Forced expression of four transcription factors are sufficient to reprogram mouse embryonic fibroblasts (MEFs) directly to induced renal tubular epithelial cells (iRECs). These cells have been characterized as tubule cells by transcriptomic, morphological and functional studies. Recently, we analyzed iRECs by untargeted metabolomics and thereby proved their cellular identity. Hence, changes by application of a common nephrotoxic agent confirmed many alterations occurring in vivo [in submission]. In this study, we investigated the impact of glucose on MEFs and iRECs by conducting an untargeted gas chromatography/mass spectrometry based profiling with high and low glucose concentrations. Whereas accumulating in MEFs used for reprogramming, glucose was efficiently metabolized by glycolysis and citric acid cycle in iRECs but also an increase in the polyol pathway was observed. The activation of this pathway and a consequent generation of reactive oxygen species is a common phenomenon in diabetic complications such as diabetic retinopathy, neuropathy and nephropathy (DN). Thus, iRECs transpired to be an excellent in vitro model for tubule damage, an aspect of DN being overshadowed by the glomerular focus. The possibility to generate iRECs also from human fibroblasts holds great potential in patient specific testing for exogenous challenges in general.

   Ceratonia siliqua L. Fabaceae, commonly known as the carob tree, is native to the eastern Mediterranean countries and its products are widely used in the diet of people living in Mediterranean Europe, Middle East and North Africa. Carobs are considered to be of high nutritional value, as they are virtually fat-free, rich in proteins, antioxidants, vitamins and contain several important minerals. Different types of carob products are available in the local market, such as carob syrup, powder, flour, snack, cream, etc. However, the potential positive health effects of carob-containing products are largely unknown and have not been extensively studied. The aim of this study was to determine significant urine and fecal metabolome alterations in 8 rats treated with carob powder for 15 days as compared to 8 non-treated ones (controls) using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and to underly specific metabolites that changed according to the treatment.
   Urine and fecal samples were collected in five time points during a 15 day period of treatment with carob powder throughout water consumption (10 g powder / L). A targeted HILIC-UPLC-MS/MS method was applied for the determination of 101 polar metabolites (sugars, amino acids, organic acids, amines, etc) in a single run of 40 min in both rat urine and feces. Chromatographic separation was performed on an Aquity BEH amide column (2.1 x 100 mm, i.d. 1.7 μm); the mobile phase was consisted of A: Acetonitrile:H₂O 95:5 v/v (+ 10 mM ammonium formate) and B: H₂O:Acetonitrile 70:30 v/v (+10 mM ammonium formate). The solvents flow rate was set at 0.5 mL/min. Mass spectrometry parameters were optimized for each of the 101 pre-selected analytes.
   Approximately 55 urinary and fecal metabolites were identified in both specimens. Data were further processed with multivariate (SIMCA 13) and univariate statistics (ANOVA). The differentiation of treated rats and controls was highlighted using discriminant multivariate models. 

Acknowledgements: The authors would like to thank the “Black Gold” project financially supported by the University of Cyprus.

The success of therapeutic proteins such as insulin has led to the massive recognition of biological medical products as highly effective clinical drugs. As the use of biologics gains popularity, in industrial biotechnology there is a push to maximise their production. The ovary cells of the Chinese hamster (CHO cells) are the most common production cell line, however - like most mammalian cells - they are very inefficient in producing desired compounds. Culture bioengineering can improve the yield, but identifying the optimal interventions is usually expensive and time-consuming. Machine learning coupled with computational modelling of CHO cells has the potential to effectively elucidate optimal bioengineering steps towards improved production of therapeutic metabolites and proteins.

In this study, we combine machine learning techniques with gene expression profiling and metabolic modelling to estimate lactate production in CHO cell cultures. We train our poly-omic method using gene expression data from varying conditions and associated reaction rates in metabolic pathways, reconstructed in silico. The poly-omic reconstruction is performed by generating a set of condition-specific metabolic models, specifically optimised for lactate export estimation. To validate our approach, we compare predicted lactate production with experimentally measured yields in a cross-validation setting. Importantly, we observe that integration of metabolic information significantly improves the predictive ability of the model when compared to gene expression alone. Our poly-omic method can therefore accurately predict whether CHO cells have optimal conditions for producing target therapeutic compounds and represents a promising tool for the optimisation of the culture engineering process.

Currently, by-products produced from the agro-industries practices could be a key source of functional and bioactive components; which could be used due to their nutritional and added- value properties. New aspects concerning the use of these wastes as by-products in production of food additives or supplements have recently gained increasing interest. In this sense, the present study describes an in-depth characterization of phytochemical compounds from hydro-methanolic extract of broad beans pods by using high-performance liquid chromatography linked with quadrupole-time-of-flight tandem mass spectrometry. The utilized analytical technique provided the tentative identification more than 140 phenolic and other phytochemical compounds in the extract, most of which have not been described up to now in broad beans pods. Thus, more than 90 phytochemicals (phenolic acids, flavonoids, iridoids, lignans, and terpenoids derivatives) are reported herein in broad beans pods for the first time. The data obtained demonstrate that vegetal by-product from the food industry could potentially be utilized as a promising source of bio-active ingredients to design new nutraceuticals and functional foods with a valuable future market. Moreover, the got data may form a basis for future bioavailability and quantitative studies, which will be a next step in our research work.

Micromeria fruticosa (Lamiaceae), also known as White micromeria, is a widely distributed dwarf perennial herb, indigenous to Palestine and the Mediterranean region. M. fruticosa is found in rocky areas, and its fresh or dried herbal tissues are commonly used as a flavoring; when crushed these tissues emit a peppermint-like odor.  The tisane/infusion of M. fruticosa leaves and areal parts are used to relieve stomachaches and heart disorders, colds, diarrhea, eye infections, weariness, wounds, hypertension, and exhaustion. M. fruticosa has also been reported to have anti-inflammatory, antioxidant, antimicrobial and gastroprotective activities. Although many attempts have studied the essential oil composition of some Micromeria species, the study of other phytochemicals and secondary metabolites is still scarce.  The aim of the present study was to investigate the metabolites in the infusion of M. fruticosa. Samples of M. fruticosa were macerated with aqueous methanol, and following centrifugation the supernatant was collected. The analysis of the phytochemicals from M. fruticosa infusion was carried out on an Agilent 1200 series LC equipped with an Agilent Zorbax C18 column. Acidic water and acetonitrile were used as mobile phases. The HPLC system was coupled to Q-TOF-MS equipped with an ESI source operated in the negative ion mode, over the range of m/z 50-1100. The analysis of the infusion of M. fruticosa leaves by means of HPLC-DAD-ESI-MS² idetified more than 160 primary and secondary metabolites (i.e., sugars, peptides, phenolic acids, flavonoids glycosides, lignans, and terpenoids derivatives), highlighting the importance of this plant as a functional food and as a promising source of bioactive phytochemicals.

Despite being well absorbed and displaying a range of biological properties, dietary terpenoids have been little studied. Better knowledge about their metabolism will help understanding the health effects of plant foods and provide information on the food metabolome. As part of the FoodBAll project we are investigating the metabolism of terpenes, identifying metabolites and biotransformations involved in their metabolism.

PhytoHub (database that compiles all known metabolites of dietary phytochemicals, including terpenes) and Nexus Meteor (in silico prediction of metabolism), were used to identify biotransformations involved in the metabolism of dietary terpenoids. Selected biotransformations were used to predict the metabolism of camphene, camphor, carvacrol, carvone, caryophyllene, 1,4-cineole, 1,8-cineole, citral, citronellal, cuminaldehyde, p-cymene, fenchone, geraniol, limonene, linalool, menthol, myrcene, nootkatone, perillyl alcohol, pinene, pulegone, terpinen-4-ol and thymol. The metabolism prediction tool “BioTransformer” that is under development at Dr. Wishart’s lab was also used to generate predicted metabolites of the mentioned compounds. The urine of Wistar rats was collected before and after 5 days of the exposure to the dietary monoterpenes (given in the dose of 0.05% of diet). Untargeted metabolomics analysis was performed in urine using high-resolution mass spectrometry (UPLC-QToF).

We identified twenty-two enzymatic reactions involved in the synthesis of terpenoid metabolites described in the literature. In average, 10 metabolites per compound were identified in rat urine, including new and known ones. Identification of metabolites was based on monoisotopic mass and formula match, presence of adducts and specific mass losses indicative of glucuronidation, sulphation and conjugation to amino acids. Validation of identification is being done using MS/MS experiments.

The combination of in silico predictions and in vivo experiment allowed the identification of known and new metabolites of different dietary terpenoids. Predicted metabolites of terpenes will be added in databases such as PhytoHub to complement the database of known metabolites. The validations of the metabolism predictions are helping the development of BioTransformer.

Funding: Grant from the Agence Nationale de la Recherche (#ANR-14-HDHL-0002-02) for FoodBAll project (JPI HDHL). JF is an AgreenSkills fellow.

Coenzymes of cellular redox reactions and cellular energy, as well as antioxidants mediate biochemical reactions fundamental to the functioning of all living cells. Conventional analysis methods lack the opportunity to evaluate these important coenzymes including coenzymes of redox reactions, oxidized/reduced nicotinamide adenine dinucleotide (NAD⁺ and NADH) and nicotinamide adenine dinucleotide phosphate (NADP⁺ and NADPH); coenzymes of energy, adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP); and antioxidants, oxidized/reduced glutathione (GSSG and GSH). As an important alternative to tissue and serum/plasma metabolomics, we show here that a simple ¹H NMR experiment can simultaneously measure the coenzymes and antioxidants in tissue and whole human blood. Owing to their unstable nature, or low concentrations, many coenzymes evade detection using established sample preparation protocols. Here we developed new methods to detect these interesting species without affecting other metabolites. Identities of the coenzymes and antioxidants in tissue and blood NMR spectra were established by combining 1D/2D NMR techniques, chemical shift databases, pH measurements and, finally, spiking with authentic compounds. Interestingly, while none of the coenzymes and antioxidants was detected in plasma, they were abundant in whole blood due to the high concentration of red blood cells. This is the first study to report identification of major coenzymes and antioxidants, and measure them, simultaneously and with high resolution, along with a vast pool of other metabolites in tissue and blood using NMR spectroscopy. Considering that the coenzymes and antioxidants represent a sensitive measure of human health and risk for numerous diseases, the presented NMR method potentially opens new opportunities in the metabolomics field.

