Counteracting gemcitabine+nab-paclitaxel induced dysbiosis in KRAS wild type and KRASG12D mutated pancreatic cancer in vivo model.

Pancreatic cancer (PC) has a very low survival rate mainly due to late diagnosis and refractoriness to therapies. The latter also cause adverse effects negatively affecting the patients' quality of life, often requiring dose reduction or discontinuation of scheduled treatments, compromising the chances of cure. We explored the effects of a specific probiotic blend on PC mice xenografted with KRAS wild-type or KRASG12D mutated cell lines alone or together with gemcitabine+nab-paclitaxel treatment to then assess tumor volume and clinical pathological variables. Beside a semi-quantitative histopathological evaluation of murine tumor and large intestine samples, histochemical and immunohistochemical analyses were carried out to evaluate collagen deposition, proliferation index Ki67, immunological microenvironment tumor-associated, DNA damage markers and also mucin production. Blood cellular and biochemical parameters and serum metabolomics were further analyzed. 16S sequencing was performed to analyze the composition of fecal microbiota. Gemcitabine+nab-paclitaxel treatment impaired gut microbial profile in KRAS wild-type and KRASG12D mice. Counteracting gemcitabine+nab-paclitaxel- induced dysbiosis through the administration of probiotics ameliorated chemotherapy side effects and decreased cancer-associated stromatogenesis. Milder intestinal damage and improved blood count were also observed upon probiotics treatment as well as a positive effect on fecal microbiota, yielding an increase in species richness and in short chain fatty acids producing- bacteria. Mice' serum metabolomic profiles revealed significant drops in many amino acids upon probiotics administration in KRAS wild-type mice while in animals transplanted with PANC-1 KRASG12D mutated all treated groups showed a sharp decline in serum levels of bile acids with respect to control mice. These results suggest that counteracting gemcitabine+nab-paclitaxel-induced dysbiosis ameliorates chemotherapy side effects by restoring a favorable microbiota composition. Relieving adverse effects of the chemotherapy through microbiota manipulation could be a desirable strategy in order to improve pancreatic cancer patients' quality of life and to increase the chance of cure.

Integrated Metabolomics, Lipidomics, and Genomics Reveal the Presence of a New Biomarker, Butanediol Glucuronide, Associated with the Activation of Liver Ketogenesis and Lipid Oxidation by Tomato-Based Sofrito in Obese Rats.

The increasing prevalence of obesity worldwide has promoted research on human metabolism and foods such as sofrito, a tomato and olive oil-based sauce from the Mediterranean diet, has shown beneficial effects on obesity and related complications. Sofrito has been associated with better cardiovascular health, metabolic syndrome, and anti-inflammatory effects. The aim of this study was to understand how sofrito intake could contribute to the control of energy metabolism in obese rats. For this purpose, integrative untargeted lipidomics, metabolomics, and targeted gene expression approaches were used in the liver and adipose tissue to identify metabolic changes and the mechanism of action promoted by sofrito intake. A new biomarker was identified in the liver, butanediol glucuronide, an indicator of ketogenic activation and lipid oxidation after the sofrito intervention. Gene expression analysis revealed an increase in the uptake and liver oxidation of lipids for energy production and ketogenesis activation as fuel for other tissues in sofrito-fed animals. Sofrito altered the lipidomic profile in the fat depots of obese rats. This multiomics study identifies a new biomarker linked to the beneficial actions of sofrito against obesity and provides further insight into the beneficial effect of the Mediterranean diet components.

Combining Feature-Based Molecular Networking and Contextual Mass Spectral Libraries to Decipher Nutrimetabolomics Profiles.

Untargeted metabolomics approaches deal with complex data hindering structural information for the comprehensive analysis of unknown metabolite features. We investigated the metabolite discovery capacity and the possible extension of the annotation coverage of the Feature-Based Molecular Networking (FBMN) approach by adding two novel nutritionally-relevant (contextual) mass spectral libraries to the existing public ones, as compared to widely-used open-source annotation protocols. Two contextual mass spectral libraries in positive and negative ionization mode of ~300 reference molecules relevant for plant-based nutrikinetic studies were created and made publicly available through the GNPS platform. The postprandial urinary metabolome analysis within the intervention of Vaccinium supplements was selected as a case study. Following the FBMN approach in combination with the added contextual mass spectral libraries, 67 berry-related and human endogenous metabolites were annotated, achieving a structural annotation coverage comparable to or higher than existing non-commercial annotation workflows. To further exploit the quantitative data obtained within the FBMN environment, the postprandial behavior of the annotated metabolites was analyzed with Pearson product-moment correlation. This simple chemometric tool linked several molecular families with phase II and phase I metabolism. The proposed approach is a powerful strategy to employ in longitudinal studies since it reduces the unknown chemical space by boosting the annotation power to characterize biochemically relevant metabolites in human biofluids.

