Using Plasma Amyloid Beta Oligomer to Screen in Alzheimer’s Disease: A Pilot Study

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In the article by Hsu, et al. the authors report a limited pilot study of the correlation between Aβ 1-40 and Aβ 1-42 and Aβ-42 oligomers (ABO) in the blood serum by ELISA in a comparison between sixteen elderly female individuals who were diagnosed with Alzheimer’s disease. While clinically interesting, the study is extremely limited in its sample size, and also lacks experimental validation on key data, i.e. absolute quantification of Aβ 1-40 and Aβ 1-42 and Aβ-42 oligomers in blood. In connection to this, the authors do not justify how an ELISA is appropriated for the quantification of a mixture of oligomers in blood, which is itself an incredibly complex mixture of proteins. This is an irreconcilable flaw in the study design. Finally, the authors failed to reference several key and seminal studies, some of which are conducted more rigorously than the manuscript by Hsu, et al. Collectively, these problems make the paper unsuitable for publication in the International Journal of Molecular Sciences. 

 

Line 36: The first word here is “Th”. This seems to be a misspelled “The”, although inclusion of a definite article here is grammatically incorrect and should be omitted.

 

Table 2 & Table 3: The data presented in these tables is perhaps better represented in a bar graph. 

 

Amyloid beta oligomer quantification: Oligomers are, by definition, disordered and an ensemble of populations, and are not characterized by one specific feature set that is detectable by antibodies (ELISA or any other immunohistochemical method). The analytical standard for detection is by atomic force microscopy (AFM) or electron microscopy (SEM/TEM). The authors do not explain how concentrations of oligomer in Table 2 are derived except that an ELISA was used (Line 216). No standard curve is provided for such a kit, and it is inconceivable that a single antibody based ELISA system has the degree of specificity or sensitivity for quantification of a disordered mixture of oligomeric species especially from unprocessed human blood. The authors must present evidence that such a kit has such a sensitivity and provide an orthogonal method of oligomer quantification such as SEM or AFM that validates these numbers. 

 

General method of biomarker analysis: There is minimal comment on the extent to which the ratio of AB1-42/AB1-40 differs with different stages of Alzheimer’s disease, or how this was normalized across the limited sample size. Additionally, other biomarkers that can be derived from flow cytometry or qRT-PCR should be included. Finally, the authors should include mass spectrometry validation of the ELISA methods used; several good examples exist in the literature of the metabolomics that can be done with high resolution MS on blood biomarkers for Alzheimer’s disease.

 

Sample population: While the mean education level is not expected to be directly causative in Alzheimer’s pathogenesis, the remarkable skew in the dataset provided suggests that the population sampled is not entirely random, and therefore it is hard to justify a conclusion that the AB1-42/AB1-40 ratio is a direct function of Alzheimer’s disease onset. Other physical characteristics of the population sample that are more physiologically relevant, such as BMI, etc. should be considered and included.

 

Correspondence emails: The authors should use institutional email addresses rather than personal Gmail accounts.

 

References: The authors fail to include several seminal studies in the field, including:

1. Hölttä, M., Hansson, O., Andreasson, U., Hertze, J., Minthon, L., Nägga, K., ... & Blennow, K. (2013). Evaluating amyloid-β oligomers in cerebrospinal fluid as a biomarker for Alzheimer’s disease. PloS one, 8(6), e66381.
2. Zhou, L., Chan, K. H., Chu, L. W., Kwan, J. S. C., Song, Y. Q., Chen, L. H., ... & Lam, K. S. L. (2012). Plasma amyloid-β oligomers level is a biomarker for Alzheimer’s disease diagnosis. Biochemical and Biophysical Research Communications, 423(4), 697-702.
3. Nabers, A., Perna, L., Lange, J., Mons, U., Schartner, J., Güldenhaupt, J., ... & Brenner, H. (2018). Amyloid blood biomarker detects Alzheimer's disease. EMBO molecular medicine, 10(5), e8763.
4. Michno, W., Blennow, K., Zetterberg, H., & Brinkmalm, G. (2021). Refining the amyloid β peptide and oligomer fingerprint ambiguities in Alzheimer’s disease: Mass spectrometric molecular characterization in brain, cerebrospinal fluid, blood, and plasma. Journal of Neurochemistry, 159(2), 234-257.

