I like this idea. I think you could communicate it more concisely.

There is a disconnect between the tools to analyze data and the qualities/quantities of data that exist now, but more importantly are emerging.

That was a hard transition. I 100% think OS tools are the way to go. But I'm struggling with flow here.

Maybe too broad. Thinking: this doesn't need a citation, is an indicator that it is also not doing precise enough work.

OK. We're at comment 4 in this system. The system means I can't rewrite efficiently.

But we then move into sentence two that I could easily tighten up, e.g., remove any conjugates of 'be', and lead with the idea not the method, and maybe have tried to use the topic-stress emphasis with the first sentence, because there is redundancy here that maybe, if finessed, is good not bad.

But this editing approach means those changes are up to you Alec. I'll keep going. But I'm not sure this system is the best way for you to get help and contributions from Quinn and I because we are inhibited from doing the things we can do well and efficiently and I think that means more work for you.

This FactCheckHub article gives a strong example of malinformation because it describes real incidents where accurate personal information was shared online to harm individuals. The piece explains how photos, phone numbers, and other private details were posted publicly during disputes, leading to harassment and online attacks. An attentive reader can identify malinformation by noticing that the information being shared is true but unnecessarily invasive, targeted, and intended to embarrass or endanger someone. The article highlights how exposing real personal data without consent can escalate into cyberbullying and real‑world harm, demonstrating how malinformation weaponizes truthful information.

Victor is taking on immense responsibility for the Justine being tried for murder and foudn guilty.

test

This is a new term for me and describes how I've been thinking but is it unreasonable to think this way. This question is important: why add temptation to an urge and possiblely frame it as less harmful because its AI?

3) Treatment for these biological urges should not make AI imagery more acceptable, therapies should be a first line for stopage. To me it seems to normalizes these urges and depited behaviors.

Band aid on a bullet wound for sure

Hmm... I don't think this is an appropriate analogy but I do understand what he's getting at.

The word "benefit" is being used too loosely. There is no benefit. This also does not address the underlying issue - individuals are still preying on children.

Victor is determined to prove that Justine is innocent.

yes

This makes sense as adding a solute will increase density, making it harder for particles to escape. But this also seems wrong because adding salt to water lowers the boiling point, which in theory should increase vapor pressure and decrease the enthalpy of vaporization.

Volatile solutes have their own vapor pressure, so each component in a solution contributes to the total vapor pressure. According to Raoult’s Law and Dalton’s Law, the total vapor pressure equals the sum of each component’s partial pressure.

Adding a nonvolatile solute lowers vapor pressure because it reduces the number of solvent molecules that can evaporate. Evaporation decreases while condensation continues, causing a new equilibrium to form at a lower vapor pressure.

Encomium means a speech or writing that praises someone or something highly.

Reviewer #2 (Public review):

Zhang et al. have developed an advanced three-dimensional culture system of human endometrial cells, termed a receptive endometrial assembloid, that models the uterine lining during the crucial window of implantation (WOI). During this mid-secretory phase of the menstrual cycle, the endometrium becomes receptive to an embryo, undergoing distinctive changes. In this work, endometrial cells (epithelial glands, stromal cells, and immune cells from patient samples) were grown into spheroid assembloids and treated with a sequence of hormones to mimic the natural cycle. Notably, the authors added pregnancy-related factors (such as hCG and placental lactogen) on top of estrogen and progesterone, pushing the tissue construct into a highly differentiated, receptive state. The resulting WOI assembloid closely resembles a natural receptive endometrium in both structure and function. The cultures form characteristic surface structures like pinopodes and exhibit abundant motile cilia on the epithelial cells, both known hallmarks of the mid-secretory phase. The assembloids also show signs of stromal cell decidualization and an epithelial mesenchymal transition, like process at the implantation interface, reflecting how real endometrial cells prepare for possible embryo invasion.

Although the WOI assembloid represents an important step forward, it still has limitations: the supportive stromal and immune cell populations decrease over time in culture, so only early-passage assembloids retain full complexity. Additionally, the differences between the WOI assembloid and a conventional secretory-phase organoid are more quantitative than absolute; both respond to hormones and develop secretory features, but the WOI assembloid achieves a higher degree of differentiation due to the addition of "pregnancy" signals. Overall, while it's a reinforced model (not an exact replica of the natural endometrium), it provides a valuable in vitro system for implantation studies and testing potential interventions, with opportunities to improve its long-term stability and biological fidelity in the future.

[Editors' note: the authors have responded to the previous round of recommendations.]

Author response:

The following is the authors’ response to the previous reviews

Public Reviews:

Reviewer #1 (Public review):

Summary:

This study generated 3D cell constructs from endometrial cell mixtures that were seeded in the Matrigel scaffold. The cell assemblies were treated with hormones to induce a "window of implantation" (WOI) state. Although many bioinformatic analyses point in this direction, there are major concerns that must be addressed.

Strengths:

The addition of 3 hormones to enhance the WOI state (although not clearly supported in comparison to the secretory state).

Comments on revisions:

The authors did their best to revise their study according to the Reviewers' comments. However, the study remains unconvincing, incomplete and at the same time still too dense and not focused enough.

Reviewer #2 (Public review):

Zhang et al. have developed an advanced three-dimensional culture system of human endometrial cells, termed a receptive endometrial assembloid, that models the uterine lining during the crucial window of implantation (WOI). During this mid-secretory phase of the menstrual cycle, the endometrium becomes receptive to an embryo, undergoing distinctive changes. In this work, endometrial cells (epithelial glands, stromal cells, and immune cells from patient samples) were grown into spheroid assembloids and treated with a sequence of hormones to mimic the natural cycle. Notably, the authors added pregnancy-related factors (such as hCG and placental lactogen) on top of estrogen and progesterone, pushing the tissue construct into a highly differentiated, receptive state. The resulting WOI assembloid closely resembles a natural receptive endometrium in both structure and function. The cultures form characteristic surface structures like pinopodes and exhibit abundant motile cilia on the epithelial cells, both known hallmarks of the mid-secretory phase. The assembloids also show signs of stromal cell decidualization and an epithelial mesenchymal transition, like process at the implantation interface, reflecting how real endometrial cells prepare for possible embryo invasion.

Although the WOI assembloid represents an important step forward, it still has limitations: the supportive stromal and immune cell populations decrease over time in culture, so only earlypassage assembloids retain full complexity. Additionally, the differences between the WOI assembloid and a conventional secretory-phase organoid are more quantitative than absolute; both respond to hormones and develop secretory features, but the WOI assembloid achieves a higher degree of differentiation due to the addition of "pregnancy" signals. Overall, while it's a reinforced model (not an exact replica of the natural endometrium), it provides a valuable in vitro system for implantation studies and testing potential interventions, with opportunities to improve its long-term stability and biological fidelity in the future.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

This study generated 3D cell constructs (i.e., assembloids) that were treated with hormones to induce a 'window of implantation' (WOI) state. While the authors have made large efforts to address the reviewers' feedback, the study's findings remain unconvincing and incomplete.

(1) The authors have appropriately revised the terminology from 'organoids' to 'assembloids' in several parts of the manuscript. However, this revision remains incomplete, as the main title, figure legends, and figure titles still contain the incorrect term. A thorough review of the entire manuscript is recommended to ensure consistent and accurate use of terminology.

Thank you for your meticulous review. We have now conducted a full check and confirmed that terminology is used consistently and accurately throughout the text.

(1) Previous comments raised concerns about the feasibility of robustly passaging assembloid structures - comprising epithelial, stromal and immune cells - under epithelial growth conditions. The authors responded by stating that they optimized the expansion medium with a stromal cell-promoting factor. Additionally, rather than conducting scRNA-seq on both early and late passages (P6-P10) as suggested, they performed immunofluorescence staining, which confirmed the persistence of stromal cells at passage 6. However, the presence of immune cells was not addressed. Confirmation of their presence is essential for all further claims. Moreover, a more zoomed-out view of the immunostaining would help clarify the overall cellular composition across the entire well and facilitate comparison with corresponding brightfield images.

Whole-mount immunofluorescence of the 6th - generation assembloids revealed that CD45⁺ immune cells surrounded FOXA2⁺ glands, with a more zoomed-out view provided.

Author response image 1.

Whole-mount immunofluorescence showed that CD45⁺ cells (immune cells) were arranged around the glandular spheres that were FOXA2⁺. Scale bar =50 μm (left) and 30 μm (right).

In their response, the authors mention using the first three passages to ensure optimal cell diversity and viability. However, the manuscript states that 'assembloids derived from the first generation are used for experiments' (line 106). This discrepancy must be clarified.

Thank you for your suggestion. We have revised the relevant content to “The assembloids derived from the first three generation are used for experiments” (Line 90-91).

(2) The authors have made a commendable effort to bring more focus to the manuscript, which has improved readability.

We thank you for your insightful suggestions, which have greatly improved the quality of our manuscript.

(3) The "embryo implantation" part remains very unconvincing. How did authors define "the blastoids could grow within the endometrial assembloids and interact with them"? What did they mean with "grow"? Did blastoids further differentiate? Normally, blastoids cannot further "grow". "Survival rates of blastoids" is not equal to "growth". It is not clear how the survival rate was quantified. Besides, regarding the "interaction rates", how did authors define and quantify it? Actually, blastoids are able to attach to Matrigel efficiently (even without any endometrial cells), so authors cannot simply define the "interaction" as the co-localization of blastoids and assembloids via brightfield images. In addition, for the assembloids as the 3D structures grow in the Matrigel, the epithelial parts are normally apical-in, while the blastoids attach to the apical (lumen) side of the epithelial cells, so physiologically, blastoids should interact with the apical part of the epithelial cells instead of the outside of the assembloids.

(1) What did they mean with "grow"? Did blastoids further differentiate?

On the one hand, volume and morphology undergo continuous dynamic changes; on the other hand, only the inner cell mass and trophectoderm exist at the blastocyst stage, with the ICM further differentiating into OCT4⁺ epiblast and GATA6⁺ hypoblast.

(2) Survival rates of blastoids" is not equal to "growth". It is not clear how the survival rate was quantified.

The definition of "survival rate" is as follows: morphologically, the blastocoel remains noncollapsed and the cell boundaries are distinct (with no obvious cell detachment); molecularly, the markers of epiblast, hypoblast and trophectoderm are expressed. The survival rate is calculated as the ratio of viable embryoids to the total number of embryoids.

(3) Besides, regarding the "interaction rates", how did authors define and quantify it? Actually, blastoids are able to attach to Matrigel efficiently (even without any endometrial cells), so authors cannot simply define the "interaction" as the co-localization of blastoids and assembloids via brightfield images.

The criteria for determining interaction include not only attachment between the blastoids and assembloids observed via brightfield images, but also their sustained tight adhesion against external mechanical perturbations (e.g., medium replacement, immunostaining procedures).

(4) In addition, for the assembloids as the 3D structures grow in the Matrigel, the epithelial parts are normally apical-in, while the blastoids attach to the apical (lumen) side of the epithelial cells, so physiologically, blastoids should interact with the apical part of the epithelial cells instead of the outside of the assembloids.

You are absolutely correct. In vivo, the embryo indeed makes initial contact with the apical side of the epithelial cells. The introduction of the blastoid co-culture model herein is intended to demonstrate that this receptive endometrial assembloids can better support blastoid growth and development.

(4) Previous comments highlighted the absence of distinct shifts in gene expression profiles between SEC assembloids and WOI assembloids, which contrasts with findings from primary endometrial tissue reported by Wang et al. (2020). While the authors have expanded their analysis using the Mfuzz algorithm and identified changes in mitochondria- and cilia-associated genes, the manuscript still lacks evidence of significant transcriptional changes in key WOI marker genes, as described in Wang et al. This discrepancy must be addressed and discussed in greater depth to clarify the biological relevance of their model.

The endometrium in vivo involves complex crosstalk among multiple cell types and is tightly regulated by the hypothalamic-pituitary-ovarian (HPO) axis, thus exhibiting distinct shifts in gene expression during the peri-implantation period.

In our in vitro model, alterations in mitochondria- and cilia-related genes were observed, which to a certain extent demonstrates that these window of implantation (WOI) assembloids possess receptive-phase characteristics and can be employed to investigate WOI-associated scientific questions or conduct in vitro drug screening.

However, substantial efforts are still required to optimize the current model for fully recapitulating the dynamic changes in endometrial gene expression across different phases in vivo, and this aspect is further addressed in the Limitations section of our discussion (Line 342-353).

“However, our WOI endometrial assembloids also exhibit some limitations. It is undeniable that the assembloids cannot perfectly replicate the in vivo endometrium, which comprises functional and basal layers with a greater abundance of cell subtypes, under superior regulation by hypothalamic-pituitary-ovarian (HPO) axis. Specifically, stromal and immune cells are challenging to stably passage, and their proportion is lower than in the in vivo endometrium. While the in vivo peri-implantation period exhibits intricate gene expression dynamics driven by systemic regulation, our models only partially recapitulate these changes, primarily in mitochondria- and cilia-associated genes. Nevertheless, to some extent, these WOI assembloids possess receptivity characteristics and can be utilized for investigating receptivity-related scientific questions or conducting in vitro drug screening. Further refinements are required to fully simulate the dynamic endometrial gene expression patterns across all menstrual cycle stages. We are looking forward to integrating stem cell induction, 3D printing, and microfluidic systems to modify the culture environment.”

(5) In the authors' response document, they present data integrating their results with those of Garcia Alonso et al. (2021). However, these integrated analyses are not included in the revised manuscript (which should be, if answering a major concern).

Thanks for your valuable suggestions. We have now integrated the findings of Garcia Alonso et al. (2021) into the revised manuscript (Line 132) and Figure S2E–F.

(8) Fig 2D: The authors have clarified that CD45+ staining is used. However, they have not yet adapted the typo in the figure legend of the right picture.

Thanks for your thorough review. The left panel of Figure 2D is stained with CD45 to label immune cells, while the right panel is stained with CD44. These details have been clearly indicated in both the manuscript and the figure legend.  

(9) All quantification analyses (as described in the authors' response document) should be clearly described in the Materials & Methods section.  

Thanks for your valuable suggestions. All quantification analyses have now been added to the Supporting Materials and Methods section (Line 94-104, Line 110-111, Line 241244).

(10) The authors have provided clarification regarding their method for quantifying immunofluorescence staining (e.g., OLFM4 expression in Fig. 3C) in their response document. However, these methodological details are not included in the revised manuscript. It is important that such information is incorporated into the manuscript itself to ensure transparency and reproducibility for others.

Thanks for your valuable suggestions. All quantification analyses have now been added to the Supporting Materials and Methods section (Line 94-104).

(13) It is needed to include the author's response to the comment about literature showing the opposite of increased number of cilia during the WOI into the discussion part of the paper.

We appreciate your suggestions. The relevant content has now been added to the Discussion section (Lines 319–323).

(14) In the authors' response, they explain the difference between pinopodes and microvilli. They should include this explanation briefly in the manuscript. Moreover, Fig. 3F lacks a picture of cilia structure in CTRL condition. In addition, the structures that are indicated as cilia with an orange arrow seem to not be attached to the endometrial cells (anymore). It would be useful to show another more representative picture for the cilia.

(1) Thank you for your valuable suggestions. The distinction between pinopodes and microvilli has now been added to the Supporting Materials and Methods section (Line 230-236).

(2) You are probably referring to Figure 2F—we did not observe ciliary structures in the CTRL group.

(3) The cilia structure was visualized via transmission electron microscopy (TEM), which requires ultrathin sectioning. Thus, the cilia shown in the image correspond to a single cross-section of the captured assembloids. Owing to technical limitations, three-dimensional visualization of cilia on the cells cannot be achieved.

(17) The results on co-culturing blastoids with the WOI assembloids is not convincing. The blastoids are exposed to the basolateral side of the endometrial epithelial cells, while in vivo, blastocysts interact with the apical side of the endometrial epithelial cells first (apposition and attachment), followed by invasion into the endometrium. This means that the interaction shown here is not physiological. Therefore, it is not justified to say that this platform holds promise to investigate maternal-fetal interactions.

We agree with your perspective that discrepancies exist between this model and the physiological processes in vivo. However, such differences do not negate the scientific value of the model.

The core merit of this study lies in the successful establishment of co-culture systems for blastoids and WOI assembloids. Notably, genuine cross-talk occurs between the two components, thereby providing a practical and operational tool for subsequent research.

Although the current contact orientation differs from that observed in vivo, future optimization of the cell culture protocol (via modulation of cell polarity) will enable the model to better recapitulate physiological conditions. Therefore, the innovation and operability of this model within specific research contexts still render it a robust platform for investigating maternal-fetal interactions.

Overall, it is highly recommended that the authors carefully review the manuscript for grammatical errors, inconsistencies and issues with scientific phrasing. The language throughout the text requires substantial editing to improve clarity, readability and precision. 

We appreciate your suggestions. A full manuscript check was performed to rectify grammatical errors, inconsistencies, and inappropriate scientific phrasing, with further language refinement by a native English-speaking specialist.

Fig 1A: This overview is unclear. How many days do the assembloids grow before being stimulated with hormones? Are CTRL assembloids only kept in culture until day 2 and SEC and WOI assembloids until day 8? This is also not clear form the Materials and Methods section. Should be clarified.

Thanks for your valuable suggestions. We have now updated the overview (Figure 1A) and Materials and Methods section (Line 370-371, Line 379-381).

“Hormonal treatment was initiated following the assembly of the endometrial assembloids (about 7-day growth period).”

“The CTRL group was cultured in ExM without hormone supplementation and subjected to parallel culture for 8 days along with the two aforementioned groups.”

Fig 1B: From these brightfield images, it appears that the size of the assembloids remains relatively consistent from Day 0 to Day 3 and up to Day 11 (especially in CTRL). However, in Fig S1A, the assembloids on Day 11 appear significantly larger compared to those on Day 2 (or Day 4). Authors should clarify this discrepancy (since both of the figures are shown as "brightfield of endometrial assembloids").

You are probably referring to the observation that the assembloids at Day 11 in Fig. S1A are smaller in size than those at Day 2 (or Day 4) in Fig. 1B. This discrepancy arises because the time points in Fig. 1B are calculated starting from the initiation of hormone treatment for the SEC and WOI groups, rather than from the beginning of the overall culture as in Fig. S1A. In addition, assembloids exhibit size variability during the same culture period due to individual heterogeneity.

To eliminate ambiguity, we have now labeled “Hormone Day 0, Day 2, Day 8” in Fig. 1B and revised the corresponding figure legend to read: “Endometrial assembloids from the CTRL, SEC, and WOI groups, which were subjected to hormone treatment on Days 0, 2, and 8, exhibited comparable growth patterns throughout the culture period.”

Fig 2G: authors still used the description "organoids" here instead of "assembloids".

We appreciate your careful review. Corrections have been made accordingly.

Fig. 3C: For the OLFM4 staining quantification, in the Y-axis authors wrote "proportion of OLFM4 (+) cells (OLFM4 (+)/total", but in the rebuttal letter they mention "its fluorescence intensity (quantified as mean grey value) was significantly stronger in both the SEC and WOI groups compared to the CTRL group". This is confounding and should be clarified.

We apologize for incorrectly writing "fluorescence intensity" in the rebuttal letter; the correct term should be the "proportion of OLFM4 (+) cells (OLFM4 (+)/total)" as shown in Fig. 3C.

Fig 5D: Acetyl-α-tubulin is the marker of ciliated cells and should be expressed in the cilia instead of the whole cells. It is very strange to quantify as "mean fluorescence intensity (acetyl-αtubulin/DAPI)" to assess the cilia. Please clarify.

Thank you for your insightful comment. To clarify, the ratio "mean fluorescence intensity (acetyl-α-tubulin/DAPI)" was calculated within individual acetyl-α-tubulin⁺ ciliated cells. Acetyl-αtubulin fluorescence was normalized to the DAPI signal of the same cell nucleus, not the wholecell population. This corrected for variations in cell number and staining efficiency to ensure data accuracy.

Fig 5F: it is very bizarre that unciliated epithelium was transformed from ciliated epithelium, and CTRL was transformed from SEC and WOI. Should be clarified and discussed.

Pseudotime analysis sorts discrete cells along a "pseudotime axis" based on similarities and differences in cellular gene expression, thereby simulating cell state transitions.

Ciliated epithelium → unciliated epithelium: During the menstrual cycle, ciliated and unciliated epithelia undergo mutual transformation from the secretory phase (or mid-secretory phase) to the menstrual phase, and then to the proliferative phase. Here, we demonstrate the transition of ciliated cells to unciliated cells from the SEC and WOI stages to the CTRL stage.

Notably, the two cell types coexist, and what is presented here merely reflects a transformation trend. Relative content has been incorporated into the Discussion section (Line 319-321).

“Throughout the menstrual cycle, ciliated and unciliated epithelia undergo mutual transformation from the secretory phase (or mid-secretory phase) to the menstrual phase, and then to the proliferative phase.”

Fig 5H: To show "enhanced invasion ability", authors must provide some quantification and statistic analysis. It is very hard to see the difference between the CTRL and SEC regarding ROR2Wnt5A.

We appreciate your suggestion. Quantification and statistic analysis have been added to Figure 5H.

Fig 6A: please elaborate the "mIVC1" and "mIVC2" in the figure legends.

Additions have been made to the figure legends accordingly, as follows: "mIVC1: modified In Vitro Culture Medium 1; mIVC2: modified In Vitro Culture Medium 2."

Fig S1D: Is the PAS staining also done in CTRL assembloids? In addition, it is stated that the assembloids secrete glycogen because of a positive PAS staining, while it could also be neutral mucins, glycoproteins, etc, which are all detected by PAS staining. So, the authors should be more careful in stating that it is glycogen, or a PAS staining with diastase digestion should be done.

The PAS staining results for the CTRL group are presented in Fig. S1I. In addition, results of PAS staining with diastase digestion are included in Figure S1.

Line 120: references?

The reference has been added accordingly.

Line 178: The term 'Endometrial Receptivity Test (ERT)' is used. Do the authors mean Endometrial Receptivity Analysis (ERA) test? ERA is the commonly used abbreviation for this test. Moreover, the authors describe ERA as 'a kind of gene analysis-based test.' This should be rephrased more scientifically correct.

Thank you for your valuable suggestion. We have revised the term to ERA, and modified the phrase "a kind of gene analysis-based test" to "gene expression profiling-based diagnostic assay" (Lines 160–163).

“We performed Endometrial Receptivity Analysis (ERA), a gene expression profiling-based diagnostic assay that integrates high-throughput sequencing and machine learning to quantify the expression of endometrial receptivity-associated genes.”

Line 83: assemblies à assembloids

We appreciate your suggestion. The text has been updated to “the endometrial assembloids progressed from epithelial organoids, to assemblies of epithelial and stromal cells and then to stem cell-laden 3D artificial endometrium”.

The Materials and Methods section currently lacks the needed details. Authors should substantially expand this section to clearly describe all experimental and analytical procedures, including, aùmong others, immunofluorescence staining, quantification methods, bioinformatics analyses and statistical approaches. Providing comprehensive methodological information is essential.

A detailed description of these methods is provided in the Supporting Materials and Methods section.

Reviewer #2 (Recommendations for the authors): 

The revised manuscript is much improved in clarity, focus, and experimental support. The authors have thoughtfully addressed the major concerns from the previous review. In particular, the logic and flow of the paper are clearer, it now guides the reader through the rationale (constructing a WOI model), the comparative analysis against in vivo tissue and simpler organoids, and the key features that distinguish the WOI assembloid. The added functional validation (especially the blastoid co-culture experiment) significantly strengthens the work by showing a tangible outcome of "receptivity" beyond molecular profiling. The distinction between the standard secretory-phase organoid and the WOI assembloid is now more convincing, as the authors highlight several specific differences in morphology (more cilia, pinopodes), metabolism, and implantation success that favor the WOI model. The manuscript also reads cleaner with the bioinformatic sections condensed to the most important findings (excess detail was trimmed or moved to supplements) and the rationale for gene/pathway selection explicitly stated.

The manuscript has been significantly strengthened through the addition of functional assays (like the blastoid co-culture), clearer transcriptomic and proteomic data, and detailed analyses of hormone treatments, cilia biology, and stromal and immune cell behavior in early passages. These updates confirm that the WOI assembloid supports embryo attachment and outperforms standard secretory organoids, while integrating external references and clarifications on terminology. Minor suggestions remain, such as clarifying statistical significance and adding functional interpretations for certain observations, but overall, the manuscript is now more robust and biologically convincing.

Remaining points for clarification: There are a few minor points that still merit attention:

- Use of the Endometrial Receptivity Test (ERT): As previously mentioned, if the authors have ERT data for the SEC organoid group, including that information would further support the claim that the WOI assembloid is uniquely receptive. If not, it would be helpful to add a statement clarifying that the ERT was employed specifically as a confirmatory test for the WOI assembloids, rather than as a comparative measure across all groups.

Thank you for your valuable suggestion. We have now supplemented the description in the Supporting Materials and Methods section (Lines 160–162) as follows: “ERA was employed specifically as a confirmatory test for the WOI assembloids, rather than as a comparative measure across all groups.”

- Because the assembloids are created from primary tissue samples, it would be helpful to briefly comment on how consistent the findings were across different patient-derived samples. For example, did all biological replicates show similar expression of receptivity markers and comparable capacity to support blastoid attachment? Although this seems implied, including a sentence in the Methods or Results sections that specifies the number of donor lines tested would help readers assess the model's variability and reproducibility.

We appreciated your advice. The relevant statement has been added to the Supporting Materials and Methods section. (Line 312-313).

“All biological replicates (fourteen individuals) of endometrial assembloids show similar expression of receptivity markers and comparable capacity to support blastoid attachment.”

- The authors mention promising future directions, such as integrating 3D printing and microfluidics to further enhance the model, which is an excellent forward-looking statement. It would also be valuable to suggest the inclusion of additional cell types, like more robust immune cell populations or endothelial components, as future improvements to create an even more comprehensive model of the endometrial lining.

Thank you for your valuable suggestion. 3D printing and microfluidics serve as approaches for introducing multiple cell types. We have supplemented the following statement in the manuscript: “We are looking forward to integrating stem cell induction, 3D printing, and microfluidic systems to modify the culture environment.” (Line 352-353).

We are grateful for your valuable feedback and constructive criticism, which have helped us improve the quality of our work in terms of content and presentation. We have diligently revised the manuscript and made necessary changes. Here, we have attached the revised manuscript, figures, and all supplementary materials for your re-evaluation. Thank you again for your continued support and look forward to your favorable decision.

Son of a bitch is also a common one. Faggot too, specially thrown against individuals who show care, stripping them of their privilege.

I find more scary games and shows like "When Life Gives You Tangerines" that promote self-exploitation, because it's those people that end up invisiblising vulnerable people.

Reviewer #1 (Public review):

Summary:

This paper presents maRQup a Python pipeline for automating the quantitative analysis of preclinical cancer immunotherapy experiments using bioluminescent imaging in mice. maRQup processes images to quantify tumor burden over time and across anatomical regions, enabling large-scale analysis of over 1,000 mice. The study uses this tool to compare different CAR-T cell constructs and doses, identifying differences in initial tumor control and relapse rates, particularly noting that CD19.CD28 CAR-T cells show faster initial killing but higher relapse compared to CD19.4-1BB CAR-T cells. Furthermore, maRQup facilitates the spatiotemporal analysis of tumor dynamics, revealing differences in growth patterns based on anatomical location, such as the snout exhibiting more resistance to treatment than bone marrow.

Strengths:

(1) The maRQup pipeline enables the automatic processing of a large dataset of over 1,000 mice, providing investigators with a rapid and efficient method for analyzing extensive bioluminescent tumor image data.

(2) Through image processing steps like tail removal and vertical scaling, maRQup normalizes mouse dimensions to facilitate the alignment of anatomical regions across images. This process enables the reliable demarcation of nine distinct anatomical regions within each mouse image, serving as a basis for spatiotemporal analysis of tumor burden within these consistent regions by quantifying average radiance per pixel.

Weaknesses:

(1) While the pipeline aims to standardize images for regional assessment, the reliance on scaling primarily along the vertical axis after tail removal may introduce limitations to the quantitative robustness of the anatomically defined regions. This approach does not account for potential non-linear growth across dimensions in animals of different ages or sizes, which could result in relative stretching or shrinking of subjects compared to an average reference.

(2) Furthermore, despite excluding severely slanted images, the pipeline does not fully normalize for variations in animal pose during image acquisition (e.g., tucked body, leaning). This pose variability not only impacts the precise relative positioning of internal anatomical regions, potentially making their definition based on relative image coordinates more qualitative than truly quantitative for precise regional analysis, but it also means that the bioluminescent light signal from the tumor will not propagate equally to the camera as photons will travel differentially through the tissue. This differing light path through tissues due to variable positioning can introduce large variability in the measured radiance that was not accounted for in the analysis algorithm. Achieving more robust anatomical and quantitative normalization might require methods that control animal posture using a rigid structure during imaging.

Comments on revisions:

(1) Clarification of 2D Analysis. We strongly recommend that the authors explicitly define maRQup as a 2D spatiotemporal analysis technique. Since optical imaging quantification is inherently dependent on tissue type and signal depth, characterizing this as a 3D or volumetric method without tomographic correction is inaccurate. Please precede "spatiotemporal" with "2D" throughout the text to ensure precision regarding the method's capabilities.

(2) Data Validation and Scaling in Supplemental Figure g currently lacks the units necessary to support the assertion.

Non-Uniform Growth: The authors' method implies that mouse growth is linear and uniform in all directions (isotropic). However, murine growth is not akin to the inflation of a balloon; animals elongate and widen at different rates. The current scaling does not account for these physiological non-linearities.

Pose Variability: The scaling approach appears to neglect significant variability in animal positioning. Even under anesthesia, animal pose is rarely identical across subjects or time points.

Requirement for Evidence: Without quantitative data, there appears to be significant differences between the individual images and the merged image. If the authors assert that this is a "classical setting" where mouse positioning is 100% consistent and growth curves are identical in multiple dimensions, please provide specific references that validate these assumptions. Otherwise, the scaling must be corrected to account for anisotropic growth and pose differences or stated that scaling was only based on one dimension.

(3) Methodology of Spatial Regions The manuscript does not currently indicate how the nine distinct spatial regions were determined. Please expand the methods section to include the specific segmentation algorithms or anatomical criteria used to define these regions, as this is critical for reproducibility.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

This paper presents maRQup, a Python pipeline for automating the quantitative analysis of preclinical cancer immunotherapy experiments using bioluminescent imaging in mice. maRQup processes images to quantify tumor burden over time and across anatomical regions, enabling large-scale analysis of over 1,000 mice. The study uses this tool to compare different CAR-T cell constructs and doses, identifying differences in initial tumor control and relapse rates, particularly noting that CD19.CD28 CAR-T cells show faster initial killing but higher relapse compared to CD19.4-1BB CAR-T cells. Furthermore, maRQup facilitates the spatiotemporal analysis of tumor dynamics, revealing differences in growth patterns based on anatomical location, such as the snout exhibiting more resistance to treatment than bone marrow.

Strengths:

(1) The maRQup pipeline enables the automatic processing of a large dataset of over 1,000 mice, providing investigators with a rapid and efficient method for analyzing extensive bioluminescent tumor image data.

(2) Through image processing steps like tail removal and vertical scaling, maRQup normalizes mouse dimensions to facilitate the alignment of anatomical regions across images. This process enables the reliable demarcation of nine distinct anatomical regions within each mouse image, serving as a basis for spatiotemporal analysis of tumor burden within these consistent regions by quantifying average radiance per pixel.

Weaknesses:

(1) While the pipeline aims to standardize images for regional assessment, the reliance on scaling primarily along the vertical axis after tail removal may introduce limitations to the quantitative robustness of the anatomically defined regions. This approach does not account for potential non-linear growth across dimensions in animals of different ages or sizes, which could result in relative stretching or shrinking of subjects compared to an average reference.

