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Authors have addressed all of the reviewers' comments and the manuscript is now ready for publication.
[# PeerJ Staff Note - this decision was reviewed and approved by Vladimir Uversky, a PeerJ Section Editor covering this Section #]
Thank you for addressing my previous comments. I appreciate the effort made in revising the manuscript
Thank you for addressing my previous comments. I appreciate the effort made in revising the manuscript
Thank you for addressing my previous comments. I appreciate the effort made in revising the manuscript.
The authors have adequately addressed the reviewers' comments in this revised review manuscript. The manuscript has been significantly improved, and it should be accepted. Thank you to the authors for their hard work.
The experimental design written is good.
The revised manuscript has been corrected in accordance with the reviewer’s comments.
The authors have adequately addressed the reviewers' comments in this revised review manuscript. The manuscript has been significantly improved, and it should be accepted.
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Peerj-108161-v1 Maunscript
Article Title
Proteomic profiling of the human amniotic stem cell-highly abundant secreted proteins
Comments to Authors:
1) It might be interesting, that the mentioned study focuses on amniotic stem cells and related secreting proteins, particularly role of ANXA2 and its implications.
Reviewer response: Authors have provided the response emphasizing the significance of this aspect in the research design
2) Human monocytic cell line- THP-1 cells were used in the study and these cells were differentiated into macrophages using PMA is relevant for the immune-related study but which passage number used for the study?
Reviewer response: Author positively clarified the reviewer question. In this study, author used the fifth passage of THP-1 cell line.
3) Study lacks the molecular mechanisms that influences macrophage activity while ANXA2 is implicated in immune regulation?
Reviewer response: Author sincerely appreciate the reviewer remarks. Author aimed to identify high-abundance proteins with potential significance with only preliminary validations. They have discussed these limitations in the conclusion and discussion sections. Regarding the in-depth molecular mechanisms, underlying ANXA2 influence on macrophage activity and immune regulation. Author plan to investigate these aspects in the future research to provide more comprehensive insights.
4) Other hub proteins were identified such as ACTG1, ANXA5, CFL1, ENO1, FLNA, FN1, GAPDH, HSP90AA1, HSP90AB1, HSPA5, HSPA8, P4HB, VCP, VIM, YWHAE, YWHAZ however Implications on ANXA2, rational behind that?
Reviewer response: Author prioritized ANXA2 for validation as this is the only protein among the identified candidates with well-established evidence of secretion in extracellular contexts. Author will do the Future studies to investigate the secretory potential of additional candidates using targeted experimental approaches.
5) The study did not show any other external validation methods such as Western blot?
Reviewer response: Author sincerely appreciate the reviewer remarks. Author acknowledges that additional external validation methods, such as Western blot, could further strengthen their findings, and they plan to incorporate them in future research for a more comprehensive analysis.
6) Exploration of GO, KEGG, and Reactome pathways make the better understanding of the identified proteins.
Reviewer response: Author positively used GO, KEGG, and Reactome pathway analyses in this study.
Please address comments of all reviewers in the revised manuscript and provide a separate response to reviewer in a point wise manner.
[# PeerJ Staff Note: It is PeerJ policy that additional references suggested during the peer-review process should *only* be included if the authors are in agreement that they are relevant and useful #]
Comments:
Language and Clarity:
The manuscript uses clear and professional English throughout, but some sentences in the Introduction and Discussion sections are overly long and complex. For example:
Introduction
• Original Sentence (Lines 23–26): "The development of secreted protein products derived from stem cells holds the potential to address the challenges associated with cell therapy, including the efficacy of stem cells when administered in situ or via intravenous injection. The development of secreted protein products derived from stem cells holds the potential to address existing challenges in cell therapy, thereby offering substantial clinical and commercial prospects."
• Issue:
o Redundant and overly complex phrasing.
• Revised Suggestion:
o "Secreted protein products from stem cells offer solutions to challenges in cell therapy, such as improving efficacy during in situ or intravenous administration. These products also hold significant clinical and commercial potential."
o Discussion
• Original Sentence (Lines 303–305): "Under pathological conditions, ANXA2 frequently functions as a pro-inflammatory factor. ANXA2 serves as a critical site for bacterial adhesion and cellular entry on the cell membrane and is instrumental in viral infection, replication, and release."
• Issue:
o Multiple roles of ANXA2 are packed into one sentence, making it dense and harder to follow.
• Revised Suggestion:
o "Under pathological conditions, ANXA2 acts as a pro-inflammatory factor. It also serves as a critical site for bacterial adhesion and cellular entry. Furthermore, ANXA2 is instrumental in viral infection, replication, and release."
• Original Sentence (Lines 306–312): "In chronic inflammatory environments, ANXA2 facilitates sustained angiogenesis, contributing to the development of diseases such as diabetic retinopathy and promoting tumor progression. Nevertheless, the precise role of ANXA2 as a secreted protein in various inflammatory or disease contexts remains inadequately understood."