Most notably, Michno et al. (J. Neurochem. 2021) presents a compelling and much more comprehensive and complex story by mass spectrometry that is not consistent with the methods chosen by the authors to quantify oligomer population. Additionally, the study presently being reviewed does not clearly differentiate its claims from what has already been established in the literature by Holtta, et al. PloS one 2013 and Zhou, et al. Biocem. Biophys. Res. Comm. 2012. Serum analysis of ABO is not novel to the authors’ work.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The manuscript has several grammatical and spelling problems, including the first word of the main text.

Author Response

We appreciate for the critical assessment of our manuscript. We have addressed the concerns below.

Comments 1: In the article by Hsu, et al. the authors report a limited pilot study of the correlation between Aβ 1-40 and Aβ 1-42 and Aβ-42 oligomers (ABO) in the blood serum by ELISA in a comparison between sixteen elderly female individuals who were diagnosed with Alzheimer’s disease. While clinically interesting, the study is extremely limited in its sample size, and also lacks experimental validation on key data, i.e. absolute quantification of Aβ 1-40 and Aβ 1-42 and Aβ-42 oligomers in blood. In connection to this, the authors do not justify how an ELISA is appropriated for the quantification of a mixture of oligomers in blood, which is itself an incredibly complex mixture of proteins. This is an irreconcilable flaw in the study design. Finally, the authors failed to reference several key and seminal studies, some of which are conducted more rigorously than the manuscript by Hsu, et al. Collectively, these problems make the paper unsuitable for publication in the International Journal of Molecular Sciences. 

Response 1: Currently there is no established correlation between plasma oligomer and the pathologically diagnosed AD. It is difficult to evaluate how useful plasma AβO is as a screening biomarker currently. Adding validation or diagnostic performance analysis would be conducted in our next study, in stead of this pilot study. However the ELISA method for the oligomer detection was commercially used in market, for which has been validated and approved by their country standards and regulations. For your reference, the detailed specifications and validation data of the ELISA kit can be found at the following link: http://www.fitfocus.top

Additionally, we have attached the product description PDF for your reference.

Comments 2: Line 36: The first word here is “Th”. This seems to be a misspelled “The”, although inclusion of a definite article here is grammatically incorrect and should be omitted.

Response 2: Thank you for pointing this out. We have corrected the typo.

Comments 3: Amyloid beta oligomer quantification: Oligomers are, by definition, disordered and an ensemble of populations, and are not characterized by one specific feature set that is detectable by antibodies (ELISA or any other immunohistochemical method). The analytical standard for detection is by atomic force microscopy (AFM) or electron microscopy (SEM/TEM). The authors do not explain how concentrations of oligomer in Table 2 are derived except that an ELISA was used (Line 216). No standard curve is provided for such a kit, and it is inconceivable that a single antibody based ELISA system has the degree of specificity or sensitivity for quantification of a disordered mixture of oligomeric species especially from unprocessed human blood. The authors must present evidence that such a kit has such a sensitivity and provide an orthogonal method of oligomer quantification such as SEM or AFM that validates these numbers. 

Response 3: As mentioned above, currently there is no established correlation between plasma oligomer and the pathologically diagnosed AD. Adding validation or diagnostic performance analysis would be conducted in our next study, in stead of this pilot study. The ELISA method for the oligomer detection was commercially used in market, for which has been validated and approved by their country standards and regulations.

Comments 4: General method of biomarker analysis: There is minimal comment on the extent to which the ratio of AB1-42/AB1-40 differs with different stages of Alzheimer’s disease, or how this was normalized across the limited sample size. Additionally, other biomarkers that can be derived from flow cytometry or qRT-PCR should be included. Finally, the authors should include mass spectrometry validation of the ELISA methods used; several good examples exist in the literature of the metabolomics that can be done with high resolution MS on blood biomarkers for Alzheimer’s disease.

Response 4: We acknowledge the suggestion regarding mass spectrometry. However, as this is a pilot study focusing on the potential relationship between ABO and other biomarkers, we plan to include more comprehensive validation methods in our future large-scale studies.