Butyrate, a postbiotic of intestinal bacteria, affects pancreatic cancer and gemcitabine response in in vitro and in vivo models.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer. The characteristic excessive stromatogenesis accompanying the growth of this tumor is believed to contribute to chemoresistance which, together with drug toxicity, results in poor clinical outcome. An increasing number of studies are showing that gut microbiota and their metabolites are implicated in cancer pathogenesis, progression and response to therapies. In this study we tested butyrate, a product of dietary fibers' bacterial fermentation, whose anticancer and anti-inflammatory functions are known. We provided in vitro evidence that, beside slowing proliferation, butyrate enhanced gemcitabine effectiveness against two human pancreatic cancer cell lines, mainly inducing apoptosis. In addition, we observed that, when administered to a PDAC mouse model, alone or combined with gemcitabine treatment, butyrate markedly reduced the cancer-associated stromatogenesis, preserved intestinal mucosa integrity and affected fecal microbiota composition by increasing short chain fatty acids producing bacteria and decreasing some pro-inflammatory microorganisms. Furthermore, a biochemical serum analysis showed butyrate to ameliorate some markers of kidney and liver damage, whereas a metabolomics approach revealed a deep modification of lipid metabolism, which may affect tumor progression or response to therapy. Such results support that butyrate supplementation, in addition to conventional therapies, can interfere with pancreatic cancer biology and response to treatment and can alleviate some damages associated to cancer itself or to chemotherapy.

Comparison of chemometric strategies for potential exposure marker discovery and false-positive reduction in untargeted metabolomics: application to the serum analysis by LC-HRMS after intake of Vaccinium fruit supplements.

Untargeted liquid chromatographic-high-resolution mass spectrometric (LC-HRMS) metabolomics for potential exposure marker (PEM) discovery in nutrikinetic studies generates complex outputs. The correct selection of statistically significant PEMs is a crucial analytical step for understanding nutrition-health interactions. Hence, in this paper, different chemometric selection workflows for PEM discovery, using multivariate or univariate parametric or non-parametric data analyses, were comparatively tested and evaluated. The PEM selection protocols were applied to a small-sample-size untargeted LC-HRMS study of a longitudinal set of serum samples from 20 volunteers after a single intake of (poly)phenolic-rich Vaccinium myrtillus and Vaccinium corymbosum supplements. The non-parametric Games-Howell test identified a restricted group of significant features, thus minimizing the risk of false-positive retention. Among the forty-seven PEMs exhibiting a statistically significant postprandial kinetics, twelve were successfully annotated as purine pathway metabolites, benzoic and benzodiol metabolites, indole alkaloids, and organic and fatty acids, and five (i.e. octahydro-methyl-ß-carboline-dicarboxylic acid, tetrahydro-methyl-ß-carboline-dicarboxylic acid, citric acid, caprylic acid, and azelaic acid) were associated to Vaccinium berry consumption for the first time. The analysis of the area under the curve of the longitudinal dataset highlighted thirteen statistically significant PEMs discriminating the two interventions, including four intra-intervention relevant metabolites (i.e. abscisic acid glucuronide, catechol sulphate, methyl-catechol sulphate, and α-hydroxy-hippuric acid). Principal component analysis and sample classification through linear discriminant analysis performed on PEM maximum intensity confirmed the discriminating role of these PEMs.

A diet enriched in omega-3 PUFA and inulin prevents type 1 diabetes by restoring gut barrier integrity and immune homeostasis in NOD mice.

