A Cross Talking between the Gut Microbiota and Metabolites of Participants in a Confined Environment

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study indicates interesting information. However, further improvement is necessary before publication. 

(1)   The most important originality (or novelty) and impact (significance) are unclear because several similar papers have been already published. 

(2)   The data of blood health parameters such as glucose, lipids, cholesterol, insulin, CRP, glucocorticoid, etc. for evaluation of health status are lacking. Without these information, deep evaluation of the modulation of intestinal microbiota and the metabolites is difficult.

(3)   References are incomplete. Authors must carefully check the recent papers closely related to this paper; At least, the following papers were important in relation to this paper.

1.       Lopez-Santamarina et al. Effects of Unconventional Work and Shift Work on the Human Gut Microbiota and the Potential of Probiotics to Restore Dysbiosis. Nutrients 2023, 15, 3070. 

2.       Bijnens et al. Controlled light exposure and intermittent fasting as treatment strategies for metabolic syndrome and gut microbiome dysregulation in night shift works. Physiol. Behav. 2023, 263, 114103. 

3.       Wollmuth et al. Microbial circadian clocks: Host-microbe interplay in diel cycles. BMC Microbiol. 2023, 23, 124.

4.       Sun et al. Sleep Deprivation and Gut Microbiota Dysbiosis: Current Understandings and Implications. Int. J. Mol. Sci. 2023, 24, 9603. 

5.       Tian et al. An important link between the gut microbiota and the circadian rhythm: Imply for treatments of circadian rhythm sleep disorder. Food Sci. Biotechnol. 2022, 31, 155–164. 

6.       Lu et al. Alterations in the intestinal microbiome and mental health status of workers in an underground tunnel environment. BMC Microbiol. 2021, 21, 7. 

7.       Rogers et al. Shiftwork, functional bowel symptoms, and the microbiome. Peer J. 2021, 9, e11406. 

Author Response

Dear Editor:

 

On behalf of all authors, I would like to thank the Nutrients editorial staff for handling and the reviewers for the detailed review and comments on this manuscript (nutrients-3009498). We have revised our manuscript as suggested by the editor and reviewers. Thank you very much for your positive comments and constructive suggestions on our manuscript. These comments and suggestions have been very helpful to improve the scientific quality of our manuscript. We have carefully studied the comments and revised the manuscript accordingly. All revisions have been indicated in red in the manuscript. We hope that the manuscript is now acceptable for the next review process. Our point-by-point responses to the comments are listed below.

Thank you again for your comments. Look forward to hearing from you.

 

Sincerely yours

 

 

Reviewer #1: This study indicates interesting information. However, further improvement is necessary before publication. 

(1)   The most important originality (or novelty) and impact (significance) are unclear because several similar papers have been already published. 

Response: Thank you very much for the constructive comments.

Specific working environments like ocean navigation and deep-sea scientific expeditions feature confined spaces for work and living, which negatively impact workers due to factors including noise and circadian rhythm disruptions. Studies have shown that confined environments significantly affect the emotional and physical well-being of individuals. However, the majority of these studies take place in semi-open settings like cargo ships on extended voyages, offering participants more room for movement and the ability to interact with the outside world.

This study simulates a closed underwater environment such as a submarine, where subjects cannot exit the simulation chamber, are unable to communicate with the outside world through mobile phones or computers, and work in frequent shifts according to a work rhythm. The impact of such a strict confined space on subjects will be more pronounced, especially on the gut microbiota and its metabolites.

Therefore, this manuscript primarily explores the effects of a strictly confined environment on the gut microbiota and metabolites in subjects. By examining the relationship between the gut microbiota and distinct metabolites, the goal is to identify potential biomarkers that may be utilized to assess or counteract the impacts of confinement.

The results indicated that residing in confined environments can significantly alter the gut microbiota and metabolites, particularly affecting lipid metabolism pathways such as glycerophospholipid metabolism. Furthermore, the alterations in potential biomarkers were consistent with changes in the gut microbiota. This suggests that modulating the gut microbiota could be an effective strategy to mitigate the impacts of confined environments. Moreover, four potential biomarkers-(1-(sn-glycero-3-phospho)-1D-myo-inositol, phosphatidylserines (20:3(8Z,11Z,14Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), ganglioside monosialic 2 (d18:0/12:0), and cardiolipins (18:2(9Z,12Z)/18:2(9Z,12Z)/18:2(9Z,12Z)/18:3(6Z,9Z,12Z)))-are suggested for assessing the impact of confined environments.