Comments 5: Sample population: While the mean education level is not expected to be directly causative in Alzheimer’s pathogenesis, the remarkable skew in the dataset provided suggests that the population sampled is not entirely random, and therefore it is hard to justify a conclusion that the AB1-42/AB1-40 ratio is a direct function of Alzheimer’s disease onset. Other physical characteristics of the population sample that are more physiologically relevant, such as BMI, etc. should be considered and included.

Response 5: Agree. We acknowledge that education, comorbidities (vascular disease, diabetes, physical characteristics), and medication history are potential confounders. Due to the small sample size (N=32), we acknowledge this as a limitation of the current study. Furthermore, as this is a cross-sectional study, the immediate impact of these factors on the specific cross-sectional profile of plasma biomarkers may be limited. Therefore, we have focused on the primary association in this initial phase. We have revised the Discussion to state that future expanded research will incorporate larger sample sizes, additional influencing factors, and more comprehensive variables to further validate and perfect this study.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript presents information on the development, refinement and verification of a rapid and accessible method for detecting amyloid beta oligomers in human plasma. The results presented may be very useful for developing effective clinical tests for the prevention of Alzheimer's disease.

However, I have some questions and comments:

- The title of Table 3 (lines 93-94) is missing.

- What does section "6. Patents" (lines 241-261) mean?

- The references in the text are not provided according to the journal's guidelines.

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted in the re-submitted files.

Comments 1: The title of Table 3 (lines 93-94) is missing.

Response 1: Thank you for pointing this out. We agree with this comment. Therefore, we have added the missing title to Table 3 in the revised manuscript.

Comments 2: What does section "6. Patents" (lines 241-261) mean?

Response 2: Thank you for the inquiry. We have revised this section and updated the headings to Author Contributions, Funding, Institutional Review Board Statement, Informed Consent Statement, Data Availability, Acknowledgments, Conflicts of Interest to properly categorize the information and strictly follow the journal's standard guidelines. 

Comments 3: The references in the text are not provided according to the journal's guidelines.

Response 3: Agree. We have, accordingly, revised and reformatted all references in the text and the bibliography to strictly adhere to the journal's guidelines.

Reviewer 3 Report

Comments and Suggestions for Authors

In this manuscript, the authors report that plasma amyloid-beta oligomers (AβO) are significantly elevated in patients with Alzheimer’s disease compared to cognitively normal controls, and that AβO levels correlate with altered plasma Aβ profiles, including increased Aβ1-40 and reduced Aβ1-42/Aβ1-40 ratio. The authors suggest that circulating AβO may serve as a promising blood-based biomarker for early Alzheimer’s disease screening, although validation in larger and more diverse cohorts is required. Following are my comments.

 

Major comments

1. The sample size is small, and all participants are female. This limits the reliability and generalizability of the results. A larger and more diverse cohort, including both sexes, is needed to support the conclusions.
2. There is no validation with established AD diagnostic tools such as CSF biomarkers or PET imaging. Without comparison to standard diagnostic methods, it is difficult to evaluate how useful plasma AβO is as a screening biomarker. Adding validation or diagnostic performance analysis would strengthen the study.
3. Key citations are missing and the authors need to add multiple citations including the following ones. Please add citations for APP/Aβ relevance to neurodegeneration (Nath et al., PMID: 39603510) and AD-related immune involvement and disease progression (Bang et al., PMID: 40818647). Furthermore, please cite papers discussing toxic amyloid oligomer formation and aggregation (Shiraz et al., PMID: 39603509).

 

Minor comments

1. Please correct minor typos throughout the manuscript. Following are some examples.

• On page 1, line 16, please insert a space, “amyloid-beta oligomers(AβO)” to “amyloid-beta oligomers (AβO)”.
• On page 1, line 1, please correct “is” to “are”.
• On page 2, line 60, please correct “biomarker” to “biomarkers”.
• On page 2, line 67, please correct “aiming” to “aims”.
• On page 3, line 98, please correct “The” to “the”.
• On page 3, line 109, please add “is” after “finding”.

 

Author Response

We appreciate the reviewer’s valuable comments. We have addressed the concerns by acknowledging the limitations of this pilot study. We have also corrected the minor errors pointed out.

Comments 1: The sample size is small, and all participants are female. This limits the reliability and generalizability of the results. A larger and more diverse cohort, including both sexes, is needed to support the conclusions.