Introduction: The integrity of the gut barrier (GB) is fundamental to regulate the crosstalk between the microbiota and the immune system and to prevent inflammation and autoimmunity at the intestinal level but also in organs distal from the gut such as the pancreatic islets. In support to this idea, we recently demonstrated that breakage of GB integrity leads to activation of islet-reactive T cells and triggers autoimmune Type 1 Diabetes (T1D). In T1D patients as in the NOD mice, the spontaneous model of autoimmune diabetes, there are alterations of the GB that specifically affect structure and composition of the mucus layer; however, it is yet to be determined whether a causal link between breakage of the GB integrity and occurrence of autoimmune T1D exists. Methods: Here we restored GB integrity in the NOD mice through administration of an anti-inflammatory diet (AID- enriched in soluble fiber inulin and omega 3-PUFA) and tested the effect on T1D pathogenesis. Results: We found that the AID prevented T1D in NOD mice by restoring GB integrity with increased mucus layer thickness and higher mRNA transcripts of structural (Muc2) and immunoregulatory mucins (Muc1 and Muc3) as well as of tight junction proteins (claudin1). Restoration of GB integrity was linked to reduction of intestinal inflammation (i.e., reduced expression of IL-1ß, IL-23 and IL-17 transcripts) and expansion of regulatory T cells (FoxP3+ Treg cells and IL-10+ Tr1 cells) at the expenses of effector Th1/Th17 cells in the intestine, pancreatic lymph nodes (PLN) and intra-islet lymphocytes (IIL) of AID-fed NOD mice. Importantly, the restoration of GB integrity and immune homeostasis were associated with enhanced concentrations of anti-inflammatory metabolites of the ω3/ω6 polyunsaturated fatty acids (PUFA) and arachidonic pathways and modifications of the microbiome profile with increased relative abundance of mucus-modulating bacterial species such as Akkermansia muciniphila and Akkermansia glycaniphila. Discussion: Our data provide evidence that the restoration of GB integrity and intestinal immune homeostasis through administration of a tolerogenic AID that changed the gut microbial and metabolic profiles prevents autoimmune T1D in preclinical models.

Assessment of Dietary Bioactive Phenolic Compounds and Agricultural Sustainability of an African Leafy Vegetable Corchorus olitorius L.

Corchorus olitorius L. is an African leafy vegetable of high nutritional interest. To assess its agricultural suitability to sustainable cultivation conditions and its potential benefits for human nutrition, its phytochemical content in response to conservation agriculture practices [i.e., no-tillage (NT) and cover crop maintenance] and low water regime were evaluated and compared with response under conventional agriculture management. Hydric stress and NT did not affect the content of antioxidant metabolites, compared to conventional agricultural practices. In both conditions, leaves were found to be a great source of phenolic compounds. The effect of these phenolic fractions was assessed on two colon cell phenotypes to evaluate putative nutraceutical properties. Polyphenol-enriched extracts (PEEs) displayed selective cytotoxic activities against tumor Caco-2 cells but not on the healthy CCD841 line. PEEs were able to trigger oxidative stress and to inhibit the activity of glutathione-independent antioxidant enzymes on Caco-2 cells. C. olitorius showed to be a promising crop for improving both agricultural sustainability and health benefits due to the great amount of antioxidant compounds in leaves, whose occurrence is not altered by stressful farming conditions. Given its high adaptability, the cultivation of this crop is therefore recommendable also in the Mediterranean Basin.

Liquid Chromatographic Quadrupole Time-of-Flight Mass Spectrometric Untargeted Profiling of (Poly)phenolic Compounds in Rubus idaeus L. and Rubus occidentalis L. Fruits and Their Comparative Evaluation.

This study provided a detailed profiling of the antioxidant and bioactive compounds occurring in three varieties of Rubus idaeus L. fruits ("Fall Gold", "Glen Ample" and "Tulameen") compared to Rubus occidentalis L. black raspberry ("Jewel" cultivar), adopting a comprehensive untargeted metabolomics approach developed with UHPLC analysis coupled with quadrupole/time-of-flight high resolution mass spectrometry, using the SWATH® acquisition protocol. The feature selection and annotation workflow, applied to the analysis of raspberry extracts in both polarities, allowed identifying 68 bioactive compounds mainly belonging to the classes of (poly)phenolic compounds. Interestingly, some of these identifications (e.g., ferulic acid glycosides and the ellagitannin-like nobotanin/malabathrin) represent the first report in raspberry fruits. Principal component analysis made possible highlighting the features more related to the expression of a genotype effect within the R. idaeus species or between the two raspberry species herein investigated. Overall, flavanols were the most discriminating features for the Fall Gold variety, whereas ellagitannins and flavonol glycosides represent more distinctive metabolic traits in Glen Ample and Tulameen fruits. Moreover, R. occidentalis Jewel variety was strongly characterized by the occurrence of anthocyanins, such as cyanidin, pelargonidin and delphinidin glycosides.

