Mulberry Leaf and Radix Astragali Regulates Differentially Expressed Genes and Proteins in the Streptozotocin-Induced Diabetic Mice Liver
Round 1
Reviewer 1 Report
The authors present genomic and proteomic changes in the liver of induced diabetic mice and investigated common regulated genes between Mulberry Leaf and Radix Astragali. These are common traditional Chinese herbs that lack serious side effects when prescribed against diabetes. The study has a sound study design, however there is a lack of description in the analyses performed. The authors suggest a possible molecular synergistic mechanism of both herbs in the reduction of the glucose in the blood.
My main concern is the lack of information in the methodology part. As it is now nobody can reproduce the results.
MAJOR
1.- Raw data: Where is the raw data deposited? how was the NGS data mapped, was it mapped at all? towards which genome (version)? which software?
2.- There is no transcriptomic analyses description. Was R used? Which package? What was the pvalue threshold used to consider a DEG.
3.- The same goes for the proteomic data
4. Are the DEGs and DPEs available? Throughout the paper, only numbers are mentioned. I think it would be more useful to the community if the genes are also provided. Although I assume you double check the comparisons, the reader should be able to check the results. It is common that numbers are mis-typed.
At least the 13-35-35 DEG/DPEs should be listed.
5. Legends description must not be written in the results section:
Line 217: Blue represented down-regulation, while red meant up-regulation., this is not a result, it should go in the figure legend.
Line 203: From “The DEGs among di…. No significant difference” is not a result. This should go in the description of the figure instead
6. For the Identification of DEGs by qRT-PCR, why were those 28 genes selected?
MINOR
Line 6: you have only one author marked with “a”, is this correct? I’ve usually seen the first author also marked
Line 60: antioxidant and hypoglycemic … what? I think a word is missing
Line 61: I think the scientific name should be in italics
Line 98: a full stop is missing
Line 99: “were measured regularly ”, what does this mean?
Line 140: Verify -> verification?
Line 174: Was -> is shown. There are several similar instances, please correct
Line 287: there is a full stop missing
Line 372: “In addition, we also found that in addition to glucose metabolism disorder, the ”, reads a little odd to have: in addition … in addition
Line 391: there are strange symbols as commas_ “Tap1、Ncoa4、Alas2，and down-regulate Fabp2, Hmox1,”. There are also other places with this strange symbols
That same sentence was hard to read: “Finally, we summed up the potential synergistic mechanism of radix astragali and 389 mulberry leaf in lowering blood glucose and speculated that it could regulate the expres-390 sion of Tap1、Ncoa4、Alas2，and down-regulate Fabp2, Hmox1, up-regulate of Hmgcr and Cyp7a1 expression, to involve in retinol metabolism pathway, cytochrome P450 metabolism pathway, affect bile acid secretion, inhibit the occurrence of iron death and other comprehensive adjustments of the body's glucose and lipid metabolism level.”
FIGURES:
Figure2. It would be helpful to add the sample name in the corresponding axis
Figure3. The boxes in the x-axis make it difficult to read the categories, maybe just leaving a space in between categories?
Figure7. It would be appreciated to have a better legend description. What is the difference between colored vs non-colored proteins?
Suppl Figure3. Good if the heatmap is sorted by DPE's based on the DD/DC group or use a clustering algorithm.
Author Response
Dear Reviewer:
Thank you for your comments for our manuscript entitled “Transcriptomics and proteomics based to dissect the anti-diabetic mechanism of the combination of Mulberry Leaf and Radix Astragali”. Those comments are very helpful for improving our paper, as well as the good guiding significance for our later research. We have read the comments carefully and made revision which we hope meet with approval.
We will reply to your comments one by one.
Q：Raw data: Where is the raw data deposited? how was the NGS data mapped, was it mapped at all? towards which genome (version)? which software?
Reply: Thank you very much. We are very sorry that we did not provide full amount of raw data and clean data in our supplementary materials in the early stage because some data may be needed to be used for patent application. The raw data obtained by the Illumina hiseqtm 2500 platform was stored in FASTQ file format. And was quality control by the FastQC(http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and NGS QC TOOLKIT v2.3.3(http://59.163.192.90:8080/ngsqctoolkit/). The clean reads which gained after filtering and removing low-quality sequences from raw data were mapped to the reference genome via TopHat 2 v2.0 (http://www.carpbase.org/download_home.php). We also added the description in our manuscript.
