Review Reports - Proteomic and Functional Characterization of Antimicrobial Peptides Derived from Fisheries Bycatch via Enzymatic Hydrolysis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this manuscript, the authors present an approach for developing antimicrobial peptides with specific activity from fisheries bycatch. While the study is of potential interest, several issues need to be addressed or clarified before the manuscript can be considered for publication.

The caption of Figure 5 does not correspond to the figure itself.
The labels “P6A” and “P6P” do not appear in the caption. Please correct the inconsistency.
Positive control data are missing in the Results section.
Section 4.4 states that standard antibiotics (ampicillin, fluconazole) were used as positive controls. However, the results (Figure 5) do not include these control data.
Without positive controls, it is difficult to interpret the actual strength and significance of the reported inhibition. Please include the positive control results.
Figure 5 does not show triplicate measurements or statistical analysis.
Although the text states that the experiments were performed in triplicate (n = 3), this is not reflected in the figure, nor is any statistical analysis (e.g., error bars, significance testing) provided.
In addition, the y-axis label overlaps with the plotted data, partially blocking the figure.
Please revise the figure to include the replicates, statistical comparisons, and improve the layout.
Figures 1–3 appear to be pasted raw images.
Please redraw these figures using scientific plotting software (e.g., OriginLab, GraphPad Prism) for publication-quality presentation.
Thousands separators should be used on the y-axis of Figure 1.
For example, “10000” should be written as “10,000.”
Appendix B (Figures S5 and S6) does not support the statements made in Section 2.1 and the Discussion.
The chromatograms of Alcalase (black) and Protamex (pink) appear almost identical, which contradicts the manuscript’s claim that the two enzymes produced clearly different peptide profiles. Please revise the analysis and discussion accordingly.
The interpretation in Section 2.2 requires correction.
According to Figure 2, a substantial number of peptides also fall within the 1000–1500 Da and 2000–3000 Da ranges, not solely 1500–2000 Da as described. Please revise the results.
Section 4.1 mentions “previously established optimal conditions,” but the specific hydrolysis parameters are not provided.
For reproducibility, please include the detailed enzymatic hydrolysis conditions (e.g., pH, temperature, enzyme-to-substrate ratio, reaction time).

Author Response

Dear Reviewer,

Thank you very much for taking the time to evaluate my manuscript. I greatly appreciate your constructive and insightful feedback. All comments have been carefully addressed, and the corresponding revisions have been implemented in the manuscript. Please find my point-by-point responses below.

Comments 1: “The caption of Figure 5 does not correspond to the figure itself.
The labels “P6A” and “P6P” do not appear in the caption. Please correct the inconsistency.”

Response 1: Figure 5 has now been updated with the corrected sample names and the inclusion of error bars. Thank you very much for drawing our attention to these points.

Comments 2: “Positive control data are missing in the Results section.
Section 4.4 states that standard antibiotics (ampicillin, fluconazole) were used as positive controls. However, the results (Figure 5) do not include these control data.
Without positive controls, it is difficult to interpret the actual strength and significance of the reported inhibition. Please include the positive control results.”

Response 2: A new graph has been added showing the results of the control group, performed using standard antibiotics (ampicillin and fluconazole) as positive controls, in order to improve the interpretation of the antimicrobial assays.

Comments 3: “Figure 5 does not show triplicate measurements or statistical analysis.
Although the text states that the experiments were performed in triplicate (n = 3), this is not reflected in the figure, nor is any statistical analysis (e.g., error bars, significance testing) provided. In addition, the y-axis label overlaps with the plotted data, partially blocking the figure. Please revise the figure to include the replicates, statistical comparisons, and improve the layout.”

Response 3: Thank you for this observation. Figure 5 has now been updated to reflect the duplicate measurements (n = 2), and the corresponding error bars have been added. The layout has also been corrected, ensuring that the y-axis label no longer overlaps with the plotted data. At this stage, the variability among replicates is clearly represented. If additional statistical significance testing is required, we would be pleased to include it.

Comments 4: “Figures 1–3 appear to be pasted raw images. Please redraw these figures using scientific plotting software (e.g., OriginLab, GraphPad Prism) for publication-quality presentation.”

Response 4: Thank you for your comment. I am currently regenerating Figure 1 directly from the original chromatographic data to ensure higher resolution and improved graphical quality in accordance with the journal’s standards. A fully revised, publication-quality version of this figure will be included in the updated manuscript. Figure 2 has been improved to enhance its clarity and overall quality. Additionally, Figure 3 has been removed from the main manuscript and is now included in the Supplementary Materials.

