Plasma Trimethylamine-N-oxide and impaired glucose regulation: Results from The Oral Infections, Glucose Intolerance and Insulin Resistance Study (ORIGINS).

Trimethylamine-N-oxide (TMAO)-a gut-microbiota metabolite-is a biomarker of cardiometabolic risk. No studies have investigated TMAO as an early biomarker of longitudinal glucose increase or prevalent impaired glucose regulation. In a longitudinal cohort study, 300 diabetes-free men and women (77%) aged 20-55 years (mean = 34±10) were enrolled at baseline and re-examined at 2-years to investigate the association between TMAO and biomarkers of diabetes risk. Plasma TMAO was measured using Ultra Performance Liquid Chromatography-Mass Spectrometry. After an overnight fast, FPG was measured longitudinally, HbA1C and insulin were measured only at baseline. Insulin resistance was defined using HOMA-IR. Multivariable generalized linear models regressed; i) FPG change (year 2 minus baseline) on baseline TMAO tertiles; and ii) HOMA-IR and HbA1c on TMAO tertiles. Multivariable relative risk regressions modeled prevalent prediabetes across TMAO tertiles. Mean values of 2-year longitudinal FPG±SE across tertiles of TMAO were 86.6±0.9, 86.7±0.9, 86.4±0.9 (p = 0.98). Trends were null for FPG, HbA1c, HOMA-IR, cross-sectionally. The prevalence ratio of prediabetes among participants in 2nd and 3rd TMAO tertiles (vs. the 1st) were 1.94 [95%CI 1.09-3.48] and 1.41 [95%CI: 0.76-2.61]. TMAO levels are associated with increased prevalence of prediabetes in a nonlinear fashion but not with insulin resistance or longitudinal FPG change.

Introduction

Type 2 diabetes is an important public health problem with over 400 million diagnosed cases globally, and in the United States the prevalence of diagnosed diabetes increased from 0.93% in 1958 to 7.40% in 2015[1]. Similarly, impaired glucose regulation (i.e., prediabetes) is also a growing public health concern. In 2012, an estimated 86 million people in the U.S. aged 20 and older had prediabetes[1], which is a strong preclinical risk factor for future type 2 diabetes. A better understanding of disease susceptibility and environmental risk factors is needed to address the growing burden of type 2 diabetes.

The microbes inhabiting the gastrointestinal tract have been hypothesized to play an etiologic role in the development of cardiometabolic diseases. Recently, Le Chatelier et al.[2] found the gut microbiome to be associated with adverse metabolic profiles both cross-sectionally and longitudinally among diabetes-free individuals, bolstering the potential for the gut microbiota to contribute to early diabetes risk, although the mechanisms remain uncertain.

Trimethylamine-N-oxide (TMAO)–a gut microbiota derived metabolite–has been hypothesized as risk factor for cardiometabolic disease. TMAO is produced primarily by the metabolism of dietary nutrients such as choline, phosphatidylcholine and L-carnitine by intestinal bacteria to produce trimethylamine which is subsequently converted to TMAO in the liver. There is strong evidence linking circulating TMAO levels to increased risk for myocardial infarction and stroke[3, 4], even in low risk individuals (e.g., age <65 years, women, low lipid levels, low C-reactive protein (CRP) levels)[5]. In regard to diabetogenesis, animal models have shown that dietary TMAO can lead to impaired glucose tolerance, increase fasting insulin levels and adipose tissue inflammation, in mice fed a high fat diet[6]. Others have demonstrated that knockdown of flavin containing monooxygenase 3 (FMO3)–which produces TMAO–in insulin resistant mice blocks the development of hyperglycemia[7]. In humans, a few studies have also shown increased TMAO levels to be associated with type 2 diabetes. Most prior studies[8-11] were cross-sectional precluding the ability to determine whether elevated TMAO preceded type 2 diabetes development or resulted from the diabetes phenotype which is often associated with alterations in host physiology, including nephropathy, which influences TMAO levels via reduced renal clearance. Additionally, health behavior changes and new pharmacological therapies occurring in response to type 2 diabetes diagnosis could contribute to intestinal dysbiosis and increased capacity for TMAO production. To our knowledge, only one longitudinal study has explored baseline TMAO as a predictor of future diabetes development [12] and, surprisingly, they report elevated TMAO levels to be related to decreased type 2 diabetes risk. Therefore, limited data are available exploring the value of TMAO in predicting type 2 diabetes development. No existing studies have explored the relationship between TMAO and early risk biomarkers linked to future type 2 diabetes development such as insulin resistance, rising longitudinal glucose levels or prediabetes.