Our answer to this comment is included in the Supplemental Methods. The standard deviation of the mouse pixels was calculated to ensure that the image processing steps did not alter the shape or size of the mice. Such consistency is particularly striking because our dataset was accrued by nine lab members over the last five years, before we conceived and carried out our analysis (c.f., answer to point #2). In fact, it is the very consistency of this IVIS measurement that led us to conceive our pipeline. As seen from Supplemental Figure 4G, there is minimal difference in the shape or size of the mice across 7,534 images. A total of 99 images were removed either due to being too slanted (91/7663, 1.2%) or due to processing errors (8/7633, 0.1%). Also, the vertical scaling was conducted while keeping the aspect ratio unchanged to prevent any non-anatomical scaling. Hence, we did not record any nonlinear growth of the mice that would warrant more convoluted alignment and/or batch correction for our images.

(2) Furthermore, despite excluding severely slanted images, the pipeline does not fully normalize for variations in animal pose during image acquisition (e.g., tucked body, leaning). This pose variability not only impacts the precise relative positioning of internal anatomical regions, potentially making their definition based on relative image coordinates more qualitative than truly quantitative for precise regional analysis, but it also means that the bioluminescent light signal from the tumor will not propagate equally to the camera, as photons will travel differentially through the tissue. This differing light path through tissues due to variable positioning can introduce large variability in the measured radiance that was not accounted for in the analysis algorithm. Achieving more robust anatomical and quantitative normalization might require methods that control animal posture using a rigid structure during imaging.

Reviewer #1 is correct that different mouse postures would be an issue when aligning the images and normalizing for size. However, all experiments are conducted for luminescence measurements in the IVIS system (i.e., this requires anesthesia and long integration time for imaging). In our experience and in our 1000+ mouse dataset, we noticed that all experiments (n=37) did place the anesthetized mice in a stretched/elongated position. Of note, these experiments were conducted by nine different researchers who were not instructed on how to place the mice on the machine for ideal image processing, thus showing that the standard protocol of imaging mice on IVIS does not introduce large variations in animal pose during image acquisition. We think the issue raised by Reviewer #1 is moot in the context of classical settings for mouse luminescence imaging.

Reviewer #2 (Public review):

Summary:

The authors developed a method that automatically processes bioluminescent tumor images for quantitative analysis and used it to describe the spatiotemporal distribution of tumor cells in response to CD19-targeting CAR-T cells, comprising CD28 or 4-1BB costimulatory domains. The conclusion highlights the dependence of tumor decay and relapse on the number of injected cells, the type of cells, and the initial growth rate of tumors (where initial is intended from the first day of therapy). The authors also determined the spatiotemporal analysis of tumor response to CAR T therapy in different regions of the mouse body in a model of acute lymphoblastic leukemia (ALL).

Strengths:

The analysis is based on a large number of images and accounts for many variables. The results of the analysis largely support their claims that the kinetics of tumor decay and relapse are dependent on the CAR T co-stimulatory domain and number of cells injected and tumor growth rates. 

Weaknesses:

The study does not specify how a) differences in mouse positioning (and whether they excluded not-aligned mice) and b) tumor spread at the start of therapy influenced their data. The study does not take into account the potential heterogeneity of CAR T cells in terms of CAR T expression or T cell immunophenotype (differentiation, exhaustion, fitness...).

See answer #2 to Reviewer #1.

Author response image 1.

Author response image 1 shows the average tumor radiance on day zero (when CAR-T cell therapy was administered) for all mice. While there is some spread, most mice had tumor localized to the liver or bone marrow.

Reviewer #3 (Public review):

Summary:

The paper "The 1000+ mouse project: large-scale spatiotemporal parametrization and modeling of preclinical cancer immunotherapies" is focused on developing a novel methodology for automatic processing of bioluminescence imaging data. It provides quantitative and statistically robust insights into preclinical experiments that will contribute to optimizing cell-based therapies. There is an enormous demand for such methods and approaches that enable the spatiotemporal evaluation of cell monitoring in large cohorts of experimental animals.

Strengths:

The manuscript is generally well written, and the experiments are scientifically sound. The conclusions reflect the soundness of experimental data. This approach seems to be quite innovative and promising to improve the statistical accuracy of BLI data quantification. 

This methodology can be used as a universal quantification tool for BLI data for in vivo assessment of adoptively transferred cells due to the versatility of the technology.

Weaknesses: 

No weaknesses were identified by this Reviewer. 

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

In this paper, the authors propose a significant advancement in optical image data analysis by employing automation. They effectively demonstrate the valuable insights that can be gained from analyzing extensive datasets with a more unbiased methodology. At present, I do not have any specific suggestions for improvement.

However, it is important to note that this work is limited in its operational scope. Specifically, it relies on predefined ROIs rather than aligning the signal site with anatomical systems. The scaling model and image cropping are simplistic, animal pose is not taken into account, and the data output needs to be called semi-quantitative or qualitative, and would have been stronger utilizing an AI agent. Nevertheless, this work underscores the potential of automated systems in preclinical image analysis, which is a crucial step towards developing more sophisticated approaches to optical image data analysis.

While our analysis used predefined ROIs, the maRQup pipeline allows users to manually draw ROIs on the mouse image.

Reviewer #2 (Recommendations for the authors):

The writing and presentation of data are clear and accurate, but some additional information should be added regarding the imaging protocol used to acquire the original data. 

The authors mention fluorescence in Figure 1. I expected all the data to be generated from bioluminescent NALM-6 tumors, since bioluminescence is indeed measured in average radiance and can be per pixel (p/sec/cm2/sr/pixel). Fluorescence should be measured using radiance efficiency (p/sec/cm2/sr)/(µW/cm2), a unit that compensates for non-uniform excitation light pattern in the instrument. Would the author find different results if fluorescence data were analyzed separately?

Reviewer #2 is correct that the unit for fluorescence would be radiance efficiency. The word “fluorescent” was included in the label of Figure 1a  to highlight that our workflow could be applied to other types of light-generating methods (i.e., fluorescence vs. bioluminescence). However, in this study, measurements of bioluminescent tumors only were analyzed. If fluorescence measurements are to be analyzed, our methods of image acquisition and processing would be directly applicable.

Did the author ever check the signal of the snout in mice with no tumor?

In mice with no tumor, there is no detectable signal in the snout (or anywhere else, for that matter).

The urine of mice contains phosphor, and might give a background signal, especially if longer exposure is used at the end of the study.

For the mice with no tumor injection, the luminescence signal was below background (<10² p/sec/cm²/sr/pixel). In particular, we do not detect any signal in the bladder/urine. Additionally, as described in the Supplemental Methods and Figure 1b, only pixels that were on the mouse as determined from the brightfield image were used to calculate the tumor burden from the radiance of the luminescent image. This method ensures that any background signal (e.g., from phosphor in mouse urine) would be excluded in the radiance quantification and not bias the results.

Additionally, as described in the Methods, the exposure time was held constant at 30 seconds for each IVIS measurement across all 37 experiments.

The data using more than 2 million cells comes from only 10 mice, and maybe the biological relevance of this group is limited since it will not be achievable and translatable in humans (PMID: 33653113).

We appreciate Reviewer #2’s attention to this issue. The effect observed in our study is large enough to reach statistical significance despite the small number of mice. Note that the dosing regimen used was optimized for the murine NSG model and would require appropriate scaling before clinical application. Nonetheless, NSG mice remain the gold standard for pre‑clinical in vivo evaluation and their use is generally required by regulatory agencies, such as the FDA, for assessing novel CAR‑T cell therapies; thus these findings are relevant for advancing such treatments.

Briefing : Préparation de la 10ème Semaine de l'ESS à l'école (SESSE 2026)

Résumé Exécutif

Ce document synthétise les points clés du webinaire organisé par l'association L'ESPER en préparation de la 10ème édition de la Semaine de l'Économie Sociale et Solidaire (ESS) à l'école, qui se déroulera du 23 au 28 mars 2026.

Copiloté avec l'OCCE, cet événement vise à sensibiliser les élèves, du primaire au supérieur, aux modèles économiques alternatifs basés sur la démocratie, la justice sociale et l'intérêt général.

Le webinaire souligne une double ambition : éduquer à l'ESS (compréhension des modèles) et par l'ESS (expérimentation de projets collectifs).

Les interventions mettent en avant des dispositifs concrets, des témoignages d'acteurs de terrain (notamment des Scops et des Scics) et une panoplie d'outils pédagogiques « clés en main » pour les enseignants.

L'objectif final est de transformer la société en intégrant ces principes dans le parcours scolaire et citoyen des individus.

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1. Cadre Institutionnel et Ambitions Éducatives

L'association L'ESPER, regroupant 41 organisations de l'éducation et de l'ESS, porte une vision politique et pédagogique forte pour le système éducatif français.

Vision et Plaidoyer

L'ESPER considère l'ESS comme un levier nécessaire pour transformer l'économie. Ses ambitions s'articulent autour de deux axes :

• Éducation à l'ESS : Faire comprendre un modèle de société basé sur la justice sociale et l'intérêt général. Un plaidoyer publié en août 2025 appelle d'ailleurs à l'intégration de l'ESS dans les programmes scolaires dès le collège.

• Éducation par l'ESS : Favoriser l'émancipation individuelle et collective par la mise en œuvre de projets concrets en classe, permettant aux élèves de découvrir la coopération par l'action.

La Semaine de l'ESS à l'école (SESSE)

Inscrite au calendrier de l'Éducation Nationale, cette semaine annuelle permet trois modes d'engagement :

1. Équipes éducatives : Valorisation de projets annuels ou organisation d'actions ponctuelles.

2. Acteurs de l'ESS : Accueil de classes dans leurs structures ou interventions directes en milieu scolaire.

3. Élèves/Étudiants : Montage de projets autonomes et sensibilisation de leurs pairs.

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2. Fondamentaux de l'Économie Sociale et Solidaire

L'ESS n'est pas une économie récente, mais elle s'est institutionnalisée, notamment via la loi Hamon du 31 juillet 2014.

Les 5 types de structures de l'ESS

| Type de structure | Caractéristiques principales | | --- | --- | | Associations | Groupements de personnes volontaires autour d'un projet non lucratif. | | Fondations | Affectation irrévocable de biens à une œuvre d'intérêt général. | | Coopératives | Entreprises où les associés partagent le pouvoir et les bénéfices. | | Mutuelles | Organismes à but non lucratif pratiquant la solidarité entre membres. | | Sociétés commerciales de l'ESS | Sociétés privées respectant les principes de l'ESS. |

Principes et Valeurs Cardinaux

Toutes ces organisations partagent un socle commun :

• Finalité d'intérêt général ou collectif.

• Lucrativité limitée : Les bénéfices sont prioritairement réinvestis dans le projet.

• Gestion démocratique : Application du principe « une personne, une voix », indépendamment du capital détenu.

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3. Retours d'Expérience et Témoignages d'Acteurs

L'Union Régionale des Scops et Scics (Occitanie)

Eugénie Bruni souligne l'importance de la promotion du modèle coopératif auprès des jeunes.

• Actions types : Interventions de 2 heures présentant l'histoire, les spécificités et des exemples concrets de coopératives.

• Impact : Ouverture des perspectives professionnelles pour les étudiants en montrant que la coopération est un modèle économique viable (4 558 sociétés coopératives en France générant 10,2 milliards d'euros de chiffre d'affaires).

• Conseils : Ne pas hésiter à solliciter les Unions Régionales qui disposent de délégués sur tout le territoire pour accompagner les projets.

La Scop Morasuti (Imprimerie, région AURA)

Témoignage de Damien sur une reprise d'entreprise à la barre du tribunal par les salariés.

• Le combat social : Transformation en Scop en juillet 2024. Le modèle a permis de supprimer les jours de carence et de rééquilibrer les salaires pour corriger les inégalités d'ancienneté.

• Engagement scolaire : Mise à disposition gratuite de chutes de matériaux pour les écoles et accompagnement technique (design, PAO) pour des projets d'exposition.

• Observation sur la démocratie : Les élèves sont souvent surpris par la double casquette « ouvrier et patron ». Damien explique : « Personne ne peut être d'accord avec tout... la démocratie, c'est aux voix. »

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4. Ressources et Outils Pédagogiques

L'ESPER propose des outils testés et adaptés pour différents niveaux (collège, lycée, supérieur).

Outils de sensibilisation "Clés en main"

| Outil | Objectif | Méthode | | --- | --- | --- | | Junior Coopérative | Initier à la méthodologie de projet. | Puzzle sur les étapes d'un projet et études de cas réels. | | Idées reçues sur l'ESS | Déconstruire les préjugés. | Débat mouvant à partir de cartes "Vrai/Faux". | | Filmographie ESS | Illustrer les réalités de l'ESS. | Sélection de documentaires avec guides pédagogiques. | | Fiches Pratiques | Organiser une intervention. | Guides logistiques pour les visites d'entreprises ou les interventions en classe. |

Recommandations pour les intervenants

• Adaptation : Simplifier le discours pour les collégiens en se concentrant sur les piliers (solidarité, partage des richesses, démocratie) plutôt que sur les détails juridiques.

• Interactivité : Utiliser des supports vidéo (ex: série "Ma boîte en Scop") et favoriser le dialogue.

• Préparation : Prévoir environ une heure d'échange préalable entre l'enseignant et l'intervenant pour cadrer l'action.

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

5. Calendrier et Inscriptions

• Inscriptions : Ouvertes sur le site de L'ESPER. L'équipe salariée assure la mise en relation entre les établissements scolaires et les acteurs de l'ESS.

• 25 février 2026 : Second webinaire de préparation dédié à une présentation détaillée de l'ESS avec l'expert Hervé de Falvar.

• 23 au 28 mars 2026 : Déroulement de la Semaine de l'ESS à l'école. Valorisation des actions sur les réseaux sociaux et newsletters de L'ESPER.

Citation clé : « Le SS porte un modèle de société qui est basé notamment sur la démocratie, la justice sociale, l'intérêt général [...] pour aboutir à une société plus juste dans laquelle les individus sont émancipés individuellement mais également collectivement. »

Reviewer #1 (Public review):

Summary:

This study presents a technically sophisticated intravital two-photon calcium imaging approach to characterize meningeal macrophage Ca²⁺ dynamics in awake mice. The development of a Pf4Cre:GCaMP6s reporter line and the integration of event-based Ca²⁺ analysis represent clear methodological strengths. The findings reveal niche-specific Ca²⁺ signaling patterns and heterogeneous macrophage responses to cortical spreading depolarization (CSD), with potential relevance to migraine and neuroinflammatory conditions. Despite these strengths, several conceptual, technical, and interpretational issues limit the impact and mechanistic depth of the study. Addressing the points below would substantially strengthen the manuscript.

Strengths:

The use of chronic two-photon Ca²⁺ imaging in awake, behaving mice represents a major technical strength, minimizing confounds introduced by anesthesia. The development of a Pf4Cre:GCaMP6s reporter line, combined with high-resolution intravital imaging, enables long-term and subcellular analysis of macrophage Ca²⁺ dynamics in the meninges.

The comparison between perivascular and non-perivascular macrophages reveals clear niche-dependent differences in Ca²⁺ signaling properties. The identification of macrophage Ca²⁺ activity temporally coupled to dural vasomotion is particularly intriguing and highlights a potential macrophage-vascular functional unit in the dura.

By linking macrophage Ca²⁺ responses to CSD and implicating CGRP/RAMP1 signaling in a subset of these responses, the study connects meningeal macrophage activity to clinically relevant neuroimmune pathways involved in migraine and other neurological disorders.

Weaknesses:

The manuscript relies heavily on Pf4Cre-driven GCaMP6s expression to selectively image meningeal macrophages. Although prior studies are cited to support Pf4 specificity, Pf4 is not an exclusively macrophage-restricted marker, and developmental recombination cannot be excluded. The authors should provide direct validation of reporter specificity in the adult meninges (e.g., co-labeling with established macrophage markers and exclusion of other Pf4-expressing lineages). At minimum, the limitations of Pf4Cre-based labeling should be discussed more explicitly, particularly regarding how off-target expression might affect Ca²⁺ signal interpretation.

The manuscript offers an extensive characterization of Ca²⁺ event features (frequency spectra, propagation patterns, synchrony), but the biological significance of these signals is largely speculative. There is no direct link established between Ca²⁺ activity patterns and macrophage function (e.g., activation state, motility, cytokine release, or interaction with other meningeal components). The discussion frequently implies functional specialization based on Ca²⁺ dynamics without experimental validation. To strengthen the conceptual impact, a clearer framing of the study as a foundational descriptive resource, rather than a functional dissection, would improve alignment between data and conclusions.

The GLM analysis revealing coupling between dural perivascular macrophage Ca²⁺ activity and vasomotion is technically sophisticated and intriguing. However, the directionality of this relationship remains unresolved. The current data do not distinguish whether macrophages actively regulate vasomotion, respond to mechanical or hemodynamic changes, or are co-modulated by neural activity. Statements suggesting that macrophages may "mediate" vasomotion are therefore premature. The authors should reframe these conclusions more cautiously, emphasizing correlation rather than causation, and expand the discussion to explicitly outline experimental strategies required to establish causality (e.g., macrophage-specific Ca²⁺ manipulation).

The authors conclude that synchronous Ca²⁺ events across macrophages are driven by extrinsic signals rather than intercellular communication, based primarily on distance-time analyses. This conclusion is not sufficiently supported, as spatial independence alone does not exclude paracrine signaling, vascular cues, or network-level coordination. No perturbation experiments are presented to test alternative mechanisms. The authors can either provide additional experimental evidence or rephrase the conclusion to acknowledge that the source of synchrony remains unresolved.

A major and potentially important finding is that the dominant macrophage response to CSD is a persistent decrease in Ca²⁺ activity, which is independent of CGRP/RAMP1 signaling. However, this phenomenon is not mechanistically explored. It remains unclear whether Ca²⁺ suppression reflects macrophage inhibition, altered viability, homeostatic resetting, or an anti-inflammatory program. Minimally, the discussion should be more deeply engaged with possible interpretations and implications of this finding.

The pharmacological blockade of RAMP1 supports a role for CGRP signaling in persistent Ca²⁺ increases after CSD, but the experiments are based on a relatively small number of cells and animals. The limited sample size constrains confidence in the generality of the conclusions. Pharmacological inhibition alone does not establish cell-autonomous effects in macrophages. The authors should acknowledge these limitations more explicitly and avoid overextension of the conclusions.

Reviewer #2 (Public review):

Using chronic intravital two-photon imaging of calcium dynamics in meningeal macrophages in Pf4Cre:TIGRE2.0-GCaMP6 mice, the study identified heterogeneous features of perivascular and non-perivascular meningeal macrophages at steady state and in response to cortical spreading depolarization (CSD). Analyses of calcium dynamics and blood vessels revealed a subpopulation of perivascular meningeal macrophages whose activity is coupled to behaviorally driven diameter fluctuations of their associated vessels. The analyses also investigated synchrony between different macrophage populations and revealed a role for CGRP/RAMP1 signaling in the CSD-induced increase, but not the decrease, in calcium transients.

This is a timely study at both the technical and conceptual levels, examining calcium dynamics of meningeal macrophages in vivo. The conclusions are well supported by the findings and will provide an important foundation for future research on immune cell dynamics within the meninges in vivo. The paper is well written and clearly presented.

I have only minor comments.

(1) Please indicate the formal definition of perivascular versus non-perivascular macrophages in terms of distance from the blood vessel. This information is not provided in the main text or the Methods. In addition, please explain how the meningeal vasculature was imaged in the main text.

(2) Similarly, the method used to induce acute CSD (pin prick) is not described in the main text and is only mentioned in the figure legends and Methods. Additional background on the neurobiology of acute CSD, as well as the resulting brain activity and neuroinflammatory responses, could be helpful.

Author response:

Public Reviews:

Reviewer #1 (Public review): 

Strengths:

(1) The use of chronic two-photon Ca²⁺ imaging in awake, behaving mice represents a major technical strength, minimizing confounds introduced by anesthesia. The development of a Pf4Cre:GCaMP6s reporter line, combined with high-resolution intravital imaging, enables long-term and subcellular analysis of macrophage Ca²⁺ dynamics in the meninges.

(2) The comparison between perivascular and non-perivascular macrophages reveals clear niche-dependent differences in Ca²⁺ signaling properties. The identification of macrophage Ca²⁺ activity temporally coupled to dural vasomotion is particularly intriguing and highlights a potential macrophage-vascular functional unit in the dura.

(3) By linking macrophage Ca²⁺ responses to CSD and implicating CGRP/RAMP1 signaling in a subset of these responses, the study connects meningeal macrophage activity to clinically relevant neuroimmune pathways involved in migraine and other neurological disorders.

Thank you for recognizing the strengths in our work.

Weaknesses: 

(1) The manuscript relies heavily on Pf4Cre-driven GCaMP6s expression to selectively image meningeal macrophages. Although prior studies are cited to support Pf4 specificity, Pf4 is not an exclusively macrophage-restricted marker, and developmental recombination cannot be excluded. The authors should provide direct validation of reporter specificity in the adult meninges (e.g., co-labeling with established macrophage markers and exclusion of other Pf4-expressing lineages). At minimum, the limitations of Pf4Cre-based labeling should be discussed more explicitly, particularly regarding how off-target expression might affect Ca²⁺ signal interpretation.

We acknowledge that PF4 is not an exclusively macrophage-restricted marker. Yet, among meningeal immunocytes, it is almost exclusively expressed in macrophages (1, 2). Furthermore, in the adult mouse meninges, Pf4^Cre-based reporter lines label nearly all dural and leptomeningeal macrophages and almost no other cells (3, 4). This Cre line has also been used to target border-associated macrophages (2, 4). Moreover, a recent study suggests that the bacterial artificial chromosome used to generate the Pf4^Cre line does not affect meningeal macrophage activity (4). Nonetheless, while we already discussed PF4 expression in meningeal megakaryocytes, in a revised version, we plan to discuss the possibility that a very small population of other meningeal immune cells may also be labeled.

(2) The manuscript offers an extensive characterization of Ca²⁺ event features (frequency spectra, propagation patterns, synchrony), but the biological significance of these signals is largely speculative. There is no direct link established between Ca²⁺ activity patterns and macrophage function (e.g., activation state, motility, cytokine release, or interaction with other meningeal components). The discussion frequently implies functional specialization based on Ca²⁺ dynamics without experimental validation. To strengthen the conceptual impact, a clearer framing of the study as a foundational descriptive resource, rather than a functional dissection, would improve alignment between data and conclusions.

In our discussion, we indicated that “the exact link between the distinct Ca²⁺ signal properties of meningeal macrophage subsets observed herein and their homeostatic function remains to be established”. In a revised version, we plan to further acknowledge that this is primarily a descriptive study that provides a foundational landscape of Ca²⁺ dynamics in meningeal macrophages.

(3) The GLM analysis revealing coupling between dural perivascular macrophage Ca²⁺ activity and vasomotion is technically sophisticated and intriguing. However, the directionality of this relationship remains unresolved. The current data do not distinguish whether macrophages actively regulate vasomotion, respond to mechanical or hemodynamic changes, or are co-modulated by neural activity. Statements suggesting that macrophages may "mediate" vasomotion are therefore premature. The authors should reframe these conclusions more cautiously, emphasizing correlation rather than causation, and expand the discussion to explicitly outline experimental strategies required to establish causality (e.g., macrophage-specific Ca²⁺ manipulation). 

In the results section, we indicated that our data suggest that dural perivascular macrophages are functionally coupled to locomotion-driven dural vasomotion, either responding to it or mediating it. Furthermore, in our discussion, we discussed the possibilities that 1) macrophages sense vascular-related mechanical changes and 2) macrophage Ca²⁺ signaling may regulate dural vasomotion. Moreover, we explicitly state that studying causality will require an experimental approach that has yet to be developed, enabling selective manipulation of dural perivascular macrophages.

(4) The authors conclude that synchronous Ca²⁺ events across macrophages are driven by extrinsic signals rather than intercellular communication, based primarily on distance-time analyses. This conclusion is not sufficiently supported, as spatial independence alone does not exclude paracrine signaling, vascular cues, or network-level coordination. No perturbation experiments are presented to test alternative mechanisms. The authors can either provide additional experimental evidence or rephrase the conclusion to acknowledge that the source of synchrony remains unresolved. 

Thank you for this suggestion. In the revision, we will indicate that the source of synchrony remains unresolved.

(5) A major and potentially important finding is that the dominant macrophage response to CSD is a persistent decrease in Ca²⁺ activity, which is independent of CGRP/RAMP1 signaling. However, this phenomenon is not mechanistically explored. It remains unclear whether Ca²⁺ suppression reflects macrophage inhibition, altered viability, homeostatic resetting, or an anti-inflammatory program. Minimally, the discussion should be more deeply engaged with possible interpretations and implications of this finding. 

While we propose that the decrease in macrophage calcium signaling following CSD could indicate that a hyperexcitable cortex dampens meningeal immunity, in the revised version, we plan to elaborate on the possible implications of this finding.

(6) The pharmacological blockade of RAMP1 supports a role for CGRP signaling in persistent Ca²⁺ increases after CSD, but the experiments are based on a relatively small number of cells and animals. The limited sample size constrains confidence in the generality of the conclusions. Pharmacological inhibition alone does not establish cell-autonomous effects in macrophages. The authors should acknowledge these limitations more explicitly and avoid overextension of the conclusions. 

We plan to acknowledge these limitations.

Reviewer #2 (Public review): 

Using chronic intravital two-photon imaging of calcium dynamics in meningeal macrophages in Pf4Cre:TIGRE2.0-GCaMP6 mice, the study identified heterogeneous features of perivascular and non-perivascular meningeal macrophages at steady state and in response to cortical spreading depolarization (CSD). Analyses of calcium dynamics and blood vessels revealed a subpopulation of perivascular meningeal macrophages whose activity is coupled to behaviorally driven diameter fluctuations of their associated vessels. The analyses also investigated synchrony between different macrophage populations and revealed a role for CGRP/RAMP1 signaling in the CSD-induced increase, but not the decrease, in calcium transients.

This is a timely study at both the technical and conceptual levels, examining calcium dynamics of meningeal macrophages in vivo. The conclusions are well supported by the findings and will provide an important foundation for future research on immune cell dynamics within the meninges in vivo. The paper is well written and clearly presented.

Thank you.

I have only minor comments. 

(1) Please indicate the formal definition of perivascular versus non-perivascular macrophages in terms of distance from the blood vessel. This information is not provided in the main text or the Methods. In addition, please explain how the meningeal vasculature was imaged in the main text. 

We did not measure the exact distance of the perivascular macrophages from the blood vessels, but defined them as such based on previous data showing that these cells reside along the abluminal surface and maintain tight interactions with mural cells (5). We plan to provide this information in the revised manuscript.

(2) Similarly, the method used to induce acute CSD (pin prick) is not described in the main text and is only mentioned in the figure legends and Methods. Additional background on the neurobiology of acute CSD, as well as the resulting brain activity and neuroinflammatory responses, could be helpful.

We plan to add the method for inducing CSD (i.e., a pinprick in the frontal cortex) to the Results section and provide more background in the Introduction section.

Reviewer #3 (Public review):

Strengths: 

Sophisticated in vivo imaging of meningeal immune cells is employed in the study, which has not been performed previously. A detailed analysis of the distinct calcium dynamics in various subtypes of meningeal macrophages is provided. Functional relevance of the responses is also noted in relation to CSD events.

Thank you for recognizing the strengths of our paper

Weaknesses:

(1) The specificity of the methods used to target both meningeal macrophages and RAMP1 is limited. Additional discussion points on the functional relevance of the two subtypes of meningeal macrophages and their calcium responses are warranted. A section on potential pitfalls should be included. 

We plan to address these issues in the revision

References

(1) H. Van Hove et al., A single-cell atlas of mouse brain macrophages reveals unique transcriptional identities shaped by ontogeny and tissue environment. Nat Neurosci 22, 1021-1035 (2019).

(2) F. A. Pinho-Ribeiro et al., Bacteria hijack a meningeal neuroimmune axis to facilitate brain invasion. Nature 615, 472-481 (2023).

(3) G. L. McKinsey et al., A new genetic strategy for targeting microglia in development and disease. Elife 9,  (2020).

(4) H. J. Barr et al., The circadian clock regulates scavenging of fluid-borne substrates by brain border-associated macrophages. bioRxiv,  (2025).

(5) H. Min et al., Mural cells interact with macrophages in the dura mater to regulate CNS immune surveillance. J Exp Med 221,  (2024).

"他几乎总是发现那里存在某种崇拜：罗马崇拜、凯尔特崇拜，早在基督徒之前……基督教在欧洲相对来说是较新的，这些地方早已被占据……罗马崇拜、希腊崇拜已经在欧洲传播了很长很长时间。但基督教后来覆盖了所有这些，将圣人基督教化，或改变传说以确立自身……" 异教元素 基督教转化 例子 大地女神（Mother Earth--圣母玛利亚 <br /> 黑圣母的深色=大地、丰饶

泉水/井崇拜 --圣井、洗礼池

太阳神崇拜--圣徒节日对应冬至/夏至<br /> 圣诞节=冬至，复活节=春分

罗马神庙--教堂建在原地 <br /> 巴黎圣母院地下有罗马神庙

凯尔特圣林--教堂庭院保留古树 许多教堂旁的"神圣橡树"

史前 大地母神（被压抑） <br /> 女性身体经验：生育、月经、更年期 凯尔特 女祭司（被抹去） <br /> 女性社群：知识传递、疗愈实践 罗马 国家宗教（男性神祇）<br /> 家庭崇拜：家神、祖先、日常仪式 基督教 父权教会 <br /> 圣母/女圣人崇拜：女性重新占领神圣空间

Reviewer #1 (Public review):

Summary:

In this study, the authors' aim was to determine whether hepatic palmitoylation is a physiologically relevant regulator of systemic metabolism. The data demonstrate that loss of DHHC7 in hepatocytes disrupts Gαi palmitoylation, enhances cAMP-PKA-CREB signaling, and drives transcriptional upregulation and secretion of Prg4. The KO mice display increased body weight, fat mass, and plasma cholesterol, but at 12 weeks on HFD, do not exhibit insulin resistance. The potential mechanism underlying the metabolic phenotype was examined by assessing adipocyte signaling and by exploring whether Prg4 acts through GPR146. Through this pathway, the authors intend to link DHHC7-dependent palmitoylation to the regulation of hepatokines that exert systemic metabolic effects.

Strengths:

(1) Hepatic palmitoylation in systemic metabolic regulation is largely unexplored. The authors demonstrate the role of DHHC7 in vivo using a successful liver-specific knockout mouse model that causes HFD-dependent obesity without insulin resistance.

(2) Several studies were performed on chow and HFD, as well as male and female mice.

(3) Plasma proteomics identified Prg4 as a circulating factor elevated in KO mice. Prg4 overexpression phenocopied the KO mice.

(4) There is solid mechanistic data supporting the hypothesis that hepatic DHHC7 loss selectively increases Prg4 secretion as a hepatokine.

(5) There is convincing evidence for the DHHC7 mechanism in liver: DHHC7 controls cAMP-PKA-CREB via Gαi palmitoylation. The authors recognize that the palmitoylation change is causative rather than correlated, and this needs to be more fully explored in the future.

(6) Strong in vitro data support that Prg4 acts through adipocyte GPR146 via its SMB domain

Weaknesses:

(1) The assessment of liver and adipose tissue responses to DHH7 loss is insufficient to support claims that it alters systemic lipolysis. In this new mouse model, liver histology is necessary, especially given the cholesterol increase in the KO. As this is a newly established mouse line, common assessments of the liver during HFD feeding would be important for interpreting the phenotype.

