Genomic instability in an interspecific hybrid of the genus Saccharomyces: a matter of adaptability.

Ancient events of polyploidy have been linked to huge evolutionary leaps in the tree of life, while increasing evidence shows that newly established polyploids have adaptive advantages in certain stress conditions compared to their relatives with a lower ploidy. The genus Saccharomyces is a good model for studying such events, as it contains an ancient whole-genome duplication event and many sequenced Saccharomyces cerevisiae are, evolutionary speaking, newly formed polyploids. Many polyploids have unstable genomes and go through large genome erosions; however, it is still unknown what mechanisms govern this reduction. Here, we sequenced and studied the natural S. cerevisiae × Saccharomyces kudriavzevii hybrid strain, VIN7, which was selected for its commercial use in the wine industry. The most singular observation is that its nuclear genome is highly unstable and drastic genomic alterations were observed in only a few generations, leading to a widening of its phenotypic landscape. To better understand what leads to the loss of certain chromosomes in the VIN7 cell population, we looked for genetic features of the genes, such as physical interactions, complex formation, epistatic interactions and stress responding genes, which could have beneficial or detrimental effects on the cell if their dosage is altered by a chromosomal copy number variation. The three chromosomes lost in our VIN7 population showed different patterns, indicating that multiple factors could explain the mechanisms behind the chromosomal loss. However, one common feature for two out of the three chromosomes is that they are among the smallest ones. We hypothesize that small chromosomes alter their copy numbers more frequently as a low number of genes is affected, meaning that it is a by-product of genome instability, which might be the chief driving force of the adaptability and genome architecture of this hybrid.

A Foodomics Approach: CE-MS for Comparative Metabolomics of Colon Cancer Cells Treated with Dietary Polyphenols.

The potential of capillary electrophoresis-mass spectrometry (CE-MS) for metabolomics is demonstrated through the analysis of metabolites from human HT29 colon cancer cells treated and non-treated with dietary polyphenols. Prior to CE-MS analysis, four different metabolite purification strategies are investigated. Namely, the results obtained after methanol deproteinization, ultrafiltration, and two solid-phase extraction methods using C18 and polymer-based cartridges are described. These generic methods can have broad applications to analyze metabolites in a large variety of matrices and fields, including the new Foodomics area.

Early Antiretroviral Therapy Preserves Functional Follicular Helper T and HIV-Specific B Cells in the Gut Mucosa of HIV-1-Infected Individuals.

HIV-1 infection is associated with B cell dysregulation and dysfunction. In HIV-1-infected patients, we previously reported preservation of intestinal lymphoid structures and dendritic cell maturation pathways after early combination antiretroviral therapy (e-ART), started during the acute phase of the infection, compared with late combination antiretroviral therapy started during the chronic phase. In this study, we investigated whether the timing of combination antiretroviral therapy initiation was associated with the development of the HIV-1-specific humoral response in the gut. The results showed that e-ART was associated with higher frequencies of functional resting memory B cells in the gut. These frequencies correlated strongly with those of follicular Th cells in the gut. Importantly, frequencies of HIV-1 Env gp140-reactive B cells were higher in patients given e-ART, in whom gp140-reactive IgG production by mucosal B cells increased after stimulation. Moreover, IL-21 release by PBMCs stimulated with HIV-1 peptide pools was greater with e-ART than with late combination antiretroviral therapy. Thus, early treatment initiation helps to maintain HIV-1-reactive memory B cells in the gut as well as follicular Th cells, whose role is crucial in the development of potent affinity-matured and broadly neutralizing Abs.

An Ultrahigh-Performance Liquid Chromatography-Time-of-Flight Mass Spectrometry Metabolomic Approach to Studying the Impact of Moderate Red-Wine Consumption on Urinary Metabolome.

Moderate red-wine consumption has been widely described to exert several benefits in human health. This is mainly due to its unique content of bioactive polyphenols, which suffer several modifications along their pass through the digestive system, including microbial transformation in the colon and phase-II metabolism, until they are finally excreted in urine and feces. To determine the impact of moderate wine consumption in the overall urinary metabolome of healthy volunteers ( n = 41), samples from a red-wine interventional study (250 mL/day, 28 days) were investigated. Urine (24 h) was collected before and after intervention and analyzed by an untargeted ultrahigh-performance liquid chromatography-time-of-flight mass spectrometry metabolomics approach. 94 compounds linked to wine consumption, including specific wine components (tartaric acid), microbial-derived phenolic metabolites (5-(dihydroxyphenyl)-γ-valerolactones and 4-hydroxyl-5-(phenyl)-valeric acids), and endogenous compounds were identified. Also, some relationships between parallel fecal and urinary metabolomes are discussed.

GC-MS based metabolomics of colon cancer cells using different extraction solvents.

The increasing incidence of colorectal cancer enforces the development of novel methodologies and protocols to deepen in the molecular mechanisms that govern disease pathophysiological events. The aim of this work is to deepen in the optimum metabolite extraction protocol from adherent mammalian cells of colon cancer for high throughput metabolomics using gas chromatography coupled to mass spectrometry (GC-MS). GC-MS results showed that metabolic information obtained from colon cancer cells was highly dependent on metabolite extraction selection, which at the same time is extremely influenced by the analytical platform. A further purpose of this investigation is to uncover an unexplored portion of HT-29 colon cancer cells metabolome, complementary to other already explored by CE-MS and LC-MS methods. At this respect, a total of 150 metabolites were identified in HT-29 colon cancer cells by GC-MS. The extraction protocol with acetonitrile-isopropanol-water was the most appropriate for fatty acids and related pathways analysis. Most of the metabolites involved in pathways of amino acids, glutathione, amino sugars and other polar metabolites were better extracted with acidified water, although water extraction showed the best overall reproducibility. Although pathways involving nitrogenous bases could be investigated using organic or aqueous extracts, a higher number of metabolites involved in these pathways were identified in the aqueous extracts. In addition, metabolite extraction protocol was observed to be crucial for the determination of potentially interesting clusters of metabolites.

Lipidomics Insights in Health and Nutritional Intervention Studies.

Lipids are among the major components of food and constitute the principal structural biomolecules of human body together with proteins and carbohydrates. Lipidomics encompasses the investigation of the lipidome, defined as the entire spectrum of lipids in a biological system at a given time. Among metabolomics technologies, lipidomics has evolved due to the relevance of lipids in nutrition and their well-recognized roles in health. Mass spectrometry advances have greatly facilitated lipidomics, but owing to the complexity and diversity of the lipids, lipidome purification and analysis are still challenging. This review focuses on lipidomics strategies, applications, and achievements of studies related to nutrition and health research.

RNAseq-based transcriptome comparison of Saccharomyces cerevisiae strains isolated from diverse fermentative environments.

Transcriptome analyses play a central role in unraveling the complexity of gene expression regulation in Saccharomyces cerevisiae. This species, one of the most important microorganisms for humans given its industrial applications, shows an astonishing degree of genetic and phenotypic variability among different strains adapted to specific environments. In order to gain novel insights into the Saccharomyces cerevisiae biology of strains adapted to different fermentative environments, we analyzed the whole transcriptome of three strains isolated from wine, flor wine or mezcal fermentations. An RNA-seq transcriptome comparison of the different yeasts in the samples obtained during synthetic must fermentation highlighted the differences observed in the genes that encode mannoproteins, and in those involved in aroma, sugar transport, glycerol and alcohol metabolism, which are important under alcoholic fermentation conditions. These differences were also observed in the physiology of the strains after mannoprotein and aroma determinations. This study offers an essential foundation for understanding how gene expression variations contribute to the fermentation differences of the strains adapted to unequal fermentative environments. Such knowledge is crucial to make improvements in fermentation processes and to define targets for the genetic improvement or selection of wine yeasts.

Plasma metabolome and skin proteins in Charcot-Marie-Tooth 1A patients.

OBJECTIVE: Charcot-Marie-Tooth 1A (CMT1A) disease is the most common inherited neuropathy that lacks of therapy and of molecular markers to assess disease severity. Herein, we have pursued the identification of potential biomarkers in plasma samples and skin biopsies that could define the phenotype of CMT1A patients at mild (Mi), moderate (Mo) and severe (Se) stages of disease as assessed by the CMT neuropathy score to contribute to the understanding of CMT pathophysiology and eventually inform of the severity of the disease. METHODS: We have used: (i) a high-throughput untargeted metabolomic approach of plasma samples in a cohort of 42 CMT1A patients and 15 healthy controls (CRL) using ultrahigh liquid chromatography coupled to mass spectrometry and (ii) reverse phase protein microarrays to quantitate the expression of some proteins of energy metabolism and of the antioxidant response in skin biopsies of a cohort of 70 CMT1A patients and 13 healthy controls. RESULTS: The metabolomic approach identified 194 metabolites with significant differences among the four groups (Mi, Mo, Se, CRL) of samples. A multivariate Linear Discriminant Analysis model using 12 metabolites afforded the correct classification of the samples. These metabolites indicate an increase in protein catabolism and the mobilization of membrane lipids involved in signaling inflammation with severity of CMT1A. A concurrent depletion of leucine, which is required for the biogenesis of the muscle, is also observed in the patients. Protein expression in skin biopsies indicates early loss of mitochondrial and antioxidant proteins in patients' biopsies. CONCLUSION: The findings indicate that CMT1A disease is associated with a metabolic state resembling inflammation and sarcopenia suggesting that it might represent a potential target to prevent the nerve and muscle wasting phenotype in these patients. The observed changes in metabolites could be useful as potential biomarkers of CMT1A disease after appropriate validation in future longitudinal studies.

Capillary Electrophoresis in Food and Foodomics.

Quality and safety assessment as well as the evaluation of other nutritional and functional properties of foods imply the use of robust, efficient, sensitive, and cost-effective analytical methodologies. Among analytical technologies used in the fields of food analysis and foodomics, capillary electrophoresis (CE) has generated great interest for the analyses of a large number of compounds due to its high separation efficiency, extremely small sample and reagent requirements, and rapid analysis. The introductory section of this chapter provides an overview of the recent applications of capillary electrophoresis (CE) in food analysis and foodomics. Relevant reviews and research articles on these topics are tabulated including papers published in the period 2011-2014. In addition, to illustrate the great capabilities of CE in foodomics the chapter describes the main experimental points to be taken into consideration for a metabolomic study of the antiproliferative effect of carnosic acid (a natural diterpene found in rosemary) against HT-29 human colon cancer cells.

Anti-HI can cause a severe delayed hemolytic transfusion reaction with hyperhemolysis in sickle cell disease patients.

BACKGROUND: Delayed hemolytic transfusion reaction (DHTR) is a life-threatening condition in sickle cell disease (SCD) patients that is frequently complicated by hyperhemolysis. Antibodies resulting from antigen disparity between donors of European ancestry and patients of African ancestry are common, but situations involving antibodies not classically of clinical significance are also encountered. Anti-HI is generally considered to be an innocuous naturally occurring antibody. STUDY DESIGN AND METHODS: We describe two cases of hyperhemolysis with anti-HI and provide details of the reported cases. RESULTS: Both SCD patients were polyimmunized and belonged to blood group B. They developed anti-HI that was reactive at 37°C, after the transfusion of group O red blood cell units matched for all known and produced antibodies classically considered to be clinically significant. Both patients developed DHTR with hyperhemolysis. In the first case, a pregnant woman, a second transfusion was unavoidable and the patient died from cardiac arrest. The state of the second patient improved without the need for further transfusion. CONCLUSION: Three other cases of DHTR with anti-HI have been described in the literature in SCD patients. The two additional cases reported here definitively demonstrate that anti-HI is dangerous in SCD patients. As a result, ABO-identical matching (including A1 status) must be considered in SCD patients with anti-HI.

Anionic metabolite profiling by capillary electrophoresis-mass spectrometry using a noncovalent polymeric coating. Orange juice and wine as case studies.

In several metabolomic studies, it has already been demonstrated that capillary electrophoresis hyphenated to mass spectrometry (CE-MS) can detect an important group of highly polar and ionized metabolites that are overseen by techniques such as NMR, LC-MS and GC-MS, providing complementary information. In this work, we present a strategy for anionic metabolite profiling by CE-MS using a cationic capillary coating. The polymer, abbreviated as PTH, is composed of a poly-(N,N,N',N'-tetraethyldiethylenetriamine, N-(2-hydroxypropyl) methacrylamide, TEDETAMA-co-HPMA (50:50) copolymer. A CE-MS method based on PTH-coating was optimized for the analysis of a group of 16 standard anionic metabolites. Separation was achieved within 12min, with high separation efficiency (up to 92,000 theoretical plates per meter), and good repeatability, namely, relative standard deviation values for migration times and peak areas were below 0.2 and 2.1%, respectively. The optimized method allowed the detection of 87 metabolites in orange juice and 142 metabolites in red wine, demonstrating the good possibilities of this strategy for metabolomic applications.

Recent advances in the application of capillary electromigration methods for food analysis and Foodomics.

This review work presents and discusses the main applications of capillary electromigration methods in food analysis and Foodomics. Papers that were published during the period February 2013-February 2015 are included following the previous review by Garcia-Cañas et al. (Electrophoresis, 2014, 35, 147-169). Analysis by CE of a large variety of food-related molecules with different chemical properties, including amino acids, hazardous amines, peptides, proteins, phenols, polyphenols, lipids, carbohydrates, DNAs, vitamins, toxins, contaminants, pesticides, residues, food additives, as well as small organic and inorganic compounds. This work includes recent results on food quality and safety, nutritional value, storage, bioactivity, as well as applications of CE for monitoring food processing. The use, among other CE developments, of microchips, CE-MS, and chiral CE in food analysis and Foodomics is also discussed.

Recent advances and applications of metabolomics to investigate neurodegenerative diseases.

Metabolomics is gaining an important role in the investigation of neurological pathologies such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis which are characterized by the absence of reliable diagnostic markers. The magnitude of emotional, physical, and financial burden related to these three devastating pathologies can be deduced considering that nearly 20 million people worldwide suffer from these three pathologies. In this chapter, an overview of the recent advances and applications of metabolomics to investigate these major neurodegenerative diseases will be presented. Metabolomics strategies are now being developed to map potential perturbations in biochemical pathways linked to neurodegeneration. Going further, there is more and more evidence supporting the concept that these pathologies can begin years or even decades before the onset of clinical symptoms, and thus, metabolomics is also increasingly being used to discover preclinical biomarkers of these neurological diseases.

The role of direct high-resolution mass spectrometry in foodomics.

Foodomics has been defined as a global discipline in which advanced analytical techniques and bioinformatics are combined to address different questions in food science and nutrition. There is a growing number of works on the development and application of non-targeted omics methods in foodomics, which reflects that this emerging discipline is already considered by the scientific community to be a valuable approach to assess food safety, quality, and traceability as well as for the study of the links between food and health. As a result, there is a clear need for more rapid, high-throughput MS approaches for developing and applying non-targeted studies. Nowadays, direct MS analysis is one of the main choices to achieve high throughput, generating a set of information from the largest possible number of samples in a fast and straightforward way. The use of high- and ultrahigh-resolution MS greatly improves the analytical performance and offers a good combination of selectivity and sensitivity. By using a range of methods for direct sample introduction/desorption/ionization, high-throughput and non-target analysis of a variety of samples can be obtained in a few seconds by HRMS analysis. In this review, a general overview is presented of the main characteristics of direct HRMS-based approaches and their principal applications in foodomics.

Potential of prodendronic polyamines with modulated segmental charge density as novel coating for fast and efficient analysis of peptides and basic proteins by CE and CE-MS.

In this work, the suitability of a new polymer family has been investigated as capillary coatings for the analysis of peptides and basic proteins by CE. This polymer family has been designed to minimize or completely prevent protein-capillary wall interactions and to modify the EOF. These coating materials are linear polymeric chains bearing as side cationizable moiety a dentronic triamine derived from N,N,N',N'-tetraethyldiethylenetriamine (TEDETA), which is linked to the backbone through a spacer (unit labeled as TEDETAMA). Four different polymers have been prepared and evaluated: a homopolymer which comprised only of those cationizable repetitive units of TEDETAMA, and three copolymers that randomly incorporate TEDETAMA together with neutral hydrosoluble units of N-(2-hydroxypropyl) methacrylamide (HPMA) at different molar percentages (25:75, 50:50 and 75:25). It has been demonstrated that the composition of the copolymers influences the EOF and therefore the separation of the investigated biopolymers. Among the novel polymers studied, poly-(TEDETAMA-co-HPMA) 50:50 copolymer was successfully applied as coating material of the inner capillary surface in CE-UV and CE-MS, providing EOF reversing together with fast and efficient baseline separation of peptides and basic proteins. Finally, the feasibility of the polymer-coated capillary was shown through the analysis of lysozyme in a cheese sample.

Metabolomics of adherent mammalian cells by capillary electrophoresis-mass spectrometry: HT-29 cells as case study.

In this work, the optimization of an effective protocol for cell metabolomics is described with special emphasis in the sample preparation and subsequent analysis of intracellular metabolites from adherent mammalian cells by capillary electrophoresis-mass spectrometry. As case study, colon cancer HT-29 cells, a human cell model to investigate colon cancer, are employed. The feasibility of the whole method for cell metabolomics is demonstrated via a fast and sensitive profiling of the intracellular metabolites HT-29 cells by capillary electrophoresis-time-of-flight mass spectrometry (CE-TOF MS). The suitability of this methodology is further corroborated through the examination of the metabolic changes in the polyamines pathway produced in colon cancer HT-29 cells by difluoromethylornithine (DFMO), a known potent ornithine decarboxylase inhibitor. The selection of the optimum extraction conditions allowed a higher sample volume injection that led to an increase in CE-TOF MS sensitivity. Following a non-targeted metabolomics approach, 10 metabolites (namely, putrescine, ornithine, gamma-aminobutyric acid (GABA), oxidized and reduced glutathione, 5'-deoxy-5'-(methylthio)adenosine, N-acetylputrescine, cysteinyl-glycine, spermidine and an unknown compound) were found to be significantly altered by DFMO (p<0.05) in HT-29 cells. In addition to the effect of DFMO on polyamine metabolism, minor modifications of other metabolic pathways (e.g., related to intracellular thiol redox state) were observed.

A fully automated method for simultaneous determination of aflatoxins and ochratoxin A in dried fruits by pressurized liquid extraction and online solid-phase extraction cleanup coupled to ultra-high-pressure liquid chromatography-tandem mass spectrometry.

According to current demands and future perspectives in food safety, this study reports a fast and fully automated analytical method for the simultaneous analysis of the mycotoxins with high toxicity and wide spread, aflatoxins (AFs) and ochratoxin A (OTA) in dried fruits, a high-risk foodstuff. The method is based on pressurized liquid extraction (PLE), with aqueous methanol (30%) at 110 °C, of the slurried dried fruit and online solid-phase extraction (online SPE) cleanup of the PLE extracts with a C18 cartridge. The purified sample was directly analysed by ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) for sensitive and selective determination of AFs and OTA. The proposed analytical procedure was validated for different dried fruits (vine fruit, fig and apricot), providing method detection and quantification limits much lower than the AFs and OTA maximum levels imposed by EU regulation in dried fruit for direct human consumption. Also, recoveries (83-103%) and repeatability (RSD < 8, n = 3) meet the performance criteria required by EU regulation for the determination of the levels of mycotoxins in foodstuffs. The main advantage of the proposed method is full automation of the whole analytical procedure that reduces the time and cost of the analysis, sample manipulation and solvent consumption, enabling high-throughput analysis and highly accurate and precise results.

Faecal metabolomic fingerprint after moderate consumption of red wine by healthy subjects.

Faecal metabolome contains information on the metabolites found in the intestine, from which knowledge about the metabolic function of the gut microbiota can be obtained. Changes in the metabolomic profile of faeces reflect, among others, changes in the composition and activity of the intestinal microorganisms. In an effort to improve our understanding of the biological effects that phenolic compounds (including red wine polyphenols) exert at the gut level, in this foodomic study we have undertaken a metabolome characterization of human faeces after moderate consumption of red wine by healthy subjects for 4 weeks. Namely, a nontargeted metabolomic approach based on the use of UHPLC-TOF MS was developed to achieve the maximum metabolite information on 82 human faecal samples. After data processing and statistical analysis, 37 metabolites were related to wine intake, from which 20 could be tentatively or completely identified, including the following: (A) wine compounds, (B) microbial-derived metabolites of wine polyphenols, and (C) endogenous metabolites and/or others derived from other nutrient pathways. After wine consumption, faecal metabolome was fortified in flavan-3-ols metabolites. Also, of relevance was the down regulation of xanthine and bilirubin-derived metabolites such as urobilinogen and stercobilin after moderate wine consumption. As far as we know, this is the first study of the faecal metabolome after wine intake.

Metabolomics of genetically modified crops.

Metabolomic-based approaches are increasingly applied to analyse genetically modified organisms (GMOs) making it possible to obtain broader and deeper information on the composition of GMOs compared to that obtained from traditional analytical approaches. The combination in metabolomics of advanced analytical methods and bioinformatics tools provides wide chemical compositional data that contributes to corroborate (or not) the substantial equivalence and occurrence of unintended changes resulting from genetic transformation. This review provides insight into recent progress in metabolomics studies on transgenic crops focusing mainly in papers published in the last decade.