Presently, we have studied the relationship between plasma TMAO and early biomarkers of type 2 diabetes risk. We hypothesize that elevated plasma TMAO-levels would be associated with markers of insulin resistance and impaired glucose regulation, cross-sectionally, as well as with rising glucose levels, longitudinally. These investigations are undertaken in a diabetes-free population without a history of cardiovascular and/or kidney disease.

Materials and methods

Study population

The Oral Infections, Glucose Intolerance and Insulin Resistance Study (ORIGINS) is a longitudinal cohort study investigating the relationship between subgingival microbial community composition, systemic inflammatory phenotype and impaired glucose metabolism[13]. The current analysis includes the first 300 participants enrolled from February 2011 to May 2013. Participants were recruited via postal mailings, email blasts, posted flyers, information sessions and word-of- mouth strategies.

Inclusion criteria were as follows: Men and women aged 20–55 years without: i) Diabetes Mellitus based on self-report physician diagnosis, fasting plasma glucose (FPG) ≥126 mg/dl or hemoglobin A1c (HbA1c)≥6.5% (48 mmol/mol); ii) self-reported history of myocardial infarction, congestive heart failure, stroke or chronic inflammatory conditions. Participants were examined at baseline and n = 297 had a TMAO assessment, and n = 241 (81%) provided fasting blood at a two-year follow-up visit (February 2013 to December 2015[13]). The Institutional Review Boards of Columbia University and The University of Minnesota approved the study. All participants provided written informed consent.

Trimethylamine-N-oxide assessment

The exposure Trimethylamine-N-oxide (TMAO) was only measured once at baseline in human plasma samples using Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS/MS) after protein precipitation using deuterated (D9)-TMAO as the internal standard[14]. UPLC-MS/MS analysis was performed on a platform comprising Eksigent ULC 100 integrated to API 4000 mass spectrometer controlled by Analyst 1.6 (ABSciex, Foster City, CA).

Laboratory measures

At baseline, FPG, serum insulin and lipids, and HbA1c were measured from blood after an overnight fast using a Cobras Integra 400 Plus (Roche Diagnostics, Indianapolis, IN, USA) as previously described[15, 16]. The Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) was used to define insulin resistance as previously defined[17, 18]. Baseline prediabetes status was defined based on either one of the below criteria being fulfilled: HbA1C of 5.7–6.4% (39–46 mmol/mol) or FPG between 100 and 125 mg/dl[19]. FPG was also measured at the year 2 follow-up visit.

Risk factors

Cardiometabolic risk factors were measured by trained research assistants in space provided by a Center for Translational Science Award (CTSA). Seated systolic and diastolic blood pressures were measured in triplicate and the last two measurements were averaged. Participant body mass index (BMI) was calculated as weight in kilograms/height in meters2. Questionnaires were administered to obtain information on: age, sex, race/ethnicity (non-Hispanic Black, non-Hispanic White, Hispanic, Other), educational level (high school completion, college or vocational training, advanced degrees), cigarette smoking (current, former or never smoking and duration/intensity of smoking). Leisure-time physical activity (LTPA) was assessed and activities were converted into metabolic equivalents (METS), further categorizing them into four LTPA categories in accordance with the 2008 Physical Activity Guidelines for Americans: no LTPA reported, low (0 to <500 MET min/wk), moderate(500 to <1,000 MET min/wk), high(≥1000 MET min/wk)[20, 21].A detailed food frequency questionnaire was administered from which The Alternative Healthy Eating Index (AHEI) score was calculated to represent diet quality based on the intake of 9 components: vegetables, fruit, nuts and soy, white or red meat, transfat, polyunsaturated or saturated fat, fiber, multivitamin use, and alcohol[22]. A higher total score of AHEI indicates a lower risk of developing chronic disease particularly chronic heart disease and diabetes[22, 23].

Statistical analysis

All statistical analyses were conducted with SAS 9.4. (SAS Institute, Cary, NC). Difference in means or prevalence of potential confounders according to TMAO levels or metabolic variables (i.e., insulin resistance, FPG and HOMA-IR) were assessed using one-way ANOVA for continuous variables and chi-square for categorical variables. Multivariable linear models regressed natural log transformed insulin resistance (due to non-normality of the original variable), FPG or HbA1c on tertiles of TMAO, in separate regression models. TMAO was divided into tertiles to relax linearity assumptions. Sequentially adjusted regression models were formed to assess the degree of confounding by specific sets of confounders. Model 1 was unadjusted. Model 2 was adjusted for age, gender, race/ethnicity, and education. Model 3 was further adjusted for BMI, systolic blood pressure and HDL. Model 4 was further adjusted for

AHEI. Tests for linear trends were performed using TMAO as a continuous variable in the aforementioned regression models. A multivariable modified Poisson regression with robust error variance was used regressed prediabetes prevalence across tertiles of TMAO; prevalence is defined as the probability of having prediabetes. Prevalence ratios and 95% confidence intervals (95%CI) are presented for the 2nd and 3rd tertiles of TMAO levels relative to the 1st tertile.

