

La autora puntualiza que las experiencias cotidianas son las que construyen la realidad social de los individuos. Su actitud se adapta al entorno social y es el factor fundamental para entender su actuar en el mundo. La subjetividad no sólo es relevante, sino que es base importante en la construcción social.

Bryan Segura - ID 977365

López argumenta que Talcott Parsons confunde los términos de subjetividad y objetividad, y expone que esta confusión puede conducir a un error interpretativo del fenómeno social puesto que se omite las experiencias vividas por los individuos.

Bryan Segura - ID 977365

Desde la introducción, al texto se puede ver como se plantea la falta de desarrollo de la subjetividad en la teoría de la acción social, al igual se subraya la confusión de lo "subjetivo" y lo "objetivo".

La autora expone que la subjetividad es importante para comprender la interpretación que los actores (individuos), le dan a las acciones sociales y el sentido que estas tienen para brindar un contexto.

La subjetividad en este contexto, se refiera las motivaciones y experiencias que cada individuo le atribuye a cada acciona que realiza, mientras que la objetividad es la relación que el observador aplica sobre estos fenómenos generando así una categorización y un análisis.

Es por eso que considero que desde el apartado del resumen la autora sugiere que la investigación sociológica debe contemplar la el valor de la subjetividad para comprender a profundidad la acción social; situación que entre el debate propuesto en el artículo, uno de los dos actores no sigue (Parsons).

Según Schutz, la crítica que hace a Parsons es que al ver los valores morales solo desde un punto de vista subjetivo, se está perdiendo el actor que siempre enfrenta un dilema cuando toma decisiones. Por un lado, tiene una meta o fin que quiere lograr, pero, por otro lado, tiene que elegir los medios o formas de llegar a esa meta, y cada elección viene con consecuencias que pueden ser buenas o malas.

Usuario: 1002556627 Nombres y Apellidos: Yessika Vanessa Gomez Gaona ID 868943 Lo que más valoro de este análisis es cómo López no solo expone las diferencias entre ambos autores, sino que también subraya la relevancia de abordar las experiencias subjetivas de las personas dentro de una sociología que aspire a explicar fenómenos sociales. La discusión sobre la tensión entre las perspectivas de Schutz y las estructurales de Personas es esclarecedora, ya que ayuda a entender cómo la subjetividad y las estructuras sociales se entrelazan en la acción social. Sin embargo, me parece que podría haber profundizado más en cómo estos debates clásicos se traducen en la práctica sociológica contemporánea, especialmente en un contexto de globalización y nuevas formas de interacción social.

Usuario: 1018485395 Nombres: Leidy Katherine Gutiérrez Viuche ID 896498

Existen implicaciones prácticas para cómo se llevan a cabo investigaciones enfocadas en los aspectos sociales, los defensores de un enfoque más subjetivo enfatizan en la importancia de capturar la complejidad de las experiencias humanas, desde el punto de vista más humano y analizando la historia detrás de cada actor, mientras que los partidarios de modelos más estructurales valoran la posibilidad de establecer reglas generales para estas investigaciones de forma objetiva.

Usuario: 1018485395 Nombres: Leidy Katherine Gutiérrez Viuche ID 896498

La autora de este texto hace una clara diferencia entre los enfoques de la investigación, pero en esta parte, nos menciona por qué Parsons le hace falta un enfoque subjetivo de las categorías del actor. Esto es importante, ya que, si bien Parsons trata de brindar el punto de vista subjetivo, lo menciona a partir de la objetividad.

Usuario: 1018485395 Nombres y Apellidos: Leidy Katherine Gutiérrez Viuche ID 896498 Considero que en esta parte se puede notar la gran diferencia entre el paradigma subjetivo y objetivo, por lo cual, puedo entender también como diferencia entre lo cualitativo y cuantitativo en ciertos aspectos. Todo dependerá del tipo de investigador, hipótesis, objetivos y metodología que se use en el trabajo.

No need to number the entries in your bibliography - the best way to format this is instead to insert a line break between each entry

Красиво!

Welcome back, this is part two of this lesson.

We're going to continue immediately from the end of part one, so let's get started.

Okay, so all three of these mount targets are now in an available state and that means we can connect into this EFS file system from any of the availability zones within the Animals for Life VPC.

So what we need to do is test out this process and we're going to interact with this file system from our EC2 instances.

So move back to the tab where we have the EC2 console open.

And at this point I want you to either, and this depends on your browser, I'll either want you to right click and duplicate this tab to open another identical copy.

If you can't do this in your browser then just open a new tab and copy and paste this URL into that tab.

You'll end up with two separate tabs open to the same EC2 screen.

So on the first tab we're going to connect to A4L-EFS instance A.

So right click and then select connect.

We're going to use instance connect.

So make sure the username is EC2-user and then click on connect.

Now right now this instance is not connected to this EFS file system and we can verify that by running a DF space-k and press enter.

You'll see that nowhere here is listed this EFS file system.

These are all volumes directly attached to the EC2 instance and of course the boot volume is provided by EBS.

Now within Linux all devices or all file systems are mounted into a folder.

So the first thing that we need to do to interact with EFS is to create a folder for the EFS file system to be mounted into.

And we can do that using this command so shudu space mkdir space-p space/efs/wp-content.

Now the hyphen p option just means that everything in this path will be created if it's not already.

So this will create forward/EFS if it doesn't already exist.

So press enter to create that folder.

So I'm going to clear the screen to keep this easy to see.

And the next thing I need to do is to install a package of tools which allows this instance or specifically the operating system to interact with the EFS product.

Now the command I'm going to use to install these tools is shudu to give us admin permissions and then DNF which is the package manager for this operating system.

And then a space hyphen y to automatically acknowledge any prompts and then a space and then install because I want to install a package and then a space.

And then the name of the tools that I want to install is amazon hyphen EFS hyphen utils.

So this is a set of tools which allows this operating system to interact with EFS.

So go ahead and press enter and that will install these tools and then we can configure the interaction between this operating system and EFS.

Again I'm going to clear the screen to keep this easy to see and I want to mount this EFS file system in that folder that we've just created.

But specifically I want it to mount every time the instance is restarted.

So of course that means we need to add it to the FSTAB file.

Now if you remember this file from elsewhere in the course it's contained within the forward/ETC folder.

So we need to move into that folder cd///ETC and then the file is called FSTAB.

So we need to run shudu to give us admin permissions and then nano which is a text editor and then the name of the file which is FSTAB.

So press enter and the file will likely have only one or two lines which is the root and/or boot volume of this instance.

So let's just move to the end because we're going to add a new line and this is contained within the lesson commands document but we're going to paste in this line.

So this line tells us that we want to mount this file system ID so file system ID colon forward/.

We want to mount that into this folder so forward/efs forward/wp-content.

We tell it that the file system type is EFS.

Remember EFS is actually based on NFS which is the network file system but this is one provided by AWS as a service and so we use a specific AWS file system which is EFS.

And the support for this has been installed by that tools package which we just installed.

Now the exact functionality of this is beyond the scope of this course but if you do want to research further then go ahead and investigate exactly what these options do.

What we need to do though is to point it at our specific EFS file system.

So this is this component of the line all the way from the start here to this forward/.

So to get the file system ID we need to go back to the EFS console and we need to copy down this full file system ID and yours will be different so make sure you copy your own file system ID into the clipboard.

Then go back here and select the colon and then delete all the way through to the start of this line.

And once you've done that paste in your file system ID what it should look like is the file system ID then a colon and then a forward/.

So at this point we need to save this file so control O to save and then enter and control X to exit.

Again I'm going to clear the screen to make it easier to see.

Then I'll run a DF space -K and this is what the file systems currently attached to this instance look like.

Then we're going to mount the EFS file system into the folder that we've created and the way that we do this is with this command.

So shudu mount and then we specify the name of the folder that we want to mount.

Now the way that this works is that this uses what we've just defined in the FSTAB file.

So we're going to mount into this folder whatever file system is defined in that file.

So that's the EFS file system and if we press enter after a few moments it should return back to the prompt and that's mounted that file system.

There we go we back at the prompt and if we do a DF space -K again we'll see that now we've got this extra line at the bottom.

So this is the EFS file system mounted into this folder.

Now to show you that this is in fact a network file system let's go ahead and move into that folder using this command.

And now that we're in that folder we're going to create a file.

So we're going to use shudu so that you have admin privileges and then we're going to use the command touch which if you remember from earlier in the course just creates an empty file.

And we're going to call this file amazing test file dot txt.

Go ahead and press enter and then do an LS space -LA and you'll see that we now have this file created within this folder.

And while we're creating it on this EC2 instance it's actually put this file on a network file system.

Now to verify that let's move back to the other tab that we have open to the EC2 console the one that's still on this running instances screen.

And now let's go ahead and connect to instance B.

So right click on instance B select connect again instance connect verify the username is as it should be and click on connect.

So now we're on instance B.

Let's do a DF space -K to verify that we don't currently have any EFS file system mounted.

Next we need to install the EFS tools package so that we can mount this file system.

So let's go ahead and install that package clear the screen to make it easier to see then we need to create the folder that we're going to be mounting this file system into.

We'll use the same command as on instance A.

Then we need to edit the FSTAB file to add this file system configuration.

So we'll do that using this command so shudu space nano space forward slash ETC forward slash FSTAB press enter.

Remember this is instance B so it won't have the line that we added on instance A.

So we need to go down to the bottom paste in this placeholder and then we need to replace the file system ID at the start with the actual file system ID.

So delete this leaving the colon and forward slash go back to the EFS console copy the file system ID into your clipboard.

Move back to this instance paste that in everything looks good.

Save that file with control O press enter exit with control X then we back at the prompt clear the screen.

We'll use the shudu mount forward slash EFS forward slash WP hyphen content command again to mount the EFS file system onto this instance and again it's using the configuration that we've just defined in the FSTAB file press enter.

After a few moments you'll be placed back at the prompt we can verify whether this is mounted with DF space hyphen K.

It has mounted by the looks of things it's at the bottom.

So now if we move into that folder so CD forward slash EFS forward slash WP hyphen content forward slash and press enter.

We now in that folder and if we do a listing so LS space hyphen LA what we'll see is the amazing test file dot txt which was created on instance A.

So this proves that this is a shared network file system where any files added on one instance are visible to all other instances.

So EFS is a multi user network based file system that can be mounted on both EC2 Linux instances as well as on premises physical or virtual servers running Linux.

Now this is a simple example of how to use EFS for now we've done everything that we need to do in this demo lesson so we just need to clean up all of the infrastructure that we've used to do that.

Go back to the EFS console we're going to go ahead and delete this file system so we should already have it selected just select delete you'll need to confirm that process by pasting in the file system ID.

So go ahead and put your file system ID and then select confirm.

Now that can take some time to delete and you'll need to wait for this process to complete.

Once it has completed we're going to go ahead and move across to the cloud formation console.

You should still have this open in a tab if you don't just type cloud formation in the search box at the top and then move to the cloud formation console.

You should still have the stack name of implementing EFS which is the stack you created at the start with the one click deployment.

Go ahead and select this stack then click on delete and confirm that deletion and once that finishes deleting that's all of the infrastructure gone that we've created in this demo lesson.

So I hope this has been a fun and enjoyable demo lesson where you've gained some practical experience of working with EFS at this point though that is everything that you need to do in this demo lesson.

So go ahead and complete the video and when you're ready I'll look forward to you joining me in the next.

Résumé de la vidéo [00:00:00][^1^][1] - [00:03:40][^2^][2]:

Le rapport annuel sur l'état de la France (RAEF) 2024, présenté par le Conseil économique, social et environnemental (CESE), se concentre sur la crise démocratique et propose des solutions pour y remédier.

Moments forts: + [00:00:00][^3^][3] Introduction du rapport * Présentation du CESE * Objectif du rapport * Thème de la démocratie + [00:00:27][^4^][4] Structure du rapport * Sondage sur le bien-être * Focus thématiques * Indicateurs pertinents + [00:01:16][^5^][5] Constatations principales * Corrélation entre inégalités et confiance * 75% des citoyens ne font pas confiance aux politiques * 25% ne se sentent pas appartenir à la société + [00:02:00][^6^][6] Approfondissement des inégalités * Analyse fine des inégalités * Importance de la proximité * Compréhension des difficultés spécifiques + [00:02:28][^7^][7] Participation citoyenne * Désir de participation aux décisions * Engagement des citoyens * Importance des sujets techniques + [00:03:01][^8^][8] Vision politique * Besoin de sortir de l'urgence * Construction d'un projet de société * Importance d'une vision à moyen et long terme

Velazquez

DOI: 10.1093/hmg/ddae119

Resource: Mutant Mouse Regional Resource Center (RRID:SCR_002953)

Curator: @AleksanderDrozdz

SciCrunch record: RRID:SCR_002953

What is this?