(2) The data show DHH7 loss causes adipose tissue dysfunction and alterations in lipid metabolism. Beyond that, I suggest not stating more regarding the phenotype of the DHH7 mice for this work. A thorough analysis would be needed to determine which factor drives the obesity and changes in energy balance in the mice. For example, the KO mice had lower oxygen consumption (but no change in CO2 production, which is also usually similarly altered), suggesting a CNS component could drive obesity. However, since the data are not normalized for lean mass and there is no information about locomotor activity, this analysis is incomplete. RER may be informative if available. A broad conservative description of the KO phenotype would be more accurate since Pgr4 has many paracrine targets and likely has autocrine signaling in the liver.

(3) Most references to lipolysis or lipolysis flux systemically would be inaccurate. To suggest a suppression of lipolysis, serum NEFA would need to be measured, and in vivo or in vitro lipolysis assays performed to test the effect of DHH7 loss or the specificity of PGR4 action on adipocytes in vivo. To demonstrate adipose tissue dysfunction, analysis of lipogenesis markers, canonical markers for insulin sensitivity, and mitochondrial dysfunction should be performed/measured.

(4) Line 179: The experiment was performed in brown adipocytes to show that Prg4 does not affect p-CREB Figure S8 under the heading: "DHHC7 controls hepatic PKA-CREB activity through Gαi palmitoylation to regulate Prg4 transcription." Unless repeated using liver lysate, the conclusions stated in the text throughout the paper should be revised.

(5) It appears that the serum and liver proteomics were only assessed for factors that increased in KO mice? Were proteins that were significantly decreased analyzed?

(6) The beige adipocyte culture method is unclear. The methods do not describe the fat pad used, and the protocol suggests the cells would be differentiated into mature white adipocytes. If they are beige cells, a reference for the method, gene expression, and cell images could support that claim.

(7) The use of tamoxifen can confound adipocyte studies, as it increases beigeing and weight gain even after a brief initiation period. Both groups were treated with Tam, but another way to induce Cre would be ideal.

(8) Evidence for the lack of the glucose phenotype is incomplete. One reason could be due to the IP route of glucose administration, which has a large impact on glucose handling during a GTT. To confirm the absence of a glucose tolerance phenotype, an OGTT should be performed, as it is more physiological. In addition, the mice should be fed for 16 weeks. Prg4 affects immune cells, changing how adipose tissue expands, and 12 weeks of HFD feeding is often not long enough to see the effects of adipose tissue inflammation spilling over into the system.

(9) There may be liver-adipose tissue crosstalk in KO mice, but this was not fully assessed in this study and would be difficult to determine in any setting, given the diverse cell types that are targets of Pdg4. The crosstalk claim is unnecessary to share the basic premises; there is the DHH7 mechanism/phenotype and the Pgr4 mechanism/phenotype, and while there is no Pgr4 adipose direct mechanism, the paper can be successfully reframed.

(10) Although the DHH7 loss on the chow diet did not result in a phenotype, did the Pgr4 increase in the KO mice on chow? This would determine whether either i) the expression of Pgr4 is dependent on HFD/obesity, or ii) circulating Pgr4 has effects only in an HFD condition. The receptors may also change on HFD, especially in adipocytes.

Impact:

This work would significantly contribute to the study of liver metabolism, provided it includes data describing the liver. The role of Pgr4 in adipocytes and other cell types is of substantial value to the field of metabolism. By reframing the paper and conducting some key experiments, its quality and impact can be increased.

Reviewer #2 (Public review):

In the current report, Sun and Colleagues sought to determine the liver-specific role that DHHC7, a DHHC palmitoyltransferase protein, plays in regulating whole-body energy balance and hepatic crosstalk with adipose tissues. The authors generated an inducible, liver-specific DHHC7 knockout mouse to determine how altered palmitoylation in hepatocytes alters hepatokine production/secretion, and in turn, systemic metabolism. The ablation of DHHC7 was found to alter the production of proteoglycan 4 (Prg4), a hepatokine previously linked to metabolic regulation. The authors propose that the change in Prg4 production is mediated by the loss of Gαi palmitoylation, due to DHHC7 ablation, thereby augmenting cAMP-PKA-CREB signaling in hepatocytes, which alleviates the 'brake' on Prg4 production. The authors further propose that Prg4 overexpression leads to excessive binding to GPR146 on adipocytes, which in turn suppresses PKA-mediated HSL activation, promoting impairments in lipolysis, leading to obesity. The report is interesting and generally well-written, but it appears to have some clear gaps in additional data that would aid in interpretation. The addition of confirmatory culture studies would be incredibly helpful for testing the hypotheses being explored. My comments, concerns, and/or suggestions are outlined below in no particular order.

(1) Figures: All data should be presented in dot-boxplot format so the reader knows how many samples were analyzed for each assay and group. n=3 for some assays/experiments is incredibly low, particularly when considering the heterogeneity in responsiveness to HFD, food intake, etc....

(2) Figure 1E-F: It is unclear when the food intake measure was performed. Mice can alter their feeding behavior based on a myriad of environmental and biological cues. It would also be interesting to show food intake data normalized to body mass over time. Mice can counterregulate anorexigenic cues by altering neuropeptide production over time. It is not clear if this is occurring in these mice, but the timing of measuring food intake is important. Additionally, the VO2 measure appears to be presented as being normalized to total body mass, when in fact, it would probably be more accurate to normalize this to lean body mass. Normalizing to total body mass provides a denominator effect due to excessive adiposity, but white fat is not as metabolically active as other high-glucose-consuming tissues. If my memory serves me right, several reports have discussed appropriate normalizations in circumstances such as this.

(3) Figure 1J-N: It is not all that surprising that fasting glucose and/or TGs were found to be similar between groups. It is well-established that mice have an incredible ability to become hyperinsulinemic in an effort to maintain euglycemia and lipid metabolism dynamics. A few relatively easy assays can be performed to glean better insights into the metabolic status of the authors' model. First, fasting insulin concentrations will be incredibly helpful. Secondly, if the authors want to tease out which adipose depot is most adversely affected by ablation, they could take an additional set of CON and KO mice, fast them for 5-6 hours, provide a bolus injection of insulin (similar to that provided during an insulin tolerance test), and then quickly harvest the animals ~15 minutes after insulin injections; followed by evaluating AKT phosphorylation. This will really tell them if these issues have impairments in insulin signaling. The gold-standard approach would be to perform a hyperinsulinemic-euglyemic clamp in the CON and KO mice. I now see GTT and ITT data, but the aforementioned assays could help provide insight.

(4) Figure 3A: This looks overexposed to me.

(5) Figures 3-4: It appears that several of these assays could be complemented with culture-based models, which would almost certainly be cleaner. The conditioned media could then be used from hepatocyte cultures to treat differentiated adipocytes.

(6) Figure 4: It is unclear how to interpret the phospho-HSL data because the fasting state can affect this readout. It needs to be made clear how the harvest was done. Moreover, insulin and glucagon were never measured, and these hormones have a significant influence over HSL activity. I suspect the KO mice have established hyperinsulinemia, which would likely affect HSL activity. This provides an example of why performing some of these experiments in a dish would make for cleaner outcomes that are easier to interpret.

Reviewer #3 (Public review):

Summary:

In the current manuscript, Sun et al aimed to determine the metabolic function of hepatocyte DHHC7, one of the key enzymes in protein palmitoylation. They generated inducible liver-specific Dhhc7 knockout mice and discovered that Dhhc7-LKO mice are more prone to gain weight and develop adipose expansion and obesity. Via unbiased proteomic analysis, they identified PRG4 as one of the top secreted factors in the liver of Dhhc7-LKO mice. Hepatic overexpression of PRG4 recapitulates the obesity phenotype observed in Dhh7-LKO mice. At the mechanistic level, PRG4, once secreted from the liver, can bind to GPR146 on adipocytes and inhibit PKA-HSL signaling and lipolysis. Taken together, their findings suggest a novel pathway by which the liver communicates with adipose tissue and impacts systemic metabolism.

Strengths:

(1) The systemic metabolic homeostasis depends on coordination among metabolically active tissues. Thus, active communication between the liver and adipose tissue when facing nutritional challenges (such as high-fat diet feeding) is crucial for achieving metabolic health. The concept that the liver can communicate with adipose tissue and impact the lipolysis process via secreted hepatokines is quite significant but remains poorly understood.

(2) Hepatocyte Dhhc7 knockout mice developed a significant obesity phenotype, which is associated with adipose expansion.

(3) Unbiased proteomic analysis identified PRG4 as one of the top secreted factors in the liver of Dhh7-LKO mice. Hepatic overexpression of PRG4 recapitulates the obesity phenotype observed in Dhh7-LKO mice.

(4) In vitro cell-based assay showed that PRG4 can bind to adipocyte GPR146, inhibit PKA-mediated HSL phosphorylation, and subsequently, the lipolysis process.

Weaknesses:

(1) Lack of a causal-effect study to generate evidence directly linking hepatocyte DHH7 and PRG4 in driving adipose expansion and obesity upon HFD feeding.

(2) Lack of direct evidence to support that PRG4 inhibits adipocyte lipolysis via GPR146. A functional assay demonstrating adipocyte lipolysis is required.

(3) The conclusion is largely based on the correlation evidence.

Author response:

Public reviews:

Reviewer #1 (Public review):

Weaknesses:

(1) The assessment of liver and adipose tissue responses to DHH7 loss is insufficient to support claims that it alters systemic lipolysis. In this new mouse model, liver histology is necessary, especially given the cholesterol increase in the KO. As this is a newly established mouse line, common assessments of the liver during HFD feeding would be important for interpreting the phenotype.

We will add the data of the liver histology in the revised version.

(2) The data show DHH7 loss causes adipose tissue dysfunction and alterations in lipid metabolism. Beyond that, I suggest not stating more regarding the phenotype of the DHH7 mice for this work. A thorough analysis would be needed to determine which factor drives the obesity and changes in energy balance in the mice. For example, the KO mice had lower oxygen consumption (but no change in CO2 production, which is also usually similarly altered), suggesting a CNS component could drive obesity. However, since the data are not normalized for lean mass and there is no information about locomotor activity, this analysis is incomplete. RER may be informative if available. A broad conservative description of the KO phenotype would be more accurate since Pgr4 has many paracrine targets and likely has autocrine signaling in the liver.

We will add the data of CO2 production, locomotor activity and RER in the revised version.

(3) Most references to lipolysis or lipolysis flux systemically would be inaccurate. To suggest a suppression of lipolysis, serum NEFA would need to be measured, and in vivo or in vitro lipolysis assays performed to test the effect of DHH7 loss or the specificity of PGR4 action on adipocytes in vivo. To demonstrate adipose tissue dysfunction, analysis of lipogenesis markers, canonical markers for insulin sensitivity, and mitochondrial dysfunction should be performed/measured.

We will measure the serum NEFA to test the effect of DHHC7. We will analyze the lipogenesis markers, canonical markers for insulin sensitivity, and mitochondrial dysfunction.

(4) Line 179: The experiment was performed in brown adipocytes to show that Prg4 does not affect p-CREB Figure S8 under the heading: "DHHC7 controls hepatic PKA-CREB activity through Gαi palmitoylation to regulate Prg4 transcription." Unless repeated using liver lysate, the conclusions stated in the text throughout the paper should be revised.

The figure S8 is to demonstrate that Prg4 has no impact on forskolin induced CREB phosphorylation at Ser133, and provide the evidence that the prg4 acts on the upstream of adenylyl cyclase. We will revise the description.

(5) It appears that the serum and liver proteomics were only assessed for factors that increased in KO mice? Were proteins that were significantly decreased analyzed?

We are analyzing the decreased proteins in the following project.

(6) The beige adipocyte culture method is unclear. The methods do not describe the fat pad used, and the protocol suggests the cells would be differentiated into mature white adipocytes. If they are beige cells, a reference for the method, gene expression, and cell images could support that claim.

We will add a reference for the method, gene expression, asn cell images.

(7) The use of tamoxifen can confound adipocyte studies, as it increases beigeing and weight gain even after a brief initiation period. Both groups were treated with Tam, but another way to induce Cre would be ideal.

We will use the Doxycycline-inducible systems in the future.

(8) Evidence for the lack of the glucose phenotype is incomplete. One reason could be due to the IP route of glucose administration, which has a large impact on glucose handling during a GTT. To confirm the absence of a glucose tolerance phenotype, an OGTT should be performed, as it is more physiological. In addition, the mice should be fed for 16 weeks. Prg4 affects immune cells, changing how adipose tissue expands, and 12 weeks of HFD feeding is often not long enough to see the effects of adipose tissue inflammation spilling over into the system.

We will perform the OGTT and feed the mice for 16 weeks in the future.

(9) There may be liver-adipose tissue crosstalk in KO mice, but this was not fully assessed in this study and would be difficult to determine in any setting, given the diverse cell types that are targets of Pdg4. The crosstalk claim is unnecessary to share the basic premises; there is the DHH7 mechanism/phenotype and the Pgr4 mechanism/phenotype, and while there is no Pgr4 adipose direct mechanism, the paper can be successfully reframed.

We will reframe the paper.

(10) Although the DHH7 loss on the chow diet did not result in a phenotype, did the Pgr4 increase in the KO mice on chow? This would determine whether either i) the expression of Pgr4 is dependent on HFD/obesity, or ii) circulating Pgr4 has effects only in an HFD condition. The receptors may also change on HFD, especially in adipocytes.

We will test the Prg4 in the KO mice on chow diet.

Reviewer #2 (Public review):

(1) Figures: All data should be presented in dot-boxplot format so the reader knows how many samples were analyzed for each assay and group. n=3 for some assays/experiments is incredibly low, particularly when considering the heterogeneity in responsiveness to HFD, food intake, etc.

We will present the data in dot-boxplot format.

(2) Figure 1E-F: It is unclear when the food intake measure was performed. Mice can alter their feeding behavior based on a myriad of environmental and biological cues. It would also be interesting to show food intake data normalized to body mass over time. Mice can counterregulate anorexigenic cues by altering neuropeptide production over time. It is not clear if this is occurring in these mice, but the timing of measuring food intake is important. Additionally, the VO2 measure appears to be presented as being normalized to total body mass, when in fact, it would probably be more accurate to normalize this to lean body mass. Normalizing to total body mass provides a denominator effect due to excessive adiposity, but white fat is not as metabolically active as other high-glucose-consuming tissues. If my memory serves me right, several reports have discussed appropriate normalizations in circumstances such as this.

We will see how to be more accurate to normalize.

(3) Figure 1J-N: It is not all that surprising that fasting glucose and/or TGs were found to be similar between groups. It is well-established that mice have an incredible ability to become hyperinsulinemic in an effort to maintain euglycemia and lipid metabolism dynamics. A few relatively easy assays can be performed to glean better insights into the metabolic status of the authors' model. First, fasting insulin concentrations will be incredibly helpful. Secondly, if the authors want to tease out which adipose depot is most adversely affected by ablation, they could take an additional set of CON and KO mice, fast them for 5-6 hours, provide a bolus injection of insulin (similar to that provided during an insulin tolerance test), and then quickly harvest the animals ~15 minutes after insulin injections; followed by evaluating AKT phosphorylation. This will really tell them if these issues have impairments in insulin signaling. The gold-standard approach would be to perform a hyperinsulinemic-euglyemic clamp in the CON and KO mice. I now see GTT and ITT data, but the aforementioned assays could help provide insight.

We have the data for evaluating AKT phosphorylation and will add it in the revised version.

(4) Figure 3A: This looks overexposed to me.

We will replace it with short exposed one.

(5) Figures 3-4: It appears that several of these assays could be complemented with culture-based models, which would almost certainly be cleaner. The conditioned media could then be used from hepatocyte cultures to treat differentiated adipocytes.

We will perform the cell culture experiments for Figures 3-4

(6) Figure 4: It is unclear how to interpret the phospho-HSL data because the fasting state can affect this readout. It needs to be made clear how the harvest was done. Moreover, insulin and glucagon were never measured, and these hormones have a significant influence over HSL activity. I suspect the KO mice have established hyperinsulinemia, which would likely affect HSL activity. This provides an example of why performing some of these experiments in a dish would make for cleaner outcomes that are easier to interpret.

We will perform some experiments in cell culture dish.

Reviewer #3 (Public review):

Weaknesses:

(1) Lack of a causal-effect study to generate evidence directly linking hepatocyte DHH7 and PRG4 in driving adipose expansion and obesity upon HFD feeding.

We will perform the causal-effect study to demonstrate the hypothesis.

(2) Lack of direct evidence to support that PRG4 inhibits adipocyte lipolysis via GPR146. A functional assay demonstrating adipocyte lipolysis is required.

We will add the direct evidence in the revised version.

(3) The conclusion is largely based on the correlation evidence.

We will perform the experiment to strengthen the conclusion base on the a causal-effect study.

Analyse de la Rhétorique Complotiste : Mécanismes, Discours et l'Allégorie du « Mouton »

Ce document de synthèse analyse les recherches et les réflexions de Loïc Massaia, vulgarisateur pour le projet Utopia, concernant la rhétorique employée dans les milieux complotistes.

Il détaille les structures argumentatives, les fonctions psychologiques du discours et l'usage spécifique de l'insulte « mouton » comme outil de distinction sociale et de clôture du débat.

Synthèse

L'analyse de la rhétorique complotiste révèle un système de communication visant moins à établir une vérité qu'à asseoir un ascendant sur l'auditoire.

Cette rhétorique se caractérise par une structure circulaire (tautologique) et un recours systématique à l'essentialisme.

L'usage de termes comme « mouton » remplit une triple fonction : une attaque ad personam pour éviter le débat de fond, une accusation de complicité passive, et un mécanisme de distinction permettant de renforcer l'estime de soi du locuteur.

En s'affranchissant des règles du « débat sain », le discours complotiste s'établit comme un système fermé où la conclusion (l'existence d'un complot) est déjà contenue dans les prémisses.

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1. Définition et Catégorisation de la Rhétorique Complotiste

Le document propose de définir la rhétorique comme l'ensemble des moyens mis en œuvre dans un discours pour convaincre, briller, manipuler ou obtenir un ascendant sur autrui.

Une définition complémentaire la décrit comme la « négociation de la différence entre les individus sur une question donnée ».

Dans le cadre du complotisme, les expressions récurrentes peuvent être classées selon quatre dimensions principales :

| Dimension | Exemples de phrases types | Objectif recherché | | --- | --- | --- | | Accusatoire | « Journalopes », « Merdias », « On ne vous dit pas tout » | Discréditer les sources d'information officielles. | | Incitatoire | « Faites vos propres recherches », « Réveillez-vous » | Pousser l'interlocuteur à adopter la même conclusion par une illusion d'autonomie. | | Négation du hasard | « Coïncidence ? Je ne crois pas », « Tout est lié » | Refuser la contingence au profit d'un dessein caché. | | Surconfiance et Distinction | « Tous des moutons », « On avait raison » | Se placer au-dessus de la « masse » ignorante. |

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2. Analyse Structurelle de l'Argumentation

Le Modèle de Toulmin

Pour évaluer la solidité d'un argument, le document mobilise le modèle de Toulmin, qui identifie les composants d'une argumentation optimale :

1. Données : Les informations de base.

2. Conclusion : Ce que l'on veut démontrer.

3. Justifications : Le lien logique entre données et conclusion.

4. Fondement : Ce qui rend la justification solide et acceptée.

5. Réfutation : L'intégration des limites et des conditions qui pourraient contredire l'argument.

La défaillance du discours complotiste

L'analyse montre que le discours complotiste omet généralement la réfutation.

Par exemple, l'argument consistant à dire que le gouvernement est une secte parce qu'il lutte contre les dérives sectaires (pour étouffer la dissidence) s'effondre si l'on introduit d'autres facteurs de distinction entre État et secte.

Circularité et Essentialisme

Le discours complotiste est décrit comme un système fermé ou une tautologie.

Il repose sur l'essentialisation : on décrète que la « nature » profonde d'une entité (le gouvernement, les élites) est malveillante.

Dès lors, toute action de cette entité, même positive en apparence, est interprétée comme une preuve supplémentaire de sa malveillance.

Le complot existe nécessairement au départ pour expliquer les faits qui servent ensuite à prouver l'existence du complot.

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3. L'Allégorie du « Mouton » : Origines et Usages

L'expression « tous des moutons » est un idiotisme animalier présent dans plusieurs langues (français, italien, anglais, polonais).

Origine Littéraire

L'image du mouton qui suit aveuglément remonte notamment à Rabelais (l'épisode des moutons de Panurge), où les animaux sautent à l'eau et meurent simplement parce que le premier a sauté.

Cela souligne une dimension « naturelle » ou essentialiste de l'animal : le besoin de suivre.

Fonctions dans le discours complotiste

1. L'identification du comploteur : S'il y a des moutons, il y a nécessairement un « berger » ou un « maître » (le comploteur).

2. L'accusation de complicité : Les non-complotistes sont jugés idiots, mais aussi complices par leur passivité.

3. Le besoin de distinction : Se déclarer « non-mouton » permet de s'extraire de la masse. Selon les travaux d'Anthony Lantian (2015), l'adhésion aux théories du complot serait un moyen de rehausser une estime de soi initialement basse en se sentant détenteur d'un savoir supérieur.

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4. La Rhétorique comme Rupture du Débat

L'usage de l'insulte « mouton » est qualifié d'argument ad personam.

Théorisée par Schopenhauer, cette tactique consiste à attaquer l'individu plutôt que ses arguments pour mettre fin à une discussion que l'on ne peut pas gagner sur le fond.

Violation des règles de la controverse honorable

En s'appuyant sur les travaux de Levi Hedge (XIXe siècle), le document identifie trois règles fondamentales d'un débat sain systématiquement violées par la rhétorique complotiste :

• Règle n°4 : Interdiction des attaques personnelles.

• Règle n°5 : Interdiction d'accuser l'adversaire de mobiles cachés.

• Règle n°7 : La vérité doit être le but, non la victoire. L'usage du ridicule ou de la raillerie (traiter l'autre de mouton) est une violation de cette règle.

Toutefois, le document souligne que ces dérives ne sont pas l'apanage des complotistes ; elles se retrouvent fréquemment dans tout débat public où l'objectif des participants est de « gagner » plutôt que de chercher la vérité.

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5. Perspectives Critiques

En conclusion, le document invite à une réflexion sur la nature même de la critique du complotisme.

Si l'on définit la rhétorique complotiste comme étant « par nature » une tautologie basée sur un essentialisme, on court le risque de produire soi-même un discours fermé et essentialiste.

Cette mise en abyme suggère que l'analyse du complotisme doit elle-même rester vigilante quant à ses propres structures argumentatives pour ne pas tomber dans les travers qu'elle dénonce.

Knowing just 'A' way of war, and which the vessels represent people, carcasses, and life bypassed for the destructive path they're now hoping another civilization takes off their hands, is very cunning indeed. But, instruction is only generational, whereas the conflict of promise eternal. The plague brought on by the explorer/settler was only poor management of mankind-like decision burdening civilization now.

Understanding the Gaspesian, or Mi'kmak tribe were the indigenous and not a part of the cultural overthrow taking place, we realize historical value to the people. Whatever matter concerning the lifestyle is reciprocated among all involved, as the documenting nature extends nearly 400 years.

Mary Smith Cranch’s letters show that women were deeply engaged in political debates even though they could not vote or hold office. Her discussions of currency policy, legal reforms, and Shays’s Rebellion reveal that she closely followed national issues and believed women had a responsibility to support the country through informed opinions and economic sacrifice.

The discussion of Shays’s Rebellion reveals how economic struggles and weak national power under the Articles of Confederation created serious unrest among farmers. While some leaders viewed the rebellion as a dangerous threat to order, others believed it represented the same revolutionary spirit that founded the country.

Great food for thought even today.

As a exploring people happening upon a entirely believed subset of life form, the white man is observed leaving clues to the woman in both their nakedness and cultural savagery by even the Natives seeking knowledge from a distance of that very place. Referencing two villages and the shady nature of moving 'skins' between them is the place the explorer was constantly in psychologically.

You need to look from ALL cases to see if it still fits together. Even if it sounds believable right off the bat, it needs to be backed up.

I like this statement because I tend to always put what ifs but this shows that you can do that without damaging your essay.

math made easy

I think that most of the stories are corrupted by high class or power people.

I feel like this is almost a perfet example of how the government really valued the Native people and how much it really cared about assimilation. The government didn't care about deadly diseases killing the future of the Native Americans, it just cared about them not being Native Americans anymore. It wanted them to act and behave like a white man, and didn't care what it took, nor if they lived or died. It generally shows the heartbreaking double standard, where something considered unspeakable and a heinous crime for White People was just ignored when it happened to Natives, that being cramming a bunch of sick and terminally ill individuals with healthy ones, without the facilities to house them separately and within quarantines.

I feel like this does emphasize a sort of knowledge that not everyone poses, that being practical knowledge. A person who grew up in the woods is likely to know a lot about herbal medicine, how to hunt animals and survive. They also would have the wisdom of experience. This seems to definitely push the narrative that you don't need to know how to do math to be intelligent and worthy of respect, but rather knowledgeable and purposeful.

Conflicts all around

With regards to Meghan's comment above and my understanding of archival silences, I cannot help but to think this shift and the eventual shift towards community is some form of reparations. How is the field paying for the crimes of its past? Can it? Should it? Is the shift towards community enough?

Be careful, prudent, thoughtful in your deliberation, do not be swayed by the ambitious trappings or in thinking this is an inconsequential decision, do no choose just anything because something must be chosen, hold the confederation/convention accountable and choose thoughfully.

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• The modern longevity movement is framed as a potential "vampire disclosure program" where scientific research into blood serves as a cover for ancient practices.
• Parabiosis, the surgical connection of two organisms' circulatory systems, was discovered in 1864 and showed that old mice could be rejuvenated by young blood.
• Prominent tech figures like Peter Thiel and Bryan Johnson are cited as primary "suspects" due to their interest in young blood transfusions and extreme life-extension protocols.
• The article suggests that historical figures like Marsilio Ficino and Elizabeth Báthory, and even Bram Stoker's Dracula, were documenting actual biological mechanisms long before modern science.
• A key scientific "twist" suggests rejuvenation doesn't come from "magic" properties in young blood, but rather from diluting the "pro-aging factors" that accumulate in old blood.
• The author posits a four-phase disclosure timeline designed to normalize vampirism as a "wellness protocol" for the public.
• Modern vampires (Thiel and Johnson) are criticized for their poor "operational security" compared to historical vampires who remained hidden.

Hacker News Discussion

• Writing Style and AI: Several users debated whether the article was AI-generated, noting a specific "LLM smell" in the structure, short sentence fragments, and repetitive "It's not X, it's Y" phrasing.
• Peter Watts' Fiction: Many commenters drew parallels to the "vampires" in Peter Watts' novels Blindsight and Echopraxia, which portray them as a distinct predatory subspecies with neurological trade-offs.
• Health Benefits of Blood Donation: There was a significant technical discussion on whether regular blood or plasma donation (bloodletting) could actually mimic these effects by diluting accumulated metabolic "junk" and microplastics.
• Satirical Takes: The thread featured high-effort roleplay, with some users pretending to be offended Transylvanian nobles and others joking that Peter Thiel is too "unsophisticated" to be a true vampire.
• Literary Origins: One commenter noted that Bram Stoker was likely influenced by Dr. John Polidori, a physician, which explains the early medical "accuracy" in vampire lore.

IPFS is Magic

Very good description of how these things often work out.

The best moat

This part explains how tariffs make foreign products more expensive so people will buy more local goods. While this can help businesses in that country, it also makes it harder for companies from other countries to compete. It shows that trade rules can protect some industries but hurt others at the same time.

There are many reason people fear globalization the main one being that millions of people have lost their jobs due to imports of production being shifted abord this cause the people to lose their job while company's profit of it because labor becomes less but at the cost of people's expense.

"I’m going to slip into econ speak. So, apologies here. But, like, they’ve already kind of, like, maximized their utility subject to constraints. And I think that’s broadly true across people. Like, obviously, when you move away from the population studied in criminal justice and look at wealthier people or more advantaged people, you know, their constraints are different. Maybe they’re less, but they’ve already kind of maximized what they can given what they’re facing. And the remaining constraints in the world tend to be, they’re sticky, you know, they’re systemic. They’re things that change slowly over time, or maybe rapidly over time, given, you know, in the rare case of a real kind of moment of social revolution or technological revolution, I guess. But they’re not something that you can just do, like an NGO, can just implement a policy and, like, fix it or something."

Seeing a lot of non-definitions throughout this episode. No one wants to define anything, but still take action

How can they say something is lacking if they cannot even agree on a definition?

this last paragraph really closed this article by specifically describing how am little change can affect someone long term in a positive way.

Festivals are sparking again and this gives some spotlight to women... could be good to mention this at some point in the paper. Many women actually headline these festivals now and are the main event that people come and see. There are still a lot of men though, but I am thinking of newer stars like Billie Eilish, Olivia Rodrigo... Taylor Swift does not do festivals but I would also like to include how her world tour influenced the industry and the economy around her.

Argues how, as wales didnt have continued poltics, had to find another way to secure basis of national history, for Edwards it was the mountains. it captured people's imagination! makes comparison to Lloyd again,but still highlights how Edwards draws distinc altho simplisitc parralels between the mountains and its people. He used it as they had both seperated and united the country, variation of landscape is also emphasised --> claims this was reason why politcal unity had been different, however the geographical character had also made it different to England.

Claims that, with Welsh history sidelined in schools,O.M. Edwards sought to make an awareness of the welsh past as it was crucial for national identity - is thisabit of a stretch?

decentralize government to mamke sure no predatorey government to minority so they dont mobilize

decentralizattion = competition :)

people learn history from all sorts of things, its fragmented and sitored based on afew key events, people and trends - but doesnt mean that it's insignificant. hiatory wasn't the only motivation for nationalists, the present was important too, but it did pla a role. nationalists were aware of history's potential to mobilize national sentiment - lots about glorious heritage and english oppression some calling it pure propaganda.

notice here that the soldiers who are racialized as black are described as "very insolent". They are acting in the same way as the white-racialized soldiers after being ordered to do so, but the term is only applied to the black-racialized.

But doesn't this risk also exist when loading on-wiki scripts maintained by others too?

Synthèse de Pratiques Pédagogiques : Créer du Lien et de la Bienveillance en Maternelle

Résumé Exécutif

Ce document synthétise les réflexions et les pratiques de Mélanie, enseignante en maternelle à Strasbourg forte de 12 ans d'expérience. Le point central de son approche est la création d'un lien affectif profond avec ses élèves, un élément qu'elle considère comme le socle indispensable à tout apprentissage. En rompant délibérément avec la distance professionnelle traditionnelle, elle préconise une « communication bienveillante » et une « fermeté bienveillante » pour instaurer un climat de confiance.

Le document détaille comment cette posture se traduit concrètement par l'abandon des systèmes de notation du comportement, l'adoption de classes à niveaux multiples pour favoriser la douceur sociale, et une organisation spatiale et pédagogique axée sur l'autonomie. L'objectif ultime est de transformer la classe en un « cocon » serein où l'enfant, apaisé et respecté dans son rythme, développe un rapport positif durable avec l'institution scolaire.

1. La Centralité du Lien Affectif

Pour Mélanie, l'attachement entre l'enseignant et l'élève n'est pas un obstacle, mais un moteur pédagogique. Cette vision a évolué au cours de sa carrière, passant d'une réserve initiale à une affirmation assumée de l'affection envers ses élèves.

Déconstruction des barrières professionnelles

• Contestation du dogme de la distance : L'enseignante s'oppose à l'idée, souvent transmise lors de la formation initiale ou par certains tuteurs, qu'il faut maintenir une distance stricte. Elle cite l'ouvrage Chagrin d'école de Daniel Pennac pour illustrer cette interdiction tacite d'aimer ses élèves.

