A Set of Proximal Regulatory Elements Contribute to the Transcriptional Activity of the Human Lipoprotein Lipase Promoter

Round 1

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

Nasmah K. Bastaki et al. investigated the role of twelve proximal regulatory elements in the transcriptional activity of the human lipoprotein lipase (LPL) promoter. By inserting full or partial LPL promoter sequences, which include or exclude these regulatory elements, into a promoterless luciferase reporter vector and testing their activity in HEK293 cells. Although the article is innovative, there are some scientific issues that need to be addressed. Specific comments are as follows:

1. The article discusses the importance of the LPL promoter and its various applications. However, there is too little experimental data to demonstrate the application of LPL in luciferase activity. Most of the data in the article involves cloning into another plasmid using PCR, but the only application characterization data is found in Figure 5. Additional control experiments are needed to validate the role of the LPL promoter in luciferase activity.

2. Are scientific terms like "proximal regulatory elements" and "luciferase assay" used consistently throughout the article?

3. Are the control groups and conditions appropriate for isolating the effects of the full and partial LPL promoter sequences on transcriptional activity?

Author Response

"Please see the attachment".

Author Response File: Author Response.pdf

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

In the article entitled: A Set of Proximal Regulatory Elements Contribute to the Transcriptional Activity of the Human Lipoprotein Lipase Promoter authors have taken into consideration an important problem from the point of human health: lipoprotein lipase. This enzyme is crucial for triglyceride metabolism which level is related to different diseases. Therefore, from the medical point this article is potentially interesting. The authors collected samples from several participants (patients) what made their studies in the medical field. The used biomolecular tchicks are adequate and correctly described. The main results indicated that the proximal regulatory elements within the LPL promotor are essential for its transcriptional activity and therefore for the level of TG in human blood.

The article is readable with a sufficient amount of tables and figures. I have found some typing mistakes so careful correction is necessary.

I did not find the bioethical commission agreement, the sentence: “The sample and medical data collection protocol and informed consent forms used were in accordance with the revised version (2000) of the 144 1975 Helsinki guidelines and were obtained with an ethical approval from the Ministry of 145 Health, Kuwait” is not satisfactory.

In my opinion, authors should underline and indicate the importance of their studies for human health in light of obesity and high processed food consumption.

 

In conclusion, in the present form, I cannot recommend the article for publication, but I believe that the authors made an effort and perform article correction.

 

Author Response

 "Please see the attachment."

Author Response File: Author Response.pdf

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

Comments and Suggestions for Authors

The authors present a study examining the influence of those regulatory elements towards the gene for lipoprotein lipase (LPL), a key molecule in the hydrolysis of plasma triglycerides. Briefly, the authors utilise plasmids to alter the genetic levels of 12 regulatory elements, and examine the impact on the gene using a renilla assay. Ultimately, the authors conclude that these elements do exert and influence, and propose them as warranting further investigation to determine their individual effects across the various tissue types of the human body.

In reviewing the manuscript I made a number of observations. The following should be considered when preparing a suitable revision.

 

1.      If possible, could the presentation of Figure 1 be improved? While the information is somewhat clear, there could be improvements made using the same information to make it easier to interpret for readers.

2.      For the most part the referencing is of a good standard, but there are a few instances where statements being made are supported by references that are quite old in age. The authors should look to improve upon the age of some references in the interest of the article and its supporting evidence.

3.      The authors mention that ‘ten DNA samples from healthy controls’ were used for the experiments. More details on the nature of these samples, from where they were obtained, and how they were obtained, are needed for context.

4.      Were the primers that were utilised for this study validated to be MQIE guidelines compliant?

5.      Overall the focus of this paper is in ways too broad in looking at ‘partial’ or ‘all’ regulatory mechanisms. The article itself refers to papers which have examined the individual influence of each regulatory mechanism, and in this way this is informative in understanding the individual effects/weight of influence each has.  The novelty of this research perhaps needs to be highlighted more in the discussion with respect to this.

6.      Was the viability of the HEK-293 cells assessed following transfection? What effect did transfection have on overall cell health?

Author Response

"Please see the attachment."

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The current manuscript is an interesting experimental study on the contribution of proximal regulatory elements to the transcriptional activity of human LPL promoter. Several relevant experiments were performed, hence I only advise on the following alterations before acceptance for publication:

- In the abstract, the identified proximal regulatory elements should be named;

- Figure 1 seems “out of place”, was it taken directly out of a specific software? Its origin should be mentioned in the figure caption, and its resolution should be improved;

-All sections and subsections should be adequately numbered;

- A schematic representation of the used methods should be added, for better understanding;

- The choice for the HEK293 cell line, over other cell lines, should be justified;

- In general, the number of references is quite short, more should be added in order to better support what is being said;

- The authors should comment on the use of their findings to support the discovery of new therapeutic targets, especially in what concerns relevant metabolic diseases;

- An abbreviation list is missing and should be added.

Author Response

"Please see the attachment."

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript presented by Bastaki and coworkers investigated the function of regulatory elements of the LPL promotor region. The transcriptional activity was quantified by coupling with the luciferase reporter. The manuscript presents a clear concept, and the experimental procedures are well-defined. However, this manuscript is very limited in its scope as it only compares the full region with 12 elements and the partial region with no elements. An increased transcriptional activity observed when all twelve regulatory elements were present is somewhat expected. However, it is possible that some of the regulatory elements may have a counteracting effect or no effect. These possibilities were not investigated. Furthermore, while the authors mentioned that ten DNA samples from healthy controls were collected, they did not even mention whether there is any difference in the DNA sequences among these samples and compare the transcriptional activity between samples. Additionally, it would be interesting to know whether there is any mutation of these regulatory elements in human DNA samples with certain diseases.  In summary, this manuscript lacks the necessary novelty and significance for publications in CIMB.

Author Response

"Please see the attachment."

Author Response File: Author Response.docx

Reviewer 4 Report

Comments and Suggestions for Authors

Previous investigations have pinpointed regulatory elements within the human LPL promoter, yet their synergistic activity had not been assessed prior to this investigation. The researchers have established that a group of twelve proximal regulatory elements play a pivotal role in enhancing the transcriptional activity at the LPL promoter. Nonetheless, the findings only confirm the anticipated influence of the cis-elements on gene expression, which does not suffice to elicit considerable academic intrigue. The data presented lack the robustness required to convincingly validate the proposed hypotheses, thus limiting the research's contribution to the academic discourse. While the diligence behind the research is recognized, a significant overhaul involving a comparative analysis of the entire set against fragmented subsets would be imperative. Such a revision would not only potentially elevate the prominence of the study but also deepen the understanding of the cis-regulatory elements associated with the LPL gene.

Author Response

"Please see the attachment."

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have made improvements to the manuscript based on the comments provided, however I do have some concerns over how broad the scope of this study is that was raised in the previous report. 

Reviewer 3 Report

Comments and Suggestions for Authors

Aside from the addition of future directions in the discussion section, there is no significant improvement in the quality of this manuscript. The luciferase reporter assay is well-established, and the cloning techniques are standard molecular biology methods. I do not see significant novelty in these protocols, and they cannot be considered as the highlights of this paper. The regulation of LPL is an interesting target; however, the main drawback of this manuscript is that it is too thin, providing only minimal results into these regulatory elements. Therefore, I will maintain my previous decision unless the authors provide additional experimental results that offer more insights into the regulation.

Reviewer 4 Report

Comments and Suggestions for Authors

The changes requested has not been reflected, thus I can't support publishing the manuscript.