Q：There is no transcriptomic analyses description. Was R used? Which package? What was the pvalue threshold used to consider a DEG.
Reply: Thank you very much. We added the transcripomic analysis description in our manuscript. A method per Fragments Per Kilobase of transcript per Million fragments (FPKM) was used to calculate the gene expression. The DEGs in the library were identified using the edger package (http://www.r-project.org/). We identified genes with foldchange2(log2FC>1) and false positive rate(FDR)<0.05 by comparing significant DEGs using this equation:
Enrichment score:
Where N is the number of genes with GO annotation in all of the genes, n is the number of genes with GO annotation in differentially expressed genes in N, M is the number of genes annotated as a particular GO term in all of the genes, and m is the annotation of a specific GO term.
Q：Are the DEGs and DPEs available? Throughout the paper, only numbers are mentioned. I think it would be more useful to the community if the genes are also provided. Although I assume you double check the comparisons, the reader should be able to check the results. It is common that numbers are mis-typed.
At least the 13-35-35 DEG/DPEs should be listed.
Reply: Thank you very much for your suggestion. We are very sorry that we did not provide full DEGS and DEPS in our supplementary materials in the early stage because some data may be needed to be used for patent application. But as your suggestion , we provided the 13-35-35 DEG/DEPs in supplementary materials table S9.
Q：Legends description must not be written in the results section:
Line 217: Blue represented down-regulation, while red meant up-regulation., this is not a result, it should go in the figure legend.
Line 203: From “The DEGs among di…. No significant difference” is not a result. This should go in the description of the figure instead
Reply: We have re-written the description of the legend in the manuscript. Thank you very much.
Q：For the Identification of DEGs by qRT-PCR, why were those 28 genes selected?
Reply: We choosed those 28 genes because our project group had some research on the differential genes and proteins of diabetes at the early stage, and we compared the common DEGs/DEPs of 13-35-35 with targets for diabetes using RS (relevancescore>28) as specific criteria were screened with the DrugBank Database. Our early research can be seen in https://doi.org/10.3389/fphys.2018.01051 DOI:10.1002/prca.201800043
Minor corrections
Q:
Line 6: you have only one author marked with “a”, is this correct? I’ve usually seen the first author also marked
Line 60: antioxidant and hypoglycemic … what? I think a word is missing
Line 61: I think the scientific name should be in italics
Line 98: a full stop is missing
Line 99: “were measured regularly ”, what does this mean?
Line 140: Verify -> verification?
Reply: We have re-written the description and checked the punctuation in our manuscript. Thank you very much.
Q:
Line 174: Was -> is shown. There are several similar instances, please correct
Line 287: there is a full stop missing
Line 372: “In addition, we also found that in addition to glucose metabolism disorder, the ”, reads a little odd to have: in addition … in addition
Line 391: there are strange symbols as commas_ “Tap1、Ncoa4、Alas2，and down-regulate Fabp2, Hmox1,”. There are also other places with this strange symbols
That same sentence was hard to read: “Finally, we summed up the potential synergistic mechanism of radix astragali and 389 mulberry leaf in lowering blood glucose and speculated that it could regulate the expres-390 sion of Tap1、Ncoa4、Alas2，and down-regulate Fabp2, Hmox1, up-regulate of Hmgcr and Cyp7a1 expression, to involve in retinol metabolism pathway, cytochrome P450 metabolism pathway, affect bile acid secretion, inhibit the occurrence of iron death and other comprehensive adjustments of the body's glucose and lipid metabolism level.”
Reply: Thank you very much. We have changed the the description of the last paragraph. And also checked the full stop and description you mentioned.
FIGURES:
Figure2. It would be helpful to add the sample name in the corresponding axis
Figure3. The boxes in the x-axis make it difficult to read the categories, maybe just leaving a space in between categories?
Figure7. It would be appreciated to have a better legend description. What is the difference between colored vs non-colored proteins?
Suppl Figure3. Good if the heatmap is sorted by DPE's based on the DD/DC group or use a clustering algorithm.
Reply: We have used the same color to represent the up and down regulated genes in the figure 2. We removed the boxes in the x-axis in figure3. We changed the figure 7 and the description of the legend. Thank you very much.
Author Response File: 
Author Response.doc
Reviewer 2 Report
In the current study, authors have investigated the combined effect of Mulberry leaf and Radix Astragali in the intervention of diabetes. Intragastric administration of Mulberry leaf and Radix Astragali in diabetic mice was followed by transcriptome and proteome analysis. The findings are noteworthy and warrants further investigation in other independent studies for reproducibility. The article is well structured into sections and subsections.
There are some concerns that needs to be addressed for improving the article.
Page 1, Lines 1-3: Title - The title can be reworded for clarity.
Page 2, Line 56: Authors have mentioned DNJ from mulberry leaves for the first time in the text. Therefore, it is suggested to use 1-Deoxynojirimycin (DNJ) instead of the direct use of acronym. The medicinal properties of DNJ, a polyhydroxy alkaloid in mulberry leaves, can be elaborated.
Page 2, Lines 67-68: The in-text referencing format is not consistent.
Page 5, Line 220: The group names in the intersection analysis for differential expression needs to be reported coherently. For instance, in figures if authors prefer to use C-DC than use the same in the text instead of using C/DC or vice-versa.
The correction is required throughout the manuscript. It is important to use a consistent nomenclature for the sample groups in the text and figures to avoid confusion to the readers.
Page 6, Lines 257-269: The section needs to be re-written. The investigation group according to Figure 3 were C/DC, C/DD, and DC/DD but in text authors describe it for C/DC, DC/DC, and again C/DC.
Page 6, Lines 287-288: Authors need to mention the sample group they are referring in this sentence. For instance, DC/DD sample group presented in the Supplementary Figure 7 is not mentioned in the text.
Page 7, Lines 343-346: The in-text referencing format is not consistent.
Page 7, Lines 389-394: Rephrasing is required to improve clarity.
Page 12, Line 533: Figure 1: In the legend, authors are recommended to use diabetic model group (DC) instead of diabete model group. In the figure, the panel labels (a) and (b) can be placed on the left instead of right.
Minor corrections:
Page 3, Line 98: Use of proper punctuation in between sentences is required for clarity (…was given to group C and DC. The body weight…).
Page 6, Line 247: Use of proper punctuation in between sentences is required for clarity (…differential proteins in proteome. In DD/DC group...).
Page 6, Line 287: Use of proper punctuation in between sentences is required for clarity (…degree of 2.46. Meanwhile, 35 co-expressed...).
Page 8, Line 382: Use of proper punctuation in between sentences is required for clarity (…oxygen species. It is concluded...).
Page 9-11: References: Authors are recommended to use a consistent referencing pattern throughout the manuscript. For instance, in some of the references, the page numbers are missing, some have journal name in uppercase, some have punctuation at the end, and some have special characters. Also, the references serial numbers are mentioned twice.
Page 12, Line 533: Figure 2: The statistical map and volcano map represent the same three sample groups. Therefore, it would be appropriate to use the same coloring scheme for both to show upregulated and downregulated genes. For instance, the upregulated genes can be depicted with same color. In the figure, the panel labels (a) and (b) can be placed on the left instead of right.
Supplementary Figure 1: In the legend, it would be appropriate to use –
The weight of the mice…, (… instead of weigh of the mice…).
… diabetic model group (DC) instead of diabete model…
Supplementary Figure 7: In the legend, “ranscriptomics” needs to be corrected to transcriptomics.
Author Response
Dear Reviewer:
Thank you for your comments for our manuscript entitled “Transcriptomics and proteomics based to dissect the anti-diabetic mechanism of the combination of Mulberry Leaf and Radix Astragali”. Those comments are very helpful for improving our paper, as well as the good guiding significance for our later research. We have read the comments carefully and made revision which we hope meet with approval.
We will reply to your comments one by one.
1.Page 1, Lines 1-3: Title - The title can be reworded for clarity.
Reply: Thank you very much. We changed the title as “Mulberry Leaf and Radix Astragali Regulates Differentially Expressed Genes and Proteins in the Streptozotocin-induced Diabetic Mice Liver”
- Page 2, Line 56: Authors have mentioned DNJ from mulberry leaves for the first time in the text. Therefore, it is suggested to use 1-Deoxynojirimycin (DNJ) instead of the direct use of acronym. The medicinal properties of DNJ, a polyhydroxy alkaloid in mulberry leaves, can be elaborated.
Reply: Thank you very much. We had revised in our manuscript. Page 2.
- Page 2, Lines 67-68: The in-text referencing format is not consistent.
Reply: Thank you. We are very sorry. The references are some mistakes. It was changed in our manuscript.
- Page 5, Line 220: The group names in the intersection analysis for differential expression needs to be reported coherently. For instance, in figures if authors prefer to use C-DC than use the same in the text instead of using C/DC or vice-versa.
The correction is required throughout the manuscript. It is important to use a consistent nomenclature for the sample groups in the text and figures to avoid confusion to the readers.
Reply: Thank you very much for your suggestion. And we are very sorry. We have revised the expressions with C-DC in all and also in the picture.
- Page 6, Lines 257-269: The section needs to be re-written. The investigation group according to Figure 3 were C/DC, C/DD, and DC/DD but in text authors describe it for C/DC, DC/DC, and again C/DC.
Page 6, Lines 287-288: Authors need to mention the sample group they are referring in this sentence. For instance, DC/DD sample group presented in the Supplementary Figure 7 is not mentioned in the text.
Reply: We have re-written the section of the group in the manuscript and the pictures, also in the supplementary materials. Thank you very much. Supplementary Figure 7 was mentioned in page 6 line 283-388, which was the PPI analysis of common genes of the three sample groups.
- Page 7, Lines 343-346: The in-text referencing format is not consistent.
Page 7, Lines 389-394: Rephrasing is required to improve clarity.
Reply: We have re-written the references in our manuscript.
And also re-writtten the last paragraph. Thank you very much.
- Page 12, Line 533: Figure 1: In the legend, authors are recommended to use diabetic model group (DC) instead of diabete model group. In the figure, the panel labels (a) and (b) can be placed on the left instead of right.
Reply: We have re-written the description of the legend. Use the diabetic model group instead of diabete model group. And we also change the panel labels as suggetted. We also changed the panel labels of figure 2. Thank you very much.
8.Minor corrections
Page 3, Line 98: Use of proper punctuation in between sentences is required for clarity (…was given to group C and DC. The body weight…).
Page 6, Line 247: Use of proper punctuation in between sentences is required for clarity (…differential proteins in proteome. In DD/DC group...).
Page 6, Line 287: Use of proper punctuation in between sentences is required for clarity (…degree of 2.46. Meanwhile, 35 co-expressed...).
Page 8, Line 382: Use of proper punctuation in between sentences is required for clarity (…oxygen species. It is concluded...).
Page 9-11: References: Authors are recommended to use a consistent referencing pattern throughout the manuscript. For instance, in some of the references, the page numbers are missing, some have journal name in uppercase, some have punctuation at the end, and some have special characters. Also, the references serial numbers are mentioned twice.
Reply: We have re-written the description and checked the punctuation in our manuscript. Also we checked up of the references. Thank you very much.
Page 12, Line 533: Figure 2: The statistical map and volcano map represent the same three sample groups. Therefore, it would be appropriate to use the same coloring scheme for both to show upregulated and downregulated genes. For instance, the upregulated genes can be depicted with same color. In the figure, the panel labels (a) and (b) can be placed on the left instead of right.
Reply: We have changed the figure 2 and added the description of the legend. Thank you very much.
Supplementary Figure 1: In the legend, it would be appropriate to use –
The weight of the mice…, (… instead of weigh of the mice…).
… diabetic model group (DC) instead of diabete model…
Supplementary Figure 7: In the legend, “ranscriptomics” needs to be corrected to transcriptomics.
Reply: We have changed the the description of the legend. And we also changed the supplementary figure 2, 5 and 6. Thank you very much.
Author Response File: Author Response.doc
Round 2
Reviewer 1 Report
I appreciate all the effort in improving the manuscript.
Author Response
Thanks