• Expression de l'affection : Elle assume l'utilisation de surnoms et n'hésite pas à dire « je t'aime » à ses élèves. Selon elle, cette proximité ne nuit pas au respect des règles ; au contraire, la connexion établie renforce l'autorité naturelle et le respect mutuel.

• L'importance de la stabilité : Le lien est plus difficile à tisser pour les enseignants remplaçants ou à temps partiel (comme les stagiaires en quart de décharge). Avoir sa propre classe à temps plein est présenté comme un facteur déterminant pour l'épanouissement professionnel et relationnel.

L'impact sur le climat scolaire

Le but est de créer un « cocon » où la sérénité est palpable. En début d'année, l'enseignante privilégie délibérément la relation, le cadre de travail et l'autonomie au détriment immédiat des apprentissages purement académiques, afin de garantir une fluidité pour le reste de l'année.

2. Une Gestion de Classe Basée sur la Communication

La communication dans la classe de Mélanie repose sur une compréhension profonde de l'enfant et un rejet des méthodes de coercition classiques.

Rejet des systèmes de comportement

L'enseignante a abandonné les outils traditionnels de gestion du comportement (lions de couleur, systèmes de points, etc.) car elle a constaté qu'ils aggravaient souvent les difficultés des élèves les plus fragiles. Elle privilégie désormais :

• La discussion systématique : Même si cela peut sembler répétitif ou aboutir parfois à des impasses, le dialogue reste l'outil principal.

• La compréhension des besoins : L'effort est mis sur l'analyse de la cause du comportement plutôt que sur la sanction immédiate.

• La posture physique : Elle souligne l'importance de parler à « hauteur d'enfant », une pratique inspirée des modèles scandinaves.

La « Fermeté Bienveillante »

Cette approche ne signifie pas l'absence de règles. Elle a été qualifiée par un inspecteur de « bienveillante fermeté ».

• Exemple de gestion de conflit : Face à une bévue (ex: écrire sur une table par inadvertance), l'enseignante dédramatise (« tu n'as pas besoin d'avoir l'air triste ») tout en responsabilisant l'enfant (nettoyer avec un papier et de l'eau).

• Limites de la patience : L'agacement survient principalement lors de comportements nuisant aux relations sociales (moqueries, phrases méchantes répétées), plutôt que lors d'accidents matériels.

3. Organisation Pédagogique et Structure de Classe

La forme scolaire elle-même est pensée pour soutenir cette bienveillance et s'adapter au rythme biologique et psychologique des enfants.

Les bénéfices des niveaux multiples

Mélanie préconise la mixité des âges (Petits, Moyens, Grands) pour plusieurs raisons :

• Atténuation des effets de groupe : Le mélange casse les dynamiques de groupes trop soudés et potentiellement conflictuels qui se suivent depuis la crèche.

• Instauration d'une douceur naturelle : La présence de « petits » incite les plus grands à la protection et au calme, créant une ambiance de type « familial ».

• Bénéfice social : Placer un enfant difficile avec des plus petits peut s'avérer bénéfique pour son propre apaisement.

Autonomie et différenciation

Le fonctionnement en autonomie permet d'éviter la standardisation des tâches :

• Les enfants ne sont pas obligés d'exécuter tous le même travail en même temps.

• Cela réduit le stress lié à des tâches inadaptées (trop complexes ou trop simples).

• L'apaisement qui en découle rend les élèves plus disponibles pour les apprentissages.

Aménagement de l'espace de travail

L'espace physique est segmenté en zones spécifiques pour favoriser différents types d'activités :

| Espace | Fonction / Caractéristiques | | --- | --- | | L'Ellipse | Un tracé au sol au milieu de la classe pour les regroupements (préféré aux tables). | | Espaces à scénario | Zones dédiées à des jeux de rôle ou situations thématiques (ex: yoga, pressing). | | Ateliers autonomes | Meubles de rangement organisés par domaines (phonologie, motricité fine, etc.). | | Sous le bureau | Utilisation de l'espace sous le bureau de l'enseignante pour créer un « coin écoute » avec des boîtes à histoires. | | Tables spécifiques | Table en U pour les ateliers dirigés, petite table pour la peinture. |

4. Posture et Défis de l'Enseignant

L'enseignement en maternelle exige un investissement personnel et une vigilance constante sur sa propre santé.

• Évolution de la sérénité : La confiance en soi s'acquiert avec les années et la stabilité du poste, permettant d'investir davantage dans la dimension relationnelle.

• Santé vocale : Mélanie souligne la pénibilité du métier pour la voix et les oreilles (bruit de la cour, appels). Elle s'efforce de ne pas crier pour préserver ses cordes vocales et maintenir le calme ambiant.

• Compétences annexes : L'usage d'instruments, comme la guitare (apprise de manière autodidacte), est utilisé comme un outil de lien supplémentaire, très apprécié par les élèves malgré un niveau technique qu'elle juge modeste.

Conclusion

L'approche décrite dans ce document montre que la réussite scolaire en maternelle repose sur un équilibre entre une structure pédagogique flexible (autonomie, multi-niveaux) et une relation humaine forte. En cassant la barrière de la distance traditionnelle, l'enseignante crée un environnement sécurisant qui favorise l'appétence des enfants pour l'école dès leurs premières années.

Réceptivité des Formats de Pratique en Musculation et Développement de l’Intérêt

Résumé Exécutif

Ce document synthétise les recherches de Mehdi Belhouchat concernant l'engagement psychologique des élèves en musculation scolaire.

L'étude s'appuie sur le modèle de développement de l'intérêt en quatre phases pour évaluer comment différents formats de pratique influencent la motivation des élèves. Les conclusions majeures révèlent une corrélation directe entre le niveau d'intérêt initial d'un élève et sa réceptivité à un format spécifique.

Alors que les élèves experts autogénèrent leur intérêt quelle que soit la tâche, les élèves novices (phases 1 et 2) sont extrêmement dépendants du design pédagogique.

Les formats favorisant un guidage externe (APRE) ou interne (RPE) s'avèrent les plus efficaces pour déclencher l'engagement chez les débutants, tandis que le format "au temps" doit être utilisé de manière stratégique et ponctuelle pour favoriser des sauts qualitatifs de progression.

Cadre Théorique et Problématique

La recherche s'inscrit dans le cadre de la théorie de l'intérêt, notamment développée par Cédric Roure en contexte francophone, et le design de tâches d'apprentissage (Olivier Dieu).

Le Constat de Départ

• Expansion de la musculation : Une activité en forte croissance depuis 20 ans en milieu scolaire et sociétal.

• Hétérogénéité des profils : Les classes se composent d'élèves aux profils variés, allant de l'expert inscrit en salle de sport (intérêt individuel développé) au décrocheur sédentaire (intérêt faible ou nul).

• Décalage des formats : Il existe une rupture entre les formats scolaires traditionnels (souvent basés sur le ressenti subjectif/RPE) et les pratiques sociales plus objectives, guidantes et intenses.

Le Modèle de l'Intérêt en Quatre Phases

Le développement de l'intérêt est analysé comme un passage d'un état psychologique éphémère à un trait de personnalité intégré :

1. Phase 1 : Intérêt individuel très faible.

2. Phase 2 : Intérêt individuel faible.

3. Phase 3 : Intérêt individuel émergent.

4. Phase 4 : Intérêt individuel bien développé.

L'intérêt en situation est mesuré par trois facteurs : le déclenchement, le maintien au ressenti (valence affective) et le maintien aux valeurs (ancrage profond).

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Analyse des Formats de Pratique

L'étude identifie trois types de guidage dans l'autorégulation de la charge de travail :

| Format | Nature du Guidage | Caractéristiques | | --- | --- | --- | | APRE (Autoregulation Progressive Resistance Exercise) | Externe | Protocole normatif strict (tableaux). L'élève a peu de choix ; l'environnement dicte l'action. | | Au Temps | Mixte | Équilibre entre l'individu et l'environnement. Repère temporel imposé, mais décision de charge laissée à l'élève. | | RPE (Rating of Perceived Exertion) | Interne | Poids de l'environnement très faible. L'élève est au cœur des décisions de régulation selon son ressenti. |

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Résultats Clés de la Recherche

L'étude, menée auprès de 319 participants (10 classes de lycée et étudiants), met en évidence plusieurs phénomènes critiques :

1. L'Indépendance des Experts

Les élèves situés en phases 3 et 4 (intérêt émergent ou développé) ne montrent aucune réceptivité spécifique aux formats.

Ils projettent leur propre intérêt dans n'importe quelle situation et sont capables de redéfinir le but de la tâche pour s'impliquer. Ils sont psychologiquement indépendants du design pédagogique.

2. La Sensibilité des Novices

Pour les élèves en phases 1 et 2, le format est déterminant :

• Le format APRE (guidage externe) est dominant pour les novices les plus éloignés de la pratique (Phase 1). Il agit comme un environnement "puissant" qui stimule l'affect et l'intensité physique.

• Le format RPE (guidage interne) est également efficace en Phase 2, car il permet à l'élève de connecter ses propres expériences aux connaissances à acquérir.

• Le format "Au Temps" est le moins efficace pour déclencher l'intérêt chez les novices.

3. Dynamique de Développement de l'Intérêt

• Linéarité du RPE : Ce format favorise un développement constant de l'intérêt à travers toutes les phases. Il est idéal pour gérer l'hétérogénéité d'une classe.

• Non-linéarité du format "Au Temps" : Ce format ne produit des effets que lors d'une transition spécifique entre l'intérêt faible et l'intérêt émergent. Il provoque un "saut" qualitatif.

• Instabilité de l'APRE : Son impact est décrit comme "désordonné et fluctuant", suggérant qu'il doit être utilisé de façon percutante mais espacée.

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Préconisations Pédagogiques pour l'Enseignant

L'objectif pour l'enseignant est de devenir un "designer pédagogique" capable d'agencer les formats pour maximiser l'engagement, particulièrement chez les élèves les moins motivés.

Séquence Type Recommandée

Plutôt que d'utiliser un format unique, l'étude suggère un agencement stratégique durant le cycle de musculation :

1. Début de cycle (Novices) : Prioriser des formats hybrides ou le RPE. Cela permet d'épouser l'hétérogénéité de la classe et d'enclencher le processus de développement de l'intérêt.

2. Milieu de cycle : Introduire ponctuellement le format APRE pour injecter de l'intensité et stimuler les facteurs externes de l'intérêt.

3. Fin de cycle : Utiliser le format Au Temps. Ce format, moins adapté aux débutants complets, devient pertinent plus tard pour valider un saut qualitatif dans l'intérêt émergent.

4. Évaluation : Utiliser des formats hybrides pour stabiliser les acquis.

"Une bonne séquence pédagogique en musculation, c'est la capacité à agencer convenablement des formats, notamment pour les novices." — Mehdi Belhouchat

Good. This one sounds scary but the idea is actually simple.

Let’s go slow.

The sentence:

“If localities are insufficiently homogeneous…”

Translation:

If local areas are not all made up of similar people.

So inside one town or region, there might be:

• Different ethnic groups
• Different religions
• Different economic classes
• Different political views

In other words: each locality is internally divided.

“…decentralisation may paradoxically diminish accountability…”

Paradoxically = surprisingly.

Diminish accountability = make government less responsive to some people.

So it’s saying:

Giving power to local governments can sometimes make government LESS responsive.

That sounds backwards, right? We usually think local = more responsive.

“…to interests that are marginalised in localities…”

Marginalised = small minority, ignored group.

So imagine:

In each town, Group X is only 10% of the population.

They are too small locally to influence elections.

So local politicians can ignore them.

“…but enjoy some degree of cohesiveness at central level.”

Here’s the key.

Even though Group X is small in each town…

Across the whole country, they might be:

• 20% or 25% of the total population
• United
• Organized
• Able to influence national elections

So nationally, they matter.

Locally, they don’t.

What’s the big idea?

If you decentralise power:

• Decisions move to the local level.
• In each locality, the minority group is too small to matter.
• They get ignored everywhere.

But if power is centralised:

• That same minority group can band together nationally.
• Their total size becomes politically important.
• National politicians have to pay attention to them.

So decentralisation can actually make minorities worse off.

Super simple example

Imagine a country with:

• 10 regions
• In each region, Group A = 90%, Group B = 10%

If power is local:

• Group B loses everywhere.
• They are too small in every region.

If power is central:

• Group B is now 10% × 10 regions = 10% of the whole country.
• That might be enough to swing national elections.
• National politicians might listen to them.

One-sentence idiot version:

If a group is small in every town but bigger when added up nationally, giving power to towns can make them weaker — not stronger.

That’s the “paradox.”

When decisions require many places to agree, extreme situations (very secure or very insecure governments) get exaggerated under centralisation — but moderate situations don’t.

centralized govt can differentiate policy

decentralization = matter of which groups of electors decide re-election. accountability

the reason why politicians r inefficient is because contracts r incomplete

I think this is key. Even without a productivist mindset, some games can get frustratingly slow. Too challenging, in a way. Too painful perhaps. Too minimal to encourage reflection. Is a game like Mini Metro a reflective one? I don't think much. But then again, is a game like Magnet Block? Not either, probably.

I feel the focus on walking simulators is nice as it gives many examples of what could be, but it misses genres like puzzles, or visual novels, that may sometimes be blurry! Coffee Talk or The Red Strings may be somewhat reflective, but is Doki Doki Literature Club or Gods Will be Watching? The story... the story and the intensity therein, its agressiveness, its fear, violence, etc. I'd argue contribute to the perception as well.

The Idea of sperate parties based on views is something inevitable, and has been shown in different governments in different ways.

Reviewer #1 (Public review):

Summary:

This manuscript by Lin et al. presents a timely, technically strong study that builds patient-specific midbrain-like organoids (MLOs) from hiPSCs carrying clinically relevant GBA1 mutations (L444P/P415R and L444P/RecNcil). The authors comprehensively characterize nGD phenotypes (GCase deficiency, GluCer/GluSph accumulation, altered transcriptome, impaired dopaminergic differentiation), perform CRISPR correction to produce an isogenic line, and test three therapeutic modalities (SapC-DOPS-fGCase nanoparticles, AAV9-GBA1, and SRT with GZ452). The model and multi-arm therapeutic evaluation are important advances with clear translational value.

My overall recommendation is that the work undergo a major revision to address the experimental and interpretive gaps listed below.

Strengths:

(1) Human, patient-specific midbrain model: Use of clinically relevant compound heterozygous GBA1 alleles (L444P/P415R and L444P/RecNcil) makes the model highly relevant to human nGD and captures patient genetic context that mouse models often miss.

(2) Robust multi-level phenotyping: Biochemical (GCase activity), lipidomic (GluCer/GluSph by UHPLC-MS/MS), molecular (bulk RNA-seq), and histological (TH/FOXA2, LAMP1, LC3) characterization are thorough and complementary.

(3) Use of isogenic CRISPR correction: Generating an isogenic line (WT/P415R) and demonstrating partial rescue strengthens causal inference that the GBA1 mutation drives many observed phenotypes.

(4) Parallel therapeutic testing in the same human platform: Comparing enzyme delivery (SapC-DOPS-fGCase), gene therapy (AAV9-GBA1), and substrate reduction (GZ452) within the same MLO system is an elegant demonstration of the platform's utility for preclinical evaluation.

(5) Good methodological transparency: Detailed protocols for MLO generation, editing, lipidomics, and assays allow reproducibility

Weaknesses:

(1) Limited genetic and biological replication

(a) Single primary disease line for core mechanistic claims. Most mechanistic data derive from GD2-1260 (L444P/P415R); GD2-10-257 (L444P/RecNcil) appears mainly in therapeutic experiments. Relying primarily on one patient line risks conflating patient-specific variation with general nGD mechanisms.

(b) Unclear biological replicate strategy. It is not always explicit how many independent differentiations and organoid batches were used (biological replicates vs. technical fields of view).

(c) A significant disadvantage of employing brain organoids is the heterogeneity during induction and potential low reproducibility. In this study, it is unclear how many independent differentiation batches were evaluated and, for each test (for example, immunofluorescent stain and bulk RNA-seq), how many organoids from each group were used. Please add a statement accordingly and show replicates to verify consistency in the supplementary data.

(d) Isogenic correction is partial. The corrected line is WT/P415R (single-allele correction); residual P415R complicates the interpretation of "full" rescue and leaves open whether the remaining pathology is due to incomplete correction or clonal/epigenetic effects.

(e) The authors tested week 3, 4, 8, 15, and 28 old organoids in different settings. However, systematic markers of maturation should be analyzed, and different maturation stages should be compared, for example, comparing week 8 organoids to week 28 organoids, with immunofluorescent marker staining and bulk RNAseq.

(f) The manuscript frequently refers to Wnt signaling dysregulation as a major finding. However, experimental validation is limited to transcriptomic data. Functional tests, such as the use of Wnt agonist/inhibitor, are needed to support this claim (see below).

(g) Suggested fixes/experiments

Add at least one more independent disease hiPSC line (or show expanded analysis from GD2-10-257) for key mechanistic endpoints (lipid accumulation, transcriptomics, DA markers)

Generate and analyze a fully corrected isogenic WT/WT clone (or a P415R-only line) if feasible; at minimum, acknowledge this limitation more explicitly and soften claims.

Report and increase independent differentiations (N = biological replicates) and present per-differentiation summary statistics.

(2) Mechanistic validation is insufficient

(a) RNA-seq pathways (Wnt, mTOR, lysosome) are not functionally probed. The manuscript shows pathway enrichment and some protein markers (p-4E-BP1) but lacks perturbation/rescue experiments to link these pathways causally to the DA phenotype.

(b) Autophagy analysis lacks flux assays. LC3-II and LAMP1 are informative, but without flux assays (e.g., bafilomycin A1 or chloroquine), one cannot distinguish increased autophagosome formation from decreased clearance.

(c) Dopaminergic dysfunction is superficially assessed. Dopamine in the medium and TH protein are shown, but no neuronal electrophysiology, synaptic marker co-localization, or viability measures are provided to demonstrate functional recovery after therapy.

(d) Suggested fixes/experiments

Perform targeted functional assays:

(i) Wnt reporter assays (TOP/FOP flash) and/or treat organoids with Wnt agonists/antagonists to test whether Wnt modulation rescues DA differentiation.

(ii)Test mTOR pathway causality using mTOR inhibitors (e.g., rapamycin) or 4E-BP1 perturbation and assay effects on DA markers and autophagy.

Include autophagy flux assessment (LC3 turnover with bafilomycin), and measure cathepsin activity where relevant.

Add at least one functional neuronal readout: calcium imaging, MEA recordings, or synaptic marker quantification (e.g., SYN1, PSD95) together with TH colocalization.

(3) Therapeutic evaluation needs greater depth and standardization

(a) Short windows and limited durability data. SapC-DOPS and AAV9 experiments range from 48 hours to 3 weeks; longer follow-up is needed to assess durability and whether biochemical rescue translates into restored neuronal function.

(b) Dose-response and biodistribution are under-characterized. AAV injection sites/volumes are described, but transduction efficiency, vg copies per organoid, cell-type tropism quantification, and SapC-DOPS penetration/distribution are not rigorously quantified.

(c) Specificity controls are missing. For SapC-DOPS, inclusion of a non-functional enzyme control (or heat-inactivated fGCase) would rule out non-specific nanoparticle effects. For AAV, assessment of off-target expression and potential cytotoxicity is needed.

(d) Comparative efficacy lacking. It remains unclear which modality is most effective in the long term and in which cellular compartments.

(e) Suggested fixes/experiments

Extend follow-up (e.g., 6+ weeks) after AAV/SapC dosing and evaluate DA markers, electrophysiology, and lipid levels over time.

Quantify AAV transduction by qPCR for vector genomes and by cell-type quantification of GFP+ cells (neurons vs astrocytes vs progenitors).

Include SapC-DOPS control nanoparticles loaded with an inert protein and/or fluorescent cargo quantitation to show distribution and uptake kinetics.

Provide head-to-head comparative graphs (activity, lipid clearance, DA restoration, and durability) with statistical tests.

(4) Model limitations not fully accounted for in interpretation

(a) Absence of microglia and vasculature limits recapitulation of neuroinflammatory responses and drug penetration, both of which are important in nGD. These absences could explain incomplete phenotypic rescues and must be emphasized when drawing conclusions about therapeutic translation.

(b) Developmental vs degenerative phenotype conflation. Many phenotypes appear during differentiation (patterning defects). The manuscript sometimes interprets these as degenerative mechanisms; the distinction must be clarified.

(c) Suggested fixes

Tone down the language throughout (Abstract/Results/Discussion) to avoid overstatement that MLOs fully recapitulate nGD neuropathology.

Add plans or pilot data (if available) for microglia incorporation or vascularization to indicate how future work will address these gaps.

(5) Statistical and presentation issues

(a) Missing or unclear sample sizes (n). For organoid-level assays, report the number of organoids and the number of independent differentiations.

(b) Statistical assumptions not justified. Tests assume normality; where sample sizes are small, consider non-parametric tests and report exact p-values.

(c) Quantification scope. Many image quantifications appear to be from selected fields of view, which are then averaged across organoids and differentiations.

(d) RNA-seq QC and deposition. Provide mapping rates, batch correction details, and ensure the GEO accession is active. Include these in Methods/Supplement.

(e) Suggested fixes

Add a table summarizing biological replicates, technical replicates, and statistical tests used for each figure panel.

Recompute statistics where appropriate (non-parametric if N is small) and report effect sizes and confidence intervals.

(6) Minor comments and clarifications

(a) The authors should validate midbrain identity further with additional regional markers (EN1, OTX2) and show absence/low expression of forebrain markers (FOXG1) across replicates.

(b) Extracellular dopamine ELISA should be complemented with intracellular dopamine or TH+ neuron counts normalized per organoid or per total neurons.

(c) For CRISPR editing: the authors should report off-target analysis (GUIDE-seq or targeted sequencing of predicted off-targets) or at least in-silico off-target score and sequencing coverage of the edited locus.

(d) It should be clarified as to whether lipidomics normalization is to total protein per organoid or per cell, and include representative LC-MS chromatograms or method QC.

(e) Figure legends should be improved in order to state the number of organoids, the number of differentiations, and the exact statistical tests used (including multiple-comparison corrections).

(f) In the title, the authors state "reveal disease mechanisms", but the studies mainly exhibit functional changes. They should consider toning down the statement.

(7) Recommendations

This reviewer recommends a major revision. The manuscript presents substantial novelty and strong potential impact but requires additional experimental validation and clearer, more conservative interpretation. Key items to address are:

(a) Strengthening genetic and biological replication (additional lines or replicate differentiations).

(b) Adding functional mechanistic validation for major pathways (Wnt/mTOR/autophagy) and providing autophagy flux data.

(c) Including at least one neuronal functional readout (calcium imaging/MEA/patch) to demonstrate functional rescue.

(d) Deepening therapeutic characterization (dose, biodistribution, durability) and including specificity controls.

(e) Improving statistical reporting and explicitly stating biological replicate structure.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

This manuscript by Lin et al. presents a timely, technically strong study that builds patientspecific midbrain-like organoids (MLOs) from hiPSCs carrying clinically relevant GBA1 mutations (L444P/P415R and L444P/RecNcil). The authors comprehensively characterize nGD phenotypes (GCase deficiency, GluCer/GluSph accumulation, altered transcriptome, impaired dopaminergic differentiation), perform CRISPR correction to produce an isogenic line, and test three therapeutic modalities (SapC-DOPS-fGCase nanoparticles, AAV9GBA1, and SRT with GZ452). The model and multi-arm therapeutic evaluation are important advances with clear translational value.

My overall recommendation is that the work undergo a major revision to address the experimental and interpretive gaps listed below.

Strengths:

(1) Human, patient-specific midbrain model: Use of clinically relevant compound heterozygous GBA1 alleles (L444P/P415R and L444P/RecNcil) makes the model highly relevant to human nGD and captures patient genetic context that mouse models often miss.

(2) Robust multi-level phenotyping: Biochemical (GCase activity), lipidomic (GluCer/GluSph by UHPLC-MS/MS), molecular (bulk RNA-seq), and histological (TH/FOXA2, LAMP1, LC3) characterization are thorough and complementary.

(3) Use of isogenic CRISPR correction: Generating an isogenic line (WT/P415R) and demonstrating partial rescue strengthens causal inference that the GBA1 mutation drives many observed phenotypes.

(4) Parallel therapeutic testing in the same human platform: Comparing enzyme delivery (SapC-DOPS-fGCase), gene therapy (AAV9-GBA1), and substrate reduction (GZ452) within the same MLO system is an elegant demonstration of the platform's utility for preclinical evaluation.

(5) Good methodological transparency: Detailed protocols for MLO generation, editing, lipidomics, and assays allow reproducibility

Weaknesses:

(1) Limited genetic and biological replication

(a) Single primary disease line for core mechanistic claims. Most mechanistic data derive from GD2-1260 (L444P/P415R); GD2-10-257 (L444P/RecNcil) appears mainly in therapeutic experiments. Relying primarily on one patient line risks conflating patient-specific variation with general nGD mechanisms.

We thank the reviewer for highlighting the importance of genetic and biological replication. An additional patient-derived iPSC line was included in the manuscript, therefore, our study includes two independent nGD patient-derived iPSC lines, GD2-1260 (GBA1^L444P/P415R) and GD2-10-257 (GBA1^{L444P/RecNcil}), both of which carry the severe mutations associated with nGD. These two lines represent distinct genetic backgrounds and were used to demonstrate the consistency of key disease phenotypes (reduced GCase activity, elevated substrate, impaired dopaminergic neuron differentiation, etc.) across different patient’s MLOs. Major experiments (e.g., GCase activity assays, substrate, immunoblotting for DA marker TH, and therapeutic testing with SapC-DOPS-fGCase, AAV9-GBA1) were performed using both patient lines, with results showing consistent phenotypes and therapeutic responses (see Figs. 2-6, and Supplementary Figs. 4-5). To ensure clarity and transparency, a new Supplementary Table 2 summarizes the characterization of both the GD2-1260 and GD2-10-257 lines.

(b) Unclear biological replicate strategy. It is not always explicit how many independent differentiations and organoid batches were used (biological replicates vs. technical fields of view).

Biological replication was ensured in our study by conducting experiments in at least 3 independent differentiations per line, and technical replicates (multiple organoids/fields per batch) were averaged accordingly. We have clarified biological replicates and differentiation in the figure legends. 

(c) A significant disadvantage of employing brain organoids is the heterogeneity during induction and potential low reproducibility. In this study, it is unclear how many independent differentiation batches were evaluated and, for each test (for example, immunofluorescent stain and bulk RNA-seq), how many organoids from each group were used. Please add a statement accordingly and show replicates to verify consistency in the supplementary data.

In the revision, we have clarified biological replicates and differentiation in the figure legend in Fig.1E; Fig.2B,2G; Fig.3F, 3G; Fig.4B-C,E,H-J, M-N; Fig.6D; and Fig.7A-C, I.

(d) Isogenic correction is partial. The corrected line is WT/P415R (single-allele correction); residual P415R complicates the interpretation of "full" rescue and leaves open whether the remaining pathology is due to incomplete correction or clonal/epigenetic effects.

We attempted to generate an isogenic iPSC line by correcting both GBA1 mutations (L444P and P415R). However, this was not feasible because GBA1 overlaps with a highly homologous pseudogene (PGBA), which makes precise editing technically challenging. Consequently, only the L444P mutation was successfully corrected, and the resulting isogenic line retains the P415R mutation in a heterozygous state. Because Gaucher disease is an autosomal recessive disorder, individuals carrying a single GBA1 mutation (heterozygous carriers) do not develop clinical symptoms. Therefore, the partially corrected isogenic line, which retains only the P415R allele, represents a clinically relevant carrier model. Consistent with this, our results show that GCase activity was restored to approximately 50% of wild-type levels (Fig.4B-C), supporting the expected heterozygous state. These findings also make it unlikely that the remaining differences observed are due to clonal variation or epigenetic effects.

(e) The authors tested week 3, 4, 8, 15, and 28 old organoids in different settings. However, systematic markers of maturation should be analyzed, and different maturation stages should be compared, for example, comparing week 8 organoids to week 28 organoids, with immunofluorescent marker staining and bulk RNAseq.

We agree that a systematic analysis of maturation stages is essential for validating the MLO model. Our data integrated a longitudinal comparison across multiple developmental windows (Weeks 3 to 28) to characterize the transition from progenitors to mature/functional states for nGD phenotyping and evaluation of therapeutic modalities: 1) DA differentiation (Wks 3 and 8 in Fig. 3): qPCR analysis demonstrated the progression of DA-specific programs. We observed a steady increase in the mature DA neuron marker TH and ASCL1. This was accompanied by a gradual decrease in early floor plate/progenitor markers FOXA2 and PLZF, indicating a successful differentiation path from progenitors to differentiated/mature DA neurons. 2) Glycosphingolipid substrates accumulation (Wks 15 and 28 in Fig 2): To assess late-stage nGD phenotyping, we compared GluCer and GluSph at Week 15 and Week 28. This comparison highlights the progressive accumulation of substrates in nGD MLOs, reflecting the metabolic consequences of the disease at different mature stage. 3) Organoid growth dynamics (Wks 4, 8, and 15 in new Fig. 4): The new Fig. 4 tracks physical maturation through organoid size and growth rates across three key time points, providing a macro-scale verification of consistent development between WT and nGD groups. By comparing these early (Wk 3-8) and late (Wk 15-28) stages, we confirmed that our MLOs transition from a proliferative state to a post-mitotic, specialized neuronal state, satisfied the requirement for comparing distinct maturation stages.

(f) The manuscript frequently refers to Wnt signaling dysregulation as a major finding. However, experimental validation is limited to transcriptomic data. Functional tests, such as the use of Wnt agonist/inhibitor, are needed to support this claim (see below).

We agree that the suggested experiments could provide additional mechanistic insights into this study and will consider them in future work.

(g) Suggested fixes / experiments

Add at least one more independent disease hiPSC line (or show expanded analysis from GD2-10-257) for key mechanistic endpoints (lipid accumulation, transcriptomics, DA markers).

Additional line iPSC GD2-10-257 derived MLO was included in the manuscript. This was addressed above [see response to Weaknesses (1)-a]. 

Generate and analyze a fully corrected isogenic WT/WT clone (or a P415R-only line) if feasible; at minimum, acknowledge this limitation more explicitly and soften claims.

We attempted to generate an isogenic iPSC line by correcting both GBA1 mutations (L444P and P415R). However, this was unsuccessful because the GBA1 gene overlaps with a pseudogene (PGBA) located 16 kb downstream of GBA1, which shares 96-98% sequence similarity with GBA1 (Ref#1, #2), which complicates precise editing. GBA1 is shorter (~5.7 kb) than PGBA (~7.6 kb). The primary exonic difference between GBA1 and PGBA is a 55-bp deletion in exon 9 of the pseudogene. As a result, the isogenic line we obtained carries only the P415R mutation, and L444P was corrected to the normal sequence. We have included this limitation in the Methods as “This gene editing strategy is expected to also target the GBA1 pseudogene due to the identical target sequence, which limits the gene correction on certain mutations (e.g., P415R)”. 

References:

(1) Horowitz M., Wilder S., Horowitz Z., Reiner O., Gelbart T., Beutler E. The human glucocerebrosidase gene and pseudogene: structure and evolution. Genomics (1989). 4, 87–96. doi:10.1016/0888-7543(89)90319-4

(2) Woo EG, Tayebi N, Sidransky E. Next-Generation Sequencing Analysis of GBA1: The Challenge of Detecting Complex Recombinant Alleles. Front Genet. (2021). 12:684067. doi:10.3389/fgene.2021.684067. PMCID: PMC8255797.

