Exploration and Clinical Verification of the Blood Co-Expression Genes of Type 2 Diabetes Mellitus and Mild Cognitive Dysfunction in the Elderly
Round 1
Reviewer 1 Report
I consider the topic very relevant with clinical interest. to be able to identify early dementia in patients with T2DM using co-expressed genes of MCI and T2DM, which may provide new therapeutic targets for the diagnosis and treatment of diseases such as dementia and Alzheimer's disease, diseases in which therapy is ineffective when initiated at such an advanced stage of the disease, it would be very important for the diagnosis to be made as early as possible.
I propose some questions and suggestions:
- Wouldn't it be important for the study to correlate the therapy that these T2DM patients were taking oral antidiabetics or insulin therapy or with their HbA1c?
- I also suggest that you remove the word “etc”. of all text eg genes (such as APOE, MPKA, APP/PS1, etc.) among others.
On materials and methods:
In the exclusion criteria:…… “Metabolic diseases “but DMT2 is a metabolic disease!In the materials and methods, they mention: ……”All patients had to undergo an evaluation of cognitive function and doctors before enrollment”. It was important to mention what this evaluation was.
As for the traditional Chinese medicines they refer “have the effect of promoting blood circulation, removing blood stagnation and exerting antioxidant effect”. the active principle of each one should be described.
Discussion:
…….have found that Chaihu Shugan pill can have a therapeutic effect on diabetic patients with depression by designing experiments, which can effectively reduce HbA1c level and depressive symptom score, improve clinical efficacy, and regulate serum 5-HT and NE levels, and is safe and reliable…….. What is the composition and active principle of Chaihu Shugan pill?
Author Response
Cover letter
Dear editor and reviewers,
On behalf of all the contributing authors, I would like to express our sincere appreciation of reviewers’ constructive comments concerning our article entitled“Shared Peripheral Blood Biomarkers for Alzheimer’s Disease, Major Depressive Disorder, and Type 2 Diabetes and Cognitive Risk Factor Analysis”(Manuscript No: HELIYON-D-22-27018). We sincerely thank the editor and all reviewers for their valuable feedback that we have used to improve the quality of our manuscript. These comments are all valuable and helpful for improving our article. According to the reviewers’ comments, we have extensively modified our manuscript. The reviewer comments are in italic font below, and specific concerns have been numbered. Our response is given in normal font. The changes/additions to the manuscript were presented in a revised format according to the journal manuscript revision requirements. Point-by-point responses to the excellent editor and three friendly reviewers are listed below this letter.
If there are any other modifications we could make, we would like very much to modify them, and we appreciate your help.
Looking forward to hearing from you soon.
With kind regards,
Sincerely yours,
Qiuyun Tu
Response to Reviewer 1 Comments
Dear professor,
We would like to convey our thanks for your insightful and constructive comments.
Accordingly, we have made corrections and highlighted in the revised version of the manuscript. Point-by-point replies are listed below.
Point-by-point responses to the reviewers’ comments
Reviewer 1
I consider the topic very relevant with clinical interest. to be able to identify early dementia in patients with T2DM using co-expressed genes of MCI and T2DM, which may provide new therapeutic targets for the diagnosis and treatment of diseases such as dementia and Alzheimer's disease, diseases in which therapy is ineffective when initiated at such an advanced stage of the disease, it would be very important for the diagnosis to be made as early as possible.
I propose some questions and suggestions:
Reviewer point #1: Wouldn't it be important for the study to correlate the therapy that these T2DM patients were taking oral antidiabetics or insulin therapy or with their HbA1c?
Author response #1: We agreed with the reviewer. In the subsection 2.9, we analyzed the oral antidiabetics or insulin therapy of patients with T2DM patients and T2DM + MCI patients. There was no significant difference in the use of hypoglycemic drugs between the two groups. In addition, we also compared the two groups of HbA 1 c, and we found that HbA 1 c in T2DM + MCI group was higher than that in T2DM group (P <0.05), but received the limitation of our sample size, this conclusion needs further verification. We have revised the above content in detail in the article.
Reviewer point #2: I also suggest that you remove the word “etc”. of all text eg genes (such as APOE, MPKA, APP/PS1, etc.) among others.
