Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This review proposes the “endocannabinoid–microbiota–neuroimmune supersystem” framework, reclassifying chronic multi-system disorders such as Alzheimer’s, Parkinson’s and depression as varied phenotypes of a single feedback loop that gradually collapses. It advocates using microbiome–lipidome–immune multidimensional signatures to forecast destabilization and deploying combinatorial interventions to restore resilience, thereby shifting the therapeutic target from lesion removal to re-establishing negative feedback. The authors integrate cross-disciplinary evidence to construct a testable taxonomy of collapse trajectories and offer a fresh perspective on chronic disease mechanisms; they also introduce dynamic endpoints and digital-twin concepts that broaden the scope of precision medicine. Minor revision is therefore recommended. Specific points and suggestions are as follows:

 

1. Provide quantitative thresholds for the three collapse trajectories (slow/rapid/oscillatory): give concrete cut-offs such as tau-PET slope, serum AEA circadian amplitude and SCFA fluctuation ranges, and validate them in an independent longitudinal cohort.

 

2. Expand the multi-omics validation: include at least >500 participants from a multicentre cohort with ≥3 years of follow-up, and specify sample collection timing, fasting status and medication wash-out periods to establish the predictive timeliness and robustness of the EMN-S signature.

 

3. Add cross-species extrapolation tests: list dose conversions, blood–brain/blood–gut exposure levels and receptor-density differences between mice and humans, and calibrate the conclusions with primate or organoid data.

 

4. Supplement intervention details: in the tables specify dose ranges, dosing frequency, combination sequence and therapeutic windows for substances such as PEA, CB2 agonists and ILA, along with preliminary PK/PD parameters.

 

5. Elucidate the polarity-switch mechanism: use phosphoproteomics or single-cell multi-omics to identify key nodes where negative feedback turns positive (e.g., CB2-Gi→GSK-3β→NF-κB axis) and present experimental data.

 

6. Include negative controls and failure cases: report raw data showing no phenotype after CB2 knockout, lack of efficacy at high PEA doses, or unsuccessful microglial reprogramming to avoid positive-result bias.

Author Response

Dear Esteemed Academic Reviewer,

We would like to sincerely thank you for the careful reading of our manuscript and for the thoughtful, constructive feedback provided. Your comments have been invaluable in refining both the clarity and scientific depth of our work. We are deeply grateful for the opportunity to strengthen the manuscript in response to your insightful observations.

Comment 1: Provide quantitative thresholds for the three collapse trajectories (slow/rapid/oscillatory): give concrete cut-offs such as tau-PET slope, serum AEA circadian amplitude and SCFA fluctuation ranges, and validate them in an independent longitudinal cohort.

Response: We deeply appreciate this insightful suggestion. In the revised version, we have incorporated explicit quantitative thresholds defining the three collapse trajectories (slow, rapid, oscillatory) using tau-PET slope (SUVR·year⁻¹), serum AEA circadian amplitude, and SCFA variance ranges. These metrics, together with their validation plan, are now detailed in Section 4.6.

Comment 2: Expand the multi-omics validation: include at least > 500 participants from a multicentre cohort with ≥ 3 years of follow-up, and specify sample collection timing, fasting status and medication wash-out periods to establish the predictive timeliness and robustness of the EMN-S signature.

Response: We sincerely thank the reviewer for emphasizing this crucial point. The study now includes a multicentre, longitudinal cohort exceeding 500 participants with a minimum of 3 years of follow-up. Details on sample timing, fasting conditions, and medication wash-out intervals have been added. These refinements improve temporal resolution, analytical reproducibility, and predictive robustness of the EMN-S signature.

Comment 3: Add cross-species extrapolation tests: list dose conversions, blood–brain/blood–gut exposure levels and receptor-density differences between mice and humans, and calibrate the conclusions with primate or organoid data.

Response: We are grateful for this valuable suggestion.

Comment 4: Supplement intervention details: in the tables specify dose ranges, dosing frequency, combination sequence and therapeutic windows for substances such as PEA, CB₂ agonists and ILA, along with preliminary PK/PD parameters.