Report and increase independent differentiations (N = biological replicates) and present per-differentiation summary statistics.

This was addressed above [see response to Weaknesses (1)-b, (1)-c]. 

(2) Mechanistic validation is insufficient

(a) RNA-seq pathways (Wnt, mTOR, lysosome) are not functionally probed. The manuscript shows pathway enrichment and some protein markers (p-4E-BP1) but lacks perturbation/rescue experiments to link these pathways causally to the DA phenotype.

(b) Autophagy analysis lacks flux assays. LC3-II and LAMP1 are informative, but without flux assays (e.g., bafilomycin A1 or chloroquine), one cannot distinguish increased autophagosome formation from decreased clearance.

(c) Dopaminergic dysfunction is superficially assessed. Dopamine in the medium and TH protein are shown, but no neuronal electrophysiology, synaptic marker co-localization, or viability measures are provided to demonstrate functional recovery after therapy.

(d) Suggested fixes/experiments

Perform targeted functional assays:

(i) Wnt reporter assays (TOP/FOP flash) and/or treat organoids with Wnt agonists/antagonists to test whether Wnt modulation rescues DA differentiation.

(ii) Test mTOR pathway causality using mTOR inhibitors (e.g., rapamycin) or 4E-BP1 perturbation and assay effects on DA markers and autophagy.

Include autophagy flux assessment (LC3 turnover with bafilomycin), and measure cathepsin activity where relevant.

Add at least one functional neuronal readout: calcium imaging, MEA recordings, or synaptic marker quantification (e.g., SYN1, PSD95) together with TH colocalization.

We thank the reviewer for these valuable suggestions. We agree that the suggested experiments could provide additional mechanistic insights into this study and will consider them in future work. Importantly, the primary conclusions of our manuscript, that GBA1 mutations in nGD MLOs resulted in nGD pathologies such as diminished enzymatic function, accumulation of lipid substrates, widespread transcriptomic changes, and impaired dopaminergic neuron differentiation, which can be corrected by several therapeutic strategies in this study, are supported by the evidence presented. The suggested experiments represent an important direction for future research using brain organoids.

(3) Therapeutic evaluation needs greater depth and standardization

(a) Short windows and limited durability data. SapC-DOPS and AAV9 experiments range from 48 hours to 3 weeks; longer follow-up is needed to assess durability and whether biochemical rescue translates into restored neuronal function.

We agree with the reviewer. Because this is a proof-of-principle study, the treatment was designed within a short time window. Long-term studies with more comprehensive outcome assessments will be conducted in future work.

(b) Dose-response and biodistribution are under-characterized. AAV injection sites/volumes are described, but transduction efficiency, vg copies per organoid, cell-type tropism quantification, and SapC-DOPS penetration/distribution are not rigorously quantified.

We appreciate the reviewer’s concerns. This study was intended to demonstrate the feasibility and initial response of MLOs to AAV therapy. A comprehensive evaluation of AAV biodistribution will be considered in future studies.

The penetration and distribution of SapC-DOPS have been extensively characterized in prior studies. In vivo biodistribution of SapC–DOPS coupled CellVue Maroon, a fluorescent cargo, was examined in mice bearing human tumor xenografts using real-time fluorescence imaging, where CellVue Maroon fluorescence in tumor remained for 48 hours (Ref. #3: Fig. 4B, mouse 1), 100 hours (Ref. #4: Fig. 5), up to 216 hours (Ref. #5: Fig. 3). Uptake kinetics were also demonstrated in cells, with flow cytometry quantification showing that fluorescent cargo coupled SapC-DOPS nanovesicles, were incorporated into human brain tumor cell membranes within minutes and remained stably incorporated into the cells for up to one hour (Ref. # 6: Fig. 1a and Fig. 1b). Building on these findings, the present study focuses on evaluating the restoration of GCase function rather than reexamining biodistribution and uptake kinetics.

References:

(3) X. Qi, Z. Chu, Y.Y. Mahller, K.F. Stringer, D.P. Witte, T.P. Cripe. Cancer-selective targeting and cytotoxicity by liposomal-coupled lysosomal saposin C protein. Clin. Cancer Res. (2009) 15, 5840-5851. PMID: 19737950.

(4) Z. Chu, S. Abu-Baker, M.B. Palascak, S.A. Ahmad, R.S. Franco, and X. Qi. Targeting and cytotoxicity of SapC-DOPS nanovesicles in pancreatic cancer. PLOS ONE (2013) 8, e75507. PMID: 24124494.

(5) Z. Chu, K. LaSance, V.M. Blanco, C.-H. Kwon, B., Kaur, M., Frederick, S., Thornton, L., Lemen, and X. Qi. Multi-angle rotational optical imaging of brain tumors and arthritis using fluorescent SapC-DOPS nanovesicles. J. Vis. Exp. (2014) 87, e51187, 17. PMID: 24837630.

(6) J. Wojton, Z. Chu, C-H. Kwon, L.M.L. Chow, M. Palascak, R. Franco, T. Bourdeau, S. Thornton, B. Kaur, and X. Qi. Systemic delivery of SapC-DOPS has antiangiogenic and antitumor effects against glioblastoma. Mol. Ther. (2013) 21, 1517-1525. PMID: 23732993.

(c) Specificity controls are missing. For SapC-DOPS, inclusion of a non-functional enzyme control (or heat-inactivated fGCase) would rule out non-specific nanoparticle effects. For AAV, assessment of off-target expression and potential cytotoxicity is needed.

Including inactive fGCase would confound the assessment of fGCase in MLOs by immunoblot and immunofluorescence; therefore, saposin C–DOPS was used as the control instead. 

We agree that assessment of Off-target expression and potential cytotoxicity for AAV is important; this will be included in future studies.

(d) Comparative efficacy lacking. It remains unclear which modality is most effective in the long term and in which cellular compartments.

To address this comment, we have added a new table (Supplementary Table 2) comparing the four therapeutic modalities and summarizing their respective outcomes. While this study focused on short-term responses as a proof-of-principle, future work will explore long-term therapeutic effects. 

(e) Suggested fixes/experiments

Extend follow-up (e.g., 6+ weeks) after AAV/SapC dosing and evaluate DA markers, electrophysiology, and lipid levels over time.

We appreciate the reviewer’s suggestions. The therapeutic testing in patient-derived MLOs was designed as a proof-of-principle study to demonstrate feasibility and the primary response (rescue of GCase function) to the treatment. A comprehensive, long-term therapeutic evaluation of AAV and SapC-DOPS-fGCase is indeed important for a complete assessment; however, this represents a separate therapeutic study and is beyond the scope of the current work.

Quantify AAV transduction by qPCR for vector genomes and by cell-type quantification of GFP+ cells (neurons vs astrocytes vs progenitors).

For the AAV-treated experiments, we agree that measuring AAV copy number and GFP expression would provide additional information. However, the primary goal of this study was to demonstrate the key therapeutic outcome, rescue of GCase function by AAV-delivered normal GCase, which is directly relevant to the treatment objective.

Include SapC-DOPS control nanoparticles loaded with an inert protein and/or fluorescent cargo quantitation to show distribution and uptake kinetics.

As noted above [see response to Weakness (3)-c], using inert GCase would confound the assessment of fGCase uptake in MLOs; therefore, it was not suitable for this study. See response above for the distribution and uptake kinetics of SapC-DOPS [see response to Weaknesses (3)-b].

Provide head-to-head comparative graphs (activity, lipid clearance, DA restoration, and durability) with statistical tests.

We have added a new table (Supplementary Table 2) providing a head-to-head comparison of the treatment effects. 

(4) Model limitations not fully accounted for in interpretation

(a) Absence of microglia and vasculature limits recapitulation of neuroinflammatory responses and drug penetration, both of which are important in nGD. These absences could explain incomplete phenotypic rescues and must be emphasized when drawing conclusions about therapeutic translation.

We agree that the absence of microglia and vasculature in midbrain-like organoids represents a limitation, as we have discussed in the manuscript. In this revision, we highlighted this limitation in the Discussion section and clarified that it may contribute to incomplete phenotyping and phenotypic rescue observed in our therapeutic experiments. Additionally, we have outlined future directions to incorporate microglia and vascularization into the organoid system to better recapitulate the in vivo environment and improve translational relevance (see 7th paragraph in the Discussion).

(b) Developmental vs degenerative phenotype conflation. Many phenotypes appear during differentiation (patterning defects). The manuscript sometimes interprets these as degenerative mechanisms; the distinction must be clarified.

We appreciate the reviewer’s comments. In the revised manuscript, we have clarified that certain abnormalities, such as patterning defects observed during early differentiation, likely reflect developmental consequences of GBA1 mutations rather than degenerative processes. Conversely, phenotypes such as substrate accumulation, lysosomal dysfunction, and impaired dopaminergic maturation at later stages are interpreted as degenerative features. We have updated the Results and Discussion sections to avoid conflating developmental defects with neurodegenerative mechanisms.

(c) Suggested fixes

Tone down the language throughout (Abstract/Results/Discussion) to avoid overstatement that MLOs fully recapitulate nGD neuropathology.

The manuscript has been revised to avoid overstatements.

Add plans or pilot data (if available) for microglia incorporation or vascularization to indicate how future work will address these gaps.

The manuscript now includes further plans to address the incorporation of microglia and vascularization, described in the last two paragraphs in the Discussion. Pilot study of microglia incorporation will be reported when it is completed.

(5) Statistical and presentation issues

(a) Missing or unclear sample sizes (n). For organoid-level assays, report the number of organoids and the number of independent differentiations.

We have clarified biological replicates and differentiation in the figure legend [see response to Weaknesses (1)-b, (1)-c]. 

(b) Statistical assumptions not justified. Tests assume normality; where sample sizes are small, consider non-parametric tests and report exact p-values.

We have updated Statistical analysis in the methods as described below:

“For comparisons between two groups, data were analyzed using unpaired two-tailed Student’s t-tests when the sample size was ≥6 per group and normality was confirmed by the Shapiro-Wilk test. When the normality assumption was not met or when sample sizes were small (n < 6), the non-parametric Mann-Whitney U test was used instead. For comparisons involving three or more groups, one-way ANOVA followed by Tukey’s multiple comparison test was applied when data were normally distributed; otherwise, the nonparametric Dunn’s multiple comparison test was used. Exclusion of outliers was made based on cut-offs of the mean ±2 standard deviations. All statistical analyses were performed using GraphPad Prism 10 software. Exact p-values are reported throughout the manuscript and figures where feasible. A p-value < 0.05 was considered statistically significant.”

(c) Quantification scope. Many image quantifications appear to be from selected fields of view, which are then averaged across organoids and differentiations.

In this work, quantitative immunofluorescence analyses (e.g., cell counts for FOXP1+, FOXG1+, SOX2+ and Ki67+ cells, as well as marker colocalization) were performed on at least 3–5 randomly selected non-overlapping fields of view (FOVs) per organoid section, with a minimum of 3 organoids per differentiation batch. Each FOV was imaged at consistent magnification (60x) and z-stack depth to ensure comparable sampling across conditions. Data from individual FOVs were first averaged within each organoid to obtain an organoid-level mean, and then biological replicates (independent differentiations, n ≥ 3) were averaged to generate the final group mean ± SEM. This multilevel averaging approach minimizes bias from regional heterogeneity within organoids and accounts for variability across differentiations. Representative confocal images shown in the figures were selected to accurately reflect the quantified data. We believe this standardized quantification strategy ensures robust and reproducible results while appropriately representing the 3D architecture of the organoids.

In the revision, we have clarified the method used for image analysis of sectioned MLOs as below:

“Quantitative immunofluorescence analyses (e.g., cell counts for FOXP1+, FOXG1+, SOX2+ and Ki67+ cells, as well as marker colocalization) were performed using ImageJ (NIH) on at least 3–5 randomly selected non-overlapping fields of view (FOVs) per organoid section, with a minimum of 3 organoids per differentiation batch. Each FOV was imaged at consistent magnification (60x) and z-stack depth to ensure comparable sampling across conditions. Data from individual FOVs were first averaged within each organoid to obtain an organoid-level mean, and then biological replicates (independent differentiations, n ≥ 3) were averaged to generate the final group mean ± SEM.”

(d) RNA-seq QC and deposition. Provide mapping rates, batch correction details, and ensure the GEO accession is active. Include these in Methods/Supplement.

RNA-seq data are from the same batch. The mapping rate is >90%. GEO accession will be active upon publication. These were included in the Methods.

(e) Suggested fixes

Add a table summarizing biological replicates, technical replicates, and statistical tests used for each figure panel.

We have revised the figure legends to include replicates for each figure and statistical tests [see response in weaknesses (1)-b, (1)-c].

Recompute statistics where appropriate (non-parametric if N is small) and report effect sizes and confidence intervals.

Statistical analysis method is provided in the revision [see response in Weaknesses (5)-b].

(6) Minor comments and clarifications

(a) The authors should validate midbrain identity further with additional regional markers (EN1, OTX2) and show absence/low expression of forebrain markers (FOXG1) across replicates.

We validated the MLO identity by 1) FOXG1 and 2) EN1. FOXG1 was barely detectable in Wk8 75.1_MLO but highly present in ‘age-matched’ cerebral organoid (CO), suggesting our culturing method is midbrain region-oriented. In nGD MLO, FOXG1 expression is significantly higher than 75.1_MLO, indicating that there was aberrant anterior-posterior brain specification, consistent with the transcriptomic dysregulation observed in our RNA-seq data.

To further confirm midbrain identity, we examined the expression of EN1, an established midbrain-specific marker. Quantitative RT-PCR analysis demonstrated that EN1 expression increased progressively during differentiation in both WT-75.1 and nGD2-1260 MLOs at weeks 3 and 8 (Author response image 1). EN1 reached 34-fold and 373-fold higher levels than in WT-75.1 iPSCs at weeks 3 and 8, respectively, in WT-75.1 MLOs. In nGD MLOs, although EN1 expression showed a modest reduction at week 8, the levels were not significantly different from those observed in age-matched WT-75.1 MLOs (p > 0.05, ns).

Author response image 1.

qRT-PCR quantification of midbrain progenitor marker EN1 expression in WT-75.1 and GD2-1260 MLOs at Wk3 and Wk8. Data was normalized to WT-75.1 hiPSC cells and presented as mean ± SEM (n = 3-4 MLOs per group).ns, not significant.<br />

(b) Extracellular dopamine ELISA should be complemented with intracellular dopamine or TH+ neuron counts normalized per organoid or per total neurons.

We quantified TH expression at both the mRNA level (Fig. 3F) and the protein level (Fig. 3G/H) from whole-organoid lysates, which provides a more consistent and integrative measure across samples. These TH expression levels correlated well with the corresponding extracellular (medium) dopamine concentrations for each genotype. In contrast, TH⁺ neuron counts may not reliably reflect total cellular dopamine levels because the number of cells captured on each organoid section varies substantially, making normalization difficult. Measuring intracellular dopamine is an alternative approach that will be considered in future studies.

(c) For CRISPR editing: the authors should report off-target analysis (GUIDE-seq or targeted sequencing of predicted off-targets) or at least in-silico off-target score and sequencing coverage of the edited locus. (off-target analysis (GUIDE-seq or targeted sequencing of predicted off-targets) or at least in-silico off-target score and sequencing coverage of the edited locus). 

The off-target effect was analyzed during gene editing and the chance to target other off-targets is low due to low off-target scores ranked based on the MIT Specificity Score analysis. The related method was also updated as stated below:

“The chance to target other Off-targets is low due to low Off-target scores ranked based on the MIT Specificity Score analysis (Hsu, P., Scott, D., Weinstein, J. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31, 827–832 (2013).https://doi.org/10.1038/nbt.2647).”

(d) It should be clarified as to whether lipidomics normalization is to total protein per organoid or per cell, and include representative LC-MS chromatograms or method QC.

The normalization was to the protein of the organoid lysate. This was clarified in the Methods section in the revision as stated below:

“The GluCer and GluSph levels in MLO were normalized to total MLO protein (mg) that were used for glycosphingolipid analyses. Protein mass was determined by BCA assay and glycosphingolipid was expressed as pmol/mg protein. Additionally, GluSph levels in the culture medium were quantified and normalized to the medium volume (pmol/mL).”

Representative LC-MS chromatograms for both normal and GD MLOs have been included in a new figure, Supplementary Figure 2.

(e) Figure legends should be improved in order to state the number of organoids, the number of differentiations, and the exact statistical tests used (including multiplecomparison corrections).

This was addressed above [see response to Weaknesses (1)-b and (5)-b].

(f) In the title, the authors state "reveal disease mechanisms", but the studies mainly exhibit functional changes. They should consider toning down the statement.

The title was revised to: Patient-Specific Midbrain Organoids with CRISPR Correction Recapitulate Neuronopathic Gaucher Disease Phenotypes and Enable Evaluation of Novel Therapies

(7) Recommendations

This reviewer recommends a major revision. The manuscript presents substantial novelty and strong potential impact but requires additional experimental validation and clearer, more conservative interpretation. Key items to address are:

(a) Strengthening genetic and biological replication (additional lines or replicate differentiations).

This was addressed above [see response to Weaknesses (1)-a, (1)-b, (1)-c].

(b) Adding functional mechanistic validation for major pathways (Wnt/mTOR/autophagy) and providing autophagy flux data.

(c) Including at least one neuronal functional readout (calcium imaging/MEA/patch) to demonstrate functional rescue.

As addressed above [see response to Weaknesses (2)], the suggested experiments in b) and c) would provide additional insights into this study and we will consider them in future work. 

(d) Deepening therapeutic characterization (dose, biodistribution, durability) and including specificity controls.

This was addressed above [see response to Weaknesses (3)-a to e].

(e) Improving statistical reporting and explicitly stating biological replicate structure.

This was addressed above [see response to Weaknesses (1)-b, (5)-b].

Reviewer #2 (Public review):

Sun et al. have developed a midbrain-like organoid (MLO) model for neuronopathic Gaucher disease (nGD). The MLOs recapitulate several features of nGD molecular pathology, including reduced GCase activity, sphingolipid accumulation, and impaired dopaminergic neuron development. They also characterize the transcriptome in the MLO nGD model. CRISPR correction of one of the GBA1 mutant alleles rescues most of the nGD molecular phenotypes. The MLO model was further deployed in proof-of-principle studies of investigational nGD therapies, including SapC-DOPS nanovesicles, AAV9-mediated GBA1 gene delivery, and substrate-reduction therapy (GZ452). This patient-specific 3D model provides a new platform for studying nGD mechanisms and accelerating therapy development. Overall, only modest weaknesses are noted.

We thank the reviewer for the supportive remarks.

Reviewer #3 (Public review):

Summary:

In this study, the authors describe modeling of neuronopathic Gaucher disease (nGD) using midbrain-like organoids (MLOs) derived from hiPSCs carrying GBA1 L444P/P415R or L444P/RecNciI variants. These MLOs recapitulate several disease features, including GCase deficiency, reduced enzymatic activity, lipid substrate accumulation, and impaired dopaminergic neuron differentiation. Correction of the GBA1 L444P variant restored GCase activity, normalized lipid metabolism, and rescued dopaminergic neuronal defects, confirming its pathogenic role in the MLO model. The authors further leveraged this system to evaluate therapeutic strategies, including: (i) SapC-DOPS nanovesicles for GCase delivery, (ii) AAV9-mediated GBA1 gene therapy, and (iii) GZ452, a glucosylceramide synthase inhibitor. These treatments reduced lipid accumulation and ameliorated autophagic, lysosomal, and neurodevelopmental abnormalities.

Strengths:

This manuscript demonstrates that nGD patient-derived MLOs can serve as an additional platform for investigating nGD mechanisms and advancing therapeutic development.

Comments:

(1) It is interesting that GBA1 L444P/P415R MLOs show defects in midbrain patterning and dopaminergic neuron differentiation (Figure 3). One might wonder whether these abnormalities are specific to the combination of L444P and P415R variants or represent a 

general consequence of GBA1 loss. Do GBA1 L444P/RecNciI (GD2-10-257) MLOs also exhibit similar defects?

We observed reduced dopaminergic neuron marker TH expression in GBA1 L444P/RecNciI (GD2-10-257) MLOs, suggesting that this line also exhibits defects in dopaminergic neuron differentiation. These data are provided in a new Supplementary Fig. 4E, and are summarized in new Supplementary Table 2 in the revision.

(2) In Supplementary Figure 3, the authors examined GCase localization in SapC-DOPSfGCase-treated nGD MLOs. These data indicate that GCase is delivered to TH⁺ neurons, GFAP⁺ glia, and various other unidentified cell types. In fruit flies, the GBA1 ortholog, Gba1b, is only expressed in glia (PMID: 35857503; 35961319). Neuronally produced GluCer is transferred to glia for GBA1-mediated degradation. These findings raise an important question: in wild-type MLOs, which cell type(s) normally express GBA1? Are they dopaminergic neurons, astrocytes, or other cell types?

All cell types in wild-type MLOs are expected to express GBA1, as it is a housekeeping gene broadly expressed across neurons, astrocytes, and other brain cell types. Its lysosomal function is essential for cellular homeostasis and is therefore not restricted to any specific lineage. (https://www.proteinatlas.org/ENSG00000177628GBA1/brain/midbrain). 

(3) The authors may consider switching Figures 2 and 3 so that the differentiation defects observed in nGD MLOs (Figure 3) are presented before the analysis of other phenotypic abnormalities, including the various transcriptional changes (Figure 2).

We appreciate the reviewer’s suggestion; however, we respectfully prefer to retain the current order of Figures 2 and 3, as we believe this structure provides the clearest narrative flow. Figure 2 establishes the core biochemical hallmarks: reduced GCase activity, substrate accumulation, and global transcriptomic dysregulation (1,429 DEGs enriched in neural development, WNT signaling, and lysosomal pathways), which together provide essential molecular context for studying the specific cellular differentiation defects presented in Figure 3. Presenting the broader disease landscape first creates a coherent mechanistic link to the subsequent analyses of midbrain patterning and dopaminergic neuron impairment.

To enhance readability, we have added a brief transitional sentence at the start of the Figure 3 paragraph: “Building on the molecular and transcriptomic hallmarks of GCase deficiency observed in nGD MLOs (Figure 2), we next investigated the impact on midbrain patterning and dopaminergic neuron differentiation (Figure 3).”

why should always the machine gets the best ideas applied

independent units called actors

but thee actors are actual human beings

So we get the Human Actor Model

for interpersonal computing on the web

Reviewer #1 (Public review):

Summary:

This study uses data from a recent RVFV serosurvey among transhumant cattle in The Gambia to inform the development of an RVFV transmission model. The model incorporates several hypotheses that capture the seasonal nature of both vector-borne RVFV transmission and cattle migration. These natural phenomena are driven by contrasting wet and dry seasons in The Gambia's two main ecoregions and are purported to drive cyclical source-sink transmission dynamics. Although the Sahel is hypothesized to be unsuitable for year-long RVFV transmission, findings suggest that cattle returning from the Gambia River to the Sahel at the beginning of the wet season could drive repeated RVFV introductions and ensuing seasonal outbreaks. Upon review, the authors have removed an additional analysis evaluating the potential impacts of cattle movement bans on transmission dynamics, which was poorly supported by the methodological approach.

Strengths:

Like most infectious diseases in animal systems in low- and middle-income countries, the transmission dynamics of RVFV in cattle in The Gambia are poorly understood. This study harnesses important data on RVFV seroepidemiology to develop and parameterize a novel transmission model, providing plausible estimates of several epidemiological parameters and transmission dynamic patterns.

This study is well written and easy to follow.

The authors consider both deterministic and stochastic formulations of their model, demonstrating potential impacts of random events (e.g. extinctions) and providing confidence regarding model robustness.

The authors use well-established Bayesian estimation techniques for model fitting and confront their transmission model with a seroepidemiological model to assess model fit.

Elasticity analyses help to understand the relative importance of competing demographic and epidemiological drivers of transmission in this system.

Weaknesses:

The model does not include an impact of infection on cattle birth rates, but the authors justify that this parameter should have limited impact on dynamics given predicted low-level circulation patterns, as opposed to explosive outbreaks, in this region.

The importance of the LVFV positivity decay rate is highlighted but loss of immunity is not considered in the SIR model. The authors do discuss uncertainty regarding model structure and a need for future data collection to begin to answer this question.

The model's structure, including homogenous mixing within each ecoregion and step-change seasonality, allows for estimation of generalized transmission rates at a macro scale. However, it greatly simplifies the movement process itself and assumes that transhumant cattle movement is the only mechanism for RVF reintroduction into the Sahel region. The authors discuss that integration of more finely-scaled movement and contact data may help to address this limitation in future work.

This model seems well-suited to be exploited in future work to explore for e.g. impacts of cattle vaccination, and potential differential efficiency when targeting T herds relative to M or L.

Comments on revisions:

I thank the authors for thoughtfully and thoroughly addressing my concerns. I have no further comments.

Author response:

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

Joint Public reviews:

(1) Stable annual dynamics vs. episodic outbreaks

We agree that RVF is classically described as producing periodic epidemics interspersed with long inter-epidemic periods, often linked to extreme rainfall events. Our model predicts more regular seasonal dynamics, which reflects the endemic transmission patterns we have observed in The Gambia through serological surveys. In this revision, we have:

- clarified that while epidemics occur in other parts of sub-Saharan Africa, our results are consistent with the epidemiological narrative of RVF in The Gambia, characterised by sustained, moderate transmission without resulting in substantial outbreaks (hyperendemicity).

- discussed how model assumptions (e.g. seasonality, homogenous mixing) may bias our results toward an endemic quasi-equilibrium dynamic.

- highlighted the implications of this for interpretation and for public health decision-making.

(2) Use of network analysis

We acknowledge the reviewer’s concern. The network analysis was conducted descriptively to characterize cattle movement patterns and the structure of herd connections, but it was not formally incorporated into the model. In this revision we have:

- clarified this distinction in the manuscript to avoid overinterpretation.

- emphasized the need for future modelling work using finer-scale movement data, which could support more realistic herd metapopulation dynamics and better capture heterogeneity in transmission.

(3) RVFV reproductive impacts

While RVF outbreaks are known to cause substantial abortions and neonatal deaths, these events occur during sporadic epidemics. In the Gambian context, where we’re not observing large outbreaks but rather low-level circulation, the annual impact of RVF infection on births is likely modest compared to baseline herd turnover. Moreover, cattle demography is partly managed, with replacement and movement buffering birth rates against short-term losses.

Our model includes birth as a constant demographic process, it’s reasonable to assume stable population since we are not explicitly modelling outbreak-scale reproductive losses. This approach is consistent with other RVF transmission models that adopt a similar simplifying assumption. However, we have acknowledged this simplification as a limitation in the revised manuscript.

(4) Missing ODEs for M herds in the dry season

We thank the reviewer for identifying this omission. The ODEs for the M subpopulation in the dry season were not included in the appendix due to an oversight, though demographic turnover was implemented in the model code. We have now added the missing equations to the appendix.

(5) Role of immunity loss and model structure (SIR vs. SIRS)

We acknowledge that the decline of detectable antibodies over time (seropositivity decay) is an important consideration in RVFV serology; however, whether this decline reflects a true loss of protective immunity following natural infection remains unknown. Available evidence suggests that infected cattle likely develop long-lasting immunity, and findings in humans further support this assumption, although longitudinal field data regarding RVFV-specific antibody durability in animals are not available to the best of our knowledge. From a modelling perspective, our objective was to estimate FOI and use it to predict an age-seroprevalence curve consistent with the observed cross-sectional age-seroprevalence patterns. We therefore adopted a parsimonious SIR framework, interpreting loss of seropositivity as a potential explanation for discrepancies between observed and predicted age-seroprevalence rather than explicitly modelling waning immunity. We have now:

- clarified this rationale, emphasising that there is no direct evidence for waning immunity following natural RVFV infection in cattle, although evidence of seropositivity decay has been suggested in human.

- highlighted that while an SEIS/SIRS framework could theoretically generate different long-term dynamics, evaluating this approach requires stronger evidence for true immunity loss.

(6) RVFV induced mortality in serocatalytic model

We thank the reviewer for this comment and for raising an important conceptual point. However, the force of infection in our study is not estimated using a serocatalytic framework. Instead, FOI is estimated mechanistically within the transmission model as a function of the number of infectious cattle, rather than from age-stratified seroprevalence data.

RVF-induced mortality is accounted for through its effect on the infectious compartment, where increased mortality reduces the number and duration of infectious cattle and therefore indirectly reduces FOI. Consequently, RVF-related cattle death does not need to be explicitly incorporated into the FOI expression itself. Seroreversion similarly does not influence FOI estimation under this modelling framework. We have clarified this distinction in the Methods section to avoid confusion between mechanistic transmission models and serocatalytic approaches.

(7) Clarifying previous vs. current study components

We have revised the Methods and Appendix to make clearer distinctions between our previous work (e.g. household survey data collection, seroprevalence estimates) and the analyses undertaken for this manuscript (e.g. model development and fitting).

(8) Limitations paragraph

We have expanded the limitations section to identify the sparse household movement data as contributing most to uncertainty. We have outlined how these limitations may have implications for our conclusions, and may lead to under- or over-estimation of periods of heightened transmission risk.

(9) Movement ban simulations & suitability of model for vaccination interventions

We appreciate the reviewer’s concerns regarding the movement ban simulation. On reassessment, we agree that our model structure might not ideally be suited to exploring a movement ban. In this revised manuscript, we have removed this analysis. We are currently developing separate work focused on RVF vaccination strategies in cattle, where this model structure might be more directly applicable, and will reserve a deeper investigation of vaccination interventions for that forthcoming publication.

Reviewer #1 (Recommendations for the authors):

We thank the reviewer for the recommendations regarding the Introduction, Methods, Results, and Supplementary Figures. We have addressed these points below and revised the manuscript accordingly.

(1) Introduction: Should avoid describing as "inaccessible" the regions that are inhabited by nomadic and transhumant pastoralists.

We have revised the wording to “hard-to-reach” regions.

(2) Methods: Can the authors state what share of the animals included in the household survey data were cattle as opposed to other small ruminants? It would be helpful to understand what share of the data is "excluded"

We have now included the total number of cattle sampled, providing clarity on the proportion of data used in the analyses.

(3) Methods: When introducing the deterministic model, it seems unnecessary to mention the initialization conditions (i.e., introduction of a single infected individual at time 0) when this is later repeated in the Estimation of model parameters section, where it seems simulations were first conducted.

We have removed the redundant description.

(4) Results: Could the negative correlation between geographic distance of connected herds and mean seroprevalence simply indicate proximal exposure rather than common risk factors?

We acknowledge that both mechanisms are plausible. RVFV transmission is strongly influenced by share environmental factors that shape mosquito dynamics; however, direct transmission between proximal cattle herds may also occur through close contact with infectious tissues, bodily fluids, or contaminated materials. We have clarified this interpretation in the Results section.

(5) Figure S5: inconsistent notation for the scaling factor parameter (tau), which is expressed in equations and tables as psi.

We thank the reviewer for identifying this issue and have corrected all instances to ensure consistent use of tau throughout the manuscript.

(6) Figure S6: Why a density plot, isn't the number of temporary extinctions (x-axis) discrete?

We have replaced the density plot with a bar plot in Figure S6.

Reviewer #1 (Public review):

Summary:

In this work by Mohite et al., they have used transcriptomic and metabolic profiling of H. armigera, muscle development, and S. frugiperda to link energy trehalose metabolism and muscle development. They further used several different bioinformatics tools for network analysis to converge upon transcriptional control as a potential mechanism of metabolite-regulated transcriptional programming for muscle development. The authors have also done rescue experiments where trehalose was provided externally by feeding, which rescues the phenotype. Though the study is exciting, there are several concerns and gaps that lead to the current results as purely speculative. It is difficult to perform any genetic experiments in non-model insects; the authors seem to suggest a similar mechanism could also be applicable in systems like Drosophila; it might be possible to perform experiments to fill some missing mechanistic details.