Author response #2: We agreed with the reviewer. We are sorry for the error in the manuscript and have revised it as requested.
Reviewer point #3: On materials and methods: In the exclusion criteria:…… “Metabolic diseases “but DMT2 is a metabolic disease!In the materials and methods, they mention: ……”All patients had to undergo an evaluation of cognitive function and doctors before enrollment”. It was important to mention what this evaluation was.
Author response #3: We have made the corrections and highlighted in the revised version of the manuscript.
Reviewer point #4: As for the traditional Chinese medicines they refer “have the effect of promoting blood circulation, removing blood stagnation and exerting antioxidant effect”. the active principle of each one should be described.
Author response #4: In the Subsection 3.6, we have supplemented the active principle of each one.
Reviewer point #5: …….have found that Chaihu Shugan pill can have a therapeutic effect on diabetic patients with depression by designing experiments, which can effectively reduce HbA1c level and depressive symptom score, improve clinical efficacy, and regulate serum 5-HT and NE levels, and is safe and reliable…….. What is the composition and active principle of Chaihu Shugan pill?
Author response #5: In the discussion, we have made the corrections and highlighted in red in the revised version of the manuscript.
Thank you again for your patient and professional advice.
Author Response File: 
Author Response.docx
Reviewer 2 Report
General and specific comments
Abstract and Materials and methods
1. Please specify in which blood cells the differentially expressed genes associated with MCI and T2DM were identified based on microarray data from GEO database?
2. Please specify in which blood cells of patients the hub genes were verified by qPCR?
Introduction
3. The appropriate reference should be added at the end of the following sentence “Studies have shown that 25-36% of people with diabetes have mild cognitive impairment (MCI)”.
4. The second sentence is a repetition of the second part of the first sentence. Please change it. “Furthermore, patients with T2DM generally show worse cognitive [10] and higher risk of dementia [11]. Compared with healthy individuals, patients with diabetes have an increased risk of dementia [12]”.
5. The appropriate reference should be added at the end of the following sentence. In addition, the abbreviation T2DM should be used since it was previously defined. “ Insulin resistance significantly increases the risk of sporadic AD [18][19], while type 2 diabetes increases the risk by 50%”.
6. Please check the phrase, it is incomprehensible. “Abnormal glucose metabolism in peripheral blood, such as hyperglycemia and hyperinsulinemia, can lead to damage of the blood-brain barrier, and then lead to abnormal glucose metabolism and cerebral insulin resistance [25]”.
7. The abbreviation NFT should be defined.
8. Please replace the word speculate with a more appropriate word in the following sentence: “In conclusion, we speculate that T2DM and MCI have a common pathogenesis, and T2DM patients with MCI are more likely to develop AD”.
Materials and methods
9. is “We searched the gene expression synthesis database (GEO) (https://www.ncbi.nlm.nih.gov/ geoprofiles/) for…”, whereas should be “We searched the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/ geo/) for…”
10. The selection criteria for microarray datasets should be clearly indicated.
11. Table 1: The GSE26168 dataset was not extracted from the GPL570 platform only from the GPL6883 platform. The GSE15932 dataset was not extracted from the GPL6883 only from the GPL570 platform.
12. Table 1: the ethnicity/race of patients should be given.
13. Table 1: PMID should be provided (if available).
14. How the authors verified the gene expression in skeletal muscle and hippocampus in respect to blood samples as tissue-specific changes are commonly observed in gene expression profiles. In addition, these tissues were taken from different patients and likely with different clinical characteristics. Therefore, it is very important to provide the selection criteria for microarray datasets (see point 10), including clinical features of patients among others.
15. What does the abbreviation MDD mean?
16. Appropriate references should be inserted into the sentence. “Inclusion criteria included meeting the diagnostic criteria for mild cognitive dysfunction and the diagnostic criteria for type 2 diabetes proposed in the China Clinical Guidelines for the Prevention and Treatment of Type 2 Diabetes (2020 edition)”. In addition, the abbreviation T2DM should be used since it was previously defined.
17. Exclusion criteria: please specify metabolic diseases in patients.
18. Whether insulin or oral hypoglycemic medications was taken by diabetic patients from the T2DM group and the MCI+T2DM group? Information on this should be included in the Subsection 2.9.