Response 1: We acknowledge the limitation regarding the small sample size and the all-female cohort. As this is a pilot study, we aimed to identify initial correlation. We have added a statement in the Discussion acknowledging that future studies will require a larger and more diverse cohort to improve generalizability. 

Comments 2: There is no validation with established AD diagnostic tools such as CSF biomarkers or PET imaging. Without comparison to standard diagnostic methods, it is difficult to evaluate how useful plasma AβO is as a screening biomarker. Adding validation or diagnostic performance analysis would strengthen the study.

Response 2: We agree with your point. This pilot study is currently in its initial phase, and our primary objective is to establish the preliminary correlation. In our future research plans, we will incorporate your suggestions and conduct comparisons with standard diagnostic tools to yield more precise results and strengthen the association with AD. 

Comments 3: Key citations are missing and the authors need to add multiple citations including the following ones. Please add citations for APP/Aβ relevance to neurodegeneration (Nath et al., PMID: 39603510) and AD-related immune involvement and disease progression (Bang et al., PMID: 40818647). Furthermore, please cite papers discussing toxic amyloid oligomer formation and aggregation (Shiraz et al., PMID: 39603509).

Response 3: We appreciate the reviewer suggesting these recent and insightful studies regarding neurodegeneration mechanisms and immune involvement. We have carefully reviewed them. However, the primary goal of our current manuscript is focused specifically on the clinical evaluation of the plasma ELISA method rather than the underlying molecular pathogenesis or immune mechanisms of AD. To maintain a concise narrative focused on diagnostic performance, we have decided not to expand the reference list to include mechanistic studies at this stage. We will certainly consider these valuable references for our future work on disease mechanisms.

Comments 4 (Minor Comments):Please correct minor typos throughout the manuscript. Following are some examples.

Response 4: We have carefully reviewed the manuscript and corrected all the typos and grammatical errors mentioned above. 

Reviewer 4 Report

Comments and Suggestions for Authors

This manuscript addresses the usage of plasma amyloid-beta oligomers (AβO) as early biomarkers for Alzheimer’s disease (AD) using a case–control design. The study contributes initial data from a Taiwanese cohort, which is relatively underrepresented in biomarker research. However, several important methodological and analytical issues limit the strength of the conclusions. 

The study includes only 32 participants (16 AD and 16 controls). This sample size is very limited for detecting reliable associations, particularly for correlation analyses involving cognitive measures. Several non-significant results such as the associations between AβO and CDR-SB or MMSE are likely due to inadequate power rather than the absence of true relationships. Please expand the discussion on how the small sample size restricts the interpretation of both group comparisons and correlation analyses.

Table 1 shows that both AD and control groups consist entirely of women. This substantially limits the generalizability of the findings, as sex differences in AD risk and biomarker profiles are well documented. Although this limitation cannot be corrected retrospectively, it should be explicitly acknowledged in the Introduction or Methods and elaborated more clearly in the Discussion. The manuscript should also address how the all-female sample may affect Aβ-related biomarker interpretation.

Education levels differ markedly between groups. This is a meaningful confounder because education influences cognitive test performance and is associated with dementia risk.

The Methods section does not report whether participants had vascular disease, diabetes, hypertension, or other conditions known to influence Aβ metabolism. Medication history, including anti-dementia treatment, is also not described. Without this information, it is difficult to determine whether group differences in plasma biomarkers are attributable to AD pathology alone. Please provide available clinical background data or clearly acknowledge this omission as a limitation.

All analyses rely on chi-square tests, Mann–Whitney U tests, and Spearman correlations. No multivariable models (e.g., logistic regression with covariate adjustment) were applied. Given the baseline imbalance in education and the known influence of age and APOE ε4 status, unadjusted analyses may yield biased results.

Author Response

Thank you very much for your detailed and constructive review. We agree that this study has limitations regarding sample size, demographics, and covariate adjustments. As this is a cross-sectional, pilot study, our primary aim is to establish the initial relationship between plasma AβO, Aβ_1-40, Aβ_1-42, Aβ_1-40/Aβ_1-42 and AD in this specific cohort. We acknowledge that other confounding factors will be crucial to include in future expanded research. We sincerely appreciate your guidance, which has provided a clear roadmap for the necessary parameters in our follow-up studies. Please find the detailed responses below.

