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The impact of diet on the microbiota composition and the role of diet in supporting optimal mental health have received much attention in the last decade. However, whether whole dietary approaches can exert psychobiotic effects is largely understudied. Thus, we investigated the influence of a psychobiotic diet (high in prebiotic and fermented foods) on the microbial profile and function as well as on mental health outcomes in a healthy human population. Forty-five adults were randomized into either a psychobiotic (n = 24) or control (n = 21) diet for 4 weeks. Fecal microbiota composition and function was characterized using shotgun sequencing. Stress, overall health and diet were assessed using validated questionnaires. Metabolic profiling of plasma, urine and fecal samples was performed. Intervention with a psychobiotic diet resulted in reductions of perceived stress (32% in diet vs. 17% in control group), but not between groups. Similarly, biological marker of stress were not affected. Additionally, higher adherence to the diet resulted in stronger decreases in perceived stress. While the dietary intervention elicited only subtle changes in microbial composition and function, significant changes in the level of 40 specific fecal lipids and urinary tryptophan metabolites were observed. Lastly, microbial volatility was linked to greater changes in perceived stress scores in those on the psychobiotic diet. These results highlight that dietary approaches can be used to reduce perceived stress in a human cohort. Using microbiota-targeted diets to positively modulate gut-brain communication holds possibilities for the reduction of stress and stress-associated disorders, but additional research is warranted to investigate underlying mechanisms, including the role of the microbiota.

Context Visceral (VAT) and subcutaneous adipose tissue (SAT) function as endocrine organs capable of influencing metabolic health across adiposity levels. Objective We aimed to investigate whether metabolites associated with VAT and SAT impact metabolic health through metabolite concentrations. Methods Analyses are based on 1790 participants from the population-based Rhineland Study. We assessed plasma levels of methionine (Met), branched-chain amino acids (BCAA), aromatic amino acids (AAA), and their metabolic downstream metabolites with liquid chromatography-mass spectrometry. VAT and SAT volumes were assessed by magnetic resonance imaging (MRI). Metabolically healthy and unhealthy phenotypes were defined using Wildman criteria. Results Metabolically unhealthy participants had higher concentrations of BCAA than metabolically healthy participants (P Conclusion Larger volumes of VAT in metabolically unhealthy individuals are associated with altered concentrations of circulating BCAA and AAA-downstream metabolites, increasing the odds of cardiometabolic risk markers. This suggests that these metabolites are involved in the mechanisms that underlie the relationship of abdominal VAT with metabolic health.

Introduction: Familial hypercholesterolemia (FH) is characterized by extremely high levels of circulating low-density lipoprotein cholesterol (LDL-C) and is caused by mutations of genes involved in LDL-C metabolism, including LDL receptor (LDLR), apolipoprotein B (APOB), or proprotein convertase subtilisin/Kexin type 9 (PCSK9). Accordingly, PCSK9 inhibitors (PCSK9i) are effective in LDL-C reduction. However, no data are available on the pleiotropic effect of PCSK9i. To this end, we performed an untargeted metabolomics approach to gather a global view on changes in metabolic pathways in patients receiving treatment with PCSK9i. Methods: Twenty-five FH patients starting treatment with PCSK-9i were evaluated by an untargeted metabolomics approach at baseline (before PCSK9i treatment) and after 12 weeks of treatment. Results: All the 25 FH subjects enrolled were on maximal tolerated lipid-lowering therapy prior to study entry. After a 12 week treatment with PCSK9i, we observed an expected significant reduction in LDL-cholesterol levels (from 201.0 ± 69.5 mg/dL to 103.0 ± 58.0 mg/dL, p &lt, 0.001). The LDL-C target was achieved in 36% of patients. After peak validation and correction, after 12 weeks of PCSK9i treatment as compared to baseline, we observed increments in creatine (p-value = 0.041), indole (p-value = 0.045), and indoleacrylic acid (p-value= 0.045) concentrations. Conversely, significant decreases in choline (p-value = 0.045) and phosphatidylcholine (p-value &lt, 0.01) together with a reduction in platelet activating factor (p-value = 0.041) were observed. Conclusions: Taking advantage of untargeted metabolomics, we first provided evidence of concomitant reductions in inflammation and platelet activation metabolites in FH patients receiving a 12 week treatment with PCSK9i.

