Inhibition of Intestinal Lipid Absorption by Cyanobacterial Strains in Zebrafish Larvae

Round 1

Reviewer 1 Report

I think that the manuscript entitled “Inhibition of Intestinal Lipid Absorption by Cyanobacterial Strains in Zebrafish Larvae” in its revised form warrants publication in Marine Drugs in the present form. All my comments have been taken into account.

Author Response

Reviewer #1

I think that the manuscript entitled “Inhibition of Intestinal Lipid Absorption by Cyanobacterial Strains in Zebrafish Larvae” in its revised form warrants publication in Marine Drugs in the present form. All my comments have been taken into account.

CIIMAR: We thank the reviewer for his comments for helping to improve the manuscript.

Reviewer 2 Report

The manuscript was improved from the previous version. The readers can understand the strategy of the authors and over all situation of the research.

I am still concerned if reported putative active compounds are really active or not. In my opinion, GNPS is a tool to select if scientists should pursue a detail. However, publication of GNPS results is author’s decision. Considering the past publication of Marine Drugs, the manuscript is at least similar level of published papers. I know the authors published good results about cyanobacterial metabolites. I hope they publish follow-up research soon.

Finally I would like to suggest adding yoshinone A in introduction or discussion. It is a very good example of anti-obesity compounds from cyanobacteria. There are several publications about it.

In the last sentence of Table 1, ppm should be parts per million, not parte per million.

Author Response

Reviewer #2

The manuscript was improved from the previous version. The readers can understand the strategy of the authors and over all situation of the research.

I am still concerned if reported putative active compounds are really active or not. In my opinion, GNPS is a tool to select if scientists should pursue a detail. However, publication of GNPS results is author’s decision. Considering the past publication of Marine Drugs, the manuscript is at least similar level of published papers. I know the authors published good results about cyanobacterial metabolites. I hope they publish follow-up research soon.

CIIMAR: We thank the reviewer for his comments to improve the quality of the manuscript. We think that the GNPS data are important for the manuscript, since the bioactivity guided molecular networking allowed us to identify 14 bioactive mass peaks (p<0.01 and correlation >0.95), whose majority revealed to be putative novel compounds, highlighting the prolific chemodiversity in cyanobacteria. Regarding the 3 putative known compounds, the observed fragment similarity by MS2 data and presence of literature references with described effects on obesity provides good evidence that identifications could be these compounds. In the future, we intend to pursue the most active strains and to isolate the responsible compounds.

Finally I would like to suggest adding yoshinone A in introduction or discussion. It is a very good example of anti-obesity compounds from cyanobacteria. There are several publications about it.

In the last sentence of Table 1, ppm should be parts per million, not parte per million.

CIIMAR: We have added yoshinone A to the introduction, and corrected the sentence of table 1.

Reviewer 3 Report

My comments is below.

The authors have appropriately addressed the comments given and questions asked.

However, the work is just repetition of many previous works, not really novel.

Author Response

The authors have appropriately addressed the comments given and questions asked.

However, the work is just repetition of many previous works, not really novel.

CIIMAR: We thank the reviewer for his comments to improve the quality of the manuscript. Regarding the novelty, we do not agree. This is the first paper showing the potential beneficial effects of cyanobacteria on intestinal lipid absorption using zebrafish larvae as physiologically relevant whole small animal model. The identification of 11 bioactive mass peaks corresponding to putative novel compounds highlights the prolific chemodiversity in cyanobacteria.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

This manuscript entitled "Inhibition of intestinal lipid absorption by cyanobacterial strains in zebrafish larvae" is interest and well-designed. However, there are some comments to be revised or added for publication. The authors have to carefully read those comments and can revise for improved manuscript.

1. The result finding may be simply speculation. The lipid deposits can be visualized easily through the Nile red and NBD-cholesterol staining in zebrafish larvae. So, I like to recommend to additional study such as Nile red and NBD-cholesterol staining.
2. What was the rationale of choosing the indicated concentration (10 μg/mL) of cyanobacterial fractions? It was not clear how the dose of treatment of the cyanobacterial fractions was decided and also analyzing the data based on only one dose is not very effective on these sorts of experiments. The authors should have used at least one more dose.
3. In Fig. 1C result showed that the lipase inhibitory effect in the 11 of these fractions (# 4, 24, 38, 65, 66, 74, 88, 190, 192, 195, 208) treated group had significant difference with that in the control group. However, TG showed significant difference only in the 66 and 74 fractions treated group in Fig. 3C. Why other fractions no significant difference when compared to control (DMSO treated group)? Also, why other lipid classes were not affected? Authors need to justify it in detail.
4. Need positive controls (e.g., ezetimibe) in lipase inhibition activity results.
5. All the chemicals and reagents used in this study should be mentioned along with the details of manufacturers/suppliers.

Reviewer 2 Report

The results of screening lack identities of active samples. Only one of samples belongs Synechococcales. We can know only fraction code numbers. The information is not enough to discuss the results. Without identities, we cannot reproduce the results.

They identified four peaks by the Dictionary of Natural Products. This is a weak evidence. They suggested just two peaks by using GNPS. There is no proof of identities of the peaks.

There is no experimental data of purified peaks.

Reviewer 3 Report

In the manuscript entitled “Inhibition of Intestinal Lipid Absorption by Cyanobacterial Strains in Zebrafish Larvae” the Authors describe the effects of cyanobacteria on intestinal lipid absorption using zebrafish larvae as an animal model. The obtained results are very interesting and clearly demonstrate that fractions displaying strong bioactivity could be used in the development of nutraceuticals to combat obesity. To reach this goal, future work is needed to identify the unknown compounds and to confirm the involvement of these new molecules, and putatively identified ones in the observed bioactivity.

As a reviewer I have only two minor remarks to the Authors of the manuscript. For ease of reading, it would be useful if the Authors give more details to the Table 1 caption (e.g., search parameters applied in the Dictionary of Natural Products). Besides the commercial source of fluorescent SCFA and LDFA (BODIPY-5 and BODIPY-C16, respectively) should be given.

In conclusion, I think that the work “Inhibition of Intestinal Lipid Absorption by Cyanobacterial Strains in Zebrafish Larvae” warrants publication in Marine Drugs after minor revision.