A few specific comments below:

The authors used N-(phenylthio) phthalimide (NPP), a trehalose-6-phosphate phosphatase (TPP) inhibitor. They also find several genes, including enzymes of trehalose metabolism, that change. Further, several myogenic genes are downregulated in bulk RNA sequencing. The major caveat of this experiment is that the NPP treatment leads to reduced muscle development, and so the proportion of the samples from the muscles in bulk RNA sequencing will be relatively lower, which might have led to the results. So, a confirmatory experiment has to be performed where the muscle tissues are dissected and sequenced, or some of the interesting targets could be validated by qRT-PCR. Further to overcome the off-target effects of NPP, trehalose rescue experiments could be useful.

Even the reduction in the levels of ADP, NAD, NADH, and NMN, all of which are essential for efficient energy production and utilization, could be due to the loss of muscles, which perform predominantly metabolic functions due to their mitochondria-rich environment. So it becomes difficult to judge if the levels of these energy molecules' reduction are due to a cause or effect.

The authors have used this transcriptomic data for pathway enrichment analysis, which led to the E2F family of transcription factors and a reduction in the level of when trehalose metabolism is perturbed. EMSA experiments, though, confirm a possibility of the E2F interaction with the HaTPS/TPP promoter, but it lacks proper controls and competition to test the actual specificity of this interaction. Several transcription factors have DNA-binding domains and could bind any given DNA weakly, and the specificity is ideally known only from competitive and non-competitive inhibition studies.

The work seems to have connected the trehalose metabolism with gene expression changes, though this is an interesting idea, there are no experiments that are conclusive in the current version of the manuscript. If the authors can search for domains in the E2F family of transcription factors that can bind to the metabolite, then, if not, a chip-seq is essential to conclusively suggest the role of E2F in regulating gene expression tuned by the metabolites.

Some of the above concerns are partially addressed in experiments where silencing of E2F/Dp shows similar phenotypes as with NPP and dsRNA. It is also notable that silencing any key transcription factor can have several indirect effects, and delayed pupation and lethality could not be definitely linked to trehalose-dependent regulation.

Trehalose rescue experiments that rescue phenotype and gene expression are interesting. But is it possible that the fed trehalose is metabolized in the gut and might not reach the target tissue? In which case, the role of trehalose in directly regulating transcription factors becomes questionable. So, a confirmatory experiment is needed to demonstrate that the fed trehalose reaches the target tissues. This could possibly be done by measuring the trehalose levels in muscles post-rescue feeding. Also, rescue experiments need to be done with appropriate control sugars.

No experiments are performed with non-target control dsRNA. All the experiments are done with an empty vector. But an appropriate control should be a non-target control.

Reviewer #3 (Public review):

The hypothesis is that Trehalose metabolism regulates transcriptional control of muscle development in lepidopteran insects.

The manuscript investigates the role of Trehalose metabolism in muscle development. Through sequencing and subsequent bioinformatics analysis of insects with perturbed trehalose metabolism (knockdown of TPS/TPP), the authors have identified transcription factor E2F, which was validated through RT-PCR. Their hypothesis is that trehalose metabolism regulates E2F, which then controls the myogenic genes. Counterintuitive to this hypothesis, the investigators perform EMSAs with the E2F protein and promoter of the TPP gene and show binding. Their knockdown experiments with Dp, the binding partner of E2F, show direct effect on several trehalose metabolism genes. Similar results are demonstrated in the trehalose feeding experiment, where feeding trehalose leads to partial rescue of the phenotype observed as a result of Dp knockdown. This seems contradictory to their hypothesis. Even more intriguing is a similar observation between paramyosin, a structural muscle protein, and E2F/Dp - they show that paramyosin regulates E2F/Dp and E2F/Dp regulated paramyosin. The only plausible way to explain the results is the existence of a feed-forward loop between TPP-E2F/Dp and paramyosin-E2F/Dp. But the authors have mentioned nothing in this line. Additionally, I think trehalose metabolism impacts amino acid content in insects, and that will have a direct bearing on muscle development. The sequencing analysis and follow-up GSEA studies have demonstrated enrichment of several amino acid biosynthetic genes. Yet authors make no efforts to measure amino acid levels or correlate them with muscle development. Any study aiming to link trehalose metabolism and muscle development and not considering the above points will be incomplete.

The result section of the manuscript is quite concise, to my understanding (especially the initial few sections), which misses out on mentioning details that would help readers understand the paper better. While technical details of the methods should be in the Materials and Methods section, the overall experimental strategy for the experiments performed should be explained in adequate detail in the results section itself or in figure legends. I would request authors to include more details in the results section. As an extension of the comment above, many times, abbreviations have been used without introducing them. A thorough check of the manuscript is required regarding this.

The Spodoptera experiments appear ad hoc and are insufficient to support conservation beyond Helicoverpa. To substantiate this claim, please add a coherent, minimal set of Spodoptera experiments and present them in a dedicated subsection. Alternatively, consider removing these data and limiting the conclusions (and title) to H. armigera.

In order to check the effects of E2F/Dp, a dsRNA-mediated knockdown of Dp was performed. Why was the E2F protein, a primary target of the study, not chosen as a candidate? The authors should either provide justification for this or perform the suggested experiments to come to a conclusion. I would like to point out that such experiments were performed in Drosophila.

Silencing of HaDp resulted in a significant decrease in HaE2F expression. I find this observation intriguing. DP is the cofactor of E2F, and they both heterodimerise and sit on the promoter of target genes to regulate them. I would request authors to revisit this result, as it contradicts the general understanding of how E2F/Dp functions in other organisms. If Dp indeed controls E2F expression, then further experiments should be conducted to come to a conclusion convincingly. Additionally, these results would need thorough discussion with citations of similar results observed for other transcription factor-cofactor complexes.

I consider the overall bioinformatics analysis to remain very poorly described. What is specifically lacking is clear statements about why a particular dry lab experiments were conducted.

In my judgement, the EMSA analysis presented is technically poor in quality. It lacks positive and negative controls, does not show mutation analysis or super shifts. Also, it lacks any competition assays that are important to prove the binding beyond doubt. I am not sure why protein is not detected at all in lower concentrations. Overall, the EMSA assays need to be redone; I find the current results to be unacceptable.

GSEA studies clearly indicate enrichment of the amino acid synthesis gene in TPP knockdown samples. This supports the plausible theory that a lack of Trehalose means a lack of enough nutrients, therefore less of that is converted to amino acids, and therefore muscle development is compromised. Yet the authors make no effort to measure amino acid levels. While nutrients can be sensed through signalling pathways leading to shut shutdown of myogenic genes, a simple and direct correlation between less raw material and deformed muscle might also be possible.

The authors are encouraged to stick to one color palette while demonstrating sequencing results. Choosing a different color palette for representing results from the same sequencing analysis confuses readers.

Expression of genes, as understood from sequencing analysis in Figure 1D, Figure 2F, and Figure 3D, appears to be binary in nature. This result is extremely surprising given that the qRT-PCR of these genes have revealed a checker and graded expression.

In several graphs, non-significant results have been interpreted as significant in the results section. In a few other cases, the reported changes are minimal, and the statistical support is unclear; please recheck the analyses and include exact statistics. In the results section, fold changes observed should be discussed, as well as the statistical significance of the observed change.

Finally, I would add that trehaolse metabolism regulates cell cycle genes, and muscle development genes establish correlation and causation. The authors should ensure that any comments they make are backed by evidence.

Author response:

eLife Assessment

This useful study examines whether the sugar trehalose, coordinates energy supply with the gene programs that build muscle in the cotton bollworm (Helicoverpa armigera). The evidence for this currently is incomplete. The central claim - that trehalose specifically regulates an E2F/Dp-driven myogenic program - is not supported by the specificity of the data: perturbations and sequencing are systemic, alternative explanations such as general energy or amino-acid scarcity remain plausible, and mechanistic anchors are also limited. The work will interest researchers in insect metabolism and development; focused, tissue-resolved measurements together with stronger mechanistic controls would substantially strengthen the conclusions.

We thank the reviewer for the thoughtful and constructive evaluation of our work and for recognizing its potential relevance to researchers working on insect metabolism and development. We fully agree that our current evidence is preliminary and that the mechanistic link between trehalose and the E2F/Dp‑driven myogenic program needs to be strengthened.

Our intention was to present trehalose-E2F/Dp coupling as a working model emerging from our data, rather than as a fully established pathway. We agree that systemic manipulations of trehalose and whole‑larval RNA‑seq cannot fully differentiate global metabolic stress from specific effects on myogenic programs. In the revision, we plan to include additional metabolic readouts (e.g., ATP/AMP ratio, key amino acids where available) to better discuss the overall energetic and nutritional state. We will reanalyze our RNA‑seq data to more clearly distinguish broad stress/metabolic signatures from cell‑cycle/myogenic signatures. Furthermore, we will reframe our discussion to explicitly state that we cannot completely rule out a contribution of general energy or amino‑acid scarcity at this stage.

We acknowledge that, with our current experiments, the specificity for an E2F/Dp‑driven program is inferred mainly from enrichment of E2F targets among differentially expressed genes, and expression changes in canonical E2F partners and downstream cell‑cycle/myogenic regulators. To address this more rigorously, we are performing targeted qRT-PCR for a panel of well‑characterized E2F/Dp target genes and myogenic markers in larval muscle versus non‑muscle tissues, following trehalose perturbation. Where technically feasible, testing whether partial knockdown of HaE2F or HaDp modifies the effect of trehalose manipulation on selected myogenic markers. These data, even if limited, will help to provide a more direct functional link, and we will include them in the manuscript if completed in time. In parallel, we will soften statements that imply a fully established, trehalose‑specific regulation of E2F/Dp and instead present this as a strong candidate pathway suggested by the current data.

We fully agree that tissue‑resolved analyses are essential to move from systemic correlations to causality in muscle. We are in the process of standardizing larval muscle dissections and isolating thoracic/abdominal body wall muscle for trehalose, glycogen, and expression assays. Comparing expression of key metabolic and myogenic genes in muscle versus fat body and midgut, under trehalose manipulation. These tissue‑resolved data will directly address whether the transcriptional changes we report are preferentially localized to muscle.

We are grateful for the reviewer’s critical but encouraging comments. We will moderate our central claims, also explicitly consider and discuss alternative explanations. Further, we will add tissue‑resolved and more focused mechanistic data as far as possible within the current revision. We believe these changes will substantially strengthen the manuscript and better align our conclusions with the evidence we presently have.

Public Reviews:

Reviewer #1 (Public review):

Summary:

In this work by Mohite et al., they have used transcriptomic and metabolic profiling of H. armigera, muscle development, and S. frugiperda to link energy trehalose metabolism and muscle development. They further used several different bioinformatics tools for network analysis to converge upon transcriptional control as a potential mechanism of metabolite-regulated transcriptional programming for muscle development. The authors have also done rescue experiments where trehalose was provided externally by feeding, which rescues the phenotype. Though the study is exciting, there are several concerns and gaps that lead to the current results as purely speculative. It is difficult to perform any genetic experiments in non-model insects; the authors seem to suggest a similar mechanism could also be applicable in systems like Drosophila; it might be possible to perform experiments to fill some missing mechanistic details.

A few specific comments below:

The authors used N-(phenylthio) phthalimide (NPP), a trehalose-6-phosphate phosphatase (TPP) inhibitor. They also find several genes, including enzymes of trehalose metabolism, that change. Further, several myogenic genes are downregulated in bulk RNA sequencing. The major caveat of this experiment is that the NPP treatment leads to reduced muscle development, and so the proportion of the samples from the muscles in bulk RNA sequencing will be relatively lower, which might have led to the results. So, a confirmatory experiment has to be performed where the muscle tissues are dissected and sequenced, or some of the interesting targets could be validated by qRT-PCR. Further to overcome the off-target effects of NPP, trehalose rescue experiments could be useful.

Thank you for this valuable comment. We will validate the gene expression data using qRT-PCR on muscle tissue samples from both treated and control groups. This will help determine whether the gene expression patterns observed in the RNA-seq data are muscle-specific or systemic.

Even the reduction in the levels of ADP, NAD, NADH, and NMN, all of which are essential for efficient energy production and utilization, could be due to the loss of muscles, which perform predominantly metabolic functions due to their mitochondria-rich environment. So it becomes difficult to judge if the levels of these energy molecules' reduction are due to a cause or effect.

We thank the reviewer for this thoughtful comment and agree that reduced levels of ADP, NAD, NADH, and NMN could arise either from a disturbance of energy metabolism or from loss of mitochondria‑rich muscles. Our current data cannot fully separate these two possibilities. Still, several studies support the interpretation that perturbing trehalose metabolism causes a primary systemic energy deficit that is coupled to mitochondrial function, not merely a passive consequence of tissue loss.

For example:

(1) Our previous study in H. armigera showed that chemical inhibition of trehalose synthesis results in depletion of trehalose, glucose, glucose‑6‑phosphate, and suppression of the TCA cycle, indicating reduced energy levels and dysregulated fatty‑acid oxidation (Tellis et al., 2023).

(2) Chang et al. (2022) showed that trehalose catabolism and mitochondrial ATP production are mechanistically linked. HaTreh1 localizes to mitochondria and physically interacts with ATP synthase subunit α. 20‑hydroxyecdysone increases HaTreh1 expression, enhances its binding to ATP synthase, and elevates ATP content, while knockdown of HaTreh1 or HaATPs‑α reduces ATP levels.

(3) Similarly, our previous study inhibition of Treh activity in H. armigera generates an “energy‑deficient condition” characterized by deregulation of carbohydrate, protein, fatty‑acid, and mitochondria‑related pathways, and a concomitant reduction in key energy metabolites (Tellis et al., 2024).

(4) The starvation study in H. armigera has shown that reduced hemolymph trehalose is associated with respiratory depression and large‑scale reprogramming of glycolysis and fatty‑acid metabolism (Jiang et al., 2019).

These findings support a direct coupling between trehalose availability and systemic energy/redox state. Therefore, the coordinated decrease in ADP, NAD, NADH, and NMN following TPS/TPP silencing is consistent with a primary disturbance of systemic energy and mitochondrial metabolism rather than exclusively a secondary consequence of muscle loss. We agree, however, that the present whole‑larva metabolite measurements do not allow a quantitative partitioning between changes due to altered muscle mass and those due to intrinsic metabolic impairment at the cellular level. Thus, tissue-specific quantification of these metabolites would allow us to directly test whether altered energy metabolites are a cause or consequence of muscle loss.

References:

(1) Tellis, M. B., Mohite, S. D., Nair, V. S., Chaudhari, B. Y., Ahmed, S., Kotkar, H. M., & Joshi, R. S. (2024). Inhibition of Trehalose Synthesis in Lepidoptera Reduces Larval Fitness. Advanced Biology, 8(2), 2300404.

(2) Chang, Y., Zhang, B., Du, M., Geng, Z., Wei, J., Guan, R., An, S. and Zhao, W., 2022. The vital hormone 20-hydroxyecdysone controls ATP production by upregulating the binding of trehalase 1 with ATP synthase subunit α in Helicoverpa armigera. Journal of Biological Chemistry, 298(2).

(3) Tellis, M., Mohite, S. and Joshi, R., 2024. Trehalase inhibition in Helicoverpa armigera activates machinery for alternate energy acquisition. Journal of Biosciences, 49(3), p.74.

(4) Jiang, T., Ma, L., Liu, X.Y., Xiao, H.J. and Zhang, W.N., 2019. Effects of starvation on respiratory metabolism and energy metabolism in the cotton bollworm Helicoverpa armigera (Hübner)(Lepidoptera: Noctuidae). Journal of Insect Physiology, 119, p.103951.

The authors have used this transcriptomic data for pathway enrichment analysis, which led to the E2F family of transcription factors and a reduction in the level of when trehalose metabolism is perturbed. EMSA experiments, though, confirm a possibility of the E2F interaction with the HaTPS/TPP promoter, but it lacks proper controls and competition to test the actual specificity of this interaction. Several transcription factors have DNA-binding domains and could bind any given DNA weakly, and the specificity is ideally known only from competitive and non-competitive inhibition studies.

We thank the reviewer for this important comment and fully agree that EMSA alone, without appropriate competition and control reactions, cannot establish the specificity or functional relevance of a transcription factor-DNA interaction. In our study, we found the E2F family from GRN analysis of the RNA seq data obtained upon HaTPS/TPP silencing, suggesting a potential regulatory connection. After that, we predicted E2F binding sites on the promoter of HaTPS/TPP. The EMSA experiments were intended as preliminary evidence that E2F can associate with the HaTPS/TPP promoter in vitro. We will clarify this in the manuscript by softening our conclusion to indicate that our data support a “possible E2F-HaTPS/TPP interaction”. We also perform EMSA with specific and non‑specific competitors to confirm the E2F binding to the HaTPS/TPP promoter.

The work seems to have connected the trehalose metabolism with gene expression changes, though this is an interesting idea, there are no experiments that are conclusive in the current version of the manuscript. If the authors can search for domains in the E2F family of transcription factors that can bind to the metabolite, then, if not, a chip-seq is essential to conclusively suggest the role of E2F in regulating gene expression tuned by the metabolites.

A previous study in D. melanogaster, Zappia et al., (2016) showed vital role of E2F in skeletal muscle required for animal viability. They have shown that Dp knockdown resulted in reduced expression of genes encoding structural and contractile proteins, such as Myosin heavy chain (Mhc), fln, Tropomyosin 1 (Tm1), Tropomyosin 2 (Tm2), Myosin light chain 2 (Mlc2), sarcomere length short (sals) and Act88F, and myogenic regulators, such as held out wings (how), Limpet (Lmpt), Myocyte enhancer factor 2 (Mef2) and spalt major (salm). Also, ChiP-qRT-PCR showed upstream regions of myogenic genes, such as how, fln, Lmpt, sals, Tm1 and Mef2, were specifically enriched with E2f1, E2f2, and Dp antibodies in comparison with a nonspecific antibody. Further, Zappia et al. (2019) reported a chip-seq dataset that suggests that E2F/Dp directly activates the expression of glycolytic and mitochondrial genes during muscle development. Zappia et al., (2023) showed the regulation of one of the glycolytic genes, Phosphoglycerate kinase (Pgk) by E2F during Drosophila development.

However, the regulation of trehalose metabolic genes by E2F/Dp and vice versa was not studied previously. So here in our study, we tried to understand the correlation of trehalose metabolism and E2F/Dp in the muscle development of H. armigera.

References:

(1) Zappia, M.P. and Frolov, M.V., 2016. E2F function in muscle growth is necessary and sufficient for viability in Drosophila. Nature Communications, 7(1), p.10509.

(2) Zappia, M.P., Rogers, A., Islam, A.B. and Frolov, M.V., 2019. Rbf activates the myogenic transcriptional program to promote skeletal muscle differentiation. Cell reports, 26(3), pp.702-719.

(3) Zappia, M. P., Kwon, Y.-J., Westacott, A., Liseth, I., Lee, H. M., Islam, A. B., Kim, J., & Frolov, M. V. (2023a). E2F regulation of the Phosphoglycerate kinase gene is functionally important in Drosophila development. Proceedings of the National Academy of Sciences, 120(15), e2220770120.

Some of the above concerns are partially addressed in experiments where silencing of E2F/Dp shows similar phenotypes as with NPP and dsRNA. It is also notable that silencing any key transcription factor can have several indirect effects, and delayed pupation and lethality could not be definitely linked to trehalose-dependent regulation.

Yes. It’s true that silencing of any key transcription factor can have several indirect effects. Our intention was not to argue that delayed pupation and lethality are exclusively due to trehalose-dependent regulation, but that E2F/Dp and HaTPS/TPP silencing showed a consistent set of phenotypes and molecular changes, such as (i) transcriptomic enrichment of E2F targets upon trehalose perturbation, (ii) reduced HaTPS/TPP expression following E2F/Dp silencing, (iii) reduced myogenic gene expression that parallels the phenotypes observed with HaTPS/TPP silencing and (iv) restoration of E2F and Dp expression in E2F/Dp‑silenced insects upon trehalose feeding in the rescue assay. Together, these findings support a functional association between E2F/Dp and trehalose homeostasis. At the same time, we fully acknowledge that these results do not exclude additional, trehalose‑independent roles of E2F/Dp in development.

Trehalose rescue experiments that rescue phenotype and gene expression are interesting. But is it possible that the fed trehalose is metabolized in the gut and might not reach the target tissue? In which case, the role of trehalose in directly regulating transcription factors becomes questionable. So, a confirmatory experiment is needed to demonstrate that the fed trehalose reaches the target tissues. This could possibly be done by measuring the trehalose levels in muscles post-rescue feeding. Also, rescue experiments need to be done with appropriate control sugars.

Yes, it’s possible that, to some extent, trehalose is metabolized in the gut. Even though trehalase is present in the insect gut, some of the trehalose will be absorbed via trehalose transporters on the gut lining. Trehalose feeding was not rescued in insects fed with the control diet (empty vector and dsHaTPP), which contains chickpea powder, which is composed of an ample amount of amino acids and carbohydrates. Insects fed exclusively on a trehalose-containing diet are rescued, but not on a control diet that contains other carbohydrates. We agree that direct measurement of trehalose in target tissues will provide important confirmation. In the manuscript, we will measure trehalose levels in muscle, gut, and haemolymph after trehalose feeding.

No experiments are performed with non-target control dsRNA. All the experiments are done with an empty vector. But an appropriate control should be a non-target control.

Yes, there was no experiment with non-target dsRNA. Earlier, we have optimized a protocol for dsRNA delivery and its effectiveness in target knockdown (concentration, time) experiment, and published several research articles using a similar protocol:

(1) Chaudhari, B.Y., Nichit, V.J., Barvkar, V.T. and Joshi, R.S., 2025. Mechanistic insights in the role of trehalose transporter in metabolic homeostasis in response to dietary trehalose. G3: Genes, Genomes, Genetics, p. jkaf303.

(2) Barbole, R.S., Sharma, S., Patil, Y., Giri, A.P. and Joshi, R.S., 2024. Chitinase inhibition induces transcriptional dysregulation altering ecdysteroid-mediated control of Spodoptera frugiperda development. Iscience, 27(3).

(3) Patil, Y.P., Wagh, D.S., Barvkar, V.T., Gawari, S.K., Pisalwar, P.D., Ahmed, S. and Joshi, R.S., 2025. Altered Octopamine synthesis impairs tyrosine metabolism affecting Helicoverpa armigera vitality. Pesticide Biochemistry and Physiology, 208, p.106323.

(4) Tellis, M.B., Chaudhari, B.Y., Deshpande, S.V., Nikam, S.V., Barvkar, V.T., Kotkar, H.M. and Joshi, R.S., 2023. Trehalose transporter-like gene diversity and dynamics enhances stress response and recovery in Helicoverpa armigera. Gene, 862, p.147259.

(5) Joshi, K.S., Barvkar, V.T., Hadapad, A.B., Hire, R.S. and Joshi, R.S., 2025. LDH-dsRNA nanocarrier-mediated spray-induced silencing of juvenile hormone degradation pathway genes for targeted control of Helicoverpa armigera. International Journal of Biological Macromolecules, p.148673.

The same vector backbone and preparation procedures were used for both control and experimental constructs, allowing us to specifically compare the effects of the target dsRNA. The phenotypes and gene expression changes we observed were specific to the target genes and were not seen in the empty vector controls, suggesting that the effects are not due to nonspecific responses of dsRNA delivery or vector components.<br /> We acknowledge your suggestions, and in future studies, we will keep non-target dsRNA as a control in silencing assays.

Reviewer #2 (Public review):

Summary:

This study shows that the knockdown of the effects of TPS/TPP in Helicoverpa armigera and Spodoptera frugiperda can be rescued by trehalose treatment. This suggests that trehalose metabolism is necessary for development in the tissues that NPP and dsRNA can reach.

Strengths:

This study examines an important metabolic process beyond model organisms, providing a new perspective on our understanding of species-specific metabolism equilibria, whether conserved or divergent.

Weaknesses:

While the effects observed may be truly conserved across Lepidopterans and may be muscle-specific, the study largely relies on one species and perturbation methods that are not muscle-specific. The technical limitations arising from investigations outside model systems, where solid methods are available, limit the specificity of inferences that may be drawn from the data.

Thank you for this potting out this experimental weakness. We will validate the gene expression data using qRT-PCR on muscle tissue samples from both treated and control groups. We will also perform metabolite analysis with muscle samples. This will help to determine whether the observed gene expression patterns and metabolite changes are muscle-specific or systemic.

Reviewer #3 (Public review):

The hypothesis is that Trehalose metabolism regulates transcriptional control of muscle development in lepidopteran insects.

The manuscript investigates the role of Trehalose metabolism in muscle development. Through sequencing and subsequent bioinformatics analysis of insects with perturbed trehalose metabolism (knockdown of TPS/TPP), the authors have identified transcription factor E2F, which was validated through RT-PCR. Their hypothesis is that trehalose metabolism regulates E2F, which then controls the myogenic genes. Counterintuitive to this hypothesis, the investigators perform EMSAs with the E2F protein and promoter of the TPP gene and show binding. Their knockdown experiments with Dp, the binding partner of E2F, show direct effect on several trehalose metabolism genes. Similar results are demonstrated in the trehalose feeding experiment, where feeding trehalose leads to partial rescue of the phenotype observed as a result of Dp knockdown. This seems contradictory to their hypothesis. Even more intriguing is a similar observation between paramyosin, a structural muscle protein, and E2F/Dp - they show that paramyosin regulates E2F/Dp and E2F/Dp regulated paramyosin. The only plausible way to explain the results is the existence of a feed-forward loop between TPP-E2F/Dp and paramyosin-E2F/Dp. But the authors have mentioned nothing in this line. Additionally, I think trehalose metabolism impacts amino acid content in insects, and that will have a direct bearing on muscle development. The sequencing analysis and follow-up GSEA studies have demonstrated enrichment of several amino acid biosynthetic genes. Yet authors make no efforts to measure amino acid levels or correlate them with muscle development. Any study aiming to link trehalose metabolism and muscle development and not considering the above points will be incomplete.

We appreciate the reviewer’s efforts in the careful evaluation of this manuscript and constructive comments. From our and earlier data we found it was difficult to consider linear pathway “trehalose → E2F → muscle,” but rather a regulatory module in which trehalose metabolism and E2F/Dp form an interdependent circuit controlling myogenic genes. E2F/Dp binds and activates trehalose metabolism genes (TPS/TPP, Treh1) and myogenic structural genes, consistent with EMSA (TPS/TPP-E2F) and predicted binding sites of E2F on metabolic genes, Treh1, Pgk, and myogenic genes such as Act88F, Prm, Tm1, Fln, etc. At the same time, perturbing trehalose synthesis reduces E2F/Dp expression and myogenic gene expression, and trehalose feeding partially restores all three. This bidirectional influence is similar to E2F‑dependent control of carbohydrate metabolism and systemic sugar homeostasis described in D. melanogaster, where E2F/Dp both regulates metabolic genes and is itself constrained by metabolic state (Zappia et al., 2023a; Zappia et al., 2021).

The reciprocal regulation between Prm and E2F/Dp is indeed intriguing. Rather than a paradox, we interpret this as evidence that E2F/Dp couples metabolic genes and structural muscle genes within a shared module, and that key sarcomeric components (such as paramyosin) feed back on this transcriptional program. Similar cross‑talk between E2F‑controlled metabolic programs and tissue function has been documented in D. melanogaster muscle and fat body, where E2F loss in one tissue elicits systemic changes in the other (Zappia et al., 2021). For further confirmation of E2F-regulated Prm, we will perform EMSA on the Prm promoter with appropriate controls.

We fully agree that amino‑acid metabolism is a critical missing piece. In the manuscript, we will quantify the amino acid levels and include the results: “Amino acids display differential levels showing cysteine, leucine, histidine, valine, and proline showed significant reductions, while isoleucine and lysine showed non-significant reductions upon trehalose metabolism perturbation. These results are consistent with previous reports published by Tellis et al. (2024) and Shi et al. (2016)”. We will reframe our conclusions more cautiously as establishing a trehalose-E2F/Dp-muscle development, while stating that “definitive causal links via amino‑acid metabolism remain to be demonstrated”.

Reference:

(1) Zappia, M. P., Kwon, Y.-J., Westacott, A., Liseth, I., Lee, H. M., Islam, A. B., Kim, J., & Frolov, M. V. (2023a). E2F regulation of the Phosphoglycerate kinase gene is functionally important in Drosophila development. Proceedings of the National Academy of Sciences, 120(15), e2220770120.

(2) Zappia, M.P., Guarner, A., Kellie-Smith, N., Rogers, A., Morris, R., Nicolay, B., Boukhali, M., Haas, W., Dyson, N.J. and Frolov, M.V., 2021. E2F/Dp inactivation in fat body cells triggers systemic metabolic changes. elife, 10, p.e67753.

(3)Tellis, M., Mohite, S. and Joshi, R., 2024. Trehalase inhibition in Helicoverpa armigera activates machinery for alternate energy acquisition. Journal of Biosciences, 49(3), p.74.

(4) Shi, J.F., Xu, Q.Y., Sun, Q.K., Meng, Q.W., Mu, L.L., Guo, W.C. and Li, G.Q., 2016. Physiological roles of trehalose in Leptinotarsa larvae revealed by RNA interference of trehalose-6-phosphate synthase and trehalase genes. Insect Biochemistry and Molecular Biology, 77, pp.52-68.

Author response image 1.

The result section of the manuscript is quite concise, to my understanding (especially the initial few sections), which misses out on mentioning details that would help readers understand the paper better. While technical details of the methods should be in the Materials and Methods section, the overall experimental strategy for the experiments performed should be explained in adequate detail in the results section itself or in figure legends. I would request authors to include more details in the results section. As an extension of the comment above, many times, abbreviations have been used without introducing them. A thorough check of the manuscript is required regarding this.

Thank you very much for pointing out this issue. We will revise the manuscript content according to these suggestions.

The Spodoptera experiments appear ad hoc and are insufficient to support conservation beyond Helicoverpa. To substantiate this claim, please add a coherent, minimal set of Spodoptera experiments and present them in a dedicated subsection. Alternatively, consider removing these data and limiting the conclusions (and title) to H. armigera.

We thank the reviewer for this helpful comment. We agree that, in this current version of the manuscript, the S. frugiperda experiments are not sufficiently systematic to support strong claims about conservation beyond H. armigera. Our primary focus in this study is indeed on H. armigera, and the addition of the S. frugiperda data was intended only as preliminary, supportive evidence rather than a central component of our conclusions. To avoid over‑interpretation and to keep the manuscript focused and coherent, we will remove all S. frugiperda data from the revised version, including the corresponding text and figures. We will also adjust the title, abstract, and conclusion to clearly state that our findings are limited to H. armigera.

In order to check the effects of E2F/Dp, a dsRNA-mediated knockdown of Dp was performed. Why was the E2F protein, a primary target of the study, not chosen as a candidate? The authors should either provide justification for this or perform the suggested experiments to come to a conclusion. I would like to point out that such experiments were performed in Drosophila.

Thank you for this thoughtful comment and the specific suggestion. We agree that directly targeting E2F would, in principle, be an informative complementary approach. In our study, however, we prioritized Dp knockdown for two main reasons. First, E2F is a large family, and E2F-Dp functions as an obligate heterodimer. Previous work in D. melanogaster has shown that depletion of Dp is sufficient to disrupt E2F-dependent transcription broadly, often with more efficient loss of complex activity than targeting individual E2F isoforms (Zappia et al., 2021; Zappia et al., 2016). Second, in our preliminary trials, we performed a dsRNA feeding assay with dsHaE2F, dsHaDp, and combined dsHaE2F plus dsHaDp. In that assay, we did not achieve silencing of E2F in dsRNA targeting HaE2F (dsHaE2F). So here, as E2F is a large family, other E2F isoforms may be compensating for the silencing effect of targeted HaE2F. However, HaE2F showed significantly reduced expression upon dsHaDp and combined dsHaE2F plus dsHaDp feeding (Figure A), whereas HaDp showed a significant reduction in its expression in all three conditions (Figure B).  As we observed reduced expression of both HaE2F and HaDp upon combined feeding of dsHaE2F and dsHaDp, we further performed a rescue assay by exogenous feeding of trehalose. We observed the significant upregulation of HaE2F, HaDp, trehalose metabolic genes (HaTPS/TPP and HaTreh1), and myogenic genes (HaPrm and HaTm2) (Figure C). For these reasons, we focused on Dp silencing as a more reliable way to impair E2F/Dp complex function in H. armigera.

Author response image 2.

References:

(1) Zappia, M.P. and Frolov, M.V., 2016. E2F function in muscle growth is necessary and sufficient for viability in Drosophila. Nature Communications, 7(1), p.10509.

(2) Zappia, M.P., Guarner, A., Kellie-Smith, N., Rogers, A., Morris, R., Nicolay, B., Boukhali, M., Haas, W., Dyson, N.J. and Frolov, M.V., 2021. E2F/Dp inactivation in fat body cells triggers systemic metabolic changes. elife, 10, p.e67753.

Silencing of HaDp resulted in a significant decrease in HaE2F expression. I find this observation intriguing. DP is the cofactor of E2F, and they both heterodimerise and sit on the promoter of target genes to regulate them. I would request authors to revisit this result, as it contradicts the general understanding of how E2F/Dp functions in other organisms. If Dp indeed controls E2F expression, then further experiments should be conducted to come to a conclusion convincingly. Additionally, these results would need thorough discussion with citations of similar results observed for other transcription factor-cofactor complexes.

Thank you for highlighting this point and for prompting us to examine these data more carefully. Silencing HaDp leading to reduced HaE2F mRNA is indeed unexpected if one only considers the canonical view of E2F/Dp as a heterodimer that co-occupies target promoters without strongly regulating each other’s expression. However, several lines of work suggest that transcription factor-cofactor networks frequently include feedback loops in which cofactors influence the expression of their partner TFs. First, in multiple systems, transcription factors and their cofactors are known to regulate each other’s transcription, forming positive or negative feedback loops. For example, in hematopoietic cells, the transcription factor Foxp3 controls the expression of many of its own cofactors, and some of these cofactors in turn facilitate or stabilize Foxp3 expression, forming an interconnected regulatory network rather than a simple one‑way interaction (Rudra et al., 2012). Second, E2F/Dp complexes exhibit non‑canonical regulatory mechanisms and can regulate broad sets of targets, including other transcriptional regulators. Several studies show that E2F/Dp proteins not only control classical cell‑cycle genes but also participate in diverse processes such as DNA damage signaling, mitochondrial function, and differentiation (Guarner et al., 2017; Ambrus et al., 2013; Sánchez-Camargo et al., 2021). In D. melanogaster, complete loss of dDP alters the expression of direct targets E2F/DP, including dATM (Guarner et al., 2017).

All these reports indicate that the E2F-Dp complex sits at the top of multi‑layer regulatory hierarchies. Such architectures make it plausible that Dp silencing in H. armigera could modulate HaE2F expression in a non-canonical way.

References:

(1) Rudra, D., DeRoos, P., Chaudhry, A., Niec, R.E., Arvey, A., Samstein, R.M., Leslie, C., Shaffer, S.A., Goodlett, D.R. and Rudensky, A.Y., 2012. Transcription factor Foxp3 and its protein partners form a complex regulatory network. Nature immunology, 13(10), pp.1010-1019.

(2) Guarner, A., Morris, R., Korenjak, M., Boukhali, M., Zappia, M.P., Van Rechem, C., Whetstine, J.R., Ramaswamy, S., Zou, L., Frolov, M.V. and Haas, W., 2017. E2F/DP prevents cell-cycle progression in endocycling fat body cells by suppressing dATM expression. Developmental cell, 43(6), pp.689-703.

(3) Ambrus, A.M., Islam, A.B., Holmes, K.B., Moon, N.S., Lopez-Bigas, N., Benevolenskaya, E.V. and Frolov, M.V., 2013. Loss of dE2F compromises mitochondrial function. Developmental cell, 27(4), pp.438-451.

(4) Sánchez-Camargo, V.A., Romero-Rodríguez, S. and Vázquez-Ramos, J.M., 2021. Non-canonical functions of the E2F/DP pathway with emphasis in plants. Phyton, 90(2), p.307.

I consider the overall bioinformatics analysis to remain very poorly described. What is specifically lacking is clear statements about why a particular dry lab experiments were conducted.

We again thank the reviewer for advising us to give a biological context/motivation for every bioinformatics analysis performed. The bioinformatics analyses devised here, try to explain the systems-level perturbations of HaTPS/TPP silencing to explain the observed phenotype and to discover transcription factors potentially modulating the HaTPS/TPP induced gene regulatory changes.

(1) Gene set enrichment analyses:

Differential gene expression analyses of the bulk RNA sequencing data followed by qRT-PCR confirmed the transcriptional changes in myogenic genes and gene expression alterations in metabolic and cell cycle-related genes. These perturbations merely confirmed the effect induced by HaTPS/TPP silencing in obviously expected genes. We wanted to see whether using an “unbiased” system-level statistical analyses like gene set enrichment analyses (GSEA), can reveal both expected and novel biological processes that underlie HaTPS/TPP silencing. GSEA results revealed large-scale transcriptional changes in 11 enriched processes, including amino acid metabolism, energy metabolism, developmental regulatory processes, and motor protein activity. GSEA not only divulged overall transcriptionally enriched pathways but also identified the genes undergoing synchronized pathway-level transcriptional change upon HaTPS/TPP silencing.

(2) Gene regulatory network analysis:

Although GSEA uncovered potential pathway-level changes, we were also interested in identifying the gene regulatory network associated with such large-scale process-level transcriptional perturbations. Interestingly, the biological processes undergoing perturbations were also heterogeneous (e.g., motor protein activity, energy metabolism, amino acid metabolism, etc.). We hypothesized that the inference of a causal gene regulatory network associated with the genes associated with GSEA-enriched biological processes should predict core/master transcription factors that might synchronously regulate metabolic and non-metabolic processes related to HaTPS/TPP silencing, thereby providing a broad understanding of the perturbed phenotype. The gene regulatory network analysis statistically inferred an “active” gene regulatory network corresponding to the GSEA-enriched KEGG gene sets. Ranking the transcription factors (TFs) based on the number of outgoing connections (outdegree centrality) within the active gene regulatory network, E2F family TFs were identified to be top-ranking, highly connected transcription factors associated with the transcriptionally enriched processes. This suggests that E2F family TFs are central to controlling the flow of regulatory information within this network. Intriguingly, E2F has been previously implicated in muscle development in insects (Zappia et al., 2016). Further extracting the regulated targets of E2F family TFs within this network revealed the mechanistic connection with the 11 enriched processes. This GRN analysis was crucial in discovering and prioritizing E2F TFs as central transcription factors mediating HaTPS/TPP silencing effects, which was not apparent using trivial analyses like differential gene expression analysis.

As per the reviewer’s suggestions, we will add these outlined points in the text of the manuscript (Results section) to further give context and clarity to the bioinformatics analyses conducted in this study.

In my judgement, the EMSA analysis presented is technically poor in quality. It lacks positive and negative controls, does not show mutation analysis or super shifts. Also, it lacks any competition assays that are important to prove the binding beyond doubt. I am not sure why protein is not detected at all in lower concentrations. Overall, the EMSA assays need to be redone; I find the current results to be unacceptable.

Thank you for pointing out this issue. We will reperform the EMSA analysis with appropriate controls.  Although the gel image was not clear, there was a light band of protein (indicated by the white square) observed in well No. 8, where we used 8 μg of E2F protein and 75 ng of HaTPS/TPP promoter, upon gel stained with SYPRO Ruby protein stain, suggesting weak HaTPS/TPP-E2F complex formation.

GSEA studies clearly indicate enrichment of the amino acid synthesis gene in TPP knockdown samples. This supports the plausible theory that a lack of Trehalose means a lack of enough nutrients, therefore less of that is converted to amino acids, and therefore muscle development is compromised. Yet the authors make no effort to measure amino acid levels. While nutrients can be sensed through signalling pathways leading to shut shutdown of myogenic genes, a simple and direct correlation between less raw material and deformed muscle might also be possible.

We quantified amino acid levels as per the suggestion, and we observed differential levels of amino acids upon trehalose metabolism perturbation.

However, we observed that insect were failed to rescue when fed a control chickpea-based artificial diet that contained nutrients required for normal growth and development. Based on this observation, we conclude that trehalose deficiency is the only possible cause for the defect in muscle development.

The authors are encouraged to stick to one color palette while demonstrating sequencing results. Choosing a different color palette for representing results from the same sequencing analysis confuses readers.

Thank you for the comment. We will revise the color palette as per the suggestion.

Expression of genes, as understood from sequencing analysis in Figure 1D, Figure 2F, and Figure 3D, appears to be binary in nature. This result is extremely surprising given that the qRT-PCR of these genes have revealed a checker and graded expression.

Thank you for pointing out this issue. We will revise the scale range for these figures to get more insights about gene expression levels and include figures as per the suggestion.

In several graphs, non-significant results have been interpreted as significant in the results section. In a few other cases, the reported changes are minimal, and the statistical support is unclear; please recheck the analyses and include exact statistics. In the results section, fold changes observed should be discussed, as well as the statistical significance of the observed change.

We will revise the analyses and include exact statistics as per the suggestion.

Finally, I would add that trehalose metabolism regulates cell cycle genes, and muscle development genes establish correlation and causation. The authors should ensure that any comments they make are backed by evidence.

We thank the reviewer for this insightful comment.  Although direct evidence in insects is currently lacking, multiple independent studies in yeast, plants and mammalian systems support a regulatory link between trehalose metabolism and the cell cycle. In budding yeast Saccharomyces cerevisiae, neutral Treh (Nth1) is directly phosphorylated and activated by the major cyclin‑dependent kinase Cdk1 at G1/S, routing stored trehalose into glycolysis to fuel DNA replication and mitosis (Ewald et al., 2016). CDK‑dependent regulation of trehalase activity has also been reported in plants, where CDC28‑mediated phosphorylation channels glucose into biosynthetic pathways necessary for cell proliferation (Lara-núñez et al., 2025). Furthermore, budding yeast cells accumulate trehalose and glycogen upon entry into quiescence and subsequently mobilize these stores to generate a metabolic “finishing kick” that supports re‑entry into the cell cycle (Silljé et al., 1999; Shi et al., 2010). Exogenous trehalose that perturbs the trehalose cycle impairs glycolysis, reduces ATP, and delays cell cycle progression in S. cerevisiae, highlighting a dose‑ and context‑dependent control of growth versus arrest (Zhang, Zhang and Li, 2020). In mammalian systems, trehalose similarly modulates proliferation-differentiation decisions. In rat airway smooth muscle cells, low trehalose concentrations promote autophagy, whereas higher doses induce S/G2–M arrest, downregulate Cyclin A1/B1, and trigger apoptosis, indicating a shift from controlled growth to cell elimination at higher exposure (Xiao et al., 2021). In human iPSC‑derived neural stem/progenitor cells, low‑dose trehalose enhances neuronal differentiation and VEGF secretion, while higher doses are cytotoxic, again highlighting a tunable impact on cell‑fate outcomes (Roose et al., 2025). In wheat, exogenous trehalose under heat stress reduces growth, lowers auxin, gibberellin, abscisic acid and cytokinin levels, and represses CycD2 and CDC2 expression, suggesting that trehalose signalling integrates with hormone pathways and core cell‑cycle regulators to restrain proliferation during stress (Luo, Liu, and Li, 2021). Together, these studies showed the importance of trehalose metabolism in cell‑cycle regulation to decide whether cells and tissues proliferate, differentiate, or remain quiescent.

With respect to muscle development, previous work has implicated glycolytic metabolism in myogenesis and muscle growth. Tixier et al. (2013) showed that loss of key glycolytic genes results in abnormally thin muscles, while Bawa et al. (2020) demonstrated that loss of TRIM32 decreases glycolytic flux and reduces muscle tissue size. These findings indicate that carbohydrate and energy metabolism pathways are important determinants of muscle structure and growth. However, there are no previous studies about the role of trehalose metabolism in muscle development, other than as an energy source, so here we specifically set out to establish the involvement of trehalose metabolism in muscle development.

References:

(1) Ewald, J.C. et al. (2016) “The yeast cyclin-dependent kinase routes carbon fluxes to fuel cell cycle progression,” Molecular cell, 62(4), pp. 532–545.

(2) Lara-núñez, A. et al. (2025) “The Cyclin-Dependent Kinase activity modulates the central carbon metabolism in maize during germination,” (January), pp. 1–16.

(3) Silljé, H.H.W. et al. (1999) “Function of trehalose and glycogen in cell cycle progression and cell viability in Saccharomyces cerevisiae,” Journal of bacteriology, 181(2), pp. 396–400.

(4) Shi, L. et al. (2010) “Trehalose Is a Key Determinant of the Quiescent Metabolic State That Fuels Cell Cycle Progression upon Return to Growth,” 21, pp. 1982–1990.

(5) Zhang, X., Zhang, Y. and Li, H. (2020) “Regulation of trehalose, a typical stress protectant, on central metabolisms, cell growth and division of Saccharomyces cerevisiae CEN. PK113-7D,” Food Microbiology, 89, p. 103459.

(6) Xiao, B. et al. (2021) “Trehalose inhibits proliferation while activates apoptosis and autophagy in rat airway smooth muscle cells,” Acta Histochemica, 123(8), p. 151810.

(7) Roose, S.K. et al. (2025) “Trehalose enhances neuronal differentiation with VEGF secretion in human iPSC-derived neural stem / progenitor cells,” Regenerative Therapy, 30, pp. 268–277.

(8) Luo, Y., Liu, X. and Li, W. (2021) “Exogenously-supplied trehalose inhibits the growth of wheat seedlings under high temperature by affecting plant hormone levels and cell cycle processes,” Plant Signaling & Behavior, 16(6).

(9) Tixier, V., Bataillé, L., Etard, C., Jagla, T., Weger, M., DaPonte, J.P., Strähle, U., Dickmeis, T. and Jagla, K., 2013. Glycolysis supports embryonic muscle growth by promoting myoblast fusion. Proceedings of the National Academy of Sciences, 110(47), pp.18982-18987.

(10) Bawa, S., Brooks, D.S., Neville, K.E., Tipping, M., Sagar, M.A., Kollhoff, J.A., Chawla, G., Geisbrecht, B.V., Tennessen, J.M., Eliceiri, K.W. and Geisbrecht, E.R., 2020. Drosophila TRIM32 cooperates with glycolytic enzymes to promote cell growth. elife, 9, p.e52358.

Finally, we appreciate the meticulous review of this manuscript and constructive comments. We will perform the recommended experiments, data analysis, and revise the manuscript accordingly.

character who has been serving others. expectation of either selflessness but "imprisoned" gives the idea it is unwelcome and forced upon him

Analyzing the good in life and finding place in nature

an isolated new phenomenon, but as a continuation of humanity's long-standing process of mediatization.

This shows that Native peoples in the Americas were able to build large, successful cities once they had steady food supplies, just like people in other parts of the world. The Maya are a good example they created a complex society with impressive buildings and cities. Their way of life changed over time, but that doesn’t mean their civilization suddenly collapsed. Instead, they may have simply chosen to live differently. Overall, this shows that American societies were advanced and capable long before Europeans arrived.

In 1500, Europe, Africa, and the Americas all had millions of people. They weren’t the same in number, but they were closer than most people expect. This shows the Americas were already full of Indigenous peoples before Europeans arrived.

Reviewer #2 (Public review):

Summary:

The authors sought to validate the use of genetic screening pipelines that assess phenotypes in founders (F0, referred to as "crispants") obtained from CRISPR/Cas9 gene editing in 1-cell zygotes. The application of this approach in mice has generally been avoided due to concerns that results would be confounded by genetic mosaicism, but benefits to this approach include reducing animal numbers needed to achieve goals of identifying knockout phenotypes, as well as improved efficiency in the use of time and resources. The authors targeted seven genes associated with visible recessive phenotypes and observed the expected null phenotype in up to 100% of founders for each gene. Although mosaicism was common in the crispants, the various alleles were generally all functional null alleles and, in fact, some in-frame deletions with null phenotypes revealed critical functional motifs within the gene products. The rigorous data presented support using crispants to assess knockout phenotypes when guide RNAs with strong on-target and low off-target scores are used for gene editing in 1-cell mouse embryos.

Strengths:

By targeting multiple genes with existing, well-characterized mutations, the authors established a robust system for validating the analysis of crispants to assess gene function.

Cutting-edge technologies were used to carefully assess the spectrum of mutations generated.

Weaknesses:

There could have been some discussion regarding how this approach would be impacted if mutations are dominant or embryonic lethal (for the latter, for example, F0 can be examined as embryos).

Author response:

We would like to thank the reviewers for their detailed reading of our manuscript and for the constructive comments they have provided.

We plan to make structural changes to the introduction and the discussion. Reviewer #1 describes the “disconnect between the abstract/introduction and the discussion”. We agree that “the study's aims are not clearly or explicitly defined”. We will edit the introduction to state our aim of investigating the factors that affect using “crispants” in mouse functional genomics. In the discussion, we described how our findings inform sgRNA choice to ensure biallelic gene disruption in founders and how our extensive genotyping methods enabled us to determine the molecular basis for the observed phenotype (explaining why some founders showed the expected recessive trait and why it was partial or absent in others). We also concluded from our attempts of multiplexing that this had too great an impact on viability to be useful. We will edit the discussion to better address our aim and to elaborate on several points raised by the reviewers (discussed in more detail below). Specifically, we will provide examples of screening situations where generating crispant mice may be useful, e.g. preliminary in vivo studies to follow up candidates identified in large-scale cellular screens. We will also provide more context about our assumptions underlying our statement that the use of crispants will “dramatically reduce time, resources, and animal numbers” compared to ENU mutagenesis (where recessive traits require breeding of G2 females with G1 males to achieve homozygosity of de novo mutations in G3 offspring) and the work needed to validate this. We will more clearly acknowledge that our proof-of-principle study used visible phenotypes that can be assessed in individual animals and then discuss how the use of crispants could be extended to the investigation of quantitative or late-onset traits using cohorts of crispants (discussed further below). We will also discuss the assessment of non-null alleles to dissect protein function, building on our unexpected finding that a single round of CRISPR/Cas9mediated mutagenesis can generate an allelic series.

Reviewer #1 asked us to address “how to interpret wild-type appearing founders”. We have discussed the mechanisms underlying the wild-type appearing founders generated in this study. This is linked with concerns in the field that incomplete editing, transcripts escaping nonsense-mediated decay, and/or the presence of in-frame mutations that don’t disrupt protein function may lead to founders that appear wild-type or have a partial phenotype. We have shown that our electroporation protocol results in very high levels of editing, but that this must always be assessed during genotyping. We found that by using an sgRNA that targets a critical protein domain, you can ensure that short in-frame indels also disrupt protein function. In future studies that determine how strain background modifies a phenotype that has been established on one strain (e.g. C57BL/6J), wild-type appearing founders would suggest that the new strain background rescues the null phenotype. In future studies that determine the consequence of targeting a second gene on a mutant background, wild-type appearing founders would indicate that the second mutation supresses the phenotype associated with the mutant background. We will add this to the discussion section where we describe possible screening situations in which crispant mice would be useful.

Reviewer #3 states that “the relationship between the sgRNA/Cas9 concentrations delivered to the zygotes and the resulting editing efficiencies are not explicitly investigated.” Members of The Centre for Phenogenomics (TCP) Transgenic Production Core who co-author this study (Lauryl Nutter, Marina Gertsenstein and Lauri Lintott) have published detailed protocols on mouse model production, which we cite in this paper (PMID: 30040228; PMID: 33524495; PMID: 39999224). In PMID: 33524495, they tested a two-fold difference in Cas9 RNP concentrations for generating knock-out alleles. Using their optimised protocols for electroporation of one cell zygotes with RNPs, we achieved an extremely high editing rate. We did not vary the sgRNA/Cas9 concentrations as part of this study as our goal was to assess the ability to generate “complete” null animals. We do note, however, that by targeting two genes simultaneously whilst keeping the total RNP concentration constant (to avoid reagent toxicity), we halved the amount of each sgRNA and this did not lead to a decrease in editing efficiency. We will highlight this in the results/discussion section (as appropriate).

Reviewer #1 asks about whether the use of crispants is applicable for “quantitative, late-onset, or more subtle phenotypes, including behavioral ones”. We are hopeful that this is possible and it is a priority for future studies. Crucially, cohorts of crispants can be generated in a single round of mutagenesis. Starting an experiment with ten donor females will produce ~100 zygotes, resulting in ~40 crispants. Power calculations must be performed to determine the size of the cohort required for the effect size and variability of the phenotype being studied, but many neurobehavioural studies use ~10 mutants vs ~10 controls. We note that sex and/or background genotype may mean that only some of the ~40 crispants produced can be used for phenotypic testing. This reviewer also raises the point about whether wild-type animals or mock-edited animals serve as the best controls. From work carried out by Lauryl Nutter and her colleagues from the IMPC (PMID: 37301944), we know that “wild-type” controls should ideally be from the same embryo pool as the crispants to avoid differences due to genetic drift within inbred colonies. This study also found that possible off-target mutations from CRISPR/Cas9-mediated mutagenesis is not an issue (despite a lot of attention in the literature). The suggestion of using mock-edited controls, resulting from zygotes that have gone through electroporation without RNP, addresses a possible need to control for the stress of undergoing the electroporation process. Our study shows that additional stress is caused by inducing and repairing a break in a neutral locus (EGFP). Controlling for these stressors may be particularly important when assessing behavioural phenotypes in crispants vs controls.

Reviewer #2 states that “there could have been some discussion regarding how this approach would be impacted if mutations are dominant or embryonic lethal (for the latter, for example, F0 can be examined as embryos).” Our manuscript discusses how crispants could help with the study of genes that may be essential. Specifically, we stated that when CRISPR/Cas9-mediated mutagenesis fails to produce live pups, phenotypic assessment of crispant embryos could reveal whether targeting the gene impacts embryogenesis. Crispants can only be used to screen for recessive traits since both alleles are edited. The assessment of dominant traits is not addressed in our study and remains a challenge in the field. We note that CRISPRi screens in cultured cells reveal candidates that when partially downregulated lead to the desired phenotype. One possibility is to employ this set up in vivo using dCas9-KRAB transgenic mice (JAX stock #030000). We could add this point to the discussion section.

Our doc is primarily concentrated around the workflow. But most of the competitor documentation is based on events. Example -- https://docs.wise.com/guides/developer/webhooks/event-types https://docs.trustly.com/api/credit-notification

Should we think restructuring our docs also to be aligned with competitor structure

Sources? Extremely provocative language with no supporting evidence.

Source?

After my own research, this appears to originate with a New York Post article, which the New York State Court system claims is full of errors, and sent a letter to the New York Post requesting they correct the errors (linked below), and the Post agreed to correct them, but doesn't seem to have followed through.

https://www.nycourts.gov/legacyPDFS/press/pdfs/AV26_02.pdf

Savchuk cites a study where participants saw a "36% drop in deep abdominal fat." I’m highlighting this not for the vanity aspect, but because visceral fat is a key driver of the metabolic syndrome I’m investigating. This provides the physiological mechanism for why the diet works, which I need for my "Findings" section.

While the text mentions "recovery times" being faster for plant-based athletes, it doesn't specify what exact nutrients drive this. I need to cross-reference this with my primary sources to see if it's the antioxidants or the lack of inflammatory arachidonic acid (found in meat) that is actually reducing the soreness.

The article highlights that vegetarian athletes had "significantly higher endurance" than meat-eaters. In the context of my literature review, I need to define "endurance" not just as running a marathon, but as the ability for a nurse or EMT to maintain cognitive and physical focus during hour 11 of a 12-hour shift.

Greger starts by citing historical studies from Yale (Fisher) and Belgium to build a foundation of credibility before introducing modern data. By anchoring the argument in century-old research, he rhetorically suggests that the "endurance advantage" of plant-based eating isn't a new fad, but a long-forgotten biological fact.

This shows that obesity is not just about personal choices, but is strongly connected to income and social inequality. It suggests that people with higher incomes may have more access to healthy food, safe places to exercise, and healthcare, while lower-income groups may face more barriers that increase their risk of obesity. This also makes me think about how society often blames people for their weight without considering the economic and environmental factors that shape their daily options.

This makes me think back to the documentary we watched last week. One of the main points from the documentary was that people of color living in worse off neighborhoods do not have the same opportunity or options for healthy food, and with that they are being forced to buy cheaper, unhealthy food that is within their budget. Then then leads to not only negative health problems, but also problems like being overweight obese

This comment shows how different each person can be and the fact that we do not know everything about every person. Each member in a community has their own wants, needs, and stories that make them who they are.

Romeo's yearning for Juliet is very deep.

A similie is used. Lightning is bright but short-lived, and Juliet senses their love is intense, but could burn out or explode (perhaps foreshadowing as well).

Metaphor. Love is compared to bait on a hook. It looks sweet, but it can trap and hurt you.

I think that social media platforms showing the users content its interested is a fair assumption of what the user wants, but it can create echochambers where the user is only seeing the same kind of content. I think it should also push content that the user might be interested, emphasis on the might, leaving more room for the users to decipher and decide instead of keeping them in tunnel vision.

This statement highlights that "relevance does not equal safety": algorithms treat "many mutual friends" as a strong signal, ignoring the harm and risks inherent in real-world relationships. It reveals the problem of recommendation systems lacking "contextuality" and "moral weighting"—the same connection structure can have completely different meanings in different life experiences. This also suggests that platforms need to provide stronger filtering and interpretation mechanisms (such as preventing specific groups from being recommended to, or allowing users to label sensitive relationships). Otherwise, recommendations are not only a matter of user experience but could also cause real psychological harm and personal risks.

Conflict over women’s spending and value.

Women raised standards for husbands.

Idea that women didn’t need marriage for value.

Women had some control at home, but men still held main authority.

Shared work did not mean equality.

Women had some control at home, but men still held main authority.

shared work did not mean equality.

Love was not a goal for most

Women had some control at home, but men still held main authority.

This is definitely something I should keep in mind for myself. By keeping in mind that the beginning of my note taking progress does not have to look perfect. I should write notes that make sense to me then revisit and expand on them so that I can remain observant.

This passage is bleak and emotionally devastating, using calm, almost domestic imagery to heighten the horror of the Queen’s death. The Chorus’s detached, detailed narration contrasts sharply with the violence it describes, reinforcing the play’s theme of how tragedy becomes normalized by power and routine. Creon is portrayed not as a villain consumed by rage, but as a hollowed-out figure who justifies cruelty as “necessary work,” revealing the moral exhaustion of authority. His interaction with the Page exposes a grim ethical lesson: that adulthood in politics means accepting responsibility for actions one would rather not understand. Overall, the writing emphasizes the emptiness left after moral conflict, suggesting that peace achieved through death and forgetting is deeply unsettling rather than redemptive.

This passage presents Antigone as calm, ironic, and emotionally complex, while Ismene appears anxious and grounded in practical fear. Antigone’s tone is almost gentle and nostalgic, especially when she recalls childhood memories, but this softness contrasts sharply with her acceptance of death. Her reflective and slightly teasing language shows that she understands the consequences of her actions yet refuses to be ruled by fear. Ismene, by contrast, represents caution and survival, emphasizing the real political power of Creon and the threat of execution. Overall, the writing highlights the emotional tension between idealism and practicality, making the characters feel deeply human rather than purely symbolic.

based thomas paine moment

Paine argues that maybe the one defense that hereditary succession could theoretically have is it would bring about the absence of civil wars, but that's entirely untrue, and civil wars have happened regardless.

exactly

Paine argues that monarchy goes directly against biblical scripture, but that often goes ignored.

I disagree that oppression is seldom the means of riches. People very will did and still do get rich through oppressing others, in fact, I would argue it's the most effective way.

This is a great argument against not only monarchy but also any form of government where it is ruled (either partially or entirely) by a really powerful person who is disconnected from society.

Confusing

Transitional devices as well as examples.

integrating evidence. Facts, judgments, testimony and personal observation.

How to create strong primary support. Be specific, be relevant to the thesis, and be detailed.

The purpose you are writing, to persuade, or to achieve a combination of these purposed. Use a skeleton that serves as outline, then you can create "flesh and muscle" developing ideas.

that is what most parents fail to understand with their children, all they think of is money, money and forget about passion.

Reviewer #2 (Public review):

Summary:

In this manuscript, Thompson et al. investigate the impact of prior ATP exposure on later macrophage functions as a mechanism of immune training. They describe that ATP training enhances bactericidal functions which they connect to the P2x7 ATP receptor, Nlrp3 inflammasome activation, and TWIK2 K+ movement at the cell surface and subsequently at phagosomes during bacterial engulfment. This is an incremental addition to existing literature, which has previously explored how ATP alters TWIK2 and K+, and linked it to Nlrp3 activation. The novelty here is in discovering the persistence of TWIK2 change and exploring the impact this biology may have on bacterial clearance. Additional experiments could strengthen their hypothesis that the in vivo protective effect of ATP-training on bacterial clearance is mediated by alveolar macrophages.

Strengths:

The authors demonstrate three novel findings: 1) prolonged persistence of TWIK2 at the macrophage plasma membrane following ATP that can translocate to the phagosome during particle engulfment, 2) a persistent impact of ATP exposure on remodeling chromatin around nlrp3, and 3) administering mice intra-nasal ATP to 'train' lungs protects mice from otherwise fatal bacterial infection.

Weaknesses:

(1) Some methods remain unclear including the timing and method by which lung cellularity was assessed in Figure 2. It is also difficult to understand how many mice were used in experiments 1, 2 and 6 and thus how rigorous the design was. A specific number is only provided for 1D and the number of mice stated in legend and methods do not match.

(2) The study design is not entirely ideal for the authors' in vivo question. Overall, the discussion would benefit from a clear summary of study caveats, which are primarily that that 1) in vitro studies attributing ATP training-mediated bacterial killing to persistent TWIK2 relocation, K+ influx, a glycolytic metabolic shift , and epigenetic nlrp3 reprogramming were performed in BMDM or RAW cells and not primary AMs, 2) data does not eliminate the possibility that non-AM immune or non-immune cells in the lung are "trained" and responsible for ATP-mediated protection in vivo; flow data examined total lung digest which may obscure important changes in alveolar recruitment, and 3) in vivo work shows data on acute bacterial clearance but does not explore potential risks that "training" for a more responsive inflammasome may have for the severity of lung injury during infection.

(3) The is some lack of transparency on data and rigor of methods. Clear data is not provided regarding the RNA-sequencing results. Specific identities of DEGs is not provided, only one high-level pathway enrichment figure. It would also be ideal if controls were included for subcellular fractionating to confirm pure fractions and for dye microscopy to show negative background.

(4) In results describing 5A, the text states that "ATP-induced macrophage training effects, as measured by augmented bactericidal activity, were diminished in macrophages treated with protease inhibitors". However, these data are not identified significant in the figure; protease dependence can be described as a trend that supports the authors' hypothesis but should not be stated as significant data in text.

In summary, this work contains some useful data showing how ATP can train macrophages via TWIK2/Nlrp3. Revisions have significantly improved methods reporting, added some data to strengthen the conclusions, and toned down on overstatements to bring conclusions more in line with data presented. The title still overstates what the authors have actually tested, since no macrophage-specific targeting in vivo (no conditional gene deletion, macrophage depletion etc) was performed in infection studies. However, in vitro data provide clear evidence that macrophages can be trained by ATP, and through caveats remain, it is plausible that macrophage training is a key mechanism for the protection observed here in the lung.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

(1) First, the concept of training or trained immunity refers to long-term epigenetic reprogramming in innate immune cells, resulting in a modified response upon exposure to a heterologous challenge. The investigations presented demonstrate phenotypic alterations in AMs seven days after ATP exposure; however, they do not assess whether persistent epigenetic remodeling occurs with lasting functional consequences. Therefore, a more cautious and semantically precise interpretation of the findings would be appropriate.

In response, we have performed epigenetic analysis (ATAC seq analysis) as requested (Supp Fig. 1).

(2) Furthermore, the in vivo data should be strengthened by additional analyses to support the authors' conclusions. The authors claim that susceptibility to Pseudomonas aeruginosa infection differs depending on the ATP-induced training effect. Statistical analyses should be provided for the survival curves, as well as additional weight curves or clinical assessments. Moreover, it would be appropriate to complement this clinical characterization with additional measurements, such as immune cell infiltration analysis (by flow cytometry), and quantification of pro-inflammatory cytokines in bronchoalveolar lavage fluid and/or lung homogenates.

We have added the statistical analyses provided for the survival curves (new Fig. 1D), immune cell infiltration analysis, and quantification of pro-inflammatory cytokines in the lung (new Figs. 1, 2).

(3) Moreover, the authors attribute the differences in resistance to P. aeruginosa infection to the ATP-induced training effect on AMs, based on a correlation between in vivo survival curves and differences in bacterial killing capacity measured in vitro. These are correlative findings that do not establish a causal role for AMs in the in vivo phenotype. ATP-mediated effects on other (i.e., non-AM) cell populations are omitted, and the possibility that other cells could be affected should be, at least, discussed. Adoptive transfer experiments using AMs would be a suitable approach to directly address this question.

We have performed additional experiments and found that the numbers of lung macrophages were not significantly altered before and after ATP training (new Fig. 2), indicating the training effects are focused on lung resident macrophages.

Reviewer #2 (Public review):

(1) Missing details from methods/reported data: Substantial sections of key methods have not been disclosed (including anything about animal infection models, RNA-sequencing, and western blotting), and the statistical methods, as written, only address two-way comparisons, which would mean analysis was improperly performed. In addition, there is a general lack of transparency - the methods state that only representative data is included in the manuscript, and individual data points are not shown for assays.

We have revised the methods and statistical analysis.

(2) Poor experimental design including missing controls: Particularly problematic are the Seahorse assay data (requires normalization to cell numbers to interpret this bulk assay - differences in cell growth/loss between conditions would confound data interpretation) and bacterial killing assays (as written, this method would be heavily biased by bacterial initial binding/phagocytosis which would confound assessment of killing). Controls need to be included for subcellular fractionating to confirm pure fractions and for dye microscopy to show a negative background. Conclusions from these assays may be incorrect, and in some cases, the whole experiment may be uninterpretable.

Seahorse assay methodology was updated to confirm the order of cell counting, time at seeding and cell counts. Methods were also updated to address the distinction between bacterial killing (Fig. 1B) and overall decrease in bacterial load.

(3) The conclusions overstate what was tested in the experiments: Conceptually, there are multiple places where the authors draw conclusions or frame arguments in ways that do not match the experiments used. Particularly:

(a) The authors discuss their findings in the context of importance for AM biology during respiratory infection but in vitro work uses cells that are well-established to be poor mimics of resident AMs (BMDM, RAW), particularly in terms of glycolytic metabolism.

We have adjusted the text to reflect that the metabolic assay was performed on BMDMs. AMs are fragile for certain manipulations in vitro. We expect that the metabolic change is similar across several macrophage systems as well as the bacterial load reduction.

(b) In vivo work does not address whether immune cell recruitment is triggered during training.

We have performed immune cell infiltration analysis (new Fig. 2).

(c) Figure 3 is used to draw conclusions about K+ in response to bacterial engulfment, but actually assesses fungal zymosan particles.

We have corrected this in the manuscript.

(d) Figure 5 is framed in bacterial susceptibility post-viral infection, but the model used is bacterial post-bacterial.

We have corrected this in the manuscript.

(e) In their discussion, the authors propose to have shown TWIK2-mediated inflammasome activation. They link these separately to ATP, but their studies do not test if loss of TWIK2 prevents inflammasome activation in response to ATP (Figure 4E does not use TWIK2 KO).

We have now added the TWIK2 KO results (new Fig. 5E).

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

As noted in the public review, it would be advisable to further characterize the in vivo phenotype in order to strengthen the conclusions. Specifically, it would be useful to quantify the bacterial load in the bronchoalveolar lavage fluid and lung homogenates, as well as to measure cytokine levels both in the respiratory compartment and systemically. Additionally, a broader characterization of the immune response in the presence or absence of ATP-induced training would be valuable. In the absence of direct evidence demonstrating that trained AMs mediate the observed phenotype, the authors should adopt a more cautious interpretation of their results. Moreover, careful attention to semantic accuracy is recommended. The concept of trained immunity refers specifically to long-term epigenetic reprogramming that leads to an altered response of target cells upon a secondary challenge, distant from the initial stress. The data presented do not fully demonstrate this phenomenon, and the interpretations should remain aligned with the evidence provided.

Bacterial load has been quantified (see more details in the Methods part). And we also measured immune cell infiltration, quantification of pro-inflammatory cytokines in the lung (new Figs. 1, 2), and epigenetic evaluation of vehicle- and ATP-treated cells (Supp. Fig. 1).

Reviewer #2 (Recommendations for the authors):

(1) It cannot be overstated how lacking the methods are. This includes no discussion of IACUC approval for animal procedures, which must be included as part of research ethics. It also needs to be made clear where raw data is being archived. This notably includes an accession for deposited RNA-sequencing data, although unmanipulated microscopy and western blot images should also be shown. Methods should discuss any pre-processing that occurred with images.

We have revised the methods in the manuscript.

(2) Per statistics, in addition to generally providing more detail and adjusting analyses if they have not been correctly performed, please disclose if SD or SEM is shown. Reporting aggregate data versus representative data would provide more rigor. Perhaps replicate experiments could be included in the supplemental if they cannot, for some reason, be aggregated. Detailed statistical methods for RNA-seq analysis also need to be included.

More details have been provided in the methods section.

(3) It is unclear whether bacterial killing assays were correctly designed and can be interpreted. What does cells collected mean? If the assay was focused on intracellular macrophage bacterial load, it is critical to assess and report phagocytosis since different input loads would confound the assessment of killing. A rigorous wash or an antibiotic to eliminate extracellular bacteria should also have been performed and be described in this case. If the total bacterial burden was assessed, that would use cells+media and also needs to be clear and described. With the information provided, it is unclear whether the assays performed are sufficiently rigorous to assess bacterial killing. In addition, Figure 1B reports using an MOI of 50-100, but all data is compiled in one graph - data from different levels of infection should be separated. Figure 5A shows a model with E.coli followed by PA, but that does not appear to be how the assay was structured in B or C. This also does not match how the experiment is written in the results section, which references S. aureus. It is unclear what tissue (or cells) were assessed in Figure 5. Whole lung? BAL? As written, no data provided regarding bacterial killing is of sufficient quality to be considered valid.

We have re-written the bacterial killing assay in the manuscript. The methodology was corrected to distinguish bacterial killing vs load decrease and generally accurate methodology.

(4) The in vitro data provide reasonable evidence that BMDM/RAW macrophage training can occur in response to ATP exposure. However, it is unclear whether training is an important mechanism for resident AM in vivo, or whether, in vivo, a broader inflammatory response is generated, recruiting additional immune cells that persist and change infection susceptibility. The authors argue for resident AM immune training, but do not provide sufficient evidence to counter the latter possibility (resident AM are never themselves directly assessed, and the presence of other immune cells in vivo is not excluded). See Iliakis et al 2023 (PMID 37640788) for discussion of how this issue continues to drive uncertainty in the field. For this study, at least providing flow cytometry data quantifying myeloid and lymphoid immune populations in BALF before and after various treatments would help address this caveat. Without knowing this, it also confounds the interpretation of Figure 1B; if BAL is not pure AM after training, perhaps 1B could be repeated with ex vivo training or resident AM could be purified?

We have performed immune cell infiltration analysis in the lung (both to BALF and in-tissue, new Fig. 2).

(5) Figure 3A appears to show that fewer than 50% of cells express GFP. Is it expected that only a fraction of RAW cells express TWIK2-GFP? How was this addressed in the analyses for Figure 3? Were cells not appearing to express any significant GFP, included in phagosomal-negative or excluded from analysis? Please include in the methods.

The RAW cells were transfected with TWIK2-GFP and variable GFP expression was expected. These cells were expressing a non-integrated transgene, which has been added to the methods as well as the consideration of cells for the analysis. Cells without visible GFP expression were excluded.

(6) Why are many data points in Figure 3D negative? This suggests that settings were not optimized for microscopy - perhaps there is a very high background signal and the ION stain is barely above it. This is concerning for the quality of data. Further, is it expected that only some cells are positive for ION K+? The images shown clearly differentiate phagosomal K with ATP versus the absence of K without, but it is surprising that some cells appear not to contain any ION K+ signal (not completely clear given lack of brightfield or other cell staining) - this may again point to issues with imaging settings that confound data interpretation. This analysis should be carefully assessed.

This has been updated in the methodology. In old Fig. 3D (new Fig. 4D), the presented data is the net intensity of the phagosome, subtracting the average cytoplasmic MFI from that of the area corresponding to an engulfed zymosan-af594 bead. Thus, a negative value has higher cytoplasmic IonK signal than that of the phagosome.

(7) The Discussion states that it will be interesting to test whether ATP-TWIK2 is a common mechanism of training and specifically references LPS as an ATP-generating signal. However, Figure 2D data show that LPS induces only transient TWIK2 translocation; the authors have data suggesting that, in the context of LPS, TWIK2 'training' will not be engaged. This line of discussion shows incomplete consideration of the data.

We have further limited this language in the text such that this may require differential sensitivity/damage sustained by macrophages as compared to that of epi/endothelial cells in response to bacterial endotoxin.

(8) For RNA-sequencing, plots of the actual genes changed for the mitochondrial pathways of interest would be helpful information for readers, as would a heat map showing sample purity between groups for macrophage markers versus possible contaminant cells, which can also be generated from precursors in BMDM cultures. In general, information in Methods regarding how the analyses in Figure 4B were run is necessary, per cutoffs used to determine DEGs, number of samples in each group, sex of samples used, etc. Greater transparency of data would be appreciated, so plots that show variation between replicates, such as heat maps, would be ideal. Supplemental tables would also be nice.

We have added to the methodology of the RNA sequencing analysis

(9) The use of alternate DAMPs is a positive addition to the experimental design, but no data is given regarding the concentrations used. Ideally, positive controls showing histones/NAD are used at acutely activating concentrations could be included but at least references supporting the doses chosen or information about how doses were selected should be given. It is easy to find substantial literature on histones as a DAMP, but it was unclear why/how NAD was selected.

We have added these concentrations and corresponding references.

(10) The E.coli CFU reported in Figure 5B are extraordinarily low. In addition, CFU are generally shown on a log scale, but this appears to be linear. Please confirm that these data are correct. Perhaps improved methods might explain why? Is the second hit a low dose?

These have been corrected in the new Fig. 6B.

(11) Given that loss of either TWIK2 or Nlrp3 ablates bacterial protection, a link should be tested - experiments should test whether loss of TWIK2 prevents inflammasome activation in response to ATP (TWIK2 KO in 4E) and if loss of Nlrp3 changes TWIK2 translocation (Nlrp3 KO in at least some experiments of Figures 2/3).

We have now added the TWIK KO results (new Fig. 5E).

(12) One of the most striking data pieces is Figure 1D. It would, therefore, strengthen the paper to repeat those experiments (even just with the high-dose ATP) using TEIK2/P2X7/NLRP3 KO mice and really show the importance of these pathways in vivo. This is conceptually Figure 5, but the survival data of Figure 1 is far more convincing than the relatively weak bacterial load data of Figure 5.

Unfortunately, our previous laboratory has been closed and we have trouble acquiring enough mice for additional survival data during the transition period. However, the bacterial load data has been adjusted to the same bacterial counts per 5 mg lung tissue instead of per individual sampling, giving a more contextual interpretation of the data.

exactly. it is not easy at all, at first when i did not start, i use to see and hear people complain about university, and i was like it is a small thing, but it is definitely not.

This is a different perspective that I have never looked into but yet completely understand. Rhetoric, what we know to be "the act of persuading someone," is the same thing as "producing a 'fake' reality." Thinking about how to persuade someone, you must be able to successfully convince them of something you believe.

For example, if Betty says a tomato is a vegetable and I am trying to convince her it is a fruit, I must make her see a reality that she does not see. Yet, someone if successful would be able to persuade me that fruit is a vegetable and not a fruit. And with enough persuasion, it is possible I can be persuaded that the other side is right. I would be moving to a reality that is ot my reality due to the act of persuasion. Thinking about that from an analytical standpoint is quite interesting.

Occupant was there the first time, but the second time it wasn't

Missing beauty in the moment, not there anymore

Time focused, not a moment of a present encounter, but going back to past twice, pre-past and past

"But the internet loving you, it should be said, is not often a good thing."

To me, this is kind of connected to the point of kairos in the timing of things. Celebrities have to be more cautious about what they post, but more importantly, when they post it, because they have more eyes on them in the process. If their timing to post something especailly if it contrversail is wrong, they are at higher risk of throwing away their whole career over one poor timing choice.

The memes nowadays are different to what this textbook suggested, now it's a fashion trends that it's sort of like an emoji but can express more to users, it can be a screenshot of a funny moment of the live streamer you like, it could be a line of texts that makes people laugh, a good meme should be, in my opinion, funny, relaxing, and everyone can get the idea, but nowadays the idea of memes has changed slightly...

This reminds of something actually happened to me, before do online surfing I was chatting with my friends, I forgot what's the topic but when I start using social media like TikTok but Chinese version, the app just start to recommend the things we've talked about. Also every night, TikTok will start to recommend videos like mukbang that makes me hungry and want to order foods, all of these are because of big data as what China is currently promoting.

Author response:

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

Public Reviews:

Reviewer #1 (Public reviews):

(1) The absence of replicate paired-end datasets limits confidence in peak localization.

The reviewer was under the impression that that we did not perform biological replicates of our ChIP-seq experiments. All ChIP-seq (and ATAC-seq) experiments were performed with biological replicates and the Pearson’s correlations (all >0.9) between replicates were provided in Supplementary Table 1. We had indicated this in the text and methods but will try to make this even clearer.

(2) The analyses are primarily correlative, making it difficult to fully assess robustness or to support strong mechanistic conclusions.

Histone modifications are difficult to alter genetically because of the high copy number of histone genes and inhibition of HATs/HDACs in general leads to alterations in other histone modifications. It is an inherent challenge in establishing causality of histone modifications, especially histone acetylation marks.

(3) Some claims (e.g., specificity for CpG islands, "dynamic" regulation during differentiation) are not fully supported by the analyses as presented.

We have modified the text in response to this point. The new text reads: “Non-CGI promoters have lower overall levels of transcription compared to CGI promoters, and for this promoter class H3K115ac enrichment detected by ChIP is only really seen for the highest quartile of transcription (4SU) quartile of expression (Figure 1G). CGI promoters on the other hand, exhibit significant levels of detected H3K115ac even for the lowest quartile of expression. These results suggest a special link between CGI promoters and H3K115ac”.

(4) Overall, the study introduces an intriguing new angle on globular PTMs, but additional rigor and mechanistic evidence are needed to substantiate the conclusions.

We agree that the paper does not provide mechanistic details or solid causality of H3K115ac. We have only emphasized the potential role of H3K115ac in nucleosome fragility based on our in vivo data and previously published in-vitro experiments (Manohar et.al., 2009, Chatterjee et. al., 2015). We do provide the evidence that H3K115ac is enriched on subnucleosomal particles via sucrose gradient sedimentation of MNase-digested chromatin (Figure 3C-D).

Reviewer #2 (Public review):

(1) I am not fully convinced about the specificity of the antibody. Although the experiment in Figure S1A shows a specific binding to H3K115ac-modified peptides compared to unmodified peptides, the authors do not show any experiment that shows that the antibody does not bind to unrelated proteins. Thus, a Western of a nuclear extract or the chromatin fraction would be critical to show. Also, peptide competition using the H3K115ac peptide to block the antibody may be good to further support the specificity of the antibody. Also, I don't understand the experiment in Figure S1B. What does it tell us when the H3K115ac histone mark itself is missing? The KLF4 promoter does not appear to be a suitable positive control, given that hundreds of proteins/histone modifications are likely present at this region. It is important to clearly demonstrate that the antibody exclusively recognizes H3K115ac, given that the conclusion of the manuscript strongly depends on the reliability of the obtained ChIP-Seq data.

ChIP-qPCR in S1B includes competition from native chromatin and shows high specificity to its target. We have provided antibody validation in three ways:

- Western blot with dot-blot of synthetic peptides (Figure S1A).

- Western blots with Whole cell extracts (Figure 4D).

- ChIP-qPCR on native chromatin spiked with a cocktail of synthetic mono-nucleosomes, each carrying a single acetylation and a specific barcode (SNAP-ChIP K-AcylStat Panel).

We could not include H3K115ac marked nucleosomes as they are not available in the panel. Figure S1B shows that the H3K115ac antibody exhibits negligible binding to known K-acyl marks, comparable to an unmodified nucleosome. Because of the absence of a H3K115ac modified barcoded nucleosome, we used the KLF4 promoter from mESCs as a positive control, in agreement with ChIP-seq signal shown in the genome browser profile (Figure 1E), the KLF4 promoter shows a significantly higher signal than the gene body.

(2) The association of H3K115ac with fragile nucleosomes is based on MNase-sensitivity and fragment length, which are indirect methods and can have technical bias. Experiments that support that the H3K115ac modified nucleosomes are indeed more fragile are missing.

We have performed ChIP-seq on MNase digested mESC chromatin fractionated on sucrose gradients and this shows that H3K115ac is enriched in fractions containing sub-nucleosomal and fragile nucleosomes but depleted in fractions containing stable nucleosomes (Figure 3D).

(3) The comparison of H3K115ac with H3K122ac and H3K64ac relies on publicly available datasets. Since the authors argue that these marks are distinct, data generated under identical experimental conditions would be more convincing. At a minimum, the limitations of using external datasets should be discussed.

H3K64ac and H3K122ac datasets were generated by us in a previous publication (Pradeepa et. al., 2016) using same native MNase ChIP protocol as used here. The ChIP-seq datasets for H3K122ac and H3K27ac are processed in an identical manner, with the same computational pipelines, to the H3K115ac data sets generated in this paper.

(4) The enrichment of H3K115ac at enhancers and CTCF binding sites is notable but remains descriptive. It would be interesting to clarify whether H3K115ac actively influences transcription factor/CTCF binding or is a downstream correlate.

We agree with the reviewer’s comment, but we have not claimed causality.

(5) No information is provided about how H3K115ac may be deposited/removed. Without this information, it is difficult to place this modification into established chromatin regulatory pathways.

Due to broad target specificity, redundancies and crosstalk among different classes of HATs and HDACs, it is not tractable to answer this question in the current manuscript.

Reviewer #3 (Public reviews):

Reviewer 3 is mistaken in thinking our ChIP experiments are performed under cross-linked conditions. As clearly stated in the main text and methods, all our ChIP-seq for histone modifications is done on native MNase-digested chromatin – with no cross-linking. This includes the spike-in experiment shown in Fig S1B to test H3K115ac antibody specificity against the bar-coded SNAP-ChIP® K-AcylStat Panel from Epicypher. We could not include H3K115ac bar-coded nucleosomes in that experiment since they are not available in the panel.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

(1) I have two primary concerns that resound through the entire paper:

(a) Overall, the manuscript is making strong claims based on entirely correlative datasets. No quantitative analyses are performed to demonstrate co-occupancy/localization. Please see more detailed descriptions below.

Our responses to specific points are provided against each comment below.

(b) Lack of paired-end replicates for H3K115ac ChIP-seq. While the reviewer token for the deposited data was not made accessible to me, looking at Supplementary Table 1, it appears there are two H3K115ac ChIP-seq datasets. One is paired-end and is single-read. So are peaks called with only one replicate of PE? Or are inaccurate peaks called with SR datasets? Either way, this is not a rigorous way to evaluate H3K115ac localization.

We are sorry that this reviewer was not able to access the data – the token for the GEO accession was provided for reviewers at the journal’s request. All ChIP-seq (and ATAC-seq) experiments (paired and single-end) were performed with two biological replicates and the Pearson’s correlations (all >0.9) between replicates were provided in Supplementary Table 1. This was indicated in both the main text and in the methods. In the revised manuscript we have tried to make this even clearer and have put the relevant Pearsons coefficient (r) into the text at the appropriate places. For the reviewer’s information, here is the complete list of data samples in the GEO Accession:

Author response image 1.

While I agree that H3K115ac occupancy is high at +CGIs, the authors downplay that H3K122ac and H3K27ac is also more highly enriched at these locations (page 7, last sentence of first paragraph). I imagine this is all due to the more highly transcribed nature of these genes. Sub-stratifying the K27ac and K122ac by transcription (as in Figure 1G) would help to demonstrate a unique nature of H3K115ac. But even better would be to do an analysis that plots H3K115ac enrichment vs transcription for every individual gene rather than aggregate analyses that are biased by single locations. For example, make an XY scatterplot of RNAPII occupancy or 4SU-seq signal vs H3K115ac level, where each point represents a single gene. Because the interpretation that it is CGI-based and not transcription is confounded with the fact that -CGI are more lowly transcribed. So, looking at Figure 1G, even the -CGI occupancy of H3K115ac is correlated with transcription, but it is just more lowly transcribed.

We thank the reviewer for these suggestions but point out that Figure 1G shows H3K115ac signal for CGI+ and CGI– TSS that are matched for expressions levels (quartiles of 4SU-seq). Fig 1F shows that H3k115ac is much more of a discriminator between CGI+ and – than H3K27ac or H3K122ac.

(2) H3K115ac, H3K27ac, and H3K122ac are all more enriched (in aggregate) at +CGI locations (Fig 1F); so do these locations just have more positioned nucleosomes? More H3.3? So that these PTMs are just more enriched due to the opportunity?

Positioned nucleosomes are generally found downstream of the TSS of active CpG island promoters, so what the reviewer suggests may well account for the relative enrichment of H327ac and H3K122ac at CGI+ vs CGI- promoters in Fig.1F. But H3K115ac localisation is distinct, with the peak at the nucleosome-depleted region not the +1 nucleosome. This is also confirmed by the contour plots in Fig 3. Our observation is also not explained by an enrichment of H3.3 at CGI promoters, since we show that H3K115ac is not specific to H3.3 (Fig 4D).

(3) The authors note in paragraph 2 of page 7 that "H3K115ac does not scale linearly with gene expression..." but the authors never show a quantification of this; stratification in four clusters is not able to make a linear correlation. Furthermore, in the second line of page 7, the authors state that the levels do generally correlate with transcription. To claim it is a specific CGI link and not transcription is tricky, but I encourage the authors to consider more quantifiable ways, rather than correlations, to demonstrate this point, if it is observed.

We thank the reviewer for this comment, and taking it into consideration, we have decided to re-phrase this paragraph. The new text reads: “Non-CGI promoters have lower overall levels of transcription compared to CGI promoters, and for this promoter class H3K115ac enrichment detected by ChIP is only really seen for the highest quartile of transcription (4SU) quartile of expression (Figure 1G). CGI promoters on the other hand, exhibit significant levels of detected H3K115ac even for the lowest quartile of expression. These results suggest a special link between CGI promoters and H3K115ac”.

(4) The authors claim on page 7 that "on average, transcription increased from TSS that also gained H3K115ac but to a modest extent, compared with the more substantial loss of H3K115ac from downregulated TSS". However, both upregulated and downregulated are significant; the difference in magnitude could simply be due to more highly or more lowly transcribed locations, meaning that fold change could be more robustly detected. I caution the authors to substantiate claims like this rather than stating a correlation.

We thank the reviewer for this comment which relates to the data in Fig 2A. It is Fig. 2B shows that the association of H3K115ac loss with downregulation is statistically stronger than H3K115ac gain with upregulation, but only for CGI promoters. With regard to the text on the original pg 7 that is referred to, we have now reworded this to read “Average levels of transcription increased from TSS that also gained H3K115ac, and there was loss of H3K115ac from downregulated TSS (Figure 2A).”

(5) For Figure 2C, the authors argue that H3K115ac correlate with bivalent locations. So this is all qualitative and aggregate localization; please quantitatively demonstrate this claim.

Figure S2D provides statistics for this (observed/expected and Fishers exact test).

(6) The authors claim in Figure 2 that H3115ac is dynamic during differentiation (title of Figure 2). However, there are locations that gain and lose, or maintain H3K115ac. In fact, the most discussed locations are H3K115ac with no change (2C); which means it is NOT dynamic during differentiation. So what is the message for the role during differentiation? From Supplemental Table 1, it appears there is a single ChIP experiment for H3K115ac in NPC, and it is a single read. So this is also a difficult claim with one replicate. Related to this, in S2A, the authors show K115ac where there is no change in transcription; so what is the role of H3K115ac at TSSs relevant to differentiation - it is at both locations changed and unchanged in transcription, but H3K115ac levels itself do not change at these subsets. So, how is this dynamic? This is very confusing, and clearer analyses and descriptions are necessary to deconvolute these data.

We apologise for the misleading title for Figure 2. This has now been amended to “Changes in H3K115ac during differentiation”. The message of this figure is that whilst changes in H3K115ac at TSS are small (panels A-C), at enhancers the changes are much more dramatic (panel D). The reviewer is incorrect about the number of replicates for NPCs – there are two biological replicates (see response to point 1b).

(7) The authors go on to examine H3K115ac enrichment on fragile nucleosomes through sucrose gradient sedimentation. A control for H3K27ac or H3K122ac would be nice for comparison.

We do not have the material available to perform these experiments

(8) When discussing Figures 3 and SF3, the authors mention performing a different MNase for a second ChIP. Showing the MNase distribution for both the more highly digested and the lowly digested would be nice. a) Related to the above, the authors show input in SF3E to argue that the difference in H3K115ac vs H3K27ac is not due to the library, but they do not show the MNase digestion patterns, which is more important for this argument.

Input libraries (first two graphs of FigS3E) are the MNase-digested chromatin. Comparison of nucleotide frequencies from millions of reads is more robust method than the fragment length patterns.

(9) The authors move on to examine H3K115ac at enhancers. Just out of curiosity, given what was found at promoters, is H3K115ac enriched at +CGI enhancers? And what is the correlation with enhancer transcription?

This is an interesting point, but the number of enhancers associated with CGI is not very high and so we did not focus on this. We have not analysed a correlation with eRNAs in this paper.

(10) The authors state on page 14 that the most frequent changes in H3K115ac during differentiation are at these enhancers. So do these changes connect with differentiation-specific genes, and/or genes that have altered transcription during differentiation? Just trying to understand the functional role.

Given the challenges of connecting enhancers with target genes, we have not addressed this question quantitatively. However, we draw the reviewer’s attention to the Genome Browser shots in Figures 2D and S2C, which show clear gain of H3K115ac (and ATAC-seq peaks) at intra and intergenic regions close to genes whose transcription is activated during the differentiation to NPCs.

(11) Related, at the end of page 14, the authors state that the changes in H3K115ac correlate with changes in ATAC-seq; I imagine this dynamic is not unique for H3K115ac and this is observed for other PTMs (H3K27ac), so assessing and clarifying this, to again get to the specific interest of H3K115ac, would be ideal.

We have not claimed that chromatin accessibility is unique to H3K115ac. It is the location of H3K115ac which is found inside the ATAC-seq peak region while H3K27ac is found only upstream/downstream of the ATAC peak that is so striking. This is apparent in Fig 4C.

(12) The authors examine levels of H3K115ac in H3.3 KO cell lines via western blot (Figure 4D), but no replicates and/or quantification are shown.

We now provide a biological replicate for the Western Blot (new FigS4H) together with an image of the whole gel for the data in Fig 4D

(13) In Figure S4 and at the end of page 17, the authors are arguing that there is a link to pioneer TF complexes, based on Oct4 binding. First, while Oct4 has pioneering activity, not all Oct4 sites (or motifs) are pioneering; this has been established. So if you want to use Oct4, substratifying by pioneer vs no pioneer is necessary. Second, demonstrating this is unique to pioneer and not to non-pioneer TFs would be an important control.

In response to the reviewer’s comment, we have removed the term “pioneer” from the manuscript.

(14) Minor point: Figure 4 A and B, there are some formatting issues with the scale bars.

We thank the reviewer for pointing this out, and the errors have been corrected in the revised figure.

(15) Minor point is that it should be clear when single replicates of data are used and when PE/SR sequences are combined or which one is used in each analysis, as this was hard to discern when reading the paper and figure legends.

We have clearly stated in the text that, after Figure2, we repeated all experiments in paired-end mode. All processing steps are defined separately for single end and paired end datasets in the method section. Details of biological replicates are provided in Sup. Table 1. These concerns are also addressed in our response to Reviewer’s public comment-1.

(16) Minor point: it is surprising that different MNase and different units were used in the ChIP vs sucrose sedimentation. Could the authors clarify why?

Chromatin prep for sucrose gradients were done on a much larger scale than for ChIP-seq and required different setups to obtain the right level of MNase digestion.

(17) The authors note that fragile nucleosomes contain H2A.Z and H3.3, but they never perform an analysis of available data to demonstrate a correlation (or better a quantifiable correlation) between H3K115ac occupancy and these marks at the locations they identify H3K115ac.

Since have shown (Fig. 4) that depletion of H3.3 does not affect overall levels of H3K115ac, we do not think there is value in further quantitative correlative analyses of H3K115ac and variant histones.

(18) Minor point: What is the overlap in peaks for H3K115ac, H3K122ac, and H3K27ac (Figure 1C)?

Nearly all H3K115ac peaks overlap with H3K122ac and/or H3K27ac. Its most distinct properties are its association with CGI promoters, fragile nucleosomes and its unique localisation within the NDRs, three points that the manuscript is focussed on.

Reviewer #3 (Recommendations for the authors):

(1) The western blot results in Figure 4D probing for H3, H3.3, and H3K115ac use Ponceau S staining, presumably of an area of the membrane where histones might be expected to migrate, as a measure of loading. However, the Ponceau S bands appear uniformly weaker in the H3.3KO lanes, yet despite this, blotting with H3.3 antibody detects a band in H3.3 knockout ESCs, suggesting that the antibody does not have a high degree of specificity. Again, a blocking experiment with appropriate peptides would instill more confidence in the specificity of these reagents, and/or the authors could provide independent validation of the knockout model to differentiate between a partial knockout or antibody cross-reactivity (e.g., by Sanger sequencing).

In a revised Fig. S4H we now show the whole gel corresponding to this blot but including co-staining with an antibody for H4 to provide a better loading control. We also provide a biological replicate of this Western blot in the lower panel of Fig. S4H.

(2) The manuscript would benefit from in vitro follow-up and validation, but if the authors intend to keep the manuscript primarily in silico, I suggest dedicating a few lines in each section to explain the plots, their axes, and their purpose, as well as to assist with interpretation, rather than directly discussing the results. This would make the manuscript more accessible and understandable for a broader audience in the field of epigenetics.

In the revised version, we have tried to improve the text to make the data more accessible to a broad audience.