We present a cellular dashboard for interactive analysis of metabolomics datasets.  The tool organizes metabolomics data biologically, into individual metabolic pathways, and groupings of related pathways.  The dashboard enables the user to easily assess the activation levels of different areas of metabolism, from gross areas such as carbohydrate degradation, to specific pathways such as proline biosynthesis.

The hierarchical organization of the dashboard is its key organizing principle.  At the highest level, the dashboard is organized into four panels: Biosynthesis, Degradation, Energy Metabolism, and Other Pathways.  Each panel consists of a series of graphs, such as for amino acid biosynthesis and cofactor biosynthesis.  Each graph depicts all measurements for metabolites within that set of metabolic pathways.  The user can click on any graph to generate a new window containing an expanded panel that includes a graph for each component of the previous graph, e.g., clicking on the graph for amino acid biosynthesis produces individual graphs showing metabolite levels within every amino acid biosynthetic pathway.

Clicking on one of those amino acid graphs drills down further to produce a graph showing the levels of each individual metabolite within the pathway.  From here, users can also view the pathway diagram overlaid with metabolite data.  In this way, the user can navigate complex metabolomics data sets to identify meaningful changes that relate to the biological events under investigation.

Salinity in soils is one of the major factors that adversely affects agriculture by inhibiting plant growth, resulting in low crop productivity. Among cereal crops (eg. rice, wheat), barley (Hordeum vulgare L.) is rated as salt-tolerant, and exhibits a considerable variation in salt tolerance amongst its cultivars. Barley is a food and brewing crop, and as a glycophyte it suffers substantial yield loss when grown under saline conditions. Relatively little is currently understood of salt stress perception and responses in plant roots, which involve complex changes at the physiological, metabolic, molecular, transcriptional, and genetic levels.

We aim to develop new tools to unravel how plants respond to the perception of salt stress. Evidence is accumulating that lipid signalling is an integral part of the complex regulatory networks that plants utilize to respond to salinity through modifications of membrane lipids.  These occur through changes in activity of such enzymes as phospholipase D and diacylglycerol kinase that produce different classes of lipid and lipid-derived messengers. In addition, abiotic stress provokes enhanced production of reactive oxygen species, resulting in lipid modifications by the oxidation of lipid species.

Lipidomics analysis using liquid chromatography mass spectrometry (LC-MS) revealed that roots from tolerant and sensitive cultivars respond differently to salt stress. To investigate the modifications of lipids leading to root responses to salinity, we are using a combination of multiple approaches, such as targeted and untargeted lipidomics of barley roots. Matrix Assisted Laser Desorption Ionisation Mass Spectrometry Imaging (MALDI-MSI) was also employed to examine the spatial distribution of lipids in barley roots grown under control and saline conditions.  The combination of LC-MS and MALDI-MSI identified a large number of metabolites and lipids with a unique spatial distribution. MSI was capable of discriminating salt vs control treated roots, identifying major lipid changes under salt treatment in a spatial manner. Non-uniform spatial distribution of metabolites was observed among different barley cultivars. Major PC lipid species and carbohydrates were identified to change the most in salt treated roots compared to control. Given the lack of fundamental knowledge of the lipids involved in signalling and metabolism under saline stress, our results provide insight into novel mechanisms of how barley roots respond to salt stress.

Metabolism is central to virtually all cellular functions and contributes to a range of diseases.  A quantitative understanding of how biochemical pathways are dysregulated in the context of diseases such as cancer and metabolic syndrome is necessary to identify new therapeutic targets.  To this end we apply stable isotope tracers, mass spectrometry, and metabolic flux analysis (MFA) to study metabolism in mammalian cells, animal models, and human patients.  Using these approaches we have characterized how proliferating and differentiated cells regulate flux of glucose and amino acids into mitochondria for maintaining redox homeostasis and lipid biosynthesis. Recently, we have developed novel methods for studying pyridine nucleotide metabolism, employing 2H tracers and mass spectrometry to quantify how specific metabolic pathways are used to regenerate NADH and NADPH. To better understand how redox pathways are regulated in the cytosol and mitochondrial matrix we have generated compartment-specific enzyme reporters that exploit the neomorphic activity of mutant isocitrate dehydrogenases (IDHs). Specifically, R132H IDH1 and R172K IDH2 produce (D)2-hydroxyglutarate (2HG) in the cytosol and mitochondria, respectively. Quantitation of labeling from specifically labeled 2H tracers provides critical insights into NAD(P)H-producing pathways in each compartment. We have employed this approach to identify redox pathway regulation under hypoxia, where oxidative pentose phosphate pathway flux is upregulated to fuel reductive carboxylation. The application of MFA to cell and animal models greatly improves our ability to characterize intracellular metabolic processes, providing a mechanistic understanding of cellular physiology and metabolic function.