Significance Thanks to their potent built-in adjuvanticity, bacterial outer membrane vesicles (OMVs) represent an attractive vaccine platform. However, their full-blown exploitation relies on the availability of efficient engineering strategies. This work provides strong experimental evidence that OMVs can be successfully decorated with heterologous antigens by channeling them to the lipoprotein transport machinery. Not only did lipidated antigens accumulate in the vesicular compartment at high levels but they also interfered with the acylation process of lipid A, thus substantially reducing the lipopolysaccharide-mediated reactogenicity, a major hurdle for vaccine applications. The approach was validated with 5 Staphylococcus aureus antigens, and mice immunized with engineered OMVs were completely protected against S. aureus challenge, paving the way for development of vaccines against this important pathogen., Bacterial outer membrane vesicles (OMVs) represent an interesting vaccine platform for their built-in adjuvanticity and simplicity of production process. Moreover, OMVs can be decorated with foreign antigens using different synthetic biology approaches. However, the optimal OMV engineering strategy, which should guarantee the OMV compartmentalization of most heterologous antigens in quantities high enough to elicit protective immune responses, remains to be validated. In this work we exploited the lipoprotein transport pathway to engineer OMVs with foreign proteins. Using 5 Staphylococcus aureus protective antigens expressed in Escherichia coli as fusions to a lipoprotein leader sequence, we demonstrated that all 5 antigens accumulated in the vesicular compartment at a concentration ranging from 5 to 20% of total OMV proteins, suggesting that antigen lipidation could be a universal approach for OMV manipulation. Engineered OMVs elicited high, saturating antigen-specific antibody titers when administered to mice in quantities as low as 0.2 μg/dose. Moreover, the expression of lipidated antigens in E. coli BL21(DE3)ΔompAΔmsbBΔpagP was shown to affect the lipopolysaccharide structure, with the result that the TLR4 agonist activity of OMVs was markedly reduced. These results, together with the potent protective activity of engineered OMVs observed in mice challenged with S. aureus Newman strain, makes the 5-combo-OMVs a promising vaccine candidate to be tested in clinics.

Converging data support the role of chronic low-grade inflammation in depressive symptomatology in obesity. One mechanism likely to be involved relies on the effects of inflammation on tryptophan (TRP) metabolism. While recent data document alterations in the indole pathway of TRP metabolism in obesity, the relevance of this mechanism to obesity-related depressive symptoms has not been investigated. The aim of this preliminary study was to assess the association between plasma levels of TRP and indole metabolites and depressive symptoms in 44 subjects with severe or morbid obesity, free of clinically relevant neuropsychiatric disorders. The interaction effect of inflammation, reflected in serum high-sensitive C-reactive protein (hsCRP) levels, and indoles on depressive symptoms was also determined. Higher serum levels of hsCRP and lower concentrations of TRP and indoles, particularly indole-3-carboxaldehyde (IAld), correlated with more severe depressive symptoms. Interestingly, the effect of high hsCRP levels in predicting greater depressive symptoms was potentiated by low IAld levels. These results comfort the link between inflammation, the indole pathway of TRP metabolism, and obesity-related depressive symptoms.

Monocyte recruitment after vascular injury and their migration through the vessel wall represent crucial events in the initiation, progression, and destabilization of atherosclerotic plaque. Circulating monocytes are exposed to stimuli that alter their physiological state, and among them, lipids play a key role. Several studies investigated the mechanisms by which lipids affect monocyte functions promoting coronary atherosclerotic plaque initiation, but information on the relationship between lipid composition and function of monocyte is scant. We aimed at studying the migration of circulating monocytes isolated from patients with acute myocardial infarction (AMI) at hospital presentation and investigating its correlation with cellular lipid profile. The migration of monocytes was tested using both fetal bovine serum (FBS) and autologous serum as chemoattractant stimuli. Monocyte lipid profile was evaluated through an untargeted lipidomics approach, using a liquid chromatography/time-of-flight mass spectrometry platform. We observed that AMI patients' monocytes showed a significant increase in FBS and autologous serum-mediated migration compared to controls. Moreover, a different monocyte lipidomic profile between the two study groups was detected. In particular, AMI patients' monocytes showed an altered composition in ceramides, with an increase in lactosylceramide and in phospholipids (ie, phosphatidylethanolamine and lisophosphatidylethanolamine). Of note, a positive correlation between lactosylceramide levels and monocyte migration was observed. Furthermore, the lactosylceramide synthase inhibition significantly reduced FBS-induced monocyte migration. Our results highlight the influence of lactosylceramide on the monocyte migration capacity, pointing out a new possible mechanism of lipids in the onset of atherothrombosis and, hence, in AMI.