Results

Baseline characteristics

Participants had a mean age of 34±10 years, and 77% were female. Median plasma TMAO level and Interquartile range (IQR) are 2.69 μM and 1.9–4.22 μM, respectively. TMAO levels were modestly associated with increased age, Hispanic ethnicity and BMI (Table 1). Interestingly, diet quality as assessed by AHEI did not differ by TMAO level. Similarly, the AHEI subscore corresponding to meat consumption was not related to TMAO levels. Additional participant characteristics are summarized in Table 1.

Table 1

Participant characteristics overall and by TMAO Tertiles: (ORIGINS) 2011–2015.

	All (N = 297)	Tertile 1 (n = 99)	Tertile 2 (n = 99)	Tertile 3 (n = 99)	P Value
TMAO (median, range)	2.69(1.90–4.22)	1.73 (1.411.90)	2.69(2.33–2.98)	5.52(4.22–7.66)	N/A
Age,years	34.06±9.86*	32.28±0.92	33.87±0.98	36.01±1.03	0.02
Female	77.10%	78.79%	77.78%	74.75%	0.78
Race					0.007
Hispanic	46.80%	49.50%	34.34%	56.57%
Non-Hispanic White	22.90%	23.23%	26.26%	19.19%
Non-Hispanic Black	16.84%	11.11%	20.20%	19.19%
Other	13.46%	16.16%	19.20%	5.05%
Education					0.5
< college	31.99%	26.26%	32.32%	37.38%
4 years of college	45.45%	51.52%	44.45%	40.40%
>college	22.56%	22.22%	23.23%	22.22%
Activity level					0.59
None	30.58%	31.26%	32.65%	27.84%
Low	12.03%	8.33%	13.27%	14.43%
Moderate	16.15%	14.58%	19.39%	14.43%
High	41.24%	45.83%	34.69%	43.30%
AHEI Score	49.05±11.88*	48.3±1.2	50.2±1.3	48.7±1.3	0.53
AHEI meat score	6.20±3.50*	6.6±0.4	6.0±0.4	6.0±0.4	0.41
BMI (kg/m2)	27.07±6.13*	26.83±0.61	26.08±0.52	28.29±0.68	0.03
Body Mass Index category					0.04
Normal	44.44%	50.51%	50.51%	32.32%
Overweight	32.33%	28.28%	31.31%	37.37%
Obese	23.23%	21.21%	18.18%	30.30%
Systolic blood pressure,mm Hg	117.75±12.45*	117 ±1.21	117±1.33	119±1.20	0.52
Diastolic blood pressure,mmHg	75.25 ±9.71*	75 ±0.91	75 ±1.05	75 ±0.96	0.98
Hypertension	97 (32.66%)	29 (29.29%)	33(33.33%)	35 (35.35%)	0.65
Prediabetes	17.85%	12.12%	21.21%	20.20%	0.19
FPG (mg/dl)	85.22±7.64*	85.23±0.82	84.15±0.69	86.28±0.77	0.15
HbA1c (%)/ mmol/mol	5.36±0.34* (35±3.7)*	5.32±0.03 (35±0.3)	5.36±0.03 (35±0.3)	5.39±0.03 (35±0.3)	0.38
Total cholesterol (mg/dl)	172.61±30.74*	173.56±3.13	174.22±3.04	170.06±3.10	0.59
LDL-cholesterol (mg/dl) **	97.98±27.86*	99.98±2.96	98.33±2.79	95.63±2.66	0.54
HDL (mg/dl)	59.05±16.06*	58.26±1.60	59.84±1.58	59.04±1.66	0.79
Chol to HDL ratio	3.12±0.05	3.18±0.11	3.10±0.09	3.08±0.09	0.75
Triglyceride (mg/dl)	77.80±45.50*	77.12±3.80	79.61±5.24	76.66±4.59	0.89
Insulin (median, 25th 75th percentile)	8.8(5.9,12.0)	8.5 (5.8,12.5)	8.0 (5.6,11.3)	9.7 (6.7,12.3)	0.14
HOMA-IR (median, 25th 75th percentile)	0.57(0.19,0.97)	0.54(0.15,1.03)	0.47(0.13,0.82)	0.70(0.31,1.04)	0.08

*Standard deviation

**n = 4 participants missing LDL-cholesterol

Cross-sectional associations between TMAO and biomarkers of diabetes risk

TMAO levels did not explain variation in FPG, HbA1c or HOMA-IR cross-sectionally, as summarized in Table 2. Results were very consistent across varying degrees of multivariable adjustment (Table 2). After full multivariable adjustment (model 4, S1 Fig), the prevalence ratio of prediabetes among participants in the 2nd and 3rd TMAO tertiles (vs. the 1st) were 1.94 [95%CI 1.09–3.48] and 1.41 [95%CI: 0.76–2.61] and results were consistent across multivariable models (S1 Fig). When combining participants in the 2nd and 3rd tertiles, the prevalence ratio for prediabetes was 1.71, p = 0.05 in crude models although results were attenuated and lost statistical significance after multivariable adjustment (S2 Fig).