19. Figure 1: in clinical correlation analysis and qPCR, the procedure for assigning patients to the appropriate study groups should be added to flowchart.
20. The version number of used software packages must be provided.
Results
21. Data visualizations in the drawings 3-9 are illegible, making it impossible to verify the described results. Additionally there is the lack of detailed descriptions of the figures.
22. For greater clarity, several dozen up- and down-regulated genes should be moved from the text to table.
23. Figure 3 could be transferred to Suppl. Mat.
24. Subsection 3.5: the AUC and 95%IC values for all hub genes are depicted in both Table 2 and text. I suggest leaving them in the table only. The AUC and 95%IC values for the two selected genes, i.e. BIRC6 and ALDH2, should be provided in the text and only for these genes ROC curves should be presented. ROC curves for other genes should be shown in Suppl. Mat.
25. In the sentence "The diagnostic efficiency of gene to disease can be speculated based on the AUC values", the word speculated should be replaced with another word.
26. The appropriate reference should be added at the end of the following sentence. “Most of these traditional Chinese medicines have the effect of promoting blood circulation, removing blood stasis, and exerting the antioxidant effect”. The next sentence should be removed (too much speculation): ”Therefore, we guess that these traditional Chinese medicines can achieve the therapeutic effect through the antioxidant effect".
27. Table 4: plant names should be written in Latin
Discussion
28. A lot of the text in the discussion can be cut.
Author Response
Cover letter
Dear editor and reviewers,
On behalf of all the contributing authors, I would like to express our sincere appreciation of reviewers’ constructive comments concerning our article entitled “Shared Peripheral Blood Biomarkers for Alzheimer’s Disease, Major Depressive Disorder, and Type 2 Diabetes and Cognitive Risk Factor Analysis” (Manuscript No: HELIYON-D-22-27018). We sincerely thank the editor and all reviewers for their valuable feedback that we have used to improve the quality of our manuscript. These comments are all valuable and helpful for improving our article. According to the reviewers’ comments, we have extensively modified our manuscript. The reviewer comments are in italic font below, and specific concerns have been numbered. Our response is given in normal font. The changes/additions to the manuscript were presented in a revised format according to the journal manuscript revision requirements. Point-by-point responses to the excellent editor and three friendly reviewers are listed below this letter.
If there are any other modifications we could make, we would like very much to modify them, and we appreciate your help.
Looking forward to hearing from you soon.
With kind regards,
Sincerely yours,
Qiuyun Tu
Response to Reviewer 2 Comments
Dear professor,
We would like to convey our thanks for your insightful and constructive comments.
Accordingly, we have made corrections and highlighted in the revised version of the manuscript. Point-by-point replies are listed below.
Point-by-point responses to the reviewers’ comments
Reviewer 2
Abstract and Materials and methods
- Please specify in which blood cells the differentially expressed genes associated with MCI and T2DM were identified based on microarray data from GEO database?
Reply: In the Subsection 2.1, we have supplemented which blood cells the differentially expressed genes associated with MCI and T2DM were identified based on microarray data from GEO database.
- Please specify in which blood cells of patients the hub genes were verified by qPCR?
Reply: In the Subsection 3.8, we have supplemented which blood cells of patients the hub genes were verified by qPCR.
Introduction
- The appropriate reference should be added at the end of the following sentence “Studies have shown that 25-36% of people with diabetes have mild cognitive impairment (MCI)”.
Reply: We have made the corrections and highlighted in the revised version of the manuscript.
- The second sentence is a repetition of the second part of the first sentence. Please change it. “Furthermore, patients with T2DM generally show worse cognitive [10] and higher risk of dementia [11]. Compared with healthy individuals, patients with diabetes have an increased risk of dementia [12]”.
Reply: We are sorry for the error in the manuscript and have revised it as requested.
- The appropriate reference should be added at the end of the following sentence. In addition, the abbreviation T2DM should be used since it was previously defined. “Insulin resistance significantly increases the risk of sporadic AD [18][19], while type 2 diabetes increases the risk by 50%”.