Data sharing in PredRet for accurate prediction of retention time: Application to plant food bioactive compounds.

Prediction of retention times (RTs) is increasingly considered in untargeted metabolomics to complement MS/MS matching for annotation of unidentified peaks. We tested the performance of PredRet (http://predret.org/) to predict RTs for plant food bioactive metabolites in a data sharing initiative containing entry sets of 29-103 compounds (totalling 467 compounds, >30 families) across 24 chromatographic systems (CSs). Between 27 and 667 predictions were obtained with a median prediction error of 0.03-0.76 min and interval width of 0.33-8.78 min. An external validation test of eight CSs showed high prediction accuracy. RT prediction was dependent on shape and type of LC gradient, and number of commonly measured compounds. Our study highlights PredRet's accuracy and ability to transpose RT data acquired from one CS to another CS. We recommend extensive RT data sharing in PredRet by the community interested in plant food bioactive metabolites to achieve a powerful community-driven open-access tool for metabolomics annotation.

Discovery of Intake Biomarkers of Lentils, Chickpeas, and White Beans by Untargeted LC-MS Metabolomics in Serum and Urine.

SCOPE: To identify reliable biomarkers of food intake (BFIs) of pulses. METHODS AND RESULTS: A randomized crossover postprandial intervention study is conducted on 11 volunteers who consumed lentils, chickpeas, and white beans. Urine and serum samples are collected at distinct postprandial time points up to 48 h, and analyzed by LC-HR-MS untargeted metabolomics. Hypaphorine, trigonelline, several small peptides, and polyphenol-derived metabolites prove to be the most discriminating urinary metabolites. Two arginine-related compounds, dopamine sulfate and epicatechin metabolites, with their microbial derivatives, are identified only after intake of lentils, whereas protocatechuic acid is identified only after consumption of chickpeas. Urinary hydroxyjasmonic and hydroxydihydrojasmonic acids, as well as serum pipecolic acid and methylcysteine, are found after white bean consumption. Most of the metabolites identified in the postprandial study are replicated as discriminants in 24 h urine samples, demonstrating that in this case the use of a single, noninvasive sample is suitable for revealing the consumption of pulses. CONCLUSIONS: The results of the present untargeted metabolomics work reveals a broad list of metabolites that are candidates for use as biomarkers of pulse intake. Further studies are needed to validate these BFIs and to find the best combinations of them to boost their specificity.

Quantification of Urinary Phenyl-γ-Valerolactones and Related Valeric Acids in Human Urine on Consumption of Apples.

Flavan-3-ols are dietary bioactive molecules that have beneficial effects on human health and reduce the risk of various diseases. Monomeric flavan-3-ols are rapidly absorbed in the small intestine and released in the blood stream as phase II conjugates. Polymeric flavan-3-ols are extensively metabolized by colonic gut microbiota into phenyl-γ-valerolactones and their related phenylvaleric acids. These molecules are the main circulating metabolites in humans after the ingestion of flavan-3-ol rich-products; nevertheless, they have received less attention and their role is not understood yet. Here, we describe the quantification of 8 phenyl-γ-valerolactones and 3 phenylvaleric acids in the urine of 11 subjects on consumption of apples by using UHPLC-ESI-Triple Quad-MS with pure reference compounds. Phenyl-γ-valerolactones, mainly as sulfate and glucuronic acid conjugates, reached maximum excretion between 6 and 12 after apple consumption, with a decline thereafter. Significant differences were detected in the cumulative excretion rates within subjects and in the ratio of dihydroxyphenyl-γ-valerolactone sulfate to glucuronide conjugates. This work observed for the first time the presence of two distinct metabotypes with regards to the excretion of phenyl-γ-valerolactone phase II conjugates.

A Dietary Intervention of Bioactive Enriched Foods Aimed at Adults at Risk of Metabolic Syndrome: Protocol and Results from PATHWAY-27 Pilot Study.