Table 2

Mean fasting plasma glucose, HbA1c, HOMA-IR across TMAO Tertiles: Cross-sectional results from ORIGINS) 2011–2015.

TMAO Tertiles	FPG (mg/dl)mean±SE	HbA1c (%)/(mmol/mol) mean±SE	HOMA-IR mean±SE
Tertile 1 (n = 99)
TMAO range (0.24–1.90)
Model 1	85.23±0.76	5.32±0.03 (35±0.3)	0.63±0.05
Model 2	85.49±0.70	5.34±0.03 (35±0.3)	0.64±0.05
Model 3	85.57±0.67	5.36±0.02 (35±0.2)	0.64±0.05
Model 4	85.68±0.71	5.33±0.02 (35±0.2)	0.66±0.05
Tertile 2 (n = 99)
TMAO range (1.91–2.69)
Model 1	84.15±0.76	5.37±0.03 (35±0.3)	0.52±0.05
Model 2	84.20±0.70	5.37±0.03 (35±0.3)	0.54±0.05
Model 3	84.12±0.67	5.36±0.02 (35±0.2)	0.56±0.05
Model 4	84.25±0.72	5.34±0.02 (35±0.2)	0.54±0.05
Tertile 3 (n = 99)
TMAO range (2.70–4.22)
Model 1	86.28±0.76a	5.39±0.03 (35±0.3)	0.70±0.05a
Model 2	85.96±0.71	5.37±0.03 (35±0.3)	0.66±0.05
Model 3	86.00±0.68	5.36±0.02 (35±0.3)	0.66±0.05
Model 4	86.01±0.74	5.35±0.03 (35±0.3)	0.66±0.05

Model 1 = unadjusted; Model 2 = age, gender, race/ethnicity, education

Model 3 = M2+ BMI, systolic blood pressure, HDL

Model 4 = M3+AHEI

ap-value for comparison of mean values between tertile 3 vs. tertile 2≤0.05

The sample size for model 4 is n = 266 for all outcomes due to missing data on AHEI.

Longitudinal association between TMAO and fasting plasma glucose

There was no statistically significant association between baseline TMAO and follow-up FPG. In unadjusted models, mean follow-up FPG across tertiles of TMAO were 86.8±1, 86.9±1, 87.1±1 mg/dL respectively. Results were consistently null in multivariable models (S3 Fig).

Discussion

We report that TMAO levels were not associated with insulin resistance, HbA1c or fasting plasma glucose cross-sectionally, or with longitudinal change in fasting plasma glucose. TMAO levels were associated with a modest increase in the prevalence of prediabetes in a nonlinear fashion such that participants with intermediate TMAO levels had a statistically significant 94% increase in prediabetes prevalence. These results were generally consistent regardless of level of risk factor adjustment although statistical significance was lost for participants in the highest TMAO tertile.

To our knowledge, the current study is the first to investigate the association between plasma TMAO and early biomarkers of diabetes risk among participants free of diabetes and clinical cardiovascular disease. Our null findings might appear at odds with several studies reporting that elevated TMAO levels predict increased risk for chronic kidney disease, myocardial infarction, stroke and heart failure[5, 24]. However, it is important to note that among studies with positive TMAO findings, the enrolled participants were generally older and had evidence of substantial pre-existing cardiovascular disease. For example, in the elegant publications by Tang and colleagues, study participants were recruited from elective diagnostic cardiac catheterization[8] which is an indication for suspected atherosclerotic coronary artery disease and an adverse cardiovascular risk profile. Accordingly, those participants had a mean FPG in the prediabetes range (102 mg/dl), 32% had diagnosed diabetes (~double the national rate) and 72% were hypertensive[5]. Participant characteristics were consistent in a second report among a similar patient population enrolled from recipients of elective cardiac catheterizations in which TMAO was predictive of all-cause mortality among patients with chronic kidney disease[24].