Figure 1. Differential metabolites analysis in metabolic set of W3_W1. (A): HMDB classi-fication of differential metabolites. (B): PLS-DA of differential metabolites in fecal samples. (C): Volcano plot of differential metabolites. (D): KEGG pathway classification of differen-tial metabolites. (E): KEGG enrichment analysis of metabolic pathways identified between W3 and W1. (F): Metabolite clustering heatmap analysis and variable importance in pro-jection (VIP) scores of differential metabolites between W3 and W1. The selected metabo-lites (VIP top 30) were those with VIP>1.0. VIP score was based on the OPLS-DA model. Significant differences were compared with each two groups (**P<0.01, ***P<0.001).

Figure 2. Metabolic markers analysis in confined environment. (A): HMDB classification of top 25 differentially expressed metabolites were sorted by VIP values. (B): Comparative analysis of the relative content of metabolites (ROC>0.95). (C): ROC curves of four lipid metabolites upregulated in W3 time point. Significant differences were compared with each two groups (***P<0.001).

 

(2)   The data of blood health parameters such as glucose, lipids, cholesterol, insulin, CRP, glucocorticoid, etc. for evaluation of health status are lacking. Without these information, deep evaluation of the modulation of intestinal microbiota and the metabolites is difficult.

Response: Thank you very much for the constructive comments.

Owing to ethical considerations, the experiment was limited to sampling and analyzing the gut microbiota and metabolic products from the subjects, without the collection of blood or other tissue samples. In the preliminary study, we administered a health survey to subjects in confined environment via questionnaires and found that they exhibited varying degrees of health issues, including insomnia, dizziness, anxiety, constipation, and anorexia. To further investigate the effects of confined environments on intestinal health, we quantified the fecal levels of LPS (Lipopolysaccharide) and MCT (mast cell trypsin) as indicators of the subjects' health status.

The results showed that after the confined environment experiment, there was a significant increase in the occurrence of health abnormalities such as constipation and insomnia among the subjects. After the confined environment experiment, there was a significant increase in the levels of LPS and MCT in the feces of subjects, indicating that the confined environment affected the gut sensitivity of the subjects. In the future, we will conduct intervention trials, measure the physiological and biochemical indicators of the subjects, and conduct an in-depth analysis of the mechanisms by which confined environment impact health.  

 

(3)   References are incomplete. Authors must carefully check the recent papers closely related to this paper; At least, the following papers were important in relation to this paper.

Response: Thank you very much for the constructive comments. The references have been cited and discussed in the manuscript.

[23] Lopez-Santamarina, A.; Mondragon, A.D.C.; Cardelle-Cobas, A.; Santos, E.M.; Porto-Arias, J.J.; Cepeda, A.; Miranda, J.M. Effects of Unconventional Work and Shift Work on the Human Gut Microbiota and the Potential of Probiotics to Restore Dysbiosis. Nutrients. 2023, 15, 3070. doi:10.3390/nu15133070.

[24] Bijnens, S.; Depoortere, I. Controlled Light Exposure and Intermittent Fasting as Treatment Strategies for Metabolic Syndrome and Gut Microbiome Dysregulation in Night Shift Workers. Physiol. Behav. 2023, 263, 114103. doi:10.1016/j.physbeh.2023.114103.

[25] Wollmuth, E.M.; Angert, E.R. Microbial Circadian Clocks: Host-Microbe Interplay in Diel Cycles. BMC Microbiol. 2023, 23, 124. doi:10.1186/s12866-023-02839-4.

[31] Sun, J.; Fang, D.; Wang, Z.; Liu, Y. Sleep Deprivation and Gut Microbiota Dysbiosis: Current Understandings and Implications. Int. J. Mol. Sci. 2023, 24, 9603. doi:10.3390/ijms24119603.

[32] Tian, Y.; Yang, W.; Chen, G.; Men, C.; Gu, Y.; Song, X.; Zhang, R.; Wang, L.; Zhang, X. An Important Link between the Gut Microbiota and the Circadian Rhythm: Imply for Treatments of Circadian Rhythm Sleep Disorder. Food Sci. Biotechnol. 2022, 31, 155-164. doi:10.1007/s10068-021-01015-6.

[33] Rogers, A.E.; Hu, Y.J.; Yue, Y.; Wissel, E.F.; Petit, R.A.; Jarrett, S.; Christie, J.; Read, T.D. Shiftwork, Functional Bowel Symptoms, and the Microbiome. Peer J 2021, 9, e11406. doi:10.7717/peerj.11406.

[39] Lu, Z.H.; Liu, Y.W.; Ji, Z.H.; Fu, T.; Yan, M.; Shao, Z.J.; Long, Y. Alterations in the Intestinal Microbiome and Mental Health Status of Workers in an Underground Tunnel Environment. BMC Microbiol. 2021, 21, 7. doi:10.1186/s12866-020-02056-3.