DOI: 10.1007/s00011-024-01936-y

Resource: None

Curator: @AleksanderDrozdz

SciCrunch record: RRID:MMRRC_050208-UCD

What is this?

DOI: 10.1007/s00011-024-01936-y

Resource: (IMSR Cat# JAX_004781,RRID:IMSR_JAX:004781)

Curator: @AleksanderDrozdz

SciCrunch record: RRID:IMSR_JAX:004781

What is this?

DOI: 10.1007/s00011-024-01936-y

Resource: (IMSR Cat# JAX_029015,RRID:IMSR_JAX:029015)

Curator: @AleksanderDrozdz

SciCrunch record: RRID:IMSR_JAX:029015

What is this?

this sort of creativity can kind of be brute forced. there's X moves possible in space Y and Z data.

Author response:

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

Public Reviews: 

Reviewer #1 (Public Review): 

Summary: 

The main goal of the paper was to identify signals that activate FLP-1 release from AIY neurons in response to H2O2, previously shown by the authors to be an important oxidative stress response in the worm. 

Strengths: 

This study builds upon the authors' previous work (Jia and Sieburth 2021) by further elucidating the gut-derived signaling mechanisms that coordinate the organism-wide antioxidant stress response in C. elegans. 

By detailing how environmental cues like oxidative stress are transduced into gut-derived peptidergic signals, this study represents a valuable advancement in understanding the integrated physiological responses governed by the gut-brain axis. 

This work provides valuable mechanistic insights into the gut-specific regulation of the FLP2 peptide signal. 

Weaknesses: 

Although the authors identify intestinal FLP-2 as the endocrine signal important for regulating the secretion of the neuronal antioxidant neuropeptide, FLP-1, there is no effort made to identify how FLP-2 levels regulate FLP-1 secretion or identify whether this regulation is occurring directly through the AIY neuron or indirectly. This is brought up in the discussion, but identifying a target for FLP-2 in this pathway seems like a crucial missing piece of information in characterizing this pathway. 

We agree that this is an important question. Specifically, identifying the FLP-2 receptor and its site of action is a major priority. Since there are at least four different receptors that have been functionally or physically linked to FLP-2 and there are at least three FLP-2 peptides, unraveling the components acting directly downstream of FLP-2 will require further investigation that we feel is beyond the scope of this current study. We have added a new panel (Fig 1E) addressing the requirements for flp-2 signaling on peroxide production in AIY. These results provide new mechanistic insight into how flp-2 impacts signaling in AIY and a new interpretation of these results has been added to the discussion.

Reviewer #2 (Public Review): 

Summary: 

The core findings demonstrate that the neuropeptide-like protein FLP-2, released from the intestine of C. elegans, is essential for activating the intestinal oxidative stress response. This process is mediated by endogenous hydrogen peroxide (H2O2), which is produced in the mitochondrial matrix by superoxide dismutases SOD-1 and SOD-3. H2O2 facilitates FLP-2 secretion through the activation of protein kinase C family member pkc-2 and the SNAP25 family member aex-4. The study further elucidates that FLP-2 signaling potentiates the release of the antioxidant FLP-1 neuropeptide from neurons, highlighting a bidirectional signaling mechanism between the intestine and the nervous system. 

Strengths: 

This study presents a significant contribution to the understanding of the gut-brain axis and its role in oxidative stress response and significantly advances our understanding of the intricate mechanisms underlying the gut-brain axis's role in oxidative stress response. By elucidating the role of FLP-2 and its regulation by H2O2, the study provides insights into the molecular basis of inter-tissue communication and antioxidant defense in C. elegans. These findings could have broader implications for understanding similar pathways in more complex organisms, potentially offering new targets for therapeutic intervention in diseases related to oxidative stress and aging. 

Weaknesses: 

(1) The experimental techniques employed in the study were somewhat simple and could benefit from the incorporation of more advanced methodologies. 

Thank you for your comment

(2) The weak identification of the key receptors mediating the interaction between FLP-2 and AIY neurons, as well as the receptors in the gut that respond to FLP-1. 

We agree that this is an important question. Specifically, identifying the FLP-2 receptor and its site of action is a major priority. Since there are at least four different receptors that have been functionally or physically linked to FLP-2 and there are at least three FLP-2 peptides, unraveling the components acting directly downstream of FLP-2 will require further investigation that we feel is beyond the scope of this current study.

(3) The study could be improved by incorporating a sensor for the direct measurement of hydrogen peroxide levels. 

We have added a new panel (Fig 1E) addressing the requirements for flp-2 signaling on peroxide production in AIY using the genetically encoded peroxide sensor HyPer7. These results provide new mechanistic insight into how flp-2 impacts signaling in AIY and a new interpretation of these results has been added to the discussion. In addition, we have used HyPer7 to measure peroxide levels in the intestinal mitochondrial matrix and outer membrane (Figs 3, 4, 5, 6)

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors): 

The major missing link in the study is how FLP-2 affects FLP-1 release from AIY: is the effect direct and does it require the previously described FLP-2 receptor FRPR-18? Although this possibility is discussed extensively (L511-528) so it is odd that the effect of an frpr-18 mutation was not tested (or if it was tested, why the results were not reported). If the authors haven't done this experiment (despite doing many less critical experiments) it would be good to know why. 

We agree that this is an important question. Specifically, identifying the FLP-2 receptor and its site of action is a major priority. Since there are at least four different receptors that have been functionally or physically linked to FLP-2 and there are at least three FLP-2 peptides, unraveling the components acting directly downstream of FLP-2 will require further investigation that we feel is beyond the scope of this current study. We have added a new panel (Fig 1E) addressing the requirements for flp-2 signaling on peroxide production in AIY. These results provide new mechanistic insight into how flp-2 impacts signaling in AIY and a new interpretation of these results has been added to the discussion.

Results:

“To address how flp-2 signaling regulates FLP-1 secretion from AIY, we examined H2O2 levels in AIY using a mitochondrially targeted pH-stable H2O2 sensor HyPer7 (mitoHyPer7, Pak et al. 2020). Mito-HyPer7 adopted a punctate pattern of fluorescence in AIY axons, and the average fluorescence intensity of axonal mito-HyPer7 puncta increased about two-fold following 10 minute juglone treatment (Fig 1E), in agreement with our previous studies using HyPer (Jia and Sieburth 2021), confirming that juglone rapidly increases mitochondrial AIY H2O2 levels. flp-2 mutations had no significant effects on the localization or the average intensity of mito-HyPer7 puncta in AIY axons either in the absence of juglone, or in the presence of juglone (Fig 1E), suggesting that flp-2 signaling promotes FLP-1 secretion by a mechanism that does not increase H2O2 levels in AIY. Consistent with this, intestinal overexpression of flp-_2 had no effect on FLP-1::Venus secretion in the absence of juglone, but significantly enhanced the ability of juglone to increase FLP-1 secretion (Fig. 1D). We conclude that both elevated mitochondrial H2O2 levels and intact _flp-2 signaling from the intestine are necessary to increase FLP-1 secretion from AIY.”

More minor comments/suggestions: 

Line 172: No justification is given as to why the authors chose to focus on flp-2 over the other potential candidates identified in their RNAi screen. 

We are currently examining the other neuropeptide hits from the screen, but we have no additional phenotypes to report.

Line 189: An explanation for the use of gDNA as opposed to cDNA should be given. 

We have changed the text in the Results section as follows:

“Expressing a flp-2 genomic DNA (gDNA), fragment (containing both the flp-2a and flp-2b isoforms that arise by alternative splicing), specifically in the nervous system failed to rescue the FLP-1::Venus defects of flp-2 mutants, whereas expressing flp-2 selectively in the intestine fully restored juglone-induced FLP1::Venus secretion to flp-2 mutants (Fig. 1D).”

Line 249-253: nlp-40 and nlp-27 were not implicated in contributing to juglone toxicity in the RNAi screen performed previously by the authors, so it is unclear why both of these peptides are investigated beyond simply being released from the intestine. Confusingly, while Figure S2D shows no overlap between NLP-40 and FLP2, NLP-27 is omitted from the analysis. 

We have clarified that these peptides are not implicated in stress responses, providing a clearer rational for why the serve as controls for specificity.

“Third, nlp-40 and nlp-27 encode neuropeptide-like proteins that are released from the intestine, but are not implicated in stress responses (Liu et al. 2023; Taylor et al. 2021; Wang et al. 2013), and juglone treatment had no detectable effects on coelomocyte fluorescence in animals expressing intestinal NLP-40::Venus or NLP-27::Venus fusion proteins (Fig. S2B and C), and NLP40::mTur2 puncta did not overlap with FLP-2::Venus puncta in the intestine (Fig. S2D).”

Line 262: A more detailed description of juglone's mechanism of action would be welcome here. Is juglone expected to act only in intestinal cells, or is its function more pervasive? 

We have added more detail:

“Juglone generates superoxide anion radicals (Ahmad and Suzuki 2019; Paulsen and Ljungman 2005) and juglone treatment of C. elegans increases ROS levels (de Castro, Hegi de Castro, and Johnson 2004) likely by promoting the global production of mitochondrial superoxide. Superoxide can then be rapidly converted into H2O2 by superoxide dismutase.”

Line 414: Justification for why expulsion frequency is used here to quantify NLP-40 secretion is required, particularly because NLP-40::Venus was already used to quantify NLP-40 secretion via the coelomocyte fluorescence method in the experiments contributing to Figure S2. 

We used expulsion frequency here because (1) it is an easier assay compared to the coelomocyte assay and (2) it is a functional assay. Defective NLP-40 exocytosis manifests as reduced exclusion frequency, therefore if NLP-40 secretion is defective in pkc-2 mutants, nlp-40 mutants should exhibit defects in expulsion frequency.

We have clarified this point:

“To determine whether pkc-2 can regulate the intestinal secretion of other peptides that are not associated with oxidative stress, we examined expulsion frequency, which is a measure of NLP-40 secretion (Mahoney et al. 2008; Wang et al. 2013).”

Line 478: The discussion of neuronally-secreted kisspeptin in this context does not seem relevant as this paper has focused on intestinal peptide secretion. 

We have removed this sentence:

In mammals, release of the RF-amide neuropeptide kisspeptin from the anteroventral periventricular nucleus (AVPV) regulates reproduction by inducing the release of gonadotropins via its stimulatory action on GnRH neurons (Han et al. 2005).

Line 526: DMSR-18 seems to be a typo. Possibly meant FRPR-8, as this is another FLP-2-activated GPCR identified in the screen (though notably, FRPR-8 is only activated by one of the two FLP-2 peptide products) On that note, DMSR-1 has two isoforms, and only one of them is activated by FLP-2 (and only one of the two FLP-2 peptides). This seems relevant to discuss. 

We have corrected the text and we have added to the discussion the number of FLP-2 peptides:

“In addition, certain FLP-2-derived peptides (of which there are at least three) can bind to the GPCRs DMSR-1, or FRPR-8 in transfected cells (Beets et al. 2023). Identifying the relevant FLP-2 peptide(s), the FLP-2 receptor and its site of action will help to define the circuit used by intestinal flp-2 to promote FLP-1 release from AIY.” 

Line 534: An explanation or speculation into why this integration might be necessary would be welcome here. 

We have edited this paragraph:

“FLP-1 release from AIY is positively regulated by H2O2 generated from mitochondria (Jia and Sieburth 2021). Here we showed that H2O2-induced FLP-1 release requires intestinal flp-2 signaling. However, flp-2 does not appear to promote FLP-1 secretion by increasing H2O2 levels in AIY (Fig 1E), and flp-2 signaling is not sufficient to promote FLP-1 secretion in the absence of H2O2 (Fig. 1D). These results point to a model whereby at least two conditions must be met in order for AIY to increase FLP-1 secretion: an increase in H2O2 levels in AIY itself, and an increase in flp-2 signaling from the intestine. Thus AIY integrates stress signals from both the nervous system and the intestine to activate the intestinal antioxidant response through FLP-1 secretion. The requirement of signals from multiple tissues for FLP-1 secretion may function to limit the activation of SKN-1, since unregulated SKN-1 activation can be detrimental to organismal health (Turner, Ramos, and Curran 2024).”

Line 569: Should specify what these candidates are. 

There are 11 proteins with thioredoxin fold domains. We modified the sentence to list one of them.

“There are several thioredoxin-domain containing proteins in addition to trx-3 in the C. elegans genome that could be candidates for this role (e.g. trx-5 and others).”

Line 660: Details about whether the M9 control had an equivalent amount of DMSO as the juglone+M9 condition is required. 