In contrast, our results from ORIGINS are consistent with recent null findings among 817 participants in the Coronary Artery Risk Development in Young Adults (CARDIA study[25] which found no association between TMAO and coronary artery calcification incidence or progression. Additionally, CARDIA also reported no association between TMAO level and cross-sectional insulin resistance. As suggested by the CARDIA investigators, the younger age (~40 years) and lower cardiovascular risk (~4% prevalent diabetes and 10% using hypertensive medications) of their cohort might explain their null finding[25]. The mean age of ORIGINS participants is similarly young (34 years) while the mean age in prior positive studies was 66 years[24] and 63 years[5]. Our observation that TMAO was modestly related to increased prediabetes prevalence supports the notion that TMAO levels might only be a predictive biomarker in populations with early or established cardiovascular risk.

Renal function might also provide some level of explanation for discordant findings in our current data as compared to other cohorts. The fact that higher renal function increases TMAO clearance raises the strong potential for confounding. Specifically, it is possible that reduced renal function causes both elevated TMAO levels and clinical cardiovascular disease (CVD) events. Our observation of increased TMAO among older participants provides indirect support for this notion. As such, our adjustment for age in multivariable models helps to mitigate confounding by renal function although future studies with assessment of renal function will be important.

It is also possible that the adverse impact of TMAO on cardiometabolic risk, only begins above a threshold of circulating TMAO that is achieved in the context of reduced renal function enabling excessive TMAO accumulation. Interestingly, Tang and colleagues found that among patients without chronic kidney disease (CKD), the predictive value of TMAO for all-cause mortality was substantially diminished and only statistically significant in the 4th TMAO quartile[24]. Moreover, the observed range of TMAO values in the non-statistically significant 1st– 3rd quartiles of TMAO were similar the ranges observed in both CARDIA and ORIGINS. Median (IQR) plasma TMAO levels in ORIGINS are 2.7 μM (1.9–4.22 μM) and in CARDIA median (IQR) values were 2.6 (1.8–4.2).[25]

Some important limitations should be noted. The ORIGINS cohort is not a nationally representative sample, limiting the generalizability of our findings. Nevertheless, the consistency in TMAO distributions between ORIGINS and CARDIA, a much larger multi-center, population-based study, suggests that results in ORIGINS are robust. Second, our sample size was small and had limited power to rule out associations of very modest magnitude. Nevertheless, the magnitude of associations observed presently are unlikely to be clinically meaningful even if larger studies identified statistically significant findings of similar magnitude. Our measure of diet quality was based on a single food frequency questionnaire which potentially mischaracterized TMAO dietary precursors proximal to the assessment of TMAO. Dietary information in the study was calculated using AHEI[22], which is based on foods and nutrients predictive of chronic disease risk generally and not TMAO specifically. Regardless, since TMAO is hypothesized as an intermediate mechanism linking diet to cardiometabolic outcomes, the dietary assessment poses minimal threat to the validity of our TMAO findings. In future studies, the precision of hypothesis tests would be enhanced by measuring gut microbiome and dietary TMAO precursors carefully, and analyzing the potential for interactions between the gut microbiome and diet on TMAO levels and subsequent cardiometabolic disease.

A strength of our study was the ability to examine the prospective association of baseline plasma TMAO levels and longitudinal changes in fasting plasma glucose among a healthy study sample which precludes potential reverse causation by the diabetes phenotype and reduces potential confounding by reduced renal function.

In a cohort of participants free of diabetes and clinical cardiovascular disease, we observed no association between TMAO levels and continuous biomarkers of early diabetes risk cross-sectionally or longitudinally. In contrast, intermediate TMAO levels were modestly associated with increased risk of prevalent prediabetes after multivariable adjustment. Future longitudinal studies are necessary to determine if TMAO increases risk for incident prediabetes and/or diabetes, despite no evident relationships with insulin resistance. Until such studies are performed, the role of TMAO as a biomarker of diabetes risk remains uncertain.

Association between Tertiles of TMAO and Prediabetes Prevalence.

Model 1 = unadjusted; Model 2 = age, gender, race/ethnicity, education; Model 3 = M2+ BMI, systolic blood pressure, HDL; Model 4 = M3+alternative healthy eating index. *p<0.05.

(TIF)

Click here for additional data file.

Association between TMAO (Tertiles 2 and 3 vs. 1) and Prediabetes Prevalence.

Model 1 = unadjusted; Model 2 = age, gender, race/ethnicity, education; Model 3 = M2+ BMI, systolic blood pressure, HDL; Model 4 = M3+ alternative healthy eating index.

(TIF)

Click here for additional data file.

Association between Baseline TMAO Tertiles and Longitudinal Fasting Plasma Glucose.

Model 1 = unadjusted; Model 2 = age, gender, race/ethnicity, education; Model 3 = M2+BMI, systolic blood pressure, HDL+ alternative healthy eating index; Model 4 = M3+baseline glucose. Y axis is centered on the mean value observed in the total population (mean = 85 mg/dL) and the range is set to twice the standard deviation of fasting glucose.