Tryptophan and its bioactive metabolites are associated with health conditions such as systemic inflammation, cardiometabolic diseases, and neurodegenerative disorders. There are dynamic interactions among metabolites of tryptophan. The interactions between metabolites, particularly those that are strong and temporally reproducible could be of pathophysiological relevance. Using a targeted metabolomics approach, the concentration levels of tryptophan and 18 of its metabolites across multiple pathways was quantified in 24-hours urine samples at 2 time-points, age 17 years (baseline) and 18 years (follow-up) from 132 (52% female) apparently healthy adolescent participants of the DOrtmund Nutritional and Anthropometric Longitudinally Designed (DONALD) Study. In sex-specific analyses, we applied 2 network approaches, the Gaussian graphical model and Bayesian network to (1) explore the network structure for both time-points, (2) retrieve strongly related metabolites, and (3) determine whether the strongly related metabolites were temporally reproducible. Independent of selected covariates, the 2 network approaches revealed 5 associations that were strong and temporally reproducible. These were novel relationships, between kynurenic acid and indole-3-acetic acid in females and between kynurenic acid and xanthurenic acid in males, as well as known relationships between kynurenine and 3-hydroxykynurenine, and between 3-hydroxykynurenine and 3-hydroxyanthranilic acid in females and between tryptophan and kynurenine in males. Overall, this epidemiological study using network-based approaches shed new light into tryptophan metabolism, particularly the interaction of host and microbial metabolites. The 5 observed relationships suggested the existence of a temporally stable pattern of tryptophan and 6 metabolites in healthy adolescent, which could be further investigated in search of fingerprints of specific physiological states. The metabolites in these relationships may represent a multi-biomarker panel that could be informative for health outcomes.

Peels are major byproducts of the juice processing industry. Citrus peels are composed of two tissues, namely the flavedo (outer layer) and the albedo (inner layer). Here, flavedo and albedo were obtained from the peel of grapefruit cv. ‘Star ruby’, lemon cv. ‘Akragas’, and sweet orange cvs. ‘Tarocco’, ‘Sanguinello’ and ‘Moro’. Freeze dried flavedo and albedo were investigated for their functional properties, and subjected to enzyme-assisted extraction with pectinase and cellulase (concentration of 6 U g−1 DW) to extract the phenolic compounds. Albedo showed superior physicochemical characteristics than flavedo. Albedo from ‘Tarocco’ exhibited the maximum water holding capacity (10.9 ± 0.48 g/g). Grapefruit albedo revealed the highest water swelling (13.4 ± 2.62 mL/g) and oil binding (6.31 ± 2.62 g/g) capacities. As regards the enzyme-assisted extraction of phytochemicals, both enzymes enhanced the extraction of phenolics (p

Amino acid metabolites in biofluids are associated with high body mass index (BMI) and cardiometabolic abnormalities. However, prospective investigations regarding these associations are few, particularly among young individuals. Moreover, little is presently known about the impact of long-term high BMI. Using data from the DOrtmund Nutritional and Anthropometric Longitudinally Designed study (111 males and 107 females), we prospectively investigated relations between repeatedly measured urinary levels of 33 metabolites and (1) previously identified long-term BMI trajectory groups from childhood into late adolescence and (2) cardiometabolic risk markers in late adolescence–young adulthood, in sex-specific linear mixed regression models. Males with long-term overweight had lower indole-3-acetic acid when compared to others. Further, methionine, isoleucine, tryptophan, xanthurenic acid, and indole-3-carboxaldehyde were negatively associated with C-reactive protein (CRP), but 5-hydroxyindole-3-acetic acid was positively associated with CRP. No associations were observed in females. Long-term overweight from childhood into late adolescence is associated with decreased urinary levels of gut bacteria-derived indole-3-acetic acid, and several urinary amino acids, including gut bacteria-derived indole-3-carboxaldehyde are associated with elevated CRP later on in life. Taken together, our data suggest that indole metabolites, and their gut bacteria producers play potentially important roles in overweight-related inflammation.