Around a quarter of the global adult population have metabolic syndrome (MetS) and therefore increased risk of cardiovascular mortality and diabetes. Docosahexaenoic acid, oat beta-glucan and grape anthocyanins have been shown to be effective in reducing MetS risk factors when administered as isolated compounds, but their effect when administered as bioactive-enriched foods has not been evaluated. OBJECTIVE: The overall aim of the PATHWAY-27 project was to evaluate the effectiveness of bioactive-enriched food consumption on improving risk factors of MetS. A pilot study was conducted to assess which of five bioactive combinations provided within three different food matrices (bakery, dairy or egg) were the most effective in adult volunteers. The trial also evaluated the feasibility of production, consumer acceptability and gastrointestinal tolerance of the bioactive-enriched food. METHOD: The study included three monocentric, parallel-arm, double-blind, randomised, dietary intervention trials without a placebo. Each recruiting centre tested the five bioactive combinations within a single food matrix. RESULTS: The study was completed by 167 participants (74 male, 93 female). The results indicated that specific bioactive/matrix combinations have effects on serum triglyceride or HDL-cholesterol level without adverse effects. CONCLUSION: The study evidenced that bioactive-enriched food offers a promising food-based strategy for MetS prevention, and highlighted the importance of conducting pilot studies.

Biomarkers of food intake for nuts and vegetable oils: an extensive literature search.

Nuts and vegetable oils are important sources of fat and of a wide variety of micronutrients and phytochemicals. Following their intake, several of their constituents, as well as their derived metabolites, are found in blood circulation and in urine. As a consequence, these could be used to assess the compliance to a dietary intervention or to determine habitual intake of nuts and vegetable oils. However, before these metabolites can be widely used as biomarkers of food intake (BFIs), several characteristics have to be considered, including specificity, dose response, time response, stability, and analytical performance. We have, therefore, conducted an extensive literature search to evaluate current knowledge about potential BFIs of nuts and vegetable oils. Once identified, the strengths and weaknesses of the most promising candidate BFIs have been summarized. Results from selected studies have provided a variety of compounds mainly derived from the fatty fraction of these foods, but also other components and derived metabolites related to their nutritional composition. In particular, α-linolenic acid, urolithins, and 5-hydroxyindole-3-acetic acid seem to be the most plausible candidate BFIs for walnuts, whereas for almonds they could be α-tocopherol and some catechin-derived metabolites. Similarly, several studies have reported a strong association between selenium levels and consumption of Brazil nuts. Intake of vegetable oils has been mainly assessed through the measurement of specific fatty acids in different blood fractions, such as oleic acid for olive oil, α-linolenic acid for flaxseed (linseed) and rapeseed (canola) oils, and linoleic acid for sunflower oil. Additionally, hydroxytyrosol and its metabolites were the most promising distinctive BFIs for (extra) virgin olive oil. However, most of these components lack sufficient specificity to serve as BFIs. Therefore, additional studies are necessary to discover new candidate BFIs, as well as to further evaluate the specificity, sensitivity, dose-response relationships, and reproducibility of these candidate biomarkers and to eventually validate them in other populations. For the discovery of new candidate BFIs, an untargeted metabolomics approach may be the most effective strategy, whereas for increasing the specificity of the evaluation of food consumption, this could be a combination of different metabolites.

Nutrimetabolomics: An Integrative Action for Metabolomic Analyses in Human Nutritional Studies.

The life sciences are currently being transformed by an unprecedented wave of developments in molecular analysis, which include important advances in instrumental analysis as well as biocomputing. In light of the central role played by metabolism in nutrition, metabolomics is rapidly being established as a key analytical tool in human nutritional studies. Consequently, an increasing number of nutritionists integrate metabolomics into their study designs. Within this dynamic landscape, the potential of nutritional metabolomics (nutrimetabolomics) to be translated into a science, which can impact on health policies, still needs to be realized. A key element to reach this goal is the ability of the research community to join, to collectively make the best use of the potential offered by nutritional metabolomics. This article, therefore, provides a methodological description of nutritional metabolomics that reflects on the state-of-the-art techniques used in the laboratories of the Food Biomarker Alliance (funded by the European Joint Programming Initiative "A Healthy Diet for a Healthy Life" (JPI HDHL)) as well as points of reflections to harmonize this field. It is not intended to be exhaustive but rather to present a pragmatic guidance on metabolomic methodologies, providing readers with useful "tips and tricks" along the analytical workflow.