(TIF)

Click here for additional data file.

16 Aug 2019

PONE-D-19-18193

Plasma Trimethylamine-N-Oxide and Impaired Glucose Regulation: Results from The Oral Infections, Glucose Intolerance and Insulin Resistance Study (ORIGINS)

PLOS ONE

Dear Dr. Demmer,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Sep 30 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Cheng Hu

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

1. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified what type of consent you obtained (for instance, written or verbal). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Roy and colleagues present the relationship between plasma TMAO and early biomarkers of type 2 diabetes risk from ORIGINS based on the participants who are men and women aged 20-55 years, diabetes-free populations without a history of cardiovascular and/or kidney disease. The results are not hugely ground-breaking. However, overall, the manuscript is technically sound and the results are well interpreted.

There are some major and minor concerns that are summarized below and the authors need to address:

The authors need to provide the rationale to use the methods and statistical analysis in the body to appreciate why they were done (eg. why do the authors do a two-year follow-up visit to measure TMAO? Why do they use UPLC-MS/MS to measure TMAO?)

It would be helpful to the reader to make sure to explain what abbreviation means the first time they use it (eg. IQR, CARDIA and so on).

I would suggest that it is important to provide all data or refer to the data availability.

It would be helpful to improve the resolution of figures.

In Table 1, how does activity level estimate?

In Figure 2, the minimum would start 0.

Reviewer #2: The manuscript explores the possibility of using Trimethylamine-N-oxide (TMAO), as an early biomarker of longitudinal glucose increase or prevalent impaired glucose regulation. Overall, the manuscript is well-written, and the study has several interesting information. But few errors with lack of validation makes the study weak. These needs to be addressed sufficiently to be considered for publication in Plos Journal.

Comments:

1.Plasma TMAO in Diabetes, whether TMAO is a cause or consequence of Diabetes (Pre-diabetes) are highly debatable. As several studies using TMAO and cardiovascular diseases provide contradicting results. Though this showed modest increase in the prevalence of prediabetes in a nonlinear fashion. Several factors which interfere with the conditions/ Pre-diabetes were lest discussed and also, Study validation is an absolute necessity.

2. There are no line numbers in the manuscript which makes it hard to pinpoint the errors.

3. 20-55 years are definitely not young population, Correct the sentences wherever needed (Eg: Last two lines of introduction).

4. TMAO levels were modestly associated with increased age. Discuss more on this point.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

27 Sep 2019

Editor comments:

The body of the manuscript has been updated to comply with the formatting guidelines. A wrriten verbal consent was obtained from participants. This has been added to the Methods section of the manuscript as well as in the online submission section.

We wish to thank the reviewers for their helpful comments, which have led to further strengthening of the manuscript. We provide a point-by-point response to reviewer comments below.

Reviewer #1 Comments

General comments: Roy and colleagues present the relationship between plasma TMAO and early biomarkers of type 2 diabetes risk from ORIGINS based on the participants who are men and women aged 20-55 years, diabetes-free populations without a history of cardiovascular and/or kidney disease. The results are not hugely ground-breaking. However, overall, the manuscript is technically sound and the results are well interpreted.

Response: We appreciate the reviewer’s comments and have responded to each specific comment below in a point-by-point fashion.

Comment #1: The authors need to provide the rationale to use the methods and statistical analysis in the body to appreciate why they were done (eg. why do the authors do a two-year follow-up visit to measure TMAO? Why do they use UPLC-MS/MS to measure TMAO?)

Response: We wish to clarify that TMAO was only measured once at baseline in this study. This is stated on page 6 (line 117) of our revised submission.

In this analysis, we were interested in investigating the following questions:

a. Is baseline plasma TMAO levels associated with insulin resistance, fasting glucose levels and HbA1c cross-sectionally

b. Is baseline plasma TMAO levels associated with longitudinal increase in fasting plasma glucose

c. Is baseline plasma TMAO levels are associated with increased prediabetes prevalence cross-sectionally

To our knowledge there are no data addressing the association between baseline TMAO and longitudinal fasting plasma glucose among generally healthy, diabetes-free population.

Traditional assays for choline metabolites such as TMAO, including radioenzymatic assays, liquid chromatography with electrochemical detection and GCMS are characterized by low specificity and tedious sample preparation with multi-step derivatization protocols. Targeted metabolomic approaches employing Liquid Chromatography-tandem mass spectrometry (UPLC-MS/MS) platforms under multiple reaction mode offer high sensitivity, specificity, accuracy, and precision for the quantitation of small molecules. The UPLC-MS/MS based assay used in the present study enabled the sensitive and accurate measurement of TMAO in the human samples. The assay has a lower limit of quantitation (LLOQ) of 0.05uM. The intra-assay accuracy and precision 98% and 2.78% while inter-assay accuracy and precision was 97% and 4.11%.