 

 

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The objectives of the study are not clearly stated nor in the abstract nor in the introduction. Please define your specific research question(s) and whether the present study achieved the necessary results to reply to that question(s). If needed, propose additional or further research work to continue research.

 

The experimental design is not clear. "During the experiment, the participants were forbidden to smoke and drink, eat normally every day". Participants were forbidden to eat and drink? Or just alcoholic drinks? And which sample did the authors use as "control"? The W1 point? I have some doubts that this is an appropriate control, at least alone. Did the authors also collect samples prior to the 7 days before the trial? These 7 days can hardly be considered as "normal" days to the 12 participants.

 

What about the diet of the participants during the trial? Is it their usual diet? If not, how can we conclude that what we see is the effect of the confinement and not of the diet or a combination of both?

Author Response

Reviewer #2:

(1) The objectives of the study are not clearly stated nor in the abstract nor in the introduction. Please define your specific research question(s) and whether the present study achieved the necessary results to reply to that question(s). If needed, propose additional or further research work to continue research.

Response: Thank you very much for the constructive comments. The specific research questions were added in the part of introduction and marked in red.

Studies have shown that confined environments significantly affect the emotional and physical well-being of individuals. However, the majority of these studies take place in semi-open settings like cargo ships on extended voyages, offering participants more room for movement and the ability to interact with the outside world.

This study simulates a closed underwater environment such as a submarine, where subjects cannot exit the simulation chamber, are unable to communicate with the outside world through mobile phones or computers, and work in frequent shifts according to a work rhythm. The impact of such a strict confined space on subjects will be more pronounced, especially on the gut microbiota and its metabolites.

Therefore, this manuscript primarily explores the effects of a strictly confined environment on the gut microbiota and metabolites in subjects. By examining the relationship between the gut microbiota and distinct metabolites, the goal is to identify potential biomarkers that may be utilized to assess or counteract the impacts of confinement.

The results indicated that residing in confined environments can significantly alter the gut microbiota and metabolites, particularly affecting lipid metabolism pathways such as glycerophospholipid metabolism. Furthermore, the alterations in potential biomarkers were consistent with changes in the gut microbiota. This suggests that modulating the gut microbiota could be an effective strategy to mitigate the impacts of confined environments. Moreover, four potential biomarkers-(1-(sn-glycero-3-phospho)-1D-myo-inositol, phosphatidylserines (20:3(8Z,11Z,14Z)/22:6 (4Z,7Z,10Z,13Z,16Z,19Z)), ganglioside monosialic 2 (d18:0/12:0), and cardiolipins (18:2(9Z,12Z)/18:2(9Z,12Z)/18:2(9Z,12Z)/18:3(6Z,9Z,12Z)))-are suggested for assessing the impact of confined environments.

Future studies will explore the impact of dietary interventions on the effects of confined environments, and validate the efficacy of the identified potential biomarkers.

 

(2) The experimental design is not clear. "During the experiment, the participants were forbidden to smoke and drink, eat normally every day". Participants were forbidden to eat and drink? Or just alcoholic drinks? And which sample did the authors use as "control"? The W1 point? I have some doubts that this is an appropriate control, at least alone. Did the authors also collect samples prior to the 7 days before the trial? These 7 days can hardly be considered as "normal" days to the 12 participants.

Response: Thank you very much for the constructive comments. We have rewritten the experimental design process and made revisions in the manuscript.

During the experiment, participants were allowed to have a regular diet, but alcohol consumption and smoking were strictly prohibited.

To mitigate the effects of dietary variations, participants will undergo a 7-day pre-adaptation period in a standard environment following recruitment. During this period, the diet of the participants will be consistent with that of the confined environment experiment, and they will be free to move and communicate with the outside world. After the adaptation period, participants will enter the confined environment simulation chamber to officially begin the experiment, which will last for 14 days. Therefore, on the first day after officially entering the simulation chamber, fecal samples from the participants are collected as the control group W1.

Figure 3 The flow chart of experimental

 

(3) What about the diet of the participants during the trial? Is it their usual diet? If not, how can we conclude that what we see is the effect of the confinement and not of the diet or a combination of both?

Response: Thank you very much for the constructive comments. We have supplemented the diet and experimental design in the manuscript.

During the experiment, the diet of the subjects was equivalent to that in a real submarine, not a special diet. To eliminate the impact caused by dietary differences, after recruitment, participants will be pre-adapted in a normal environment for 7 days. During this period, the diet of the participants will be consistent with that of the confined environment experiment, and they will be free to move and communicate with the outside world. Thus, the differences in gut metabolites and gut microbiota are not caused by diet, but by the confined environment.

Author Response File: Author Response.pdf