Comments 5: “Thousands separators should be used on the y-axis of Figure 1.
For example, “10000” should be written as “10,000.”

Response 5: Thank you for pointing this out. Once the figure is regenerated in higher resolution, I will also revise the y-axis formatting to include the appropriate thousands of separators (e.g., “10,000” instead of “10000”). The updated version will be included in the revised manuscript.

Comments 6: “Appendix B (Figures S5 and S6) does not support the statements made in Section 2.1 and the Discussion. The chromatograms of Alcalase (black) and Protamex (pink) appear almost identical, which contradicts the manuscript’s claim that the two enzymes produced clearly different peptide profiles. Please revise the analysis and discussion accordingly.”

Response 6: Thank you for this valuable observation. After revisiting the chromatograms in Figures S5 and S6, I agree that the visual differences between the Alcalase and Protamex hydrolysates are less pronounced than originally described. In light of this, I have revised the corresponding statements in Section 2.1 and in the Discussion to ensure that they accurately reflect the chromatographic data. The text now emphasizes the subtle differences observed and avoids overstating the distinction between the two enzymatic profiles.

If needed, I can also refine the chromatogram presentation to further improve clarity.

Comments 7: “The interpretation in Section 2.2 requires correction. According to Figure 2, a substantial number of peptides also fall within the 1000–1500 Da and 2000–3000 Da ranges, not solely 1500–2000 Da as described. Please revise the results.”

Response 7: Thank you for this important observation. I have revisited Section 2.2 and revised the text to accurately reflect the peptide mass distribution shown in Figure 2. The revised description now includes the additional mass ranges observed (1000–1500 Da and 2000–3000 Da), ensuring that the results are consistent with the data presented in the figure.

Comments 8: “Section 4.1 mentions “previously established optimal conditions,” but the specific hydrolysis parameters are not provided. For reproducibility, please include the detailed enzymatic hydrolysis conditions (e.g., pH, temperature, enzyme-to-substrate ratio, reaction time).”

Response 8: The full enzymatic hydrolysis parameters have now been included in Section 4.1 to ensure reproducibility. The revised text specifies the pH, temperature, enzyme-to-substrate ratio, and reaction time used for both Alcalase and Protamex hydrolyses. The phrase “previously established optimal conditions” was also clarified to explicitly describe these parameters. Thank you for pointing this out.

We hope that these revisions satisfactorily address all concerns. Please let us know if further modifications are required, we would be pleased to implement them.

Thank you once again for your insightful comments and valuable contribution to improving our manuscript.

Sincerely,

Vicky Blumen.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript entitled „Proteomic and Functional Characterization of Antimicrobial Peptides Derived from Fisheries Bycatch via Enzymatic Hydrolysis“ by Blumen Galendi et al. describes the identification of antimicrobial peptides from fisheries bycatch by enzymatic digestion. The work is interesting and it is of relevance to identify useful agents from bycatch.

There are a few comments regarding the data presentation. In general, the results section would benefit from a more careful and detailed description of the presented results.

- The Authors show SDS-PAGE of enzymatically digested products in the Supplementary data to confirm the protease digestion but lack undigested samples on the gels for comparison. Additionally, since the SDS-PAGE data are not shown in the main text, the paragraph SDS-PAGE in section 4.2. of Materials & Methods should be moved from the main text to the supplementary file.

- Section 2.2.: for clarification, the main text should indicate that MALDI-TOF analysis shown in Figure 2 is from an Alcalase-treated Hepatus pudibundus sample (HPA).

- Section 2.2.: the main text refers to the supplementary data but does not refer to Figure 3 of the main text. The use of Hepatus pudibundus Protamex-treated (HPP) sample should be mentioned in the main text.

- Section 2.3. it should be explained, how the samples are scored and OAC/ALC should be defined

- The four peptides shown in Table 1 are identical for PBA and HPP, two different samples treated with two different enzymes – is that correct?

- The BLAST search should be better described and there should be a link and reference to the NCBI database. It is not clear, which selection of peptides is shown, why these sequences were chosen as query for a BLAST search. This section requires a better explanation. Table 2 also contains BLAST search of peptides twice, such as LKYPLE and LEEEELKLF. No BLAST results are shown for the other peptides in Table 1.