Untargeted Metabolomics Analytical Strategy Based on Liquid Chromatography/Electrospray Ionization Linear Ion Trap Quadrupole/Orbitrap Mass Spectrometry for Discovering New Polyphenol Metabolites in Human Biofluids after Acute Ingestion of Vaccinium myrtillus Berry Supplement.

In this work, liquid chromatography, coupled with an electrospray ionization hybrid linear ion trap quadrupole/Orbitrap mass spectrometry, has been used to accurately identify polyphenol metabolites in human serum and urine after acute ingestion of a V. myrtillus berry supplement. The supplement was obtained by cryo-milling of bilberries, which were freeze-dried within 1 week after their harvesting, to maintain the berry native composition. Thirty-six derivatives of benzoic acids, hydroxyhippuric acids, cinnamic acids, phenylpropionic acids, phenylvaleric acids, phenylpentenoic acids and abscisic acid, together with two berry-native anthocyanins, one flavonol metabolite and two catechol derivatives were putatively identified in the investigated biofluids. The annotated compounds included 13 metabolites, among glucuronides and sulphates of phenylvaleric and phenylpentenoic acids, which have been identified for the first time in human biofluids after ingestion of V. myrtillus berries. It should be emphasized that the presence of phenylvaleric and phenylpentenoic acid derivatives is in agreement with their origin from fruit native flavanol monomers and oligomers, which are widely distributed in Vaccinium berries, but usually overlooked in metabolomics studies regarding bilberry. The identification of these compounds confirmed the key-role of untargeted metabolomics approach in the discovery of new metabolites which could be biologically active. Graphical Abstract.

Food intake biomarkers for apple, pear, and stone fruit.

Fruit is a key component of a healthy diet. However, it is still not clear whether some classes of fruit may be more beneficial than others and whether all individuals whatever their age, gender, health status, genotype, or gut microbiota composition respond in the same way to fruit consumption. Such questions require further observational and intervention studies in which the intake of a specific fruit can be precisely assessed at the population and individual levels. Within the Food Biomarker Alliance Project (FoodBAll Project) under the Joint Programming Initiative "A Healthy Diet for a Healthy Life", an ambitious action was undertaken aiming at reviewing existent literature in a systematic way to identify validated and promising biomarkers of intake for all major food groups, including fruits. This paper belongs to a series of reviews following the same BFIRev protocol and is focusing on biomarkers of pome and stone fruit intake. Selected candidate biomarkers extracted from the literature search went through a validation process specifically developed for food intake biomarkers.

Evolution of gut microbiota composition from birth to 24 weeks in the INFANTMET Cohort.

BACKGROUND: The gut is the most extensively studied niche of the human microbiome. The aim of this study was to characterise the initial gut microbiota development of a cohort of breastfed infants (n = 192) from 1 to 24 weeks of age. METHODS: V4-V5 region 16S rRNA amplicon Illumina sequencing and, in parallel, bacteriological culture. The metabolomic profile of infant urine at 4 weeks of age was also examined by LC-MS. RESULTS: Full-term (FT), spontaneous vaginally delivered (SVD) infants' microbiota remained stable at both phylum and genus levels during the 24-week period examined. FT Caesarean section (CS) infants displayed an increased faecal abundance of Firmicutes (p < 0.01) and lower abundance of Actinobacteria (p < 0.001) after the first week of life compared to FT-SVD infants. FT-CS infants gradually progressed to harbouring a microbiota closely resembling FT-SVD (which remained stable) by week 8 of life, which was maintained at week 24. The gut microbiota of preterm (PT) infants displayed a significantly greater abundance of Proteobacteria compared to FT infants (p < 0.001) at week 1. Metabolomic analysis of urine at week 4 indicated PT-CS infants have a functionally different metabolite profile than FT (both CS and SVD) infants. Co-inertia analysis showed co-variation between the urine metabolome and the faecal microbiota of the infants. Tryptophan and tyrosine metabolic pathways, as well as fatty acid and bile acid metabolism, were found to be affected by delivery mode and gestational age. CONCLUSIONS: These findings confirm that mode of delivery and gestational age both have significant effects on early neonatal microbiota composition. There is also a significant difference between the metabolite profile of FT and PT infants. Prolonged breastfeeding was shown to have a significant effect on the microbiota composition of FT-CS infants at 24 weeks of age, but interestingly not on that of FT-SVD infants. Twins had more similar microbiota to one another than between two random infants, reflecting the influence of similarities in both host genetics and the environment on the microbiota..