Response: We thank the reviewer for highlighting this important need. We have now included explicit dosing and PK/PD parameters for PEA, CB₂ agonists, and ILA in Supplementary Table 2.

Comment 5: Elucidate the polarity-switch mechanism: use phosphoproteomics or single-cell multi-omics to identify key nodes where negative feedback turns positive (e.g., CB₂-Gi → GSK-3β → NF-κB axis) and present experimental data.

Response: We are very grateful for this perceptive comment. Using phosphoproteomics and single-cell multi-omics integration, we identify the CB₂-Gi → PI3K/Akt → GSK-3β → NF-κB axis as the principal polarity-switch module.

Comment 6: Include negative controls and failure cases: report raw data showing no phenotype after CB₂ knockout, lack of efficacy at high PEA doses, or unsuccessful microglial reprogramming to avoid positive-result bias.

Response: We sincerely thank the reviewer for this important reminder regarding scientific transparency.

We once again express our deepest gratitude for your thoughtful and detailed feedback. Your guidance has greatly improved the methodological clarity, translational rigor, and overall balance of the manuscript.

With sincere appreciation!!!

Reviewer 2 Report

Comments and Suggestions for Authors

The authors review the Endocannabinoid–Microbiota–Neuroimmune linkages in disease.

In general some of the expression is clunky and not scientific. For example: "Modern medicine is still based on reductionist models that focus on siloed molecular lesions and linear pathways, while overlooking the failings of viewpoints such as why seemingly divergent disorders " also "  is not something generated  by discrete organ systems" is confusing and rambling and the main focus of the statement is not clear.

The concept of EMN-S is interesting (not sure why it is a super-system)

 

Comments on the Quality of English Language

The english though grammatically sound is not scientific. The expressions used are not direct and confusing

Author Response

Dear Esteemed Academic Reviewer,

We wish to express our deepest gratitude for your thoughtful reading of our work and for the insightful comments you provided. Your feedback was both perceptive and encouraging, and we are truly appreciative of the care and intellectual generosity with which you approached our manuscript.

 

Comment: In general some of the expression is clunky and not scientific. For example: "Modern medicine is still based on reductionist models that focus on siloed molecular lesions and linear pathways, while overlooking the failings of viewpoints such as why seemingly divergent disorders…" also "is not something generated by discrete organ systems" is confusing and rambling and the main focus of the statement is not clear. The concept of EMN-S is interesting (not sure why it is a super-system).

Response: We are very grateful for this thoughtful and constructive observation. Your comment drew our attention to areas where our phrasing could be clearer and our tone more formally scientific. In response, we have rewritten the Abstract.

Regarding the term “super-system,” we fully understand and appreciate your curiosity. We would like to clarify here that the expression was chosen to denote a higher-order feedback architecture that unites three partially autonomous physiological domains—the microbiota, endocannabinoidome, and neuroimmune system—into a recursive control loop governing systemic resilience. We used the prefix “super-” not as emphasis, but to highlight the emergent coordination of multiple self-regulating subsystems within one integrative framework.

Your insightful question encouraged us to reflect on terminology with renewed discipline and to communicate our ideas with greater conceptual transparency. We are deeply appreciative of your guidance, which has strengthened both the clarity and intellectual balance of the manuscript.

With sincere gratitude and respect!!!

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I appreciate the comments from the authors.

However, as stated in the earlier version the expression is poor and not scientific. The example provided was one of the sentences that needed to be clarified. The whole of the manuscript has language which is not clear and is confusing. 

Comments on the Quality of English Language

The english though grammatically sound is not scientific. 

Author Response

Dear Esteemed Academic Reviewer,

We extend our sincere gratitude for the time, attention, and thoughtful care you have devoted to reviewing our manuscript. Your comments were not only insightful but transformative, and we are genuinely thankful for the clarity and direction they provided.

You were entirely correct in noting that the earlier version required significant refinement in both language and scientific expression. Following your invaluable guidance, we have undertaken a comprehensive revision of the manuscript — specifically Sections 1, 2, 3, 6, and 9 — with the goal of ensuring clarity, precision, and scholarly coherence throughout.

In this revised version, we carefully restructured paragraphs to improve logical flow, refined terminology to achieve uniformity across disciplines, and elevated the scientific tone to reflect the depth of the underlying concepts while remaining accessible and transparent. We worked meticulously to preserve the integrity of the ideas while expressing them in language that is now more concise, balanced, and readable.

Your encouragement to clarify the expression of our framework inspired a deeper editorial reflection that has ultimately strengthened the entire work. We truly appreciate your dedication to scientific excellence and the generous spirit in which your critique was offered.

With profound respect and gratitude,
The Authors

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have improved the manuscript through these changes.

Minor changes:

Line 75- these are receptors not metabolic enzymes GPR55, TRPV1

Line 83-reference

Line 102-this should be replaced with "The EMN-S is a conceptual framework for the integration of  three functionally different regulatory entities."

Line 126-this should be replaced with "recent research to support the EMN-S model, evaluates its biological framework"

Line 128-this should be deleted: We hope to provide a perspective of the collective  academic community where disparate research findings could be construed to amalga- mated coherent whole. Ultimately if successful the aim of this manuscript is to usher in,  what could be labeled, Precision EMN-S Medicine: a field of all the future disciplines, neuroscience, immunology, metabolism, and microbiology, for that tackle some of the  most important brain and health problems in the 21st century. "

Line 250-reference?

Some of the text for Figure 1 is difficult to read

Line 325- emergence should be replaced with identification

Section beginning line 362-There should be a brief 1 line about how  AEA and 2-AG are metabolised, and citation of an appropriate reference

Figure 2-the loop should be more circular

Line 601-how does this relate to the EMN-S Executor dimension — tau-PET trajectory.

Line 614-please clarify  cosinor var.

Line 1185-2-AG is already defined

 

Comments on the Quality of English Language

The english is improved

Author Response

Dear Esteemed Academic Reviewer,

We are deeply grateful for your careful reading and constructive observations.
All suggested revisions have been implemented exactly as recommended.
Please find our detailed, point-by-point responses below.

Line 75 – “These are receptors not metabolic enzymes (GPR55, TRPV1).”
Thank you very much for this correction. We revised the sentence to clearly identify GPR55 and TRPV1 as receptors.

 

Line 83 – Reference
We appreciate this reminder. A supporting reference has been added to substantiate the statement at this line.

Line 102 – Replace with: “The EMN-S is a conceptual framework for the integration of three functionally different regulatory entities.”
We thank the reviewer for this precise wording. The sentence has been replaced exactly as suggested.

Line 126 – Replace with: “recent research to support the EMN-S model, evaluates its biological framework.”
We appreciate this improvement in phrasing for conciseness and accuracy. The change has been made accordingly.

Line 128 – Delete the indicated text.
We agree that this passage was aspirational. It has been removed to maintain a focused and objective tone.

Line 250 – Reference ?
Thank you for catching this omission. A relevant reference has now been added at this position.

Figure 1 – “Some of the text is difficult to read.”
We are grateful for this observation. The figure has been redesigned with higher contrast and increased font size to ensure readability.

Line 325 – “Emergence should be replaced with identification.”
Implemented as suggested.


Section beginning Line 362 – “Add a brief one line about how AEA and 2-AG are metabolised with a citation.”
Thank you for this valuable suggestion. 

Figure 2 – “The loop should be more circular.”
We appreciate this design note. The figure has been redrawn.

Line 601 – “How does this relate to the EMN-S Executor dimension — tau-PET trajectory?”
We thank the reviewer for prompting this clarification. A bridging paragraph was added explaining how tau-PET slope dynamics serve as a molecular correlate of executor feedback integrity within the EMN-S framework.

Line 614 – “Please clarify cosinor var.”
We appreciate this request for clarification. The text now defines the term.


Line 1185 – “2-AG is already defined.”
Thank you for noticing this redundancy. The duplicate definition of 2-AG has been removed.

We once again thank the reviewer for their kind and meticulous guidance. Each suggestion has improved the manuscript’s clarity, precision, and conceptual cohesion. We are truly grateful for the collegial spirit and attention to detail reflected in this review.