- How were the frequencies of the bioactivities determined? This should be explained in the text. How were the results obtained, e.g., how were ACE inhibitors identified? This is missing from the text. The BioPep database should be cited as a reference, and the corresponding link should be provided. Without that information, the results of the database search cannot be verified. Only two of four top scored peptides from Table 1 are shown in this analysis, why have the others not been included? Peptides form PBA and HPP are shown twice, as is LEEEELKLF, but with different results.

- Figure 5: this figure needs to be revised. It is not clear why samples are now differently named, what the numbers in MP3 and MP5 mean, and what P6P, P6A are? Error bars are missing in the figure.

Overall, the study is interesting, but the manuscript has qualitative shortcomings that require a comprehensive revision.

Author Response

Dear reviewer,

Thank you very much for taking the time to review my manuscript. I greatly appreciate your constructive feedback and have addressed the comments carefully. I hope the revisions meet your expectations. If any additional adjustments are required, please let me know, and I will be glad to make further changes.

Point-by-point response to Comments and Suggestions for Authors.

Comments 1: “The Authors show SDS-PAGE of enzymatically digested products in the Supplementary data to confirm the protease digestion but lack undigested samples on the gels for comparison. Additionally, since the SDS-PAGE data are not shown in the main text, the paragraph SDS-PAGE in section 4.2. of Materials & Methods should be moved from the main text to the supplementary file.”

Response 1: Thank you for pointing that out. With regard to the SDS-PAGE analysis using non-hydrolyzed samples, we would like to provide a brief methodological clarification. The native muscle extract represents a highly concentrated and complex protein matrix, which limits uniform protein migration in electrophoretic separation systems and often results in poorly resolved gel profiles. While SDS-PAGE is commonly used to visualize protein hydrolysis, under these conditions the crude extract does not yield well-resolved electrophoretic profiles using standard SDS-PAGE protocols.

For this reason, enzymatic hydrolysis was performed prior to SDS-PAGE analysis, allowing improved protein and peptide separation and clearer electrophoretic visualization. In this context, the success of enzymatic hydrolysis was more reliably confirmed using complementary analytical approaches, including degree of hydrolysis measurements, chromatographic profiling, and mass spectrometry. Accordingly, only the hydrolyzed samples were included in the SDS-PAGE analysis presented in the manuscript. In addition, the primary focus of the study is the identification and characterization of the most relevant proteins and peptides generated after hydrolysis, rather than the intact muscle protein composition.

Regarding the relocation of the SDS-PAGE from Section 4.2 to the Supplementary Materials, this adjustment has already been completed.

Comments 2: “Section 2.2.: for clarification, the main text should indicate that MALDI-TOF analysis shown in Figure 2 is from an Alcalase-treated Hepatus pudibundus sample (HPA). Section 2.2.: the main text refers to the supplementary data but does not refer to Figure 3 of the main text. The use of Hepatus pudibundus Protamex-treated (HPP) sample should be mentioned in the main text.”

Response 2: This point is very well noted. I have already updated the main text regarding the analyses described in Section 2.2. The MALDI-TOF figure has also been removed from the main manuscript, as it is now included in the Supplementary Materials.

Comments 3: “Section 2.3. it should be explained, how the samples are scored and OAC/ALC should be defined.”

Response 3: The description of how the samples were scored, as well as the definitions of OAC and ALC, has been clarified in the revised manuscript. However, if any additional modifications are needed, I would be happy to address them.

Comments 4: “The four peptides shown in Table 1 are identical for PBA and HPP, two different samples treated with two different enzymes – is that correct?”

Response 4: No, that was not correct. My apologies for the confusion. The table formatting had been altered, which consequently affected the peptide sequences. This issue has now been fully corrected. Thank you very much for bringing it to my attention.

Comments 5: “The BLAST search should be better described and there should be a link and reference to the NCBI database. It is not clear, which selection of peptides is shown, why these sequences were chosen as query for a BLAST search. This section requires a better explanation. Table 2 also contains BLAST search of peptides twice, such as LKYPLE and LEEEELKLF. No BLAST results are shown for the other peptides in Table 1.”

Response 5: The description of the BLAST procedure has been expanded and now includes a direct reference and link to the NCBI database. The criteria used for peptide selection have also been clarified. Because the two peptides in question originate from different organisms, we considered it relevant to illustrate how the same sequence can display distinct bioactivity patterns in Teleostei versus Crustacea. However, if you believe it would be more appropriate to replace one of these entries with an additional peptide to avoid duplication, I would be happy to revise the table accordingly.

Comments 6: “How were the frequencies of the bioactivities determined? This should be explained in the text. How were the results obtained, e.g., how were ACE inhibitors identified? This is missing from the text. The BioPep database should be cited as a reference, and the corresponding link should be provided. Without that information, the results of the database search cannot be verified. Only two of four top scored peptides from Table 1 are shown in this analysis, why have the others not been included? Peptides form PBA and HPP are shown twice, as is LEEEELKLF, but with different results.”

Response 6: Thank you for this observation. We have now clarified the procedure in the manuscript: each peptide sequence was queried individually using the BIOPEP-UWM “Profiles of Potential Biological Activity” tool (https://biochemia.uwm.edu.pl/biopep-uwm/) and the database has been added to the References. Frequencies in Figure 3 correspond to the number of times a given bioactivity was annotated across all peptides returned by BIOPEP-UWM. ACE-inhibitory peptides were flagged when BIOPEP-UWM reported a match classified as “ACE inhibitory peptides” (we followed the database annotation criteria). Regarding Table 2, all peptides from Table 1 were evaluated, but only those yielding significant BLASTp alignments (e-value < 1 and identity ≥ 60%) were retained for reporting; peptides without meaningful matches were excluded. Finally, duplicated entries (e.g., LEEEELKLF, LKYPLE) are intentional: identical peptide sequences can map to precursor proteins from different taxonomic groups (Teleostei vs Crustacea) and therefore appear with distinct BLAST/annotation results; a clarifying sentence was added to the manuscript to explain this.

Comments 7: “Figure 5: this figure needs to be revised. It is not clear why samples are now differently named, what the numbers in MP3 and MP5 mean, and what P6P, P6A are? Error bars are missing in the figure.”

Response 7: Figure 5 has now been updated with the corrected sample names and the inclusion of error bars. Thank you very much for drawing our attention to these points.

We hope that these revisions satisfactorily address all concerns. Please let us know if further modifications are required—we would be pleased to implement them.

Thank you once again for your insightful comments and valuable contribution to improving our manuscript.

Sincerely,

Vicky Blumen.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have now submitted a revised version of the manuscript. However, the manuscript still has some shortcomings. Although various corrections have been made, some points remain unresolved. Furthermore, some of the newly added data raise additional questions.

Response to Comment 3:

- The scoring is now explained, but there is still the reference for BIOPEP-UWM database missing: Minkiewicz P, Iwaniak A, Darewicz M. BIOPEP-UWM Database of Bioactive Peptides: Current Opportunities. International Journal of Molecular Sciences. 2019; 20(23):5978. doi: 10.3390/ijms20235978.

- The BIOPEP-UWM website should be also added at first appearance – page 4.

- The BIOPEP-UWM database should also be mentioned in line 142. The database should be also consistently named BIOPEP-UWM (currently, there is either BioPep or BIOPEP-UWM).

- Response to Comment 5: The Blastp search is still not clear to me and may not be clear to other readers as well. The authors state, that the Blast search was done only from top-scored sequences from Table 1: “Only the top scoring peptides from Table 1, based on their highest OAC and ALC values, were selected for BLAST analysis, as these sequences exhibited the strongest predicted bioactivities. Peptides that did not yield significant alignments (e-value > 1 or identity < 60%) were excluded from Table 2. Although all peptides listed in Table 1 were examined, only those with meaningful BLAST matches were retained.“ However, Table 2 contains different sequences than Table 1 and shows 5 sequences for each sample for the Blastp search while in Table 1 only 4 sequences per sample are shown. Several of the sequences in Table 2 do not appear in Table 1. This is very confusing and requires a better explanation about the selected sequences than currently provided in the text.

- Figure 4, 5 and 6 show mean + SD? Please add this information to the figure caption. How many replicates were performed? What are the CLSI guidelines, is there a reference?

- Error bars are quite high in some cases, what is the statistical significance of inhibition?