We have performed toxicity assay and neuropeptide release assays comparing M9 DMSO, and Juglone treatment and we have included this new data in Fig S1C, D and S2E. Methods: 

“A stock solution of 50mM juglone in DMSO was freshly made on the same day of liquid toxicity assay. 120μM  working solution of juglone in M9 buffer was prepared using stock solution before treatment. Around 60-80 synchronized adult animals were transferred into a 1.5mL Eppendorf tube with fresh M9 buffer and washed three times, and a final wash was done with either the working solution of juglone with or M9  DMSO at the concentrations present in juglone-treated animals does not contribute to toxicity since DMSO treatment alone caused no significant change in survival compared to M9-treated controls (Fig. S1C).

For coelomocyte imaging, L4 stage animals were transferred in fresh M9 buffer on a cover slide, washed six times with M9 before being exposed to 300μM juglone in M9 buffer (diluted from freshly made 50mM stock solution), 1mM H2O2 in M9 buffer, or M9 buffer. DMSO at the concentrations present in juglone-treated animals does not alter neuropeptide secretion since DMSO treatment alone caused no significant change in FLP-1::Venus or FLP-2::Venus coelomocyte fluorescence compared to M9-treated controls.  (Fig. S1D and S2E).”

Line 1191: Should be FLP-1:Venus in AIY, not the intestine  

Corrected.

In general, the significance of reporting in the figures is very unclear. "a, b, c" to report statistical analysis is confusing in the figure legends, and also unnecessary when they denote non-significance. There are some cases where it is reported that a symbol (eg. ***) denotes statistical significance, but there is no indication of what level of statistical significance the symbol represents (for example, in Figures 2C and 2D) 

Levels of significance was summarized in the end of legend for each figure unless indicated for specific symbols (for example Fig. 1C), we have edited this figure legend: 

“E Representative images and quantification of fluorescence of matrix-targeted HyPer7 in the axon of AIY following M9 or juglone treatment for 10min. Arrowheads denote puncta marked by MLS::HyPer7 fusion proteins (Excitation: 500 and 400nm; emission: 520nm). Ratio of images taken with 500nM (GFP) and 400nM (CFP) for excitation was used to measure H2O2 levels. Unlined *** and ns denote statistical analysis compared to “wild type”. n = 25, 25, 25, 25 independent animals. Scale bar: 10μM.

F Representative images and quantification of average fluorescence in the posterior region of transgenic animals expressing P_gst-4::gfp_ after 4h vehicle M9 or juglone exposure. Asterisks mark the intestinal region used for quantification. P_gst-4::gfp_ expression in the body wall muscles, which appears as fluorescence on the edge animals in some images, was not quantified. Unlined *** and ns denote statistical analysis compared to “wild type”; unlined ## and ### denotes statistical analysis compared to “wild type+juglone”. n = 25, 26, 25, 25, 25, 25, 25, 25 independent animals. Scale bar: 10μM.”

Figure 2C: It is unclear which conditions have H2O2 treatment (as described in the legend). There is also no mention of what ### indicates. 

Levels of significance for ### was summarized in the end of legend, No H2O2 treatment was performed in this assay, we have edited this figure legend: 

“C. Representative images and quantification of average coelomocyte fluorescence of the indicated mutants expressing FLP-2::Venus fusion proteins in the intestine following M9 or juglone treatment for 10min. Unlined *** and ns denote statistical analysis compared to “wild type”. n = 29, 25, 24, 30, 23, 30, 25, 25, 25 independent animals. Scale bar: 5μM.”

Figure 2D: It is not previously mentioned that M9 condition contains DMSO, as implied by the legend. 

We have edited this figure legend:

“D. Quantification of average coelomocyte fluorescence of transgenic animals expressing FLP-2::Venus fusion proteins in the intestine following treatment of fresh M9 buffer or the indicated stressors for 10min. Unlined *** denotes statistical analysis compared to “M9”. n = 23, 25, 25 independent animals.”  

Figure 3J: The y-axis label should more clearly describe the ratio being measured. 

We have updated the panel and this figure legend: 

“J. Schematic, representative images and quantification of fluorescence in the posterior region of the indicated transgenic animals co-expressing mitochondrial matrix targeted HyPer7 (matrix-HyPer7) or mitochondrial outer membrane targeted HyPer7 (OMMHyPer7) with TOMM-20::mCherry following M9 juglone or H2O2 treatment. Ratio of images taken with 500nM (GFP) and 400nM (CFP) for excitation and 520nm for emission was used to measure H2O2 levels. Unlined *** and ns denote statistical analysis compared to “wild type; unlined ## denotes statistical analysis compared to “wild type+juglone”. (top) n = 20, 20, 18, 20, 19, 19, 20, 20 independent animals.

(bottom) n = 20, 20, 19, 20, 20, 20, 20, 20 independent animals. Scale bar: 5μM.” 

Figure S3A: *** is mislabelled. It should be a comparison to wildtype. 

We have edited this figure legend: 

“A. Quantification of average coelomocyte fluorescence of the indicated mutants expressing FLP-2::Venus fusion proteins in the intestine following M9 or juglone treatment for 10min. Unlined *** denotes statistical analysis compared to “wild type”; ### and ns denote statistical analysis compared to “wild type+juglone”. n = 29, 27, 29, 27, 25, 26, 24 independent animals.”  

Reviewer #2 (Recommendations For The Authors): 

(1) The localization experiments could benefit from the application of ultra-high-resolution fluorescence microscopy. This would allow for a more detailed analysis of the spatial distribution of SOD-1/3::GFP in relation to mitochondria-targeted TOMM-20::mCherry fusion proteins in the posterior intestinal region of transgenic animals. 

We agree that high resolution microscopy would be a great way to more precisely localize SOD proteins relative to the mitochondria, and this would enhance understanding of the source of peroxide in this system. We do not conduct this type of microcopy in the lab, so this approach would require a collaboration with a lab that is set up for this. Thus we feel that this is beyond the scope of the current study.  

(2) The paper may note the challenge of directly measuring mitochondrial H2O2 concentrations. However, advancements in chemical or fluorescent sensors for H2O2 detection within mitochondria could provide more direct evidence of its role in FLP-2 secretion. 

We have considered using chemical sensors, but many are either not efficiently taken up by worms (the skin is largely impermeable to all but the most hydrophobic molecules), or they would label peroxide indiscriminately in all tissues making detection specifically in the intestine challenging. We have had good luck with genetically encoded peroxide sensors since they provide tissue specificity and good spatial resolution depending on where we target them. We have added imaging results for HyPer7 in the AIY neuron to Figure 1E. 

Results:

“To address how flp-2 signaling regulates FLP-1 secretion from AIY, we examined H2O2 levels in AIY using a mitochondrially targeted pH-stable H2O2 sensor HyPer7 (mitoHyPer7, Pak et al. 2020). Mito-HyPer7 adopted a punctate pattern of fluorescence in AIY axons, and the average fluorescence intensity of axonal mito-HyPer7 puncta increased about two-fold following 10 minute juglone treatment (Fig 1E), in agreement with our previous studies using HyPer (Jia and Sieburth 2021), confirming that juglone rapidly increases mitochondrial AIY H2O2 levels. flp-2 mutations had no significant effects on the localization or the average intensity of mito-HyPer7 puncta in AIY axons either in the absence of juglone, or in the presence of juglone (Fig 1E), suggesting that flp-2 signaling promotes FLP-1 secretion by a mechanism that does not increase H2O2 levels in AIY. Consistent with this, intestinal overexpression of flp-_2 had no effect on FLP-1::Venus secretion in the absence of juglone, but significantly enhanced the ability of juglone to increase FLP-1 secretion (Fig. 1D). We conclude that both elevated mitochondrial H2O2 levels and intact _flp-2 signaling from the intestine are necessary to increase FLP-1 secretion from AIY.” 

(3) To confirm the activation of AIY neurons by FLP-2, measuring calcium activity in these neurons may be a robust approach. It would be beneficial to determine if synthetic FLP-2 can activate AIY neurons and subsequently induce an intestinal antioxidant response. 

This is a great idea. We have begun to examine GCaMP fluorescence in AIY and we see responses to oxidative stressors. We think that this data is too preliminary at the moment to include here.  

(4) The identification of the key receptors mediating the interaction between FLP-2 and AIY neurons, as well as the receptors in the gut that respond to FLP-1, would complete the signaling pathway and strengthen the study's conclusions. 

We agree that this is an important question. Specifically, identifying the FLP-2 receptor and its site of action is a major priority. Since there are at least four different receptors that have been functionally or physically linked to FLP-2 and there are at least three FLP-2 peptides, unraveling the components acting directly downstream of FLP-2 will require further investigation that we feel is beyond the scope of this current study.  

(5) Investigating whether direct manipulation of AIY neurons, through methods such as optogenetic activation or inhibition, can trigger the gut's antioxidant response would provide insight into the functional relevance of this neuronal activity. 

Also an excellent idea. We previously published that Channelrhodopsin activation specifically in AIY indeed increases FLP-1 secretion, but we have not yet examined its effects on antioxidant responses in the intestine.  This may require a more sustained activation of AIY than Channelrhodopsin can provide.

(6) For the analysis of intestinal Pges-1::GFP fluorescence, specifying the region of interest would enhance the precision of the data and the reproducibility of the results. 

We analyze fluorescence intensity of a 16-pixel diameter circle in the posterior intestine (as indicated by the asterisks) and we have added this to the methods, we edited this paragraph:

“or transcriptional reporter imaging, young adult animals with indicated genotype were transferred into a 1.5mL Eppendorf tube with M9 buffer, washed three times and incubated in M9 buffer or 60uM working solution of juglone for 1h in dark on rotating mixer before recovering on fresh NGM plates with OP50 for 3h in dark at 20°C. The posterior end of the intestine was imaged with the 60x objective and quantification for average fluorescence intensity of a 16-pixel diameter circle in the posterior intestine was calculated using Metamorph.”

(7) Assessing the potential for pharmacological modulation of FLP-2 or H2O2 levels could provide valuable insights into therapeutic strategies aimed at enhancing the oxidative stress response. 

Agreed.

(8) For improved clarity, it is suggested that the schematic currently presented in Figure S1A be integrated into Figure 2C, as this would facilitate the reader's comprehension of the experimental design and findings. 

Moved.

Résumé de la vidéo [00:00:03][^1^][1] - [00:27:07][^2^][2]:

Cette vidéo explore comment le racisme peut influencer les décisions de vote, souvent contre nos propres intérêts. Samah Karaki discute des mécanismes psychologiques et sociaux derrière ce phénomène.

Moments forts: + [00:00:03][^3^][3] Introduction * Présentation de Laeticia et de sa situation familiale * Stress et angoisse liés à son fils de 5 ans * Difficultés quotidiennes rencontrées par la famille + [00:04:00][^4^][4] Burnout parental * Augmentation du phénomène en France * Témoignages de parents épuisés * Solutions proposées, comme le relais parental + [00:10:00][^5^][5] Analyse psychologique * Intervention de Lilian Olstein, psychanalyste * Importance des limites dans l'éducation des enfants * Conséquences du manque de structure + [00:15:00][^6^][6] Campagne en Belgique * Initiatives belges pour prévenir l'épuisement parental * Témoignages de parents en thérapie * Stratégies pour gérer le stress parental + [00:20:00][^7^][7] Groupes de parole * Création de groupes de soutien pour parents * Témoignages de participants * Importance de briser le tabou autour de l'épuisement parental

Résumé de la vidéo [00:27:09][^1^][1] - [00:53:49][^2^][2]:

Cette vidéo explore les défis et les réalités de la parentalité moderne, en mettant en lumière les expériences de plusieurs parents qui jonglent entre vie professionnelle et familiale.

Moments forts: + [00:27:09][^3^][3] Alba et la parentalité positive * Désir d'avoir une famille nombreuse * Pratique de l'éducation positive * Équilibre entre vie professionnelle et familiale + [00:31:02][^4^][4] La charge mentale des mères actives * Stress et organisation quotidienne * Impact sur la santé mentale * Témoignages de mères surmenées + [00:37:01][^5^][5] Le relais parental pour parents épuisés * Services offerts par le relais parental * Témoignages de parents en burnout * Importance du soutien professionnel + [00:44:01][^6^][6] Les défis des parents d'enfants hyperactifs * Gestion des crises et des comportements impulsifs * Stratégies d'adaptation des parents * Impact sur la vie familiale + [00:51:00][^7^][7] Burnout parental et soutien familial * Témoignages de parents en détresse * Importance du soutien familial * Stratégies pour surmonter le burnout

Résumé de la vidéo [00:53:52][^1^][1] - [01:22:25][^2^][2]:

Cette vidéo aborde les défis du burnout parental et les solutions pour y faire face. Elle suit plusieurs parents, notamment Laeticia et David, dans leur parcours pour retrouver un équilibre familial.

Moments forts: + [00:53:52][^3^][3] Défis parentaux modernes * Parents se sentent fragiles * Burnout parental fréquent * Importance de la parole parentale + [00:55:00][^4^][4] Expériences personnelles * Témoignages de parents * Difficultés de communication * Impact sur la relation parent-enfant + [00:57:00][^5^][5] Groupes de soutien * Importance des groupes de parole * Stratégies pour reprendre pied * Exercice de réflexion sur les besoins personnels + [01:00:00][^6^][6] Adaptation des enfants * Enfants testant les limites * Importance de l'autorité bienveillante * Activités structurées comme le yoga + [01:06:00][^7^][7] Temps pour soi * Importance de prendre du temps pour soi * Activités pour se détendre * Impact positif sur la santé mentale

Résumé de la vidéo [01:22:27][^1^][1] - [01:37:07][^2^][2]:

Cette vidéo explore comment le racisme peut influencer les décisions de vote, même contre nos propres intérêts. Elle met en lumière des exemples concrets et des analyses psychologiques pour expliquer ce phénomène.

Moments forts: + [01:22:27][^3^][3] Impact du bien-être parental sur les enfants * Les parents en bonne santé mentale influencent positivement leurs enfants * Les mamans célibataires prennent plus de temps à se remettre de l'épuisement parental * Importance de la santé mentale des parents + [01:23:00][^4^][4] Stratégies pour gérer l'hyperactivité * Utilisation de caméras pour analyser les crises familiales * Mise en place de stratégies pour surmonter les crises * Importance de la communication et de la compréhension mutuelle + [01:26:00][^5^][5] Neurofeedback dynamique pour l'hyperactivité * Nouvelle méthode venue du Canada * Utilisation de capteurs pour rééduquer l'activité cérébrale * Résultats positifs observés chez l'enfant + [01:29:27][^6^][6] Retour au travail après un burnout parental * Difficultés rencontrées par Alba après son retour * Stratégies pour gérer le stress et la fatigue * Importance du soutien au travail et à la maison + [01:34:43][^7^][7] Changement de comportement chez un enfant difficile * Impact positif du relais parental * Importance de la distance émotionnelle pour les parents * Amélioration du comportement de l'enfant et du bien-être familial

Author response:

eLife Assessment

This valuable study uses consensus-independent component analysis to highlight transcriptional components (TC) in high-grade serous ovarian cancers (HGSOC). The study presents a convincing preliminary finding by identifying a TC linked to synaptic signaling that is associated with shorter overall survival in HGSOC patients, highlighting the potential role of neuronal interactions in the tumor microenvironment. This finding is corroborated by comparing spatially resolved transcriptomics in a small-scale study; a weakness is in being descriptive, non-mechanistic, and requiring experimental validation.

We sincerely thank the editors for the valuable and constructive feedback. We appreciate the recognition of our findings and the significance of identifying transcriptional components in high-grade serous ovarian cancers. We acknowledge the insightful point on our study's descriptive nature and limited mechanistic depth. While further experimental validation would indeed enhance our conclusions, such work extends beyond the current scope of this manuscript. However, we would like to highlight that mechanistic studies demonstrating the impact of tumor-infiltrating nerves on disease progression are emerging (Zahalka et al., 2017; Allen et al., 2018; Balood et al., 2022; Jin et al., 2022; Globig et al., 2023; Restaino et al., 2023; Darragh et al., 2024). Importantly, members of our group have contributed to these findings. These studies, including in vitro and in vivo work in head and neck squamous cell carcinoma as well as high-grade serous ovarian carcinoma, demonstrate that substance P released from tumor-infiltrating nociceptors potentiates MAP kinase signaling in cancer cells, thereby influencing disease progression. This effect can be mitigated in vivo by blocking the substance P receptor (Restaino et al., 2023). Our present work identifies a transcriptional component that aligns with the presence of functional nerves within malignancies. These published mechanistic studies support our findings and suggest that this transcriptional component could serve as a potential screening tool to identify innervated tumors. Such information is clinically relevant, as patients with innervated tumors may benefit from more aggressive therapy.

Reviewer #1 (Public review):

This manuscript explores the transcriptional landscape of high-grade serous ovarian cancer (HGSOC) using consensus-independent component analysis (c-ICA) to identify transcriptional components (TCs) associated with patient outcomes. The study analyzes 678 HGSOC transcriptomes, supplemented with 447 transcriptomes from other ovarian cancer types and noncancerous tissues. By identifying 374 TCs, the authors aim to uncover subtle transcriptional patterns that could serve as novel drug targets. Notably, a transcriptional component linked to synaptic signaling was associated with shorter overall survival (OS) in patients, suggesting a potential role for neuronal interactions in the tumor microenvironment. Given notable weaknesses like lack of validation cohort or validation using another platform (other than the 11 samples with ST), the data is considered highly descriptive and preliminary.

Strengths:

(1) Innovative Methodology:

The use of c-ICA to dissect bulk transcriptomes into independent components is a novel approach that allows for the identification of subtle transcriptional patterns that may be overshadowed in traditional analyses.

We sincerely thank the reviewer for recognizing the strengths and novelty of our study. We appreciate the positive feedback on our use of consensus-independent component analysis (c-ICA) to decompose bulk transcriptomes, which we believe allowed us to detect subtle transcriptional signals often overlooked in traditional analyses.

(2) Comprehensive Data Integration:

The study integrates a large dataset from multiple public repositories, enhancing the robustness of the findings. The inclusion of spatially resolved transcriptomes adds a valuable dimension to the analysis.

Thank you for recognizing the robustness of our study through comprehensive data integration. We appreciate the acknowledgment of our efforts to leverage a large, multi-source dataset, as well as the additional insights gained from spatially resolved transcriptomes. We believe this integrative approach enhances the depth of our analysis and contributes to a more nuanced understanding of the tumor microenvironment.

(3) Clinical Relevance:

The identification of a synaptic signaling-related TC associated with poor prognosis highlights a potential new avenue for therapeutic intervention, emphasizing the role of the tumor microenvironment in cancer progression.

We appreciate the reviewer’s recognition of the clinical implications of our findings. The identification of a synaptic signaling-related transcriptional component associated with poor prognosis underscores the potential for novel therapeutic targets within the tumor microenvironment. We agree that this insight could open new avenues for intervention and further highlights the role of neuronal interactions in cancer progression.

Weaknesses:

(1) Mechanistic Insights:

While the study identifies TCs associated with survival, it provides limited mechanistic insights into how these components influence cancer progression. Further experimental validation is necessary to elucidate the underlying biological processes.

We appreciate the reviewer’s point regarding the limited mechanistic insights provided in our study. We agree that further experimental validation would enhance our understanding of how the biology captured by these transcriptional components influence cancer progression. However, we respectfully note that such validation is beyond the current scope of this article.   Our current analyses are done on publicly available expression array and spatial transcriptomic array datasets. For future studies, we therefore intend to combine spatial transcriptomic data with immunohistochemical analysis of the same tumors for validation purposes. We have started with setting up in vitro cocultures of neurons and ovarian cancer cells to obtain mechanistic insight in how genes with a large weight in TC121 regulate synaptic signaling and how that affects ovarian cancer cells.

(2) Generalizability:

The findings are primarily based on transcriptomic data from HGSOC. It remains unclear how these results apply to other subtypes of ovarian cancer or different cancer types.

In Figure 5, we present the activity of TC121 across various cancer types, demonstrating broader applicability. However, due to limited treatment response data, we were unable to assess associations between TC activity scores and patient response. Additionally, transcriptomic and survival data specific to other ovarian cancer subtypes beyond HGSOC are currently not available, limiting our ability to generalize these findings to those groups. We intend to leverage survival data from TCGA to explore associations between TC activity scores and overall survival of patients with other cancer types. Nonetheless, we recognize limitations with TCGA survival data, as outlined in this article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8726696/.

(3) Innovative Methodology:

Requires more validation using different platforms (IHC) to validate the performance of this bulk-derived data. Also, the lack of control over data quality is a concern.

We acknowledge the reviewer’s suggestion to validate our results with alternative platforms, such as IHC; however, we regret that such validation is beyond the scope of this article. Regarding data quality control, we implemented a series of checks:

• Bulk Transcriptional Profiles: We applied principal component analysis (PCA) on the sample Pearson product-moment correlation matrix, focusing on the first principal component (PCqc), which accounted for approximately 80-90% of the variance, primarily reflecting technical rather than biological variability  (Bhattacharya et al., 2020). Samples with a correlation below 0.8 with PCqc were removed as outliers. Additionally, we generated unique MD5 hashes for each CEL file to identify and exclude duplicate samples. Per gene, expression values were standardized to a mean of zero and a variance of one across the GEO, CCLE, GDSC, and TCGA datasets to minimize probeset- or gene-specific variability.

• Spatial Transcriptional Profiles: We used PCA for quality control here as well, retained samples only if their loading factors for the first principal component showed consistent signs across all profiles (i.e., all profiles had either positive or negative loading factors for the first PC) from that individual spatial transcriptomic sample. Samples that did not meet this criterion were excluded from analyses.

(4) Clinical Application:

Although the study suggests potential drug targets, the translation of these findings into clinical practice is not addressed. Probably given the lack of some QA/QC procedures it'll be hard to translate these results. Future studies should focus on validating these targets in clinical settings.

While this study is exploratory in nature, we agree that future studies should focus on validating these potential drug targets in clinical settings. As suggested, QA/QC procedures were integral to our analyses. We applied rigorous quality control, including PCA-based checks and duplicate removal across datasets, to ensure data integrity (detailed in our previous response).

In terms of clinical application, which we partially discussed in the manuscript, we will discuss additional strategies to prevent synaptic signaling and neurotransmitter release in the tumor microenvironment (TME). Drugs such as ifenprodil and lamotrigine are used in treating neuronal disorders to block glutamate release responsible for subsequent synaptic signaling, whereas the vesicular monoamine transporter (VMAT) inhibitor reserpine can block the formation of synaptic vesicles (Reid et al., 2013; Williams et al., 2001). Previous in vitro studies with HGSOC cell lines showed a significant effect of ifenprodil alone on cancer cell proliferation, whereas reserpine seemed to trigger apoptosis in cancer cells (North et al., 2015; Ramamoorthy et al., 2019). Such strategies could potentially be used to inhibit synaptic neurotransmission in the TME.

Reviewer #2 (Public review):

Summary:

Consensus-independent component analysis and closely related methods have previously been used to reveal components of transcriptomic data that are not captured by principal component or gene-gene coexpression analyses.

Here, the authors asked whether applying consensus-independent component analysis (c-ICA) to published high-grade serous ovarian cancer (HGSOC) microarray-based transcriptomes would reveal subtle transcriptional patterns that are not captured by existing molecular omics classifications of HGSOC.

Statistical associations of these (hitherto masked) transcriptional components with prognostic outcomes in HGSOC could lead to additional insights into underlying mechanisms and, coupled with corroborating evidence from spatial transcriptomics, are proposed for further investigation.

This approach is complementary to existing transcriptomics classifications of HGSOC.

The authors have previously applied the same approach in colorectal carcinoma (Knapen et al. (2024) Commun. Med).

Strengths:

(1) Overall, this study describes a solid data-driven description of c-ICA-derived transcriptional components that the authors identified in HGSOC microarray transcriptomics data, supported by detailed methods and supplementary documentation.

We thank the reviewer for acknowledging the strength of our data-driven approach and the use of consensus-independent component analysis (c-ICA) to identify transcriptional components within HGSOC microarray data. We aimed to provide comprehensive methodological detail and supplementary documentation to support the reproducibility and robustness of our findings. We believe this approach allows for the identification of subtle transcriptional signals that might be overlooked by traditional analysis methods.

(2) The biological interpretation of transcriptional components is convincing based on (data-driven) permutation analysis and a suite of analyses of association with copy-number, gene sets, and prognostic outcomes.

We appreciate the reviewer’s positive feedback on the biological interpretation of our transcriptional components. We are pleased that our approach, which includes data-driven permutation testing and analyses of associations with copy-number alterations, gene sets, and prognostic outcomes, was found convincing. These analyses were integral to enhancing the robustness and biological relevance of our findings.

(3) The resulting annotated transcriptional components have been made available in a searchable online format.

Thank you for acknowledging the availability of our annotated transcriptional components in a searchable online format.

(4) For the highlighted transcriptional component which has been annotated as related to synaptic signalling, the detection of the transcriptional component among 11 published spatial transcriptomics samples from ovarian cancers appears to support this preliminary finding and requires further mechanistic follow-up.

Thank you for acknowledging the accessibility of our annotated transcriptional components. We prioritized making these data available in a searchable online format to facilitate further research and enable the community to explore and validate our findings.

Weaknesses:

(1) This study has not explicitly compared the c-ICA transcriptional components to the existing reported transcriptional landscape and classifications for ovarian cancers (e.g. Smith et al Nat Comms 2023; TCGA Nature 2011; Engqvist et al Sci Rep 2020) which would enable a further assessment of the additional contribution of c-ICA - whether the c-ICA approach captured entirely complementary components, or whether some components are correlated with the existing reported ovarian transcriptomic classifications.

We appreciate the reviewer’s insightful suggestion to compare our c-ICA-derived transcriptional components with previously reported ovarian cancer classifications, such as those from Smith et al. (2023), TCGA (2011), and Engqvist et al. (2020). To address this, we will incorporate analyses comparing the activity scores of our transcriptional components with these published landscapes and classifications, particularly focusing on any associations with overall survival. Additionally, we plan to evaluate correlations between gene signatures from these studies and our identified TCs, enhancing our understanding of the unique contributions of the c-ICA approach.

(2) Here, the authors primarily interpret the c-ICA transcriptional components as a deconvolution of bulk transcriptomics due to the presence of cells from tumour cells and the tumour microenvironment. However, c-ICA is not explicitly a deconvolution method with respect to cell types: the transcriptional components do not necessarily correspond to distinct cell types, and may reflect differential dysregulation within a cell type. This application of c-ICA for the purpose of data-driven deconvolution of cell populations is distinct from other deconvolution methods that explicitly use a prior cell signature matrix.

Thank you for highlighting this nuanced aspect of c-ICA interpretation. We acknowledge that c-ICA, unlike traditional deconvolution methods, is not specifically designed for cell-type deconvolution and does not rely on a predefined cell signature matrix. While we explored the transcriptional components in the context of tumor and microenvironmental interactions, we agree that these components may not correspond directly to distinct cell types but rather reflect complex patterns of dysregulation, potentially within individual cell populations.

Our goal with c-ICA was to uncover hidden transcriptional patterns possibly influenced by cellular heterogeneity. However, we recognize these patterns may also arise from regulatory processes within a single cell type. To investigate further, we plan to use single-cell transcriptional data (~60,000 cell-types annotated profiles from GSE158722) and project our transcriptional components onto these profiles to obtain activity scores, allowing us to assess each TC’s behavior across diverse cellular contexts after removing the first principal component to minimize background effects.

References

Allen JK, Armaiz-Pena GN, Nagaraja AS, Sadaoui NC, Ortiz T, Dood R, Ozcan M, Herder DM, Haemerrle M, Gharpure KM, Rupaimoole R, Previs R, Wu SY, Pradeep S, Xu X, Han HD, Zand B, Dalton HJ, Taylor M, Hu W, Bottsford-Miller J, Moreno-Smith M, Kang Y, Mangala LS, Rodriguez-Aguayo C, Sehgal V, Spaeth EL, Ram PT, Wong ST, Marini FC, Lopez-Berestein G, Cole SW, Lutgendorf SK, diBiasi M, Sood AK. 2018. Sustained adrenergic signaling promotes intratumoral innervation through BDNF induction. Cancer Res 78:canres.1701.2016.

Balood M, Ahmadi M, Eichwald T, Ahmadi A, Majdoubi A, Roversi Karine, Roversi Katiane, Lucido CT, Restaino AC, Huang S, Ji L, Huang K-C, Semerena E, Thomas SC, Trevino AE, Merrison H, Parrin A, Doyle B, Vermeer DW, Spanos WC, Williamson CS, Seehus CR, Foster SL, Dai H, Shu CJ, Rangachari M, Thibodeau J, Rincon SVD, Drapkin R, Rafei M, Ghasemlou N, Vermeer PD, Woolf CJ, Talbot S. 2022. Nociceptor neurons affect cancer immunosurveillance. Nature 611:405–412.

Bhattacharya A, Bense RD, Urzúa-Traslaviña CG, Vries EGE de, Vugt MATM van, Fehrmann RSN. 2020. Transcriptional effects of copy number alterations in a large set of human cancers. Nat Commun 11:715.

Darragh LB, Nguyen A, Pham TT, Idlett-Ali S, Knitz MW, Gadwa J, Bukkapatnam S, Corbo S, Olimpo NA, Nguyen D, Court BV, Neupert B, Yu J, Ross RB, Corbisiero M, Abdelazeem KNM, Maroney SP, Galindo DC, Mukdad L, Saviola A, Joshi M, White R, Alhiyari Y, Samedi V, Bokhoven AV, John MSt, Karam SD. 2024. Sensory nerve release of CGRP increases tumor growth in HNSCC by suppressing TILs. Med 5:254-270.e8.

Globig A-M, Zhao S, Roginsky J, Maltez VI, Guiza J, Avina-Ochoa N, Heeg M, Hoffmann FA, Chaudhary O, Wang J, Senturk G, Chen D, O’Connor C, Pfaff S, Germain RN, Schalper KA, Emu B, Kaech SM. 2023. The β1-adrenergic receptor links sympathetic nerves to T cell exhaustion. Nature 622:383–392.

Jin M, Wang Y, Zhou T, Li W, Wen Q. 2022. Norepinephrine/β2-adrenergic receptor pathway promotes the cell proliferation and nerve growth factor production in triple-negative breast cancer. J Breast Cancer 26:268–285.

!!!

Es muy interesante que el gobierno da el dinero a las madres y las mujeres jefas de hogar, especialmente porque a la desigualdad de género en America Latin. Pero es muy bueno y es una forma de empoderamiento de las mujeres.

Esto es uno critico que tiene verdad, pero pienso que el dinero es demasiado pequeño para crear la dependencia especialmente para las personas a sobrevivir sin otros fuentes de dinero

Esta es una idea muy buena porque muchos otros programas sociales de otros países excluyen las personas sin residencias en el proceso del programa. Entonces, me parece este enfoque es muy interesante e impactante

Es muy bueno que este programa va dirigido los niños y las madres que son más vulnerables a la pobreza

consider using a piece of punctuation here to indicate that the reader should take a breath here (since this is a clause-marker "and", not a listing "and")

Say something about what you'd charge to get to 250k ARR. Something like:

"We'll charge $x, expect to convert y% of them, and aim to reach at least $z ARR by June next year."

Focus on the most recent launch, rather than the number of times you've launched.

"We launched our latest version x weeks ago and have made $500 in revenue from y users. We're working towards an update in z weeks based on feedback received from the launch."

(It's not a problem if y is small.)

Say something specific about why you would move to SF. Say for talent, proximity to customers, or something else.

Also it's a bit clearer to say you're considering it than "you understand the need".

"We're considering staying in SF after YC for x and y."

.h2

DOI: 10.1038/s41598-024-78442-y

Resource: (NCI-DTP Cat# IGR-OV1, RRID:CVCL_1304)

Curator: @evieth

SciCrunch record: RRID:CVCL_1304

What is this?

DOI: 10.1038/s41598-024-78442-y

Resource: (NCI-DTP Cat# OVCAR-8, RRID:CVCL_1629)

Curator: @scibot

SciCrunch record: RRID:CVCL_1629

What is this?

DOI: 10.1038/s41598-024-78442-y

Resource: (CLS Cat# 300342/p657_SK-OV-3, RRID:CVCL_0532)

Curator: @scibot

SciCrunch record: RRID:CVCL_0532

What is this?

DOI: 10.1007/s12195-024-00817-y

Resource: (CLS Cat# 300342/p657_SK-OV-3, RRID:CVCL_0532)

Curator: @evieth

SciCrunch record: RRID:CVCL_0532

What is this?

DOI: 10.1007/s12195-024-00817-y

Resource: South Dakota State University Functional Genomics Core Facility (RRID:SCR_023786)

Curator: @scibot

SciCrunch record: RRID:SCR_023786

What is this?

DOI: 10.1007/s12195-024-00817-y

Resource: (RRID:CVCL_1345)

Curator: @scibot

SciCrunch record: RRID:CVCL_1345

What is this?

DOI: 10.1186/s12964-024-01917-y

Resource: (Santa Cruz Biotechnology Cat# sc-2054, RRID:AB_631748)

Curator: @scibot

SciCrunch record: RRID:AB_631748

What is this?

DOI: 10.1186/s12964-024-01917-y

Resource: (Thermo Fisher Scientific Cat# ICN623411, RRID:AB_2334981)

Curator: @scibot

SciCrunch record: RRID:AB_2334981

What is this?

DOI: 10.1186/s12964-024-01917-y

Resource: None

Curator: @scibot

SciCrunch record: RRID:AB_1720366

What is this?

DOI: 10.1186/s12964-024-01917-y

Resource: (RRID:CVCL_0419)

Curator: @scibot

SciCrunch record: RRID:CVCL_0419

What is this?

DOI: 10.1186/s12964-024-01917-y

Resource: (Cell Signaling Technology Cat# 89326, RRID:AB_2721818)

Curator: @scibot

SciCrunch record: RRID:AB_2721818

What is this?

DOI: 10.1186/s12964-024-01917-y

Resource: (ATCC Cat# CRL-4010, RRID:CVCL_3383)

Curator: @scibot

SciCrunch record: RRID:CVCL_3383

What is this?

DOI: 10.1186/s12964-024-01917-y

Resource: (Cell Signaling Technology Cat# 14997, RRID:AB_2721812)

Curator: @scibot

SciCrunch record: RRID:AB_2721812

What is this?

DOI: 10.1186/s12964-024-01917-y

Resource: (Cell Signaling Technology Cat# 8457, RRID:AB_10950489)

Curator: @scibot

SciCrunch record: RRID:AB_10950489

What is this?

DOI: 10.1186/s12964-024-01917-y

Resource: (ECACC Cat# 90112714, RRID:CVCL_0035)

Curator: @scibot

SciCrunch record: RRID:CVCL_0035

What is this?

DOI: 10.1186/s12964-024-01917-y

Resource: (NCI-DTP Cat# MCF7, RRID:CVCL_0031)

Curator: @scibot

SciCrunch record: RRID:CVCL_0031

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how is her grandmother able to have such a forgiving attitude?

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langley virginia cia

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AI Overview

Learn more...

The Central Intelligence Agency (CIA) headquarters is located in Langley, Virginia, in the George Bush Center for Intelligence: 

• Location

  The headquarters is in the unincorporated community of Langley, in Fairfax County, Virginia, near Washington, D.C. 

The area where the CIA is located was originally known as Langley before the town of McLean was founded in 1910. The name "Langley" is still used to describe the McLean neighborhood where the CIA is located. 

• CIA Museum - CIA

  CIA

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Explore CIA Headquarters

CIA (.gov)

https://www.cia.gov › legacy › headquarters

](https://www.cia.gov/legacy/headquarters/)

Located in Langley, Virginia, CIA Headquarters includes the Original Headquarters Building, the New Headquarters Building, and the Grounds. Since 1961, this ...

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George Bush Center for Intelligence

Wikipedia

https://en.wikipedia.org › wiki › George_Bush_Center_...

](https://en.wikipedia.org/wiki/George_Bush_Center_for_Intelligence)

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](https://en.wikipedia.org/wiki/George_Bush_Center_for_Intelligence)

The George Bush Center for Intelligence is the headquarters of the Central Intelligence Agency, located in the unincorporated community of Langley in Fairfax ...

‎Name - ‎History - ‎Location and facilities

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Central Intelligence Agency

Wikipedia

https://en.wikipedia.org › wiki › Central_Intelligence_A...

](https://en.wikipedia.org/wiki/Central_Intelligence_Agency)

The agency is headquartered in the George Bush Center for Intelligence in Langley, Virginia. Central Intelligence Agency. Seal of the Central Intelligence ...

‎George Bush Center - ‎Langley, Virginia - ‎Organizational structure of the...

[

50 Years in Langley: Recollections of the Construction ...

CIA (.gov)

https://cia.gov › resources › csi › books-monographs

](https://cia.gov/resources/csi/books-monographs/50-years-in-langley-recollections-of-the-construction-of-cias-original-headquarters-building/)

In late 1961, CIA employees began relocating from a disparate collection of buildings in Washington, DC, to a newly constructed headquarters complex in Langley, ...

[

CIA Museum

CIA (.gov)

https://www.cia.gov › legacy › museum

](https://www.cia.gov/legacy/museum/)

Although the CIA Museum is not open to the public because of its location at CIA Headquarters in Langley, Virginia, we can do the next best thing and open ...

‎Exhibits - ‎Artifacts - ‎Radio Receiver Concealment - ‎Concealed Compass

[

People often ask if #CIA Headquarters is in Langley or ...

Instagram - cia

4.9K+ likes - 11 months ago

](https://www.instagram.com/cia/p/Czt3MPFsmsA/?hl=en)

The answer? Technically, both! The town of McLean was founded in 1910, but before then, the area where CIA HQ is located was known as Langley.

[

50 Years in Langley

CIA (.gov)

https://www.cia.gov › resources › csi › static › 5...

](https://www.cia.gov/resources/csi/static/f4b6cf75ca9efcabacc393400eb01dd8/50-Years-in-Langley.pdf)

PDF

In late 1961, CIA employees began relocating from a disparate collection of buildings in Washington,. DC, to a newly constructed headquarters complex in ...

30 pages

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Aerial view of the CIA Headquarters, Langley, Virginia

Library of Congress (.gov)

https://www.loc.gov › item

](https://www.loc.gov/item/2011632954/)

Aerial view of the CIA Headquarters, Langley, Virginia. View Enlarged Image [ digital file from original ] Download: JPEG (286x188px)

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CIA Headquarters in Langley, Virginia, is known as the ...

Instagram - cia

8.2K+ likes - 1 year ago

](https://www.instagram.com/cia/p/Cx-5rHHMM4k/)

CIA Headquarters in Langley, Virginia, is known as the home to one of the world's top spy agencies. However, you might be surprised to learn that our campus ...

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https://www.identiv.com/resources/blog/the-worlds-...

](https://www.identiv.com/resources/blog/the-worlds-most-secure-buildings-cia-headquarters-in-langley-virginia)

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Identiv

https://www.identiv.com › resources › blog › the-worlds-...

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1000 colonial farm road, langley, fairfax county, virginia

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• Buildings

  The headquarters is made up of the Original Headquarters Building (OHB) and the New Headquarters Building (NHB). 

  • Security

  The CIA campus is one of the world's most secure buildings. The CIA's security practices are top secret, but some details are known, such as the use of top security clearance for architects involved in the project. 

  • Courtyard

  The courtyard is a popular place for lunch, breaks, and special events. It features a grassy lawn, fishpond, and flowering plants and trees. 

  • Museum

  The CIA Museum has artifacts such as a robotic bug, a makeup compact with a secret message, and an A-12 OXCART reconnaissance aircraft. The museum also features exhibits about the hunt for Bin Ladin, the Berlin Tunnel, and Project AZORIAN. 

  George Bush Center for Intelligence - Wikipedia

  The George Bush Center for Intelligence is the headquarters of the Central Intelligence Agency, located in the unincorporated comm...

  Wikipedia

  Central Intelligence Agency - Wikipedia

  The Central Intelligence Agency (CIA /ˌsiː.aɪˈeɪ/), known informally as the Agency, metonymously as Langley and historically as th...

  Wikipedia

Author response:

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

Public Reviews: 

Reviewer #1 (Public Review): 

Summary: 

This study explores the therapeutic potential of KMO inhibition in endometriosis, a condition with limited treatment options. 

Strengths: 

KNS898 is a novel specific KMO inhibitor and is orally bioavailable, providing a convenient and non-hormonal treatment option for endometriosis. The promising efficacy of KNS898 was demonstrated in a relevant preclinical mouse model of endometriosis with pathological and behavioural assessments performed. 

Weaknesses: 

(1) The expression of KMO in human normal endometrium and endometrial lesions was not quantified. Western blot or quantification of IHC images will provide valuable insight.

Given the differential expression of KMO in luminal epithelial cells lining the endometrial glands compared to the other parts of the endometrium, a general endometrial Western Blot prep is not going to be additionally helpful or accurate in addressing this question, without e.g. laser capture microdissection or single cell quantitative proteomics. Furthermore, KMO is a flavin-dependent monooxygenase and the activity, especially generating the oxidative stressor product 3-hydroxykynurenine is far more dependent on kynurenine substrate availability than it is on actual enzyme abundance - although it is important to show (as we have done), that KMO is present in the human endometrial glands and in human distended endometrial gland-like structures (DEGLS).

If KMO is not overexpressed in diseased tissues i.e. it may have homeostatic roles, and inhibition of KMO may have consequences on general human health and wellbeing.

KMO certainly does have important homeostatic roles, for example as key step in the repletion of NAD+ through de novo synthesis. Although with good nutrition and sufficient NAD+ precursors in the diet e.g. niacin, that specific role may be partially redundant. KMO knockout mice exhibit normal fertility and fecundity and do not show a survival deficit compared to littermate wildtype controls (e.g. Mole et al Nature Medicine 2016). To further develop KNS898 towards clinical use, preclinical GLP safety and toxicology studies and human Phase 1 clinical trials will of course need to be completed, but that is standard for the development of any new drug

In addition, KMO expression in control mice was not shown or quantified.

Control mice that were not inoculated intraperitoneally with endometrial fragments did not develop DEGLS and therefore there is nothing to show or quantify.

Images of KMO expression in endometriosis mice with treatments should be shown in Figure 4.

We have now included a representative KMO immunohistochemistry image from each endometriosis group and included all KMO immunohistochemistry images in Supplementary Information.

The images showing quantification analysis (Figure 4A-F) can be moved to supplementary material.

This recommendation contradicts the emphasis placed by the same reviewer earlier regarding quantification, so we have elected to keep it where it is.

(2) Figure 1 only showed representative images from a few patients. A description of whether KMO expression varies between patients and whether it correlates with AFS stages/disease severity will be helpful. Images from additional patients can be provided in supplementary material. 

We have added extra information to the Figure legend to clarify the disease stage of the superficial peritoneal lesions which were illustrated (Stage I/II) and to link them to the information in supplementary Table S1. In total we examined 11 peritoneal lesions and 5 ovarian lesions (stage III/IV) – in every sample examined immunopositive staining was most intense in epithelial cells lining gland-like structures. Sections illustrated were chosen to illustrate this key finding.

(3) For Home Cage Analysis, different measurements were performed as stated in methods including total moving distance, total moving time, moving speed, isolation/separation distance, isolated time, peripheral time, peripheral distance, in centre zones time, in centre zones distance, climbing time, and body temperature. However, only the finding for peripheral distance was reported in the manuscript. 

This was indeed a large amount of output, which we rationalised for the benefit of a concise paper. The paper now includes a description of which parameters showed a difference with drug treatment.

(4) The rationale for choosing the different dose levels of KNS898 - 0.01-25mg/kg was not provided. What is the IC50 of a drug? 

KNS898 dosing has been extensively characterised by us in multiple species, and the pIC50 has already been published (e.g. Hayes et al Cell Reports 2023 and elsewhere). We now include the pIC50 in the present manuscript to save the reader from having to search through another reference.

(5) Statistical significance: 

(a) Were stats performed for Fig 3B-E?

Now included, thank you.

(b) Line 141 - 'P = 0.004 for DEGLS per group' 

However, statistics were not shown in the figure. 

Thanks, now displayed on figure.

(c) Line 166 - 'the mechanical allodynia threshold in the hind paw was statistically significantly lower compared to baseline for the group' 

However, statistics were not shown in the figure. 

(d) Line 170 - 'Two-way ANOVA, Group effect P = 0.003, time effect P < 0.0001' The stats need to be annotated appropriately in Figure 5A as two separate symbols. 

Arguably the far more important comparison in this figure is whether there is any effect of treatment, and to mark multiple statistical comparisons on the figure would make it difficult to understand. Instead, the figure legend and results text have been clarified on this point.

(e) Figure 5B - multiple comparisons of two-way ANOVA are needed. G4 does not look different to G3 at D42. 

Multiple comparison testing (Dunnett’s T3) was done and the results have been clarified in the text and figure legends.

(f) Line 565 - 'non-significant improvement in KNS898 treated groups'. However, ** was annotated in Figure 5A. 

Thank you. This is an error that has been checked and corrected.

(6) Discussion is very light. No reference to previous publications was made in the discussion. Discussion on potential mechanistic pathways of KYR/KMO in the pathogenesis of endometriosis will be helpful, as the expression and function of KMO and/or other metabolites in endometrial-related conditions. 

The discussion is deliberately concise and focussed. The paper has 21 references to previous publications. A speculative discussion is generally not favoured by us.

The findings in this study generally support the conclusion although some key data which strengthen the conclusion eg quantification of KMO in normal and diseased tissue is lacking.

We differ from the reviewer here and do not think that those data would materially affect the likelihood of KMO inhibition being efficacious in human endometriosis in Phase 2/3 clinical trials.

Before KMO inhibitors can be used for endometriosis, the function of KMO in the context of endometriosis should be explored eg KMO knockout mice should be studied. 

We take the view that before KMO inhibitors can be used for endometriosis in patients there are multiple other regulatory and clinical development steps that are required that would be a priority. While using a KMO knockout mouse might be an interesting scientific experiment, it would not impact on the critical path in a material way.

Reviewer #2 (Public Review): 

Summary: 

The authors aim to address the clinical challenge of treating endometriosis, a debilitating condition with limited and often ineffective treatment options. They propose that inhibiting KMO could be a novel non-hormonal therapeutic approach. Their study focuses on: 

• Characterising KMO expression in human and mouse endometriosis tissues. 

• Investigating the effects of KMO inhibitor KNS898 on inflammation, lesion volume, and pain in a mouse model of endometriosis. 

• Demonstrating the efficacy of KMO blockade in improving histological and symptomatic features of endometriosis. 

Strengths: 

• Novelty and Relevance: The study addresses a significant clinical need for better endometriosis treatments and explores a novel therapeutic target. 

• Comprehensive Approach: The authors use both human biobanked tissues and a mouse model to study KMO expression and the effects of its inhibition. 

• Clear Biochemical Outcomes: The administration of KNS898 reliably induced KMO blockade, leading to measurable biochemical changes (increased kynurenine, increased kynurenic acid, reduced 3-hydroxykynurenine). 

Weaknesses: 

• Limited Mechanistic Insight: The study does not thoroughly investigate the mechanistic pathways through which KNS898 affects endometriosis. Specifically, the local vs. systemic effects of KMO inhibition are not well differentiated. 

While we agree that this is not a comprehensive mechanistic analysis, given that the ultimate therapy would be almost certainly a once daily oral dosing i.e. systemic administration, we do not consider differentiating local vs systemic effects of KMO inhibition to be critical to therapeutic development in this scenario.

• Statistical Analysis Issues: The choice of statistical tests (e.g., two-way ANOVA instead of repeated measures ANOVA for behavioral data) may not be the most appropriate, potentially impacting the validity of the results. 

The selection of two-way ANOVA (time and group) is sufficient and correct for this experimental analysis and its use does not invalidate the results. We agree that repeated measures ANOVA could be a valid alternative.

• Quantification and Comparisons: There is insufficient quantitative comparison of KMO expression levels between normal endometrium and endometriosis lesions,

Please see response above to quantification question raised by Reviewer 1.

and the systemic effects of KNS898 are not fully explored or quantified in various tissues. 

Please see earlier responses. KNS898 has been thoroughly explored in multiple tissues, species and experimental models, but those data do not need rehearsed here.

• Potential Side Effects: The systemic accumulation of kynurenine pathway metabolites raises concerns about potential side effects, which are not addressed in the study. 

As discussed above (response to Reviewer 1), KMO knockout mice exhibit normal fertility and fecundity and do not show a survival deficit compared to littermate wildtype controls (e.g. Mole et al Nature Medicine 2016). To further develop KNS898 towards clinical use, preclinical GLP safety and toxicology studies and human Phase 1 clinical trials will naturally need to be completed, but this is standard for the development of any new drug.

Achievement of Aims: 

• The authors successfully demonstrated that KMO is expressed in endometriosis lesions and that KNS898 can induce KMO blockade, leading to biochemical changes and improvements in endometriosis symptoms in a mouse model. 

Support of Conclusions: 

• While the data supports the potential of KMO inhibition as a therapeutic strategy, the conclusions are somewhat overextended given the limitations in mechanistic insights and statistical analysis. The study provides promising initial evidence but requires further exploration to firmly establish the efficacy and safety of KNS898 for endometriosis treatment. 

We do not agree that the conclusions are overextended based on the data presented, as expanded in the reply to the eLife editorial assessment at the beginning of this response. It is clear that additional preclinical, regulatory and clinical development work, and human clinical trials will be required to firmly establish the efficacy and safety of KN898 for endometriosis treatment.

Impact on the Field: 

• The study introduces a novel therapeutic target for endometriosis, potentially leading to non-hormonal treatment options. If validated, KMO inhibition could significantly impact the management of endometriosis. 

Utility of Methods and Data: 

• The methods used provide a foundation for further research, although they require refinement. The data, while promising, need more rigorous statistical analysis and deeper mechanistic exploration to be fully convincing and useful to the community. 

We believe that the data are a) convincing, and b) useful to the community. To be advanced effectively towards patients, KNS898 needs to follow the critical development path outlined above.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors): 

(1) Change 'hyperalgia' to hyperalgesia throughout the manuscript including the title. 

Done

(2) Line 69 - write '3-HK' in full. 

Done

(3) Line 85 - the findings of the study include 'define the preclinical efficacy of KNS898 in reducing inflammation'. The inflammatory profile was not studied. 

Changed to “disease”

(4) Line 259 - write 'EPHect' in full. 

Done

(5) Line 260 - write 'AFS' in full. Also, abbreviate 'AFS' in the caption of Table S1. 

Done

(6) 20 patients were listed in Table S1 but only 19 were accounted for in the methods section. 

Apologies there was an error and has now been corrected in the methods section as one of the endometrial samples had not been included. Table S1 has also been changed to make it clear which samples were eutopic endometrium to differentiate them from the lesions.

(7) The location from which the endometrial lesion tissues were obtained should be provided in Table S1. 

Table S1 has been changed to make it clear that the subtypes of lesions examined were classified as Stage I/II – superficial peritoneal subtype and Stage III/IV – endometrioma. The methods section has also been updated to reflect these subtypes (lines 272-277).

(8) Table S2 - G5 should be given compound 'A' not 'B'. 

Thank you. Corrected.

(9) Figure 2E was not referenced in the text and no figure legend was provided. 

Now referenced and the figure legend updated.

(10) Figure 3A - font needs to be enlarged. HCA baseline recording was annotated as performed twice in the protocol. When is the baseline taken and on what day was the Week 12 measurement taken (refer to Figures 5C and D)? 

Font has been enlarged as requested. The second HCA baseline annotation in Fig 3A is a cut-and-paste error, now rectified and the time of second measurement annotated.

(11) Line 133 - 'In KNS898-treated group G4 (endometriosis + treatment from Day 19), DEGLS formed in 4 of 15 mice (26.7%) and in G5 (Endo + treatment start on Day 26) in 6 of 15 mice (40%) (Fig. 3f).'. The aforementioned data is not reflected in Figure 3F. 

Thank you. This has been rectified.

(12) Line 137 - 'Mice with endometriosis receiving KNS898 from the time of inoculation (G4) had an average of 2.0 DEGLS per animal with DEGLS (total = 8 DEGLS in 4 mice in G4) and those receiving KNS898 1 week after inoculation (G5) had an average of 1.8 DEGLS per animal (total = 11 DEGLS in 6 mice in G5) (Figs. 3g and 3h).' 

The aforementioned data is not reflected in Figure 3G. There is no Figure 3H shown. 

Rectified as above.

(13) Provide a discussion of why KA levels were significantly lower in Figure 3E compared to Figure 2C. 

(14) Figure legend for Figure 3 - G1 and G2 were noted as n=8. However, Figure S1 and Table S2 noted both groups as n=10. 

Thank you. This is a typographical error. The legend for Fig 3 should indeed read n=10 for G1 and G2 and has been corrected.

(15) Line 181 - 'compared to non-operated and sham-operated control groups'. Only the sham group was shown in Figures 5C and D. 

This text has been clarified to refer only to the data shown.

(16) Figure 1 images need scalebars. Same for Figure 4. 

Now added

(17) Figure 3B - y-axis is fold change? 

Relative concentration. Legend has been clarified.

(18) Figures 5A and B - are the last Von Frey measurements taken on Day 40 (as per Figure 3A) or 42?

Taken on Day 42. Fig 3A (the prospective protocol figure) has been clarified to reflect what actually happened (D42) as opposed to what was planned (D40) to pre-empt any further confusion.

(19) Symbols in Figure S1 need to be explained in the Figure legend. 

Done

(20) Figures 2A and 2D should not be plotted in log scale to match the description of results in Line 106 and Line 118. 

These particular results are plotted on a log scale to allow the reader to visualise that detectable levels of drug are measurable at very low doses and that there is no significant pharmacodynamic effect at that low dose. We choose to retain the present format.

Reviewer #2 (Recommendations For The Authors): 

Comments and queries 

Introduction/aims section: 

Line 82 - 87: Clarify in the proposal aims what is being accessed and analysed in humans and/or in animal models (mice). Specifically state clearly the correlations with KMO expression. Were the correlations between KMO expression with features of inflammation performed only in mice or also in humans? 

Thank you for this comment. The aims have been clarified in the Introduction.

Section - KMO is expressed in human eutopic endometrium and human endometriosis tissue lesions: 

Was any quantitative or semi-quantitative method used to quantify the KMO expression in human tissues? Although the authors claimed that "KMO was strongly immunopositive in human peritoneal endometriosis lesions" by the representative figures it is not clear if KMO expression is similar, higher or lower between normal endometrium and peritoneal endometriosis lesions. 

We have added extra information to the legend of Figure 1 to identify the PIN number of the superficial lesions illustrated. The key finding from the immunostaining with the antibody which had been previously validated as specific for KMO was that the most intense immunopositive response was in glandular epithelial cells and the samples illustrate this result.

Section - Oral KNS898 inhibits KMO in mice: 

The authors clearly confirmed the target engagement of KNS898 in inhibiting KMO activity and, therefore, affecting upstream and downstream metabolites systemically in (peripheral fluid/ plasma) mice. Whether KNS898 effect is broad and targets systemic immune cells and whole body cells and tissue was not explored. It was also not explored if KNS898 is able to specifically inhibit KMO locally at the endometrium tissue by targeting epithelial and/or infiltrated immune cells, for example. 

That is correct.

It would be interesting to measure (or if it was measured to report in this section and also in Figure 2) the levels of KYN, KA and 3HK in naïve animals that did not receive KNS898. It would help to understand the net effect of KNS898 on the levels of kynurenine pathway metabolites and, therefore, justify the dose chosen.

These data are already presented in Fig 3B-E, control group.

Perhaps then the chosen dose could be lower considering the possible substantial changes in kynurenine pathway metabolites levels, which are reported to exert an effect in many cells, tissues and systems and could, therefore, precipitate side effects. Even more considering that the values for these metabolites are expressed as ng/ml, which hinders the comparison of the metabolite levels with the one reported for naïve animals in the literature. I would also suggest expressing the metabolite levels as nM/L. 

This is not a relevant method of determining dose-limiting toxicity or safety pharmacology/toxicology, either non-GLP or GLP. There are international guidelines on the proper conduct of those studies. This is also why it is important not to make claims about the safety or otherwise of an experimental compound in an in vivo setting that has not explicitly complied with those regulatory standards. With regard to the units recommendation, accepted units are ng/mL or nM, not usually nM/L.

Section - KMO blockade reduces endometrial gland-like lesion burden in experimental endometriosis in mice: 

Line 130: It would be better to replace "blockade of 3HK production" with "reduction of 3HK production" to better reflect the results. 

Changed to “inhibition of 3HK production”.

Line 140: In G5 (treatment starting at Day 26/ 1 week after inoculation), is the experimental model of endometriosis already established with all pathological and phenotypic features? 

This was not specifically tested in this experiment.

Lines 146 - 148: It would be better to specify that "Overall, there was no significant difference IN BODY WEIGHT between G3 and the KNS898 treatment groups G4 and G5 (endometriosis + treatment from Day 26)". Otherwise, this last sentence might be interpreted as the overall conclusion of this result sub-section. 

Thank you, a good point and has been corrected.

The authors demonstrated with an experimental approach that KMO blockade reduces a pathological measure of endometriosis i.e., endometrial gland-like lesion burden, in experimental endometriosis in mice when both administrated concomitant but also after the disease development. Although mechanistic insights about how reduced KMO activity can reduce the developed distended endometrial gland-like structures were not explored. Therefore, it remains to be investigated which (and how ) kynurenine pathway metabolites are directly linked to the beneficial effects of KMO blockade in the experimental model of endometriosis.

We agree.

Although the beneficial effects on the pathological measures are evident, Figure 3 shows an exorbitant accumulation of KYN and KA and also a substantial reduction in 3HK after the treatment with KNS898, which then raises concerns about tolerability and side effects. Would this effective KNS898 dose be viable and translational as a therapeutic approach? 

Please refer to comments above at multiple junctures about safety pharmacology and the clinical development critical path.

Section - KMO is expressed in experimental endometriosis in mice: 

By histological examination, the authors confirm that the treatment with KNS898 specifically reduced the KMO expression intensity in the DEGLS from mice. Therefore, the effect exerted by KNS898 locally on the KMO expression at the DEGLS could be, at least, partially responsible for the beneficial effects observed in Figure 3 i.e., the reduction of pathological measures. Although remains to be explored whether the effect of KNS898 in other cells or tissues could also be accountable for the beneficial effects exerted by KNS898 on the animal model of endometriosis. 

This is correct.

From a logical experimental point of view, I would suggest switching the order of the result subsection "KMO blockade reduces endometrial gland-like lesion burden in experimental endometriosis in mice" and "KMO is expressed in experimental endometriosis in mice" as well as the respective Figures 3 and 4. 

We do not agree. Fig 3 (and section) is the macroscopic enumeration of DEGLS, Fig 4 (and section) is the microscopic and immunohistochemical evaluation of the lesions introduced in Fig 3. The sequence as originally presented is the more logical.

Sections - KMO inhibition reduces mechanical allodynia in experimental endometriosis - and - KMO inhibition reduces mechanical allodynia in experimental endometriosis: 

The authors suggested that the KMO inhibition with KNS898 exerts beneficial effects on behavioural paradigms related to the experimental model of endometriosis. Based on the statistical analysis performed for the author, KMO inhibition with KNS898 reduces mechanical allodynia, as well as rescues, impaired cage exploration behaviour and mobility in mice with endometriosis. However, I believe that the most indicated statistical tests for Von Frey (allodynia behaviour) and Home cage (illness behaviour) analyses over time would be repeated measures ANOVA and paired t-test, respectively (and not two-way ANOVA as performed). Therefore for a more trustful analysis and interpretation of this data set, I would suggest the authors modify the statistical analysis and report the corresponding interpretation of these tests. 

The selection of two-way ANOVA (time and group) is suitable for this experimental analysis and its use does not invalidate the results. We agree that repeated measures ANOVA could be a valid alternative.

Overall, the authors present a solid and useful case for KMO inhibition as a potential therapeutic strategy for endometriosis. However, the study would benefit from more detailed mechanistic insights, appropriate statistical analyses, and an evaluation of potential side effects. With these improvements, the research could have a significant impact on the field and pave the way for new treatment modalities for endometriosis. 

We thank the reviewer for the positive comments and we have responded to the criticisms above.

Specific recommendations for improvement: 

• Mechanistic Studies: Conduct detailed studies to understand the local vs. systemic effects of KMO inhibition and its specific impacts on different cell types and tissues. If not feasible here, the authors could include in the discussion section a detailed overview of the possible mechanisms implicated. 

While we agree that this is not a comprehensive mechanistic analysis, given that the ultimate therapy would be almost certainly a once daily oral dosing i.e. systemic administration, we do not consider differentiating local vs systemic effects of KMO inhibition to be critical to therapeutic development in this scenario. We do not think speculation about possible mechanisms that is not supported by experimental data should be included. Furthermore, that notion (of statements not supported by data) has been given as a criticism by the reviewers, and therefore consistency on this point must be preferable.

• Quantitative Analysis: Include more robust quantitative methods to compare KMO expression levels in different tissues and assess the correlation between KNO expression and pathological and behavioural changes. 

As discussed above, the pathophysiological importance of KMO is in its enzymatic activity, not in its abundance as a protein, and 3HK production is far more dependent on kynurenine substrate availability rather than KMO protein abundance.

• Appropriate Statistics: Use the most suitable statistical tests for behavioural and other repeated measures data to ensure accurate interpretation. 

As discussed above

• Side Effect Evaluation: Investigate potential side effects of systemic KMO inhibition, particularly focusing on the long-term implications of altered kynurenine pathway metabolites. If not feasible here, the authors could include in the discussion section a detailed overview of the possible side effects associated as well as inform if KNS898 can cross the BBB and its implications. 

For a novel small molecule therapeutic compound in preclinical/clinical development, there are strictly regulated preclinical and clinical development standards that need to be met. It would not be responsible to publish or make claims about safety and potential adverse effect profiles without conducting the proper panel of tests within a suitable regulatory framework.

Si j'ai bien compris, la différence entre pays vient ici des seuls différences dans la réponse des taux à l'habitat au taux de la banque centrale. Mais pourquoi supposer qu'il y a une hétérogénéité dans la réponse des taux à l'habitat au taux d'intérêt (puisque pour les pays de la ze, le choc de taux est commun) mais pas dans les réponses de l'investissement et des prix de l'immo aux taux ? Réaliser l'exercice avec les équations estimées pays par pays serait plus cohèrent non ?

C'est vraiment beaucoup -0.72. C'est pas qu'il retourne plus rapidement à l'équilibre de long terme, c'est qu'il y retourne quasi instantanément (enfin au bout de 2 trimestres)

privilégiés, il y a rarement eu un tel écart entre les deux adversaires..

La respiración externa se compone de dos funciones: 1. La ventilación (inhalar y exhalar) 2. El intercambio degases que ocurre entre el aire y la sangre

se compone de 2 funciones: 1. El intercambio de gases entre la sangre y otros tejidos. 2. La utilización del oxigeno por los tejidos

Reviewer #2 (Public review):

The manuscript "Spatial frequency adaptation modulates population receptive field sizes" is a heroic attempt to untangle a number of visual phenomena related to spatial frequency using a combination of psychophysical experiments and functional MRI. While the paper clearly offers an interesting and clever set of measurements supporting the authors' hypothesis, my enthusiasm for its findings is somewhat dampened by the small number of subjects, high noise, and lack of transparency in the report. Despite several of the methods being somewhat heuristically and/or difficult to understand, the authors do not appear to have released the data or source code nor to have committed to doing so, and the particular figures in the paper and supplements give a view of the data that I am not confident is a complete one. If either data or source code for the analyses and figures were provided, this concern could be largely mitigated, but the explanation of the methods is not sufficient for me to be anywhere near confident that an expert could reproduce these results, even starting from the authors' data files.

Major Concerns:

I feel that the authors did a nice job with the writing overall and that their explanation of the topic of spatial frequency (SF) preferences and pRFs in the Introduction was quite nice. One relatively small critique is that there is not enough explanation as to how SF adaptation would lead to changes in pRF size theoretically. In a population RF, my assumption is that neurons with both small and large RFs are approximately uniformly distributed around the center of the population. (This distribution is obviously not uniform globally, but at least locally, within a population like a voxel, we wouldn't expect the small RFs to be on average nearer the voxel's center than the voxel's edges.) Why then would adaptation to a low SF (which the authors hypothesize results in higher relative responses from the neurons with smaller RFs) lead to a smaller pRF? The pRF size will not be a function of the mean of the neural RF sizes in the population (at least not the neural RF sizes alone). A signal driven by smaller RFs is not the same as a signal driven by RFs closer to the center of the population, which would more clearly result in a reduction of pRF size. The illustration in Figure 1A implies that this is because there won't be as many small RFs close to the edge of the population, but there is clearly space in the illustration for more small RFs further from the population center that the authors did not draw. On the other hand, if the point of the illustration is that some neurons will have large RFs that fall outside of the population center, then this ignores the fact that such RFs will have low responses when the stimulus partially overlaps them. This is not at all to say that I think the authors are wrong (I don't) - just that I think the text of the manuscript presents a bit of visual intuition in place of a clear model for one of the central motivations of the paper.

The fMRI methods are clear enough to follow, but I find it frustrating that throughout the paper, the authors report only normalized R2 values. The fMRI stimulus is a very interesting one, and it is thus interesting to know how well pRF models capture it. This is entirely invisible due to the normalization. This normalization choice likely leads to additional confusion, such as why it appears that the R2 in V1 is nearly 0 while the confidence in areas like V3A is nearly 1 (Figure S2). I deduced from the identical underlying curvature maps in Figures 4 and S2 that the subject in Figure 4 is in fact Participant 002 of Figure S2, and, assuming this deduction is correct, I'm wondering why the only high R2 in that participant's V1 (per Figure S2) seems to correspond to what looks like noise and/or signal dropout to me in Figure 4. If anything, the most surprising finding of this whole fMRI experiment is that SF adaptation seems to result in a very poor fit of the pRF model in V1 but a good fit elsewhere; this observation is the complete opposite of my expectations for a typical pRF stimulus (which, in fairness, this manuscript's stimulus is not). Given how surprising this is, it should be explained/discussed. It would be very helpful if the authors showed a map of average R2 on the fsaverage surface somewhere along with a map of average normalized R2 (or maps of each individual subject).

On page 11, the authors assert that "Figure 4c clearly shows a difference between the two conditions, which is evident in all regions." To be honest, I did not find this to be clear or evident in any of the highlighted regions in that figure, though close inspection leads me to believe it could be true. This is a very central point, though, and an unclear figure of one subject is not enough to support it. The plots in Figure 5 are better, but there are many details missing. What thresholding was used? Could the results in V1 be due to the apparently small number of data points that survive thresholding (per Figure S2)? I would very much like to see a kernel density plot of the high-adapted (x-axis) versus low-adapted (y-axis) pRF sizes for each visual area. This seems like the most natural way to evaluate the central hypothesis, but it's notably missing.

Regarding Figure 4, I was curious why the authors didn't provide a plot of the difference between the PRF size maps for the high-adapted and low-adapted conditions in order to highlight these apparent differences for readers. So I cut the image in half (top from bottom), aligned the top and bottom halves of the figure, and examined their subtraction. (This was easy to do because the boundary lines on the figure disappear in the difference figure when they are aligned correctly.) While this is hardly a scientific analysis (the difference in pixel colors is not the difference in the data) what I noticed was surprising: There are differences in the top and bottom PRF size maps, but they appear to correlate spatially with two things: (1) blobs in the PRF size maps that appear to be noise and (2) shifts in the eccentricity maps between conditions. In fact, I suspect that the difference in PRF size across voxels correlates very strongly with the difference in eccentricity across voxels. Could the results of this paper in fact be due not to shifts in PRF size but shifts in eccentricity? Without a better analysis of the changes in eccentricity and a more thorough discussion of how the data were thresholded and compared, this is hard to say.

While I don't consider myself an expert on psychophysics methods, I found the sections on both psychophysical experiments easy to follow and the figures easy to understand. The one major exception to this is the last paragraph of section 4.1.2, which I am having trouble following. I do not think I could reproduce this particular analysis based on the text, and I'm having a hard time imagining what kind of data would result in a particular PSE. This needs to be clearer, ideally by providing the data and analysis code.

Overall, I think the paper has good bones and provides interesting and possibly important data for the field to consider. However, I'm not convinced that this study will replicate in larger datasets - in part because it is a small study that appears to contain substantially noisy data but also because the methods are not clear enough. If the authors can rewrite this paper to include clearer depictions of the data, such as low- and high-adapted pRF size maps for each subject, per visual-area 2D kernel density estimates of low- versus high-adapted pRF sizes for each voxel/vertex, clear R2 and normalized-R2 maps, this could be much more convincing.

La documentation en lien indique que cela correspond à n'importe quel caractère alphanumérique, y compris le tiret bas. C'est équivalent à [A-Za-z0-9_].

Résumé de la vidéo [00:00:00][^1^][1] - [00:28:36][^2^][2]:

Ce documentaire traite du harcèlement scolaire et des méthodes pour y faire face. Il présente des témoignages d'enfants harcelés et des stratégies pour les aider à se défendre.

Moments forts: + [00:00:00][^3^][3] Introduction au harcèlement scolaire * Exemples de moqueries et insultes * Importance de l'autodérision * Préparation des élèves à affronter le harcèlement + [00:02:16][^4^][4] Thérapie brève pour les victimes * Témoignage d'un parent sur la crise de panique de son enfant * Explication de la thérapie brève * Importance de se concentrer sur le présent + [00:07:00][^5^][5] Stratégies de défense * Techniques pour répondre aux insultes * Importance de l'attitude et de la posture * Exemples de réponses pour déstabiliser le harceleur + [00:17:01][^6^][6] Comprendre les harceleurs * Témoignage d'un ancien harceleur * Sentiment de supériorité et de toute-puissance * Difficulté à arrêter le harcèlement + [00:22:12][^7^][7] Rôle des adultes et des institutions * Importance de la prise en charge de la souffrance * Témoignage d'un parent et d'un enfant harcelé * Stratégies pour rendre le harcèlement visible et y mettre fin

Résumé de la vidéo [00:28:38][^1^][1] - [00:53:45][^2^][2]:

Cette partie du documentaire traite des méthodes et des expériences pour lutter contre le harcèlement scolaire, en mettant en avant des témoignages et des stratégies pour aider les victimes à se défendre et à surmonter leurs traumatismes.

Points forts : + [00:28:38][^3^][3] Recherche de solutions * Formation en dehors de l'éducation nationale * Importance de parler sans peur des représailles * Nécessité de gronder les agresseurs + [00:30:20][^4^][4] Difficulté de parler * Peur et douleur de revivre les événements * Violence silencieuse de l'exclusion * Importance de l'écoute et du soutien + [00:34:01][^5^][5] Outils pour les victimes * Apprendre à négocier avec les personnalités difficiles * Importance de l'autodérision * Stratégies pour répondre aux harceleurs + [00:42:52][^6^][6] Rôle des parents et enseignants * Ne pas priver l'enfant de sa victoire * Risques de l'escalade de la violence * Importance de l'accompagnement et de l'outillage + [00:49:22][^7^][7] Témoignages de victimes * Expériences de harcèlement et de violence * Impact sur la confiance en soi * Importance de parler et de chercher du soutien

Résumé de la vidéo [00:53:47][^1^][1] - [00:57:30][^2^][2]:

Cette partie du documentaire aborde les progrès réalisés par les enfants victimes de harcèlement scolaire grâce à des séances de soutien. Les enfants apprennent à se défendre et à changer de posture, ce qui réduit le harcèlement et améliore leur bien-être général.

Points forts : + [00:53:47][^3^][3] Progrès des enfants * Les enfants montrent des signes de progrès * Ils deviennent plus autonomes * Leur confiance en eux augmente + [00:54:26][^4^][4] Changements positifs * Les enfants ne craignent plus les harceleurs * Ils se sentent plus épanouis * Les parents constatent des améliorations + [00:55:58][^5^][5] Réduction du harcèlement * Les harceleurs embêtent moins les enfants * Les enfants apprennent à se défendre * Le soutien des séances est efficace + [00:56:15][^6^][6] Autonomie et responsabilité * Les enfants deviennent acteurs de leur solution * Ils prennent en charge leur propre défense * Les séances les rendent plus autonomes + [00:57:02][^7^][7] Rôle des éducateurs * Les éducateurs épaulent les enfants * Ils ne résolvent pas le problème seuls * Ils sèment des graines de changement pour l'avenir

del

Pour le graphique 3, cela serait bien de d'abord montrer de l'économie totale avec une échelle différente (un graphique séapéré) puis de la décomposition sectorielle. Il y a beaucoup d'information dans ce graphe. On pourrait noter que la disctincion PGF investissement TIC devient floue. Un investissement TIC est probablement demand un changement d'organisaation, que l'on peut identifier comme du progrès "technique" au sens de Solow. Je retiens que sur les 0,5 pp de différence entre France et US, 0,3 est lié aux nouvelles technologies.

Patógenos para tiña de piel lampiña y tiña de pies

Diagnóstico de la tiña, laboratorio

DOI: 10.1038/s41419-023-05982-y

Resource: None

Curator: @AleksanderDrozdz

SciCrunch record: RRID:IMSR_JAX:007750

What is this?

DOI: 10.1038/s44318-024-00233-y

Resource: (Thermo Fisher Scientific Cat# A-11012, RRID:AB_2534079)

Curator: @scibot

SciCrunch record: RRID:AB_2534079

What is this?

DOI: 10.1038/s44318-024-00233-y

Resource: (Jackson ImmunoResearch Labs Cat# 111-035-006, RRID:AB_2337936)

Curator: @scibot

SciCrunch record: RRID:AB_2337936

What is this?

DOI: 10.1038/s44318-024-00233-y

Resource: (Thermo Fisher Scientific Cat# A-11005, RRID:AB_2534073)

Curator: @scibot

SciCrunch record: RRID:AB_2534073

What is this?

DOI: 10.1038/s44318-024-00233-y

Resource: None

Curator: @scibot

SciCrunch record: RRID:AB_2338500

What is this?

DOI: 10.1038/s44318-024-00233-y

Resource: (Thermo Fisher Scientific Cat# A-11001, RRID:AB_2534069)

Curator: @scibot

SciCrunch record: RRID:AB_2534069

What is this?

DOI: 10.1038/s44318-024-00233-y

Resource: (Santa Cruz Biotechnology Cat# sc-40, RRID:AB_627268)

Curator: @scibot

SciCrunch record: RRID:AB_627268

What is this?

DOI: 10.1038/s44318-024-00233-y

Resource: (Thermo Fisher Scientific Cat# A-11008, RRID:AB_143165)

Curator: @scibot

SciCrunch record: RRID:AB_143165

What is this?

DOI: 10.1038/s44318-024-00233-y

Resource: None

Curator: @scibot

SciCrunch record: RRID:AB_2861464

What is this?

DOI: 10.1038/s44318-024-00233-y

Resource: (Santa Cruz Biotechnology Cat# sc-23948, RRID:AB_628410)

Curator: @scibot

SciCrunch record: RRID:AB_628410

What is this?

DOI: 10.1038/s44318-024-00233-y

Resource: (Sigma-Aldrich Cat# F1804, RRID:AB_262044)

Curator: @scibot

SciCrunch record: RRID:AB_262044

What is this?