One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficient material in a consistent and effective way using in vitro cell models. Although the production of EVs in bioreactors maximizes EV yield in comparison to conventional cell cultures, the impact of their cell growth conditions on EVs has not yet been established. In this study, we grew two prostate cancer cell lines, PC-3 and VCaP, in conventional cell culture dishes and in two-chamber bioreactors to elucidate how the growth environment affects the EV characteristics. Specifically, we wanted to investigate the growth condition-dependent differences by non-targeted metabolite profiling using liquid chromatography-mass spectrometry (LC-MS) analysis. EVs were also characterized by their morphology, size distribution, and EV protein marker expression, and the EV yields were quantified by NTA. The use of bioreactor increased the EV yield >100 times compared to the conventional cell culture system. Regarding morphology, size distribution and surface markers, only minor differences were observed between the bioreactor-derived EVs (BR-EVs) and the EVs obtained from cells grown in conventional cell cultures (C-EVs). In contrast, metabolomic analysis revealed statistically significant differences in both polar and non-polar metabolites when the BR-EVs were compared to the C-EVs. The results show that the growth conditions markedly affected the EV metabolite profiles and that metabolomics was a sensitive tool to study molecular differences of EVs. We conclude that the cell culture conditions of EV production should be standardized and carefully detailed in publications and care should be taken when EVs from different production platforms are compared with each other for systemic effects.

Metabolomics analysis generates vast arrays of data, necessitating comprehensive workflows involving expertise in analytics, biochemistry and bioinformatics in order to provide coherent and high-quality data that enable discovery of robust and biologically significant metabolic findings. In this protocol article, we introduce notame, an analytical workflow for non-targeted metabolic profiling approaches, utilizing liquid chromatography–mass spectrometry analysis. We provide an overview of lab protocols and statistical methods that we commonly practice for the analysis of nutritional metabolomics data. The paper is divided into three main sections: the first and second sections introducing the background and the study designs available for metabolomics research and the third section describing in detail the steps of the main methods and protocols used to produce, preprocess and statistically analyze metabolomics data and, finally, to identify and interpret the compounds that have emerged as interesting.

Alterations in the gut microbiota composition have been associated with a range of neurodevelopmental, neurodegenerative, and neuropsychiatric disorders. The gut microbes transform and metabolize dietary- and host-derived molecules generating a diverse group of metabolites with local and systemic effects. The bi-directional communication between brain and the microbes residing in the gut, the so-called gut–brain axis, consists of a network of immunological, neuronal, and endocrine signaling pathways. Although the full variety of mechanisms of the gut–brain crosstalk is yet to be established, the existing data demonstrates that a single metabolite or its derivatives are likely among the key inductors within the gut–brain axis communication. However, more research is needed to understand the molecular mechanisms underlying how gut microbiota associated metabolites alter brain functions, and to examine if different interventional approaches targeting the gut microbiota could be used in prevention and treatment of neurological disorders, as reviewed herein. Abbreviations: 4-EPS 4-ethylphenylsulfate; 5-AVA(B) 5-aminovaleric acid (betaine); Aβ Amyloid beta protein; AhR Aryl hydrocarbon receptor; ASD Autism spectrum disorder; BBB Blood–brain barrier; BDNF Brain-derived neurotrophic factor; CNS Central nervous system; GABA ɣ-aminobutyric acid; GF Germ-free; MIA Maternal immune activation; SCFA Short-chain fatty acid; 3M-4-TMAB 3-methyl-4-(trimethylammonio)butanoate; 4-TMAP 4-(trimethylammonio)pentanoate; TMA(O) Trimethylamine(- N -oxide); TUDCA Tauroursodeoxycholic acid; ZO Zonula occludens proteins

Background Accumulating evidence is supporting the protective effect of whole grains against several chronic diseases. Simultaneously, our knowledge is increasing on the impact of gut microbiota on our health and on how diet can modify the composition of our bacterial cohabitants. Herein, we studied C57BL/6 J mice fed with diets enriched with rye bran and wheat aleurone, conventional and germ-free C57BL/6NTac mice on a basal diet, and the colonic fermentation of rye bran in an in vitro model of the human gastrointestinal system. We performed 16S rRNA gene sequencing and metabolomics on the study samples to determine the effect of bran-enriched diets on the gut microbial composition and the potential contribution of microbiota to the metabolism of a novel group of betainized compounds. Results The bran-enriched study diets elevated the levels of betainized compounds in the colon contents of C57BL/6 J mice. The composition of microbiota changed, and the bran-enriched diets induced an increase in the relative abundance of several bacterial taxa, including Akkermansia , Bifidobacterium , Coriobacteriaceae , Lactobacillus , Parasutterella , and Ruminococcus , many of which are associated with improved health status or the metabolism of plant-based molecules. The levels of betainized compounds in the gut tissues of germ-free mice were significantly lower compared to conventional mice. In the in vitro model of the human gut, the production of betainized compounds was observed throughout the incubation, while the levels of glycine betaine decreased. In cereal samples, only low levels or trace amounts of other betaines than glycine betaine were observed. Conclusions Our findings provide evidence that the bacterial taxa increased in relative abundance by the bran-based diet are also involved in the metabolism of glycine betaine into other betainized compounds, adding another potential compound group acting as a mediator of the synergistic metabolic effect of diet and colonic microbiota. Electronic supplementary material The online version of this article (10.1186/s40168-019-0718-2) contains supplementary material, which is available to authorized users.

Novel betainized compounds in humans are associated with diets rich in whole grains, and they improve insulin resistance and insulin secretion. These results suggest that these novel compounds may contribute to the beneficial effects of whole grain-rich diets. The studies were registered at clinicaltrials.gov as NCT00945854 (Naples) and NCT00573781 (Kuopio).