- New Figure 6: The text does not match the data in figure 6: “The fraction eluted at 10% ACN exhibited minimal antimicrobial activity, with inhibition values of approximately 2% for S. aureus, 1% for E. coli, and 1% for C. albicans. In contrast, the 50% ACN fraction showed the highest activity among all chromatographic fractions, inhibiting S. aureus by nearly 80%, C. albicans by approximately 50%, and E. coli by ~2%. The fraction eluted at 100% ACN displayed intermediate activity (≈30% for S. aureus, 10% for E. coli, and 15% for C. albicans), whereas the non-retained fraction showed low but detectable inhibition (≈20%, 5%, and 10%, respectively)”. However, Candida albicans (magenta) is not visible in the figure at all while the text states a 50% inhibition of the 50% ACN fraction against Candida, and 80% inhibition of S. aureus which is also not seen in the figure. In addition, the 10% ACN fraction shows partial inhibition of E. coli and not of the others. Overall, this figure needs to be revised.

- Minor comment: some error bars are shown on the top only while some are shown in both directions. The appearance should be the same in all figures. The positive controls of Figure 5 could be also incorporated into Figure 4.

Author Response

Dear Reviewer,

Comments 1: “- The scoring is now explained, but there is still the reference for BIOPEP-UWM database missing: Minkiewicz P, Iwaniak A, Darewicz M. BIOPEP-UWM Database of Bioactive Peptides: Current Opportunities. International Journal of Molecular Sciences. 2019; 20(23):5978. doi: 10.3390/ijms20235978.

- The BIOPEP-UWM website should be also added at first appearance – page 4.

- The BIOPEP-UWM database should also be mentioned in line 142. The database should be also consistently named BIOPEP-UWM (currently, there is either BioPep or BIOPEP-UWM).”

Response 1: We thank the reviewer for pointing this out. The reference for the BIOPEP-UWM database (Minkiewicz et al., 2019) has now been added to the reference list. In addition, the BIOPEP-UWM website has been included at its first appearance in the manuscript. The database is now consistently referred to as “BIOPEP-UWM” throughout the text, including the section around line 142. These corrections ensure consistency, transparency, and proper attribution of the database throughout the manuscript.

Comments 2: “The Blastp search is still not clear to me and may not be clear to other readers as well. The authors state, that the Blast search was done only from top-scored sequences from Table 1: “Only the top scoring peptides from Table 1, based on their highest OAC and ALC values, were selected for BLAST analysis, as these sequences exhibited the strongest predicted bioactivities. Peptides that did not yield significant alignments (e-value > 1 or identity < 60%) were excluded from Table 2. Although all peptides listed in Table 1 were examined, only those with meaningful BLAST matches were retained.“ However, Table 2 contains different sequences than Table 1 and shows 5 sequences for each sample for the Blastp search while in Table 1 only 4 sequences per sample are shown. Several of the sequences in Table 2 do not appear in Table 1. This is very confusing and requires a better explanation about the selected sequences than currently provided in the text.”

Response 2: We thank the reviewer for this important observation and and acknowledge that this point required further clarification in the previous version of the manuscript. The BLASTp analysis has now been thoroughly clarified in the Results section.

While Table 1 presents peptides selected based on their highest predicted bioactivity scores (OAC and ALC), the BLASTp analysis was intentionally conducted on a broader subset of confidently identified peptide sequences. This approach was adopted to avoid restricting sequence similarity analysis exclusively to the top-ranked bioactive peptides and to better determine the most likely parent proteins and biological origins of the identified peptides.

All confidently identified peptides were initially screened using BLASTp against the NCBI non-redundant protein database. However, only peptides meeting the predefined significance thresholds (e-value ≤ 1 and sequence identity ≥ 60%) were retained and reported in Table 2. Consequently, some peptides included in Table 2 do not appear in Table 1, as inclusion criteria for BLASTp analysis were based on alignment quality rather than bioactivity ranking alone.

The number of sequences per sample shown in Table 2 reflects the peptides that yielded the most informative and significant BLAST alignments, rather than a fixed correspondence with Table 1. Accordingly, Table 2 reports up to five peptide sequences per sample, corresponding to those with the most informative and statistically significant BLAST alignments. This explains why Table 2 may contain a different number and composition of sequences compared to Table 1.

These clarifications have been explicitly incorporated into the revised manuscript to ensure transparency, internal consistency, and ease of interpretation for readers.

Comments 3: “Figure 4, 5 and 6 show mean + SD? Please add this information to the figure caption. How many replicates were performed? What are the CLSI guidelines, is there a reference?”.

