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Article type: News and Views: Groundbreaking Discoveries in ImmunologyTitle: Uncovering the influence of diet and gut microbiota in human serum metabolomeAuthors: David Obeso1,2‡, Elisa Zubeldia-Varela1,2‡ and Alma Villasenor11Instituto de Medicina Molecular Aplicada (IMMA), Departamento de Ciencias Medicas Basicas. Facultad de Medicina. Universidad San Pablo-CEU, CEU Universities, Madrid, Espana.2Centro de Metabolomica y Bioanalisis (CEMBIO), Facultad de Farmacia, Universidad San Pablo-CEU, CEU Universities, Urbanizacion Monteprincipe, Madrid, Espana.‡: These Authors have equally contributedCorrespondence: Alma Villasenor, Centro de Metabolomica y Bioanalisis (CEMBIO), Facultad de Farmacia, Universidad San Pablo-CEU, CEU Universities, Urbanizacion Monteprincipe, Madrid, EspanaEmail address:alma.villasenor@ceu.esKeywords: Metabolomics, Diet, Microbiota, Allergy, Serum, MetabolomeAbbreviations: MS, mass spectrometry; LC, liquid chromatography; short chain fatty acids (SCFA)Acknowledgement: We would like to thank Anna Globinska for her assistance in generating the graphical figure. AV Gratefully acknowledges the Martin-Carbajo family for supporting her scientific development through the X Mario Martin Velamazan award.Author contributions: D.O., E.Z-V. and A.V contributed to the writing of the manuscript and have given approval to the manuscript. A.V. supervised the final version.

Obesity has reached an epidemic level worldwide, and bariatric surgery (BS) has been proven to be the most efficient therapy to reduce severe obesity-related comorbidities. Given that the gut microbiota plays a causal role in obesity development and that surgery may alter the gut environment, investigating the impact of BS on the microbiota in the context of severe obesity is important. Although, alterations at the level of total gut bacteria, total gene content and total metabolite content have started to be disentangled, a clear deficit exists regarding the analysis of the active fraction of the microbiota, which is the fraction that is most reactive to the BS. Here, active gut microbiota and associated metabolic functions were evaluated using shotgun proteomics and metabolomics in 40 severely obese volunteers. Samples from each volunteer were obtained under basal conditions, after a short high protein and calorie-restricted diet, and 1 and 3 months after BS, including laparoscopic surgery through Roux-en-Y Gastric Bypass or Sleeve Gastrectomy. The results revealed for the first time the most active microbes and metabolic flux distribution pre- and post-surgery and deciphered main differences in the way sugars and short-fatty acids are metabolized, demonstrating that less energy-generating and anaerobic metabolism and detoxification mechanisms are promoted post-surgery. A comparison with non-obese proteome data further signified different ways to metabolize sugars and produce short chain fatty acids and deficiencies in proteins involved in iron transport and metabolism in severely obese individuals compared to lean individuals., This work was funded by grants SAF2015-65878-R, BIO2017-85522-R, PID2019-105969GB-I00 and RTI2018-095166-B-I00 from the Ministry of Science, Innovation and Universities, by the Ministry of Science and Innovation, by the Instituto de Salud Carlos III (projects PIE14/00045 and AC17/00022), Fundación Agencia Española contra el Cáncer and Instituto de Salud Carlos III(projects ERA NET TRANSCAN-2 AC17/00022 and AECC 2017-1485), Generalitat Valenciana (project Prometeo/2018/A/133) and co-financed by the European Regional Development Fund (ERDF). The proteomic analysis was performed in the Proteomics Facility of The Spanish National Center for Biotechnology (CNB-CSIC) that belongs to ProteoRed, PRB3-ISCIII, supported by grant PT17/0019.

The connection between gut microbiota and human health is becoming increasingly relevant and the number of groups working in this field is constantly growing. In this context, from high-throughput gene sequencing to metabolomics analysis, the omics technologies have contributed enormously to unveil the secret crosstalk between us and our microbes. All the omics technologies produce a great amount of information, and processing this information is time-consuming and expensive. For this reason, a correct experimental design and a careful pre-analytical planning are crucial. To study the human gut microbiota, faeces are the sample of choice. Faecal material is complex, and procedures for collecting and preserving faeces are not well-established. Furthermore, increasing evidence suggests that multiple confounding factors, such as antibiotics consumption, mode of delivery, diet, aging and several diseases and disorders can alter the composition and functionality of the microbiota. This review is focused on the discussion of critical general issues during the pre-analytical planning, from patient handling to faeces sampling, including collection procedures, transport, storage conditions and possible pre-treatments, which are critical for a successful research in omics with a special attention to metabolomics and gene sequencing. We also point out that the adoption of standard operating procedures in the field is needed to guarantee accuracy and reproducibility of results.

Composition of the gut microbiota changes during ageing, but questions remain about whether age is also associated with deficits in microbiome function and whether these changes occur sharply or progressively. The ability to define these deficits in populations of different ages may help determine a chronological age threshold at which deficits occur and subsequently identify innovative dietary strategies for active and healthy ageing. Here, active gut microbiota and associated metabolic functions were evaluated using shotgun proteomics in three well‐defined age groups consisting of 30 healthy volunteers, namely, ten infants, ten adults and ten elderly individuals. Samples from each volunteer at intervals of up to 6 months (n = 83 samples) were used for validation. Ageing gradually increases the diversity of gut bacteria that actively synthesize proteins, that is by 1.4‐fold from infants to elderly individuals. An analysis of functional deficits consistently identifies a relationship between tryptophan and indole metabolism and ageing (p < 2.8e−8). Indeed, the synthesis of proteins involved in tryptophan and indole production and the faecal concentrations of these metabolites are directly correlated (r2 > .987) and progressively decrease with age (r2 > .948). An age threshold for a 50% decrease is observed ca. 11–31 years old, and a greater than 90% reduction is observed from the ages of 34–54 years. Based on recent investigations linking tryptophan with abundance of indole and other “healthy” longevity molecules and on the results from this small cohort study, dietary interventions aimed at manipulating tryptophan deficits since a relatively “young” age of 34 and, particularly, in the elderly are recommended., S.R. was recipient of a postdoctoral “Sara Borrell” contract from the Instituto de Salud Carlos III. This work was funded by grants SAF2015‐65878‐R and RTI2018‐095166‐B‐I00 from the Ministry of Science, Innovation and Universities with the cofunds of the European Regional Development Fund (ERDF) and the Agencia Estatal de Investigación (AEI), grant PIE14/00045 from the Instituto de Salud Carlos III, grant AC17/00022 from the Instituto de Salud Carlos III and Fundación Agencia Española contra el Cáncer (AECC) within the ERA NET TRANSCAN‐2, grant 2017‐1485 from the AECC and grant Prometeo/2018/A/133 from the Generalitat Valenciana. M.F. acknowledges the support of BIO2017‐85522‐R, from the Ministry of Science, Innovation and Universities, with the cofunds of the ERDF and the Agencia Estatal de Investigación (AEI). The proteomic analysis was performed in the Proteomics Facility of The Spanish National Center for Biotechnology (CNB‐CSIC) that belongs to ProteoRed, PRB3‐ISCIII, supported by grant PT17/0019. The funding bodies did not have a role in the design or conduct of the study, the analysis and interpretation of the results, the writing of the report, or the decision to publish.