Comment #2: It would be helpful to the reader to make sure to explain what abbreviation means the first time they use it (eg. IQR, CARDIA and so on).

Response: We have now clarified abbreviations of IQR on page 8 (line 180) and CARDIA on page 14 (line 267) are spelled out as it first appears in the paper.

Comment #3: I would suggest that it is important to provide all data or refer to the data availability.

Response: All relevant data are within the manuscript and a limited data set is privately available on Figshare and a doi has been issued (10.6084/m9.figshare.9913667)

Comment #4: It would be helpful to improve the resolution of figures.

Response: The resolution of figures is now improved and revised figures are uploaded.

Comment #5: In Table 1, how does activity level estimate?

Response: The methods used to quantify Leisure-time physical activity are described on page 7 (line number 145) of the revision.

Comment #6: In Figure 2, the minimum would start 0.

Response: Since a value of zero is arbitrary relative to the mean values of fasting glucose and the variability of the measure, we have chosen to center the figure Y axis on the mean value observed in the total population (mean=85 mg/dL) and to set the range as twice the standard deviation of fasting glucose (14) in this sample. This gives the reader a better perspective of glucose variability across TMAO groups relative to the size of glucose variability overall. This is now clarified in the figure legend.

Reviewer # 2 Comments

General comments: The manuscript explores the possibility of using Trimethylamine-N-oxide (TMAO), as an early biomarker of longitudinal glucose increase or prevalent impaired glucose regulation. Overall, the manuscript is well-written, and the study has several interesting information. But few errors with lack of validation makes the study weak. These needs to be addressed sufficiently to be considered for publication in Plos Journal.

Response: We appreciate the reviewer’s comments and have addressed each of the comments below.

Comments # 1: Plasma TMAO in Diabetes, whether TMAO is a cause or consequence of Diabetes (Pre-diabetes) are highly debatable. As several studies using TMAO and cardiovascular diseases provide contradicting results. Though this showed modest increase in the prevalence of prediabetes in a nonlinear fashion. Several factors which interfere with the conditions/ Pre-diabetes were lest discussed and also, Study validation is an absolute necessity.

Response: We agree that results from previous studies have mixed results in the association of TMAO and cardiovascular disease. We were able to replicate the null findings that were observed between TMAO and cross-sectional insulin resistance in the CARDIA study by Meyer et al*, the latter being a generally healthy population similar to ORIGINS. While our data cannot prove causality, they help to characterize TMAO in relation to early cardiometabolic risk which will help to inform the design and premise of future studies that can address causal associations with greater validity.

*Meyer KA, Benton TZ, Bennett BJ, Jacobs DR, Jr., Lloyd-Jones DM, Gross MD, et al. Microbiota-Dependent Metabolite Trimethylamine N-Oxide and Coronary Artery Calcium in the Coronary Artery Risk Development in Young Adults Study (CARDIA). Journal of the American Heart Association. 2016;5(10).

Comment # 2: There are no line numbers in the manuscript which makes it hard to pinpoint the errors.

Response: Line numbers are now added to facilitate the review

Comment # 3: 20-55 years are definitely not young population, Correct the sentences wherever needed (Eg: Last two lines of introduction).

Response: Reference to 20-55 years as young population was removed from the following lines (line 92 on page 5, line 318 and line 321 on page 16).

Comment #4: TMAO levels were modestly associated with increased age. Discuss more on this point.

Response: Table 1 shows the mean age of those in Tertile 3 is 36.01±1.03 compared to Tertile 1 which was 32.28±0.92. The reasons for this relationship are not entirely clear but one possibility is that older participants have decreased renal function leading to increased TMAO. In response to the reviewer’s comments, we have added more discussion on this point to the manuscript on page 14 as follows: “Renal function might also provide some level of explanation for discordant findings in our current data as compared to other cohorts. The fact that higher renal function increases TMAO clearance raises the strong potential for confounding. Specifically, it is possible that reduced renal function causes both elevated TMAO levels and clinical CVD events. Our observation of increased TMAO among older participants provides indirect support for this notion. As such, our adjustment for age in multivariable models helps to mitigate confounding by renal function although future studies with assessment of renal function will be important.”

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

26 Nov 2019

PONE-D-19-18193R1

Plasma Trimethylamine-N-Oxide and Impaired Glucose Regulation: Results from The Oral Infections, Glucose Intolerance and Insulin Resistance Study (ORIGINS)

PLOS ONE

Dear Dr. Demmer,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Jan 10 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Cheng Hu

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I would suggest that the manuscript needs to provide all abbreviations used.

Page 3 line 6 CRP -> C-reactive protein

Page 5 line 105 FPG -> fasting plasma glucose (FPG)

Page 5 line 106 HbA1c -> hemoglobin A1c (HbA1c)

Page 6 line 124 fasting plasma glucose (FPG) -> FPG

Page 8 line 169 (PR) -> Delete

Page 8 line 182 Trimethylamine-N-oxide (TMAO) -> TMAO

Page 8 line 183 The Oral Infections, Glucose intolerance and Insulin Resistance Study (ORIGNS) -> ORIGINS

Page 11 line 211 hemoglobin A1c -> HbA1c

Page 11 line 212 Trimethylamine-N-oxide (TMAO) -> TMAO

Page 11 line 213 The Oral Infections, Glucose intolerance and Insulin Resistance Study (ORIGNS) -> ORIGINS

Page 14 line 276 CVD -> cardiovascular disease

Page 15 line 284 CKD -> chronic kidney disease

Page 15 line 301 Alternative Healthy Eating Index (AHEI) -> AHEI

It would be helpful to the reader to provide the rationale in the figure 2 legend which the authors explained in the response of comment 6.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

27 Nov 2019

We wish to thank the reviewer for the helpful comments, which have led to further strengthening of the manuscript.

Reviewer #1 Comments

Reviewer #1: I would suggest that the manuscript needs to provide all abbreviations used.

Page 3 line 6 CRP -> C-reactive protein

Page 5 line 105 FPG -> fasting plasma glucose (FPG)

Page 5 line 106 HbA1c -> hemoglobin A1c (HbA1c)

Page 6 line 124 fasting plasma glucose (FPG) -> FPG

Page 8 line 169 (PR) -> Delete

Page 8 line 182 Trimethylamine-N-oxide (TMAO) -> TMAO

Page 8 line 183 The Oral Infections, Glucose intolerance and Insulin Resistance Study (ORIGNS) -> ORIGINS

Page 11 line 211 hemoglobin A1c -> HbA1c

Page 11 line 212 Trimethylamine-N-oxide (TMAO) -> TMAO

Page 11 line 213 The Oral Infections, Glucose intolerance and Insulin Resistance Study (ORIGNS) -> ORIGINS

Page 14 line 276 CVD -> cardiovascular disease

Page 15 line 284 CKD -> chronic kidney disease

Page 15 line 301 Alternative Healthy Eating Index (AHEI) -> AHEI

It would be helpful to the reader to provide the rationale in the figure 2 legend which the authors explained in the response of comment 6.

Response: We appreciate the reviewer’s comments and have provided the expansion/acronyms as appropriate in the corresponding pages as noted below:

Page 3 line 68 CRP now reads as C-reactive protein (CRP)

Page 5 line 105 FPG now reads as fasting plasma glucose (FPG)

Page 5 line 106 HbA1c now reads as hemoglobin A1c (HbA1c)

Page 6 line 124 fasting plasma glucose is deleted and now reads as FPG

Page 8 line 169 (PR) is deleted

Page 8 line 182 Trimethylamine-N-oxide (TMAO) now reads as TMAO

Page 8 line 183 The Oral Infections, Glucose intolerance and Insulin Resistance Study (ORIGNS) now reads as ORIGINS

Page 11 line 211 hemoglobin A1c now reads as HbA1c

Page 11 line 212 Trimethylamine-N-oxide (TMAO) now reads as TMAO

Page 11 line 213 The Oral Infections, Glucose intolerance and Insulin Resistance Study (ORIGNS) now reads as ORIGINS

Page 14 line 276 CVD now reads as cardiovascular disease

Page 15 line 284 CKD now reads as chronic kidney disease

Page 15 line 301 Alternative Healthy Eating Index (AHEI) now reads as AHEI

The rationale that was explained in the comment 6 has been added to the figure 2. legend in the revised manuscript text.

Submitted filename: Response to Reviewers_11_27_19.docx

Click here for additional data file.

20 Dec 2019

Plasma Trimethylamine-N-Oxide and Impaired Glucose Regulation: Results from The Oral Infections, Glucose Intolerance and Insulin Resistance Study (ORIGINS)

PONE-D-19-18193R2

Dear Dr. Demmer,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Cheng Hu

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

3 Jan 2020

PONE-D-19-18193R2

Plasma Trimethylamine-N-Oxide and Impaired Glucose Regulation: Results from The Oral Infections, Glucose Intolerance and Insulin Resistance Study (ORIGINS)

Dear Dr. Demmer:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Cheng Hu

Academic Editor

PLOS ONE