Reply: We are sorry for the error in the manuscript and have revised it as requested.
- Please check the phrase, it is incomprehensible. “Abnormal glucose metabolism in peripheral blood, such as hyperglycemia and hyperinsulinemia, can lead to damage of the blood-brain barrier, and then lead to abnormal glucose metabolism and cerebral insulin resistance [25]”.
Reply: We are sorry for the error in the manuscript and have revised it as requested.
- The abbreviation NFT should be defined.
Reply: We have added the Abbreviations at the end of the article.
- Please replace the word speculate with a more appropriate word in the following sentence: “In conclusion, we speculate that T2DM and MCI have a common pathogenesis, and T2DM patients with MCI are more likely to develop AD”.
Reply: We have made the corrections and highlighted in the revised version of the manuscript.
Materials and methods
- is “We searched the gene expression synthesis database (GEO) (https://www.ncbi.nlm.nih.gov/ geoprofiles/) for…”, whereas should be “We searched the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/) for…”
Reply: We are sorry for the error in the manuscript and have revised it as requested.
- The selection criteria for microarray datasets should be clearly indicated.
Reply: In the Subsection 2.1, we have supplemented the selection criteria for microarray datasets.
- Table 1: The GSE26168 dataset was not extracted from the GPL570 platform only from the GPL6883 platform. The GSE15932 dataset was not extracted from the GPL6883 only from the GPL570 platform.
Reply: We tried to obtain all the dataset information from the same pllatform, but the datasets within the GEO database were limited and could not meet our needs. Therefore, we chose different datasets. However, we performed batch correction on all the datasets to eliminate the differences between the different platforms. The above content is mentioned in the limitations of the article.
- Table 1: the ethnicity/race of patients should be given.
Reply: We have made the corrections and highlighted in the revised version of the manuscript.
- Table 1: PMID should be provided (if available).
Reply: We have made the corrections and highlighted in the revised version of the manuscript.
- How the authors verified the gene expression in skeletal muscle and hippocampus in respect to blood samples as tissue-specific changes are commonly observed in gene expression profiles. In addition, these tissues were taken from different patients and likely with different clinical characteristics. Therefore, it is very important to provide the selection criteria for microarray datasets (see point 10), including clinical features of patients among others.
Reply: In the Subsection 2.1, we have supplemented the selection criteria for microarray datasets.We substituted the obtained coexpressed genes into validation data sets to verify whether these coexpressed genes were expressed in tissues.
- What does the abbreviation MDD mean?
Reply: We agreed with the reviewer. We are sorry for the error in the manuscript and have removed this error.
- Appropriate references should be inserted into the sentence. “Inclusion criteria included meeting the diagnostic criteria for mild cognitive dysfunction and the diagnostic criteria for type 2 diabetes proposed in the China Clinical Guidelines for the Prevention and Treatment of Type 2 Diabetes (2020 edition)”. In addition, the abbreviation T2DM should be used since it was previously defined.
Reply: We have made the corrections and highlighted in the revised version of the manuscript.
- Exclusion criteria: please specify metabolic diseases in patients.
Reply: We have made the corrections and highlighted in the revised version of the manuscript.
- Whether insulin or oral hypoglycemic medications was taken by diabetic patients from the T2DM group and the MCI+T2DM group? Information on this should be included in the Subsection 2.9.
Reply: We agreed with the reviewer. In the subsection 2.9, we analyzed the oral antidiabetics or insulin therapy of patients with T2DM patients and T2DM + MCI patients. There was no significant difference in the use of hypoglycemic drugs between the two groups. But received the limitation of our sample size, this conclusion needs further verification. We have revised the above content in detail in the article.
- Figure 1: in clinical correlation analysis and qPCR, the procedure for assigning patients to the appropriate study groups should be added to flowchart.
Reply: We agreed with the reviewer and have made the corrections.
- The version number of used software packages must be provided.
Reply: We have made the corrections and displayed in the revision in review mode.
Results
- Data visualizations in the drawings 3-9 are illegible, making it impossible to verify the described results. Additionally there is the lack of detailed descriptions of the figures.
Reply: We are very sorry that the resolution of the picture is too low because the resolution of the picture was obviously compressed when we uploaded the picture to the WORD document. We re-adjusted the resolution of the images and uploaded clear original images to the submission system.
- For greater clarity, several dozen up- and down-regulated genes should be moved from the text to table.
Reply: We agreed with the reviewer, we transferred the genes from the text to the table, however, because the table was larger, we placed them in the appendix.
- Figure 3 could be transferred to Suppl. Mat.
Reply: We agreed with the reviewer, and we have transferred Figure 3 to Suppl. Mat.
- Subsection 3.5: the AUC and 95%IC values for all hub genes are depicted in both Table 2 and text. I suggest leaving them in the table only. The AUC and 95%IC values for the two selected genes, i.e. BIRC6 and ALDH2, should be provided in the text and only for these genes ROC curves should be presented. ROC curves for other genes should be shown in Suppl. Mat.
Reply: We agreed with the reviewer. We readjusted this section to merely show BIRC6 and ALDH2 ROC curves. We present the ROC curves for all genes in Suppl. Mat.
- In the sentence "The diagnostic efficiency of gene to disease can be speculated based on the AUC values", the word speculatedshould be replaced with another word.
Reply: We have made the corrections and displayed in the revision in review mode.
- The appropriate reference should be added at the end of the following sentence. “Most of these traditional Chinese medicines have the effect of promoting blood circulation, removing blood stasis, and exerting the antioxidant effect”. The next sentence should be removed (too much speculation): ”Therefore, we guess that these traditional Chinese medicines can achieve the therapeutic effect through the antioxidant effect".
Reply: We have made the corrections and highlighted in red in the revised version of the manuscript. We have added references in this section and removed unnecessary statements.
- Table 4: plant names should be written in Latin.
Reply: We agreed with the reviewer. We are sorry for the error in the manuscript and have revised it as requested.
Discussion
- A lot of the text in the discussion can be cut.
Reply: We have made the corrections and displayed in the revision in review mode.
In addition, we also revised our English language and style.
Thank you again for your patient and professional advices.
Author Response File: Author Response.docx
Reviewer 3 Report
The authors of the manuscript analyzed data on gene expression in the blood of elderly people with type 2 diabetes mellitus and patients with mild cognitive impairment from the GEO database. Genes that are differentially expressed when compared with control groups of patients have been identified. Then an intensive bioinformatics analysis was carried out and genes were identified, the expression of which changes in the two studied pathologies in a similar way. The bioinformatics data were partly supplemented by their own experimental results obtained by the qRT-PCR method. As a result, 9 genes were selected that may be involved in the pathogenetic disorders of the two pathologies (LNX2, BIRC6, ANKRD46, IRS1, TGFB1, APOA1, PSEN1, NPY and ALDH2). However, these results should be considered very preliminary, requiring confirmation using cellular and animal models. A statistical analysis of the choice of hypoglycemic drugs, mainly used in traditional Chinese medicine, was also carried out in two groups of patients (T2DM and T2DM + MCI). No statistically significant differences were found. This part of the work is the least convincing and may be omitted from the manuscript. Another drawback of the manuscript is the overload of the Methods section with materials that can be transferred to supplementary material.
Author Response
Cover letter
Dear editor and reviewers,
On behalf of all the contributing authors, I would like to express our sincere appreciation of reviewers’ constructive comments concerning our article entitled "Shared Peripheral Blood Biomarkers for Alzheimer’s Disease, Major Depressive Disorder, and Type 2 Diabetes and Cognitive Risk Factor Analysis”(Manuscript No: HELIYON-D-22-27018). We sincerely thank the editor and all reviewers for their valuable feedback that we have used to improve the quality of our manuscript. These comments are all valuable and helpful for improving our article. According to the reviewers’ comments, we have extensively modified our manuscript. The reviewer comments are in italic font below, and specific concerns have been numbered. Our response is given in normal font. In the manuscript, we used the revision mode for revision, and the revision part is marked in red.
If there are any other modifications we could make, we would like very much to modify them, and we appreciate your help.
Looking forward to hearing from you soon.
With kind regards,
Sincerely yours,
Qiuyun Tu
Response to Reviewer 3 Comments
Dear professor,
We would like to convey our thanks for your insightful and constructive comments.
Accordingly, we have made corrections and highlighted in the revised version of the manuscript. Point-by-point replies are listed below.
Point-by-point responses to the reviewers’ comments
Reviewer 3
The authors of the manuscript analyzed data on gene expression in the blood of elderly people with type 2 diabetes mellitus and patients with mild cognitive impairment from the GEO database. Genes that are differentially expressed when compared with control groups of patients have been identified. Then an intensive bioinformatics analysis was carried out and genes were identified, the expression of which changes in the two studied pathologies in a similar way. The bioinformatics data were partly supplemented by their own experimental results obtained by the qRT-PCR method. As a result, 9 genes were selected that may be involved in the pathogenetic disorders of the two pathologies (LNX2, BIRC6, ANKRD46, IRS1, TGFB1, APOA1, PSEN1, NPY and ALDH2). However, these results should be considered very preliminary, requiring confirmation using cellular and animal models. A statistical analysis of the choice of hypoglycemic drugs, mainly used in traditional Chinese medicine, was also carried out in two groups of patients (T2DM and T2DM + MCI). No statistically significant differences were found. This part of the work is the least convincing and may be omitted from the manuscript. Another drawback of the manuscript is the overload of the Methods section with materials that can be transferred to supplementary material.
Reviewer point #1: However, these results should be considered very preliminary, requiring confirmation using cellular and animal models.
Author response #1: We strongly agree with the reviewer. We mainly conducted an exploratory study preliminarily to find potential hub genes. We have obtained only a preliminary conclusion, and further verification of cell and animal models needs to be improved. In order to verify the reliability of these hub genes' diagnosis of diseases and the pathogenic mechanism of these hub genes, we will design cell and animal models in future experiments to provide more evidence for early diagnosis and early treatment of diseases. We have included this in the limitations of the article, which will be the direction of our future research.
Reviewer point #2: A statistical analysis of the choice of hypoglycemic drugs, mainly used in traditional Chinese medicine, was also carried out in two groups of patients (T2DM and T2DM + MCI). No statistically significant differences were found. This part of the work is the least convincing and may be omitted from the manuscript.
Author response #2: We agreed with the reviewer. In the subsection 2.9, we analyzed the oral antidiabetics or insulin therapy of patients with T2DM patients and T2DM + MCI patients. There was no significant difference in the use of hypoglycemic drugs between the two groups. As your suggestion says, this part lacks conviction, and we have cut this part.
Reviewer point #3: Another drawback of the manuscript is the overload of the Methods section with materials that can be transferred to supplementary material.
Author response #3: Thank you very much for reviewing your comments. Indeed, we used much space to explain the Materials and Methods section, but only some things were essential. To ensure the article's integrity, we transferred the cuts to Appendix 1.
Thanks for your suggestion. For English grammar-related problems, we used a paid editing service to help polish the language in the revised manuscript. Moreover, we hope the revised manuscript could be acceptable to you. Thank you again for your patience and professional advice.
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
The authors satisfied the comments and re-proposed a version that can be accepted for publication.
Author Response
Cover letter
Dear editor and reviewers,
On behalf of all the contributing authors, I would like to express our sincere appreciation of reviewers’ constructive comments concerning our article entitled "Shared Peripheral Blood Biomarkers for Alzheimer’s Disease, Major Depressive Disorder, and Type 2 Diabetes and Cognitive Risk Factor Analysis” (Manuscript No: HELIYON-D-22-27018). We sincerely thank the editor and all reviewers for their valuable feedback that we have used to improve the quality of our manuscript. These comments are all valuable and helpful for improving our article. According to the reviewers’ comments, we have extensively modified our manuscript. In the manuscript, we used the revision mode for revision, and the revision part is marked in red.
If there are any other modifications we could make, we would like very much to modify them, and we appreciate your help.
Looking forward to hearing from you soon.
With kind regards,
Sincerely yours,
Qiuyun Tu
Response to Reviewer 2 Comments
Dear professor,
Thanks for your suggestion. For English grammar-related problems, we used a paid editing service to help polish the language in the revised manuscript. Moreover, we hope the revised manuscript could be acceptable to you. Thank you again for your patience and professional advice.