• Issue:
o Combines unrelated ideas (angiogenesis, specific diseases, and lack of understanding of ANXA2's broader role) in a single sentence.
• Revised Suggestion:
o "In chronic inflammatory environments, ANXA2 facilitates sustained angiogenesis, which contributes to diseases like diabetic retinopathy and tumor progression. However, the precise role of ANXA2 as a secreted protein in various inflammatory and disease contexts remains unclear."
Literature references:
Comments for the Authors:
1. On Gaps in Literature:
"The manuscript provides a solid overview of ADSCs' therapeutic potential but would benefit from a deeper discussion on the challenges of standardizing secretome production for clinical use. For instance, citing recent studies on GMP-compliant production and quality control measures could enhance this section."
2. On Recent Advancements:
"The authors could include recent literature that demonstrates the translational potential of secretomes, such as their use in neurodegenerative diseases or ischemic injury models, to broaden the manuscript's clinical perspective."
3. On Scalability:
"To strengthen the discussion, it would be helpful to address the practical aspects of secretome scalability and storage for clinical applications, referencing relevant studies on this topic."
Suggestions for Enhancing the Literature Context:
1. Expand on Quality Control Challenges:
The manuscript could reference studies that address the need for standardized methods in secretome research and production. Suggested references include:
o Silini AR et al., 2021
o "CM from intact hAM: an easily obtained product with relevant implications for translation in regenerative medicine."
DOI: 10.1186/s13287-021-02607-z
o Papait A et al., 2022
o "Comparison of EV-free fraction, EVs, and total secretome of amniotic mesenchymal stromal cells for their immunomodulatory potential: a translational perspective."
DOI: 10.3389/fimmu.2022.960909
2. Incorporate Recent Advancements in ADSC Therapies:
Highlight studies demonstrating the application of ADSC-derived secretomes in diseases like fibrosis, neurodegeneration, and immune disorders. Suggested references:
• Lin FH et al., 2023
"Amniotic membrane mesenchymal stromal cell-derived secretome in the treatment of acute ischemic stroke: A case report."
DOI: 10.12998/wjcc.v11.i27.6543
• Peng ZQ et al., 2024
"Human amniotic mesenchymal stem cells-derived conditioned medium and exosomes alleviate oxidative stress-induced retinal degeneration by activating PI3K/Akt/FoxO3 pathway."
DOI: 10.1016/j.exer.2024.109919
3. Discuss Scalability and Clinical Translation:
Secreted protein products' storage, transportation, and scalability are vital for real-world therapeutic use. Suggested references:
• Ragni E et al., 2021
"Anti-inflammatory and regenerative features of amniotic mesenchymal stromal cells for musculoskeletal tissues."
DOI: 10.1002/sctm.20-0390
• Silini AR et al., 2021
"Comparison of secretome components for clinical-scale production of amniotic mesenchymal stromal cells."
DOI: 10.1186/s13287-021-02506-3
[# PeerJ Staff Note: It is PeerJ policy that additional references suggested during the peer-review process should *only* be included if the authors are in agreement that they are relevant and useful #]
Figures: Key Observations: Figure legends need to be improved. Please expand figure legends to provide detailed explanations of clustering methods, color scales, and the biological relevance of results. This will improve clarity for readers unfamiliar with the data."
Figure 1: Heatmap of Quantified Proteins
1. The heatmap lacks a clear explanation of the Z score -/+.
2. The clustering method used to group proteins is not mentioned.
Figure 2: Venn Diagram of High-Abundance Proteins
1. The Venn diagram is visually clear but does not explain the biological significance of the overlaps.
2. There’s no explanation of how shared proteins were prioritized for downstream analysis.
Figure 3: Functional Enrichment Analysis (GO and KEGG Pathways)
We need better figure legends that explain the number of genes and pathways, along with their corresponding p-values, in Figure B.
Figure 4: Protein-Protein Interaction (PPI) Network
1. Criteria for selecting hub proteins are not explained.
2. The visual network could benefit from color-coding to indicate protein importance (e.g., hub proteins in red). Use visual enhancements such as color-coding or node sizes in the PPI network to indicate protein importance or abundance levels. This would make the data presentation more intuitive.
Comment for the Author:
Originality and Scope:
1."The manuscript addresses an important research question about the proteomic profiling of amniotic stem cell secretomes and their therapeutic potential. However, to better emphasize the study’s originality, consider discussing how your findings build upon or differ from prior work. For example, highlight any unique approaches, datasets, or discoveries in comparison to similar studies."
Clarity of Experimental Steps
The methodology for identifying "highly abundant proteins" and hub proteins is insufficiently described.
Verified Suggestions:
1. Include explicit thresholds for defining "highly abundant proteins" (e.g., intensity, spectral counts, or statistical criteria).
Example addition: "Highly abundant proteins were defined as those with a normalized intensity > 2-fold compared to background levels and identified with at least three unique peptides."
2. Describe the algorithm used for hub protein selection in the PPI network. For example:"Hub proteins were identified using the STRING database (v11.5) and ranked based on betweenness centrality and degree using the MCODE plugin."
3. Describe controls used in functional assays, such as treating macrophages without ANXA2 as a negative control.
4. Details on software and analysis parameters are insufficient. "Include specifics on the software and settings used for pathway enrichment and PPI analyses (e.g., STRING version, statistical thresholds). This will make the bioinformatics methodology more reproducible."
Comment for the Author:
1. Expand on limitations regarding the functional characterization of ANXA2, particularly its pro-inflammatory versus immunosuppressive roles.
2. Consider discussing potential future experiments to explore ANXA2's role in in vivo models.
3. Include a dedicated paragraph in the Discussion outlining specific next steps, such as testing ANXA2's function in disease models like arthritis or fibrosis.
1. The focus on ANXA2 as a hub protein is intriguing, but a comparison with other proteins (e.g., FLNA or HSP90AA1) would make the study more balanced. Why was ANXA2 prioritized over these proteins for further validation?
2. The findings have significant translational potential, particularly for regenerative medicine and immune modulation therapies. However, the manuscript could better articulate the potential clinical applications of these results.
3. The lack of in vivo validation of ANXA2’s role is a notable limitation. While the study makes important strides in characterizing the secretome, including more functional assays would strengthen the conclusions.
Thank you for the opportunity to review the manuscript for the journal PeerJ Life & Environment. I have included my comments below.
The manuscript titled "Proteomic Profiling of Highly Abundant Secreted Proteins from Human Amniotic Stem Cells" by Dan et al. focuses on characterizing secreted proteins from amniotic mesenchymal stem cells (AMSCs) and amniotic epithelial cells (AECs). The authors employed mass spectrometry-based proteomics to identify hub proteins and explore their associated biological functions. Their findings were supported by techniques including LC-MS/MS, quantitative PCR (qPCR), and bioinformatic analysis.
The manuscript's English requires thorough revision to eliminate grammatical and typographical errors and overstatement.
Some irrelevant references do not support this study (Lines# 52-61). The authors need to review their references and ensure their relevance to this study.
Lines# 48-51 state that no abbreviations should be used in the text. Authors must include the correct abbreviations for scientific terms.
Molecular studies: The authors aim to identify highly abundant proteins in adipose-derived stem cells (ADSCs). They report that the secreted protein ANXA2 is highly expressed in their ADSCs and enhances the expression of inflammatory factors in macrophages. However, they did not employ additional molecular or histopathological techniques to confirm the protein expression of the cytokines beyond using only quantitative PCR (qPCR).
The investigators' use of a fixed concentration of 10 ng of Annexin A2 in the treatment and time model does not effectively demonstrate the optimal effect of Annexin A2 in this study. To validate the molecular results, the investigators should optimize their approach by varying the model conditions.
This manuscript inadequately describes some testing procedures. The authors should provide a detailed account of the testing methods, such as how peptide samples were analyzed using LC-MS/MS.
Replication. Although the investigators used an N = 3 in their groups to Validate the expression of inûammatory factors mRNA in macrophages, the "model" treatment was only done once. The readers have no idea of the variability when the "model" treatment is repeated.
The authors identified highly abundant secreted proteins, particularly FLNA, TAGLN2, and Col3a1; however, no molecular or pathological studies examining their pathophysiological functions were conducted.
ANXA2 was observed to enhance the expression of pro-inflammatory cytokines in macrophages, but the molecular mechanism was not clearly described in the discussion.
Lines# 39-41 this information is insufficient for the start of the introduction.
However, the rationale, design, and interpretation of the data have many significant weaknesses, and the manuscript requires many changes before it can be accepted for publication.
no comment
no comment
no comment
Peerj-108161 Maunscript
Article Title
Proteomic profiling of the human amniotic stem cell-highly abundant secreted proteins
Comments to Authors:
1) It might be interesting, that the mentioned study focuses on amniotic stem cells and related secreting proteins, particularly role of ANXA2 and its implications
2) Human monocytic cell line- THP-1 cells were used in the study and these cells were differentiated into macrophages using PMA is relevant for the immune-related study but which passage number used for the study?
3) Study lacks the molecular mechanisms that influences macrophage activity while ANXA2 is implicated in immune regulation?
4) Other hub proteins were identified such as ACTG1, ANXA5, CFL1, ENO1, FLNA, FN1, GAPDH, HSP90AA1, HSP90AB1, HSPA5, HSPA8, P4HB, VCP, VIM, YWHAE, YWHAZ however Implications on ANXA2, rational behind that?
5) The study did not show any other external validation methods such as Western blot?
6) Exploration of GO, KEGG, and Reactome pathways make the better understanding of the identified proteins
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