Response 3: We thank the reviewer for this comment. The figure captions of Figures 4, 5, and 6 have been revised to explicitly state that the results are expressed as mean percentage inhibition ± standard deviation (SD). The number of experimental replicates has also been clarified, with antimicrobial assays performed in duplicate (n = 2).

In addition, the antimicrobial assays were conducted following the general principles of the Clinical and Laboratory Standards Institute (CLSI) guidelines. The corresponding CLSI reference has now been added to the Materials and Methods section and cited in the relevant figure captions.

Comments 4: “Error bars are quite high in some cases, what is the statistical significance of inhibition?”

Response 4: We thank the reviewer for this important observation. The relatively high error bars observed in some samples reflect the biological variability inherent to antimicrobial screening assays performed with complex peptide hydrolysates. As clarified in the revised manuscript, these experiments were conducted in duplicate (n = 2) as an initial screening to identify fractions and hydrolysates with relevant antimicrobial potential.

At this stage, the primary objective was to compare general activity trends among enzymes, fractions, and microorganisms rather than to establish definitive statistical significance. Therefore, no inferential statistical analyses were applied at this stage. This has now been clarified in the Results section and figure captions.

We agree that increasing the number of replicates and performing statistical analyses will be essential in future studies. Accordingly, this limitation and the need for dose–response assays and expanded replication have been explicitly acknowledged in the “Limitations and Future Directions” section of the Discussion.

Comments 5: “New Figure 6: The text does not match the data in figure 6: “The fraction eluted at 10% ACN exhibited minimal antimicrobial activity, with inhibition values of approximately 2% for S. aureus, 1% for E. coli, and 1% for C. albicans. In contrast, the 50% ACN fraction showed the highest activity among all chromatographic fractions, inhibiting S. aureus by nearly 80%, C. albicans by approximately 50%, and E. coli by ~2%. The fraction eluted at 100% ACN displayed intermediate activity (≈30% for S. aureus, 10% for E. coli, and 15% for C. albicans), whereas the non-retained fraction showed low but detectable inhibition (≈20%, 5%, and 10%, respectively)”. However, Candida albicans (magenta) is not visible in the figure at all while the text states a 50% inhibition of the 50% ACN fraction against Candida, and 80% inhibition of S. aureus which is also not seen in the figure. In addition, the 10% ACN fraction shows partial inhibition of E. coli and not of the others. Overall, this figure needs to be revised.”

Response 5: We thank the reviewer for carefully identifying the inconsistencies between the text and the previous version of Figure 6. We agree that the mismatch between the graphical data and its description could lead to confusion.

Accordingly, Figure 6 has been fully revised and is now presented as Figure 5 in the revised manuscript. The updated figure accurately reflects the antimicrobial inhibition values observed for each chromatographic fraction against Staphylococcus aureus, Escherichia coli, and Candida albicans, including the visibility of C. albicans inhibition where applicable. Error bars are now consistently displayed in both directions for all datasets.

In parallel, the Results text describing this figure has been rewritten to ensure full consistency with the revised data. The description now correctly reflects the relative activities of the non-retained, 10%, 50%, and 100% ACN fractions, as well as the distribution of antimicrobial activity across different hydrophobicity ranges.

These revisions resolve the discrepancies noted by the reviewer and improve the clarity and interpretability of the chromatographic fractionation results.

Comments 6: “Minor comment: some error bars are shown on the top only while some are shown in both directions. The appearance should be the same in all figures. The positive controls of Figure 5 could be also incorporated into Figure 4.”

Response 6: We thank the reviewer for this helpful suggestion. All figures have been revised to ensure consistent presentation of error bars, which are now displayed symmetrically in both directions across all datasets.

In addition, the positive control data previously shown in Figure 5 “have been incorporated into Figure 4 in the revised manuscript, allowing for clearer visual comparison between samples and controls. These adjustments improve figure consistency and overall clarity.

We hope that these revisions satisfactorily address all concerns. We would be pleased to address any further comments if needed.

Thank you once again for your insightful comments and valuable contribution to improving our manuscript.

Sincerely,

Vicky Blumen.

Author Response File: Author Response.pdf

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have now submitted a third manuscript version that addressed all comments. However, the authors are encouraged to improve the quality of the presentation in future works and to carefully proofread manuscripts before submission, as several errors were found in tables and figures. After revision of the text and newly added data, as well as the provision of extensive supplementary data, the manuscript is, in my opinion, now suitable for publication.