Comments 1: The study includes only 32 participants (16 AD and 16 controls). This sample size is very limited for detecting reliable associations, particularly for correlation analyses involving cognitive measures. Several non-significant results such as the associations between AβO and CDR-SB or MMSE are likely due to inadequate power rather than the absence of true relationships. Please expand the discussion on how the small sample size restricts the interpretation of both group comparisons and correlation analyses.

Table 1 shows that both AD and control groups consist entirely of women. This substantially limits the generalizability of the findings, as sex differences in AD risk and biomarker profiles are well documented. Although this limitation cannot be corrected retrospectively, it should be explicitly acknowledged in the Introduction or Methods and elaborated more clearly in the Discussion. The manuscript should also address how the all-female sample may affect Aβ-related biomarker interpretation.

Response 1: Agree. We fully acknowledge that the small sample size and the all-female cohort are major limitations. Accordingly, we have significantly expanded the Discussion section to address how these factors constrain the statistical power and generalizability of our findings, specifically noting the influence of biological sex on AD pathology.

Comments 2: Education levels differ markedly between groups. This is a meaningful confounder because education influences cognitive test performance and is associated with dementia risk.

The Methods section does not report whether participants had vascular disease, diabetes, hypertension, or other conditions known to influence Aβ metabolism. Medication history, including anti-dementia treatment, is also not described. Without this information, it is difficult to determine whether group differences in plasma biomarkers are attributable to AD pathology alone. Please provide available clinical background data or clearly acknowledge this omission as a limitation.

All analyses rely on chi-square tests, Mann–Whitney U tests, and Spearman correlations. No multivariable models (e.g., logistic regression with covariate adjustment) were applied. Given the baseline imbalance in education and the known influence of age and APOE ε4 status, unadjusted analyses may yield biased results.

Response 2: Agree. We acknowledge that education, comorbidities (vascular disease, diabetes), and medication history are potential confounders. Due to the small sample size (N=32), we were unable to conduct regression analyses to avoid overfitting and instability, and we acknowledge this as a limitation of the current study. Furthermore, as this is a cross-sectional study, the immediate impact of these factors on the specific cross-sectional profile of plasma biomarkers may be limited. Therefore, we have focused on the primary association in this initial phase. We have revised the Discussion to state that future expanded research will incorporate larger sample sizes, additional influencing factors, and more comprehensive variables to further validate and perfect this study.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have unfortunately failed to address the fundamental scientific flaws that undergird this study. 

ELISA: The attached product documentation is not in English, and has no QC or cross validation. The authors should not assume the reviewers are fluent in non-English languages. Additionally, simply because a kit is commercially available does not indicate that the kit or the experimental platform provides validated data. As mentioned in my original comments, it is not theoretically or fundamentally possible for a single antibody to recognize a mixture of disordered proteins, and hence ELISA cannot be used to quantify oligomer content. If the authors can demonstrate a linear response on the ELISA kit used against a known and validated standard set of oligomer concentrations standardized by another metric (e.g. AFM) this could be used, but in the absence of validation, the data is meaningless. 

The authors have responded that validation is a subject for future study. This is an egregious oversight of science in this manuscript - How can an experiment that has never been validated be used to report new "results"?

Sample size: The limited and biased sample size is a fatal flaw in the fundamental research design. The authors should limit their claims accordingly such that other factors are more clearly acknowledged to have been potentially operative in AB40/AB42 ratio / oligomer formation as a major study limitation. 

Data represented in tables: The data and corresponding standard deviations summarized in Table 2 can be better represented in a bar graph.

Comments on the Quality of English Language

The manuscript has several grammatical and spelling problems, including the first word of the main text.

Author Response

Dear Reviewer,

Thank you for your further comments and for the opportunity to clarify the methodology and scientific design of our study. We appreciate the rigor with which you have evaluated our work. Below are our responses to the specific concerns raised.

Comment 1: The attached product documentation is not in English, and has no QC or cross validation. The authors should not assume the reviewers are fluent in non-English languages. Additionally, simply because a kit is commercially available does not indicate that the kit or the experimental platform provides validated data. As mentioned in my original comments, it is not theoretically or fundamentally possible for a single antibody to recognize a mixture of disordered proteins, and hence ELISA cannot be used to quantify oligomer content. If the authors can demonstrate a linear response on the ELISA kit used against a known and validated standard set of oligomer concentrations standardized by another metric (e.g. AFM) this could be used, but in the absence of validation, the data is meaningless.

The authors have responded that validation is a subject for future study. This is an egregious oversight of science in this manuscript - How can an experiment that has never been validated be used to report new "results"?

Response 1:  Yes, we fully agree with the reviewer’s insightful comment. To address this important concern, we have added the following description to the revised manuscript to further clarify and support the validation and clinical utility of the ELISA amyloid oligomers kit used in this study.

''A previous clinical trial registered at www.chictr.org.cn with its clinical trial number: ChiCTR2100046054 and IRB: ChiCTR2100046054, has shown the clinical efficacy of using this ELISA oligomers kit in screening and monitoring Alzheimer’s disease.

In that clinical trial that has recruited 70 patients with mild to moderate AD who were diagnosed and treated with donepezil (10 mg/day) for over 12 months. Baseline demographic data, hippocampal MRI imaging, serum biomarker levels of amyloid beta oligomer (AβO) and P-Tau181, and Mini-Mental State Examination (MMSE) scores were analyzed. Its results have shown that after 12 months of treatment follow-up, the MMSE scores in the entire cohort significantly decreased from baseline (17.0 ± 7.5) to (14.3 ± 8.5) (P<0.001). Higher baseline serum AβO levels, regarded as AβO >110pg/m, were associated with faster cognitive decline during subsequent treatment, 4.72 score decline in MMSE within 12 months. Oppositely the lower AβO levels, regarded as AβO ≤ 110pg/m, were associated with less cognitive decline, 1.45 score decline in MMSE within 12 months. That original data is preparing for submission.''

Comment 2: The limited and biased sample size is a fatal flaw in the fundamental research design. The authors should limit their claims accordingly such that other factors are more clearly acknowledged to have been potentially operative in AB40/AB42 ratio / oligomer formation as a major study limitation. 

Response 2: Agree. We fully acknowledge that the small sample size are major limitations. Accordingly, we have significantly expanded the Discussion section to address how limited sample size constrains the statistical power and generalizability of our findings.

Comment 3: The data and corresponding standard deviations summarized in Table 2 can be better represented in a bar graph.

Author Response: Thank you for this constructive suggestion. We have converted the data and p value previously listed in Table 2 into a new bar graph (now Figure [2]) to improve visual clarity and facilitate comparison between groups.

 

We hope that these clarifications and the additional clinical evidence provided address your concerns. We remain at your disposal for any further questions.

Author Response File: Author Response.docx

Reviewer 4 Report

Comments and Suggestions for Authors

The author addressed the comments well.

Author Response

Comments 1: [The author addressed the comments well.]

Reviews 1: [Thank you very much for your time and effort in re-evaluating our manuscript. We are pleased to learn that our revisions have addressed your previous concerns effectively. We also appreciate your positive feedback . Your constructive suggestions during the initial review process have significantly improved the quality and rigor of our work. We have noted that you have chosen to sign your review report and have indicated that the English quality is now satisfactory. We are honored by your professional support of this research. Thank you again for your valuable guidance throughout the peer-review process.]

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have not addressed what the reviewer had indicated were two fundamental flaws in the study design that cannot be rectified with simply changing the language of the text:

1. ELISA for "oligomers": ELISA fundamentally relies on high specificity of primary antibody binding, and oligomers are by definition non-discrete targets. Therefore, it is inconceivable that this can be detected by ELISA. The clinical trial referenced also does not provide validation data. Validation data means cross-checking the assay method with a known independent assay method such as SEM, AFM, SEC-MALS, etc. The authors still did not provide such data.
2. The extreme bias in the sample population makes it impossible to assess conclusions drawn by any degree of certainty. The authors are studying two biased groups who evidently come from different biophysiological and social/demographic backgrounds. The authors must correct this and re-perform this study with a larger, more diverse, and more equitable sample population.

Because the authors have failed to produce additional data that rectifies fundamental flaws in the research design as indicated above, this manuscript should be rejected.

Comments on the Quality of English Language

The manuscript has several grammatical and spelling problems, including the first word of the main text.