Background & aims: Patients with cystathionine β-synthase deficiency (CBSD) exhibit high circulating levels of homocysteine and enhanced lipid peroxidation. We have characterized the plasma lipidome in CBSD patients and related lipid abnormalities with reactions underlying enhanced homocysteine levels. Methods and results: Using an ultra-high-performance liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry method, plasma lipids were determined with an untargeted lipidomics approach in 11 CBSD patients and 11 matched healthy subjects (CTRL). Compared to CTRL, CBSD patients had a higher medium and long-chain polyunsaturated fatty acids (PUFA) content in phosphatidylethanolamine (PE) and lysophosphatidylethanolamine (LPE) species (p < 0.02), and depletion of phosphatidylcholine (PC; p = 0.02) and of lysophosphatidylcholine (LPC; p = 0.003) species containing docosahexaenoic acid (DHA), suggesting impaired phosphatidylethanolamine-N-methyltransferase (PEMT) activity. PEMT converts PE into PC using methyl group by S-adenosylmethionine (SAM) thus converted in S-adenosylhomocysteine (SAH). Whole blood SAM and SAH concentrations by liquid chromatography tandem mass spectrometry were 1.4-fold (p = 0.015) and 5.3-fold (p = 0.003) higher in CBSD patients than in CTRL. A positive correlation between SAM/SAH and PC/PE ratios (r = 0.520; p = 0.019) was found. Conclusions: A novel biochemical abnormality in CBSD patients consisting in depletion of PC and LPC species containing DHA and accumulation of PUFA in PE and LPE species is revealed by this lipidomic approach. Changes in plasma SAM and SAH concentrations are associated with such phospholipid dysregulation. Given the key role of DHA in thrombosis prevention, depletion of PC species containing DHA in CBSD patients provides a new direction to understand the poor cardiovascular outcome of patients with homocystinuria.

Given to its ability to irreversibly acetylate the platelet cyclooxygenase-1 enzyme, acetylsalicylic acid (ASA) is successfully employed for the prevention of cardiovascular disease. Recently, an antitumoral effect of ASA in colorectal cancer has been increasingly documented. However, the molecular and metabolic mechanisms by which ASA exerts such effect is largely unknown. Using a new, untargeted liquid chromatography&ndash, mass spectrometry approach, we have analyzed urine samples from seven healthy participants that each ingested 100 mg of ASA once daily for 1 week. Of the 2007 features detected, 25 metabolites differing after ASA ingestion (nominal p &lt, 0.05 and variable importance in projection (VIP) score &gt, 1) were identified, and pathway analysis revealed low levels of glutamine and of metabolites involved in histidine and purine metabolisms. Likewise, consistent with an altered fatty acid &beta, oxidation process, a decrease in several short- and medium-chain acyl-carnitines was observed. An abnormal &beta, oxidation and a lower than normal glutamine availability suggests reduced synthesis of acetyl-Co-A, as they are events linked to one another and experimentally related to ASA antiproliferative effects. While giving an example of how untargeted metabolomics allows us to explore new clinical applications of drugs, the present data provide a direction to be pursued to test the therapeutic effects of ASA&mdash, e.g., the antitumoral effect&mdash, beyond cardiovascular protection.

An interesting population of ulvacean green algae was collected from a linmo-rheocrenic, thermal, mineral (chloride, sodium, sulphate) spring known as "Ain Abu Sherouf" in the Siwa Oasis, the Western Desert of Egypt. A detailed combined investigation on its morphotaxonomy, autecology, and a multilocus sequence data set including the chloroplast-encoded rbcL gene and the nuclear-encoded nrDNA SSU and ITS allowed us to identify this population as Ulva ftexuosa subsp. paradoxa (syn. Ulva paradoxa, Ulvales, Chlorophyta), although the algal thalli were in average narrower than usual in this taxon. Analysis of the molecular rbcL-ITS sequencing data demonstrated a close phylogenetic relationship of the studied population to two Ulva isolates from Japan and China and their taxonomic status was further discussed. The pigment profiling confirmed chlorophylls and carotenoids typical of the Ulvaceae (lutein, alpha/beta-carotenes, violaxanthin, and neoxanthin). Lipidomic analysis revealed the presence of the monogalactosyl diacylglycerols (MGDG), digalactosyl diacylglycerols (DGDG), sulfoquinovosyl diacylglycerols (SQDG), and diacylglyceryl N,N,N-trimethylhomoserine (DGTS) lipid classes. Presence of the plasma membrane DGTS lipids in remarkable proportion might be an adaptation to this nutrient-poor mineral spring habitat, with relatively low phosphorus values. The lower unsaturation index (UI) values of the plastidial DGDG and SQDG lipids are likely to reflect the need to maintain adequate membrane fluidity in this thermal mineral spring system. This polyphasic study not only extended our little knowledge on the distribution, autecology, and adaptive mechanisms of U. flexuosa subsp. paradoxa in the Saharan habitats of Africa, but also re-assessed and refined the taxonomic and phylogenetic affiliation of previously sampled close relatives from East Asia.

Westiellopsis prolifica, a true-branching cyanobacterium isolated from the hyperarid desert soil habitats of El-Farafra Oasis (Western Desert of Egypt), was investigated with a polyphasic approach. The main morphological characteristics of our material, together with its habitat properties, corresponded (with minor discrepancies in the dimensions) to descriptions in the literature. On the basis of molecular data of the 16S ribosomal RNA gene, the strain of W. prolifica isolated from the El-Farafra Oasis clustered with available isolates of Westiellopsis and other genera of Hapalosiphonaceae. Bio-organic screening showed the presence of thylakoid lipids characterized by low unsaturation index and short fatty acyl chains, features presumably ensuring membrane functionality at high temperatures. Carotenoid and xanthophyll composition were typical of cyanobacteria (α/β carotene 38.1%, echinenone 14.3%, zeaxanthin 13.4%, and myxoxanthophyll 8.8%). This study improved the knowledge on the distribution...

The present work reports a case study for the industrialization of a biorefinery aimed at valorizing fish processing residues in order to obtain ω-3 concentrates for the nutraceutical sector. The biorefinery includes a series of processes: oil extraction from fish waste, fish oil transesterification with ethanol, ω-3 concentration based on supercritical CO2 fractionation. The biorefinery is at first designed and then modeled by Aspen Plus™: this allows for a comprehensive picture (mass and energy flows, processing costs and costs for investment) of the overall process. In accordance with the zero-waste concept, all the biorefinery by-products are valorized: fish proteins as fishmeal, and glycerol, saturated fatty acids and short chain unsaturated fatty acids as liquid biofuel. In addition, fish oil transesterification with ethanol is also experimentally tested in different operational conditions in order to maximize fish oil conversion to fatty acid ethyl esters. A trout processing company located in Trentino Province, Italy, represents the case study. The company produces 870 t/year of fish waste. Mass balances allow estimating a production of 26.6 t/year of ω-3 rich oil (income: 1.3 M€), 160 t/year of fish proteins (income: 0.27 M€), and 160 t/year of liquid biofuel. The biofuel, fed to a 100 kWel CHP unit, allows to produce 720 MWhe/year and 870 MWhth/year, covering the total electricity consumption of the plant and providing more than 45% of the thermal energy needs. This translates into a reduction in utility costs of about 0.12 M€ and avoids the emission of 416 t/year of CO2. Finally, the total investment costs for the whole biorefinery amount to about 3.34 M€. In conclusion, this study demonstrates that the production of ω-3 rich fish oils in a bio-refinery concept could become a good opportunity for valorizing fish by-products and increasing the competitiveness of the fish processing industry.

Molecular imprinted poly(acrylamido)-derivative nanogels have shown their selectivity to bind the protein human serum transferrin (HTR) and also showed their capability for instantaneous solvent-induced modification upon the addition of acetonitrile. Integrated to matrix-assisted laser desorption/ionization time-of-flight mass analysis the HTR-imprinted solvent-responsive nanogels permitted the determination of HTR straight from serum and offered novel perspectives in targeted protein analysis.

It is known that the freshwater heterotrich ciliate Blepharisma japonicum uses five pigments called blepharismins, stored in its extrusive pigment granules, for both light perception and chemical defense against predators. In this work we focused our attention on the defensive strategies of two additional pigmented species of Blepharisma, B. stoltei and B. undulans. In particular: (1) we observed the predator–prey interactions of B. stoltei or B. undulans against one multicellular and two unicellular predators; (2) we clarified the nature of B. stoltei and B. undulans pigments by means of High Performance Liquid Chromatography (HPLC) and Liquid Chromatography Mass Spectrometry (LC-MS); and (3) we demonstrated and compared the toxicity of the purified pigments on a panel of ciliated protists, and against one metazoan predator. The results indicate that the chemical defense mechanism present in B. stoltei and B. undulans is mediated by the same five blepharismins previously characterized for B. japonicum, although produced in different proportions.

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-&gamma, 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-&gamma, 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-&gamma, 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-&gamma, 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-&gamma, valerolactone phase II conjugates.

Tryptophan and tyrosine metabolism has a major effect on human health, and disorders have been associated with the development of several pathologies. Recently, gut microbial metabolism was found to be important for maintaining correct physiology. Here, we describe the development and validation of a UHPLC-ESI-MS/MS method for targeted quantification of 39 metabolites related to tryptophan and tyrosine metabolism, branched chain amino acids and gut-derived metabolites in human plasma and urine. Extraction from plasma was optimised using 96-well plates, shown to be effective in removing phospholipids. Urine was filtered and diluted ten-fold. Metabolites were separated with reverse phase chromatography and detected using triple quadrupole MS. Linear ranges (from ppb to ppm) and correlation coefficients (r2 &gt, 0.990) were established for both matrices independently and the method was shown to be linear for all tested metabolites. At medium spiked concentration, recovery was over 80% in both matrices, while analytical precision was excellent (CV &lt, 15%). Matrix effects were minimal and retention time stability was excellent. The applicability of the methods was tested on biological samples, and metabolite concentrations were found to be in agreement with available data. The method allows the analysis of up to 96 samples per day and was demonstrated to be stable for up to three weeks from acquisition.

Rationale Electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are both used to generate ions for the analysis of metabolites by liquid chromatography/mass spectrometry (LC/MS). We compared the performance of these methods for the analysis of Corvina grapevine berry methanolic extracts, which are complex mixtures of diverse metabolites. Methods Corvina berries representing three ripening stages (veraison, early-ripening and full-ripening) were collected during two growing seasons, powdered and extracted with methanol. Untargeted metabolomic analysis was carried out by LC/ESI-MS and LC/APCI-MS. Processed data files were assembled into a data matrix for multivariate statistical analysis. The limits of detection (LODs), limits of quantification (LOQs), linear ranges, and matrix effects were investigated for strongly polar metabolites such as sucrose and tartaric acid and for moderately polar metabolites such as caftaric acid, epicatechin and quercetin 3-O-glucoside. Results Multivariate statistical analysis of the 608 features revealed that APCI was particularly suitable for the ionization of strongly polar metabolites such as sugars and organic acids, whereas ESI was more suitable for moderately polar metabolites such as flavanols, flavones and both glycosylated and acylated anthocyanins. APCI generated more fragment ions whereas ESI generated more adducts. ESI achieved lower LODs and LOQs for sucrose and tartaric acid but featured narrower linear ranges and greater matrix effects. Conclusions ESI and APCI are not complementary ion sources. Indeed, ESI can be exploited to analyze moderately polar metabolites, whereas APCI can be used to investigate weakly polar/non-polar metabolites and, as demonstrated by our results, also strongly polar metabolites. ESI and APCI can be used in parallel, exploiting their strengths to cover the plant metabolome more broadly than either method alone. Copyright © 2016 John Wiley & Sons, Ltd.

The aim of this study was a combined and multifaceted characterization (morphological, molecular, lipid, pigment, and ecological data) of a Lemanea (freshwater red alga) population from the south-eastern Alps, exploring its adaptive strategies to the montane habitat, (turbulent, very-cold glacial stream with extremely low-conductivity). Although the thalli were small (only up to 1 cm), the morphology was within the current circumscription of Lemanea fluviatilis. The molecular data placed this population within a clade of specimens identified as L. fluviatilis and L. fucina. This L. fluviatilis population was determined to possess lipid classes, especially phosphatidylcholine and monogalactosyldiacylglycerol with high unsaturation index (UI) and long acyl chains, which are typical adaptations for maintaining adequate membrane fluidity and consequently all the metabolic processes associated to the plasma membrane. The carotenoids profile revealed that, besides α /β-carotene, there are significant amounts of zeaxanthin and lutein. This study further demonstrated that red algae are a rich source of important food web w-3 fatty acids and may play an important role in the diets of grazers. L. fluviatilis is reported from one of the highest elevations (2,170 m a.s.l.) known for the genus Lemanea and this species. This study confirms the presence of L. fluviatilis in a cold, unpolluted, turbulent stream and this type of stream may be its preferred habitat.

Three new secondary metabolites (named erythrolactones A2, B2 and C2), that are characterized by a central 4-hydroxy-unsaturated ? lactone ring bearing an alkyl saturated chain at C(2) and a butyl-benzenoid group at C(5), together with their respective sulfate esters (erythrolactones A1, B1 and C1), have been isolated from cell cultures of Pseudokeronopsis erythrina, clone TL-1. The structures are assigned on the basis of extensive spectroscopic measurements (1D and 2D NMR, UV, IR and HR-MALDI-TOF). A plausible biogenetic route for their formation is also suggested. Cold-shock treatment was performed in order to induce the discharge of the metabolites contained in pigment granules lying on the ciliary organelles of this microorganism. HPLC-ESI-MS analysis of this granule discharge reveals that erythrolactones A2-C2 are actually therein contained, strongly suggesting a possible role for these metabolites in the chemical defence strategy of P. erythrina. Three new molecules, erythrolactones A2, B2 and C2, and their respective sulfate esters (A1, B1, C1) were isolated from cell cultures of Pseudokeronopsis erythrina and their structures elucidated by NMR and MS analyses. The release of pigment granule contents by cold-shock and subsequent analyses revealed that only non-sulfonated molecules are stored. A possible biogenetic route is described.

A biomimetic route towards assisted folding was explored. Molecularly imprinted polymeric nanoparticles (MIP NPs), i.e. biomimetics with entailed molecular recognition properties made by a template assisted synthesis, were prepared to target a structured epitope: the cystine containing peptide CC9ox, which corresponds to the apical portion of the β-hairpin hormone Hepcidin-25. The structural selection was achieved by the MIP NPs; moreover, the MIP NPs demonstrated favouring the folding of the linear random peptide (CC9red) into the structured one (CC9ox), anticipating the future role of the MIP NPs as in situ nanomachines to counteract folding defects.

Molecularly imprinted polymers (MIPs) were combined to MALDI-TOF-MS to evaluate a selective enrichment (SE) method for the determination of clinically relevant biomarkers from complex biological samples. The concept was proven with the myocardial injury marker Troponin I (cTnI). In a first part, MIP materials entailed for the recognition of cTnI epitopes (three peptides selected) were prepared and characterized in dimensions (0.7-2μm), dissociation constants (58-817 nM), kinetics of binding (5-60 min), binding capacity (ca. 1.5 µg/mg polymer), imprinting factors (3 > IF > 5) and selectivity for the peptide epitope. Then, the MIPs, incubated with cTnI peptides and spotted on the target with the DHB matrix, were assayed for the desorption of the peptides in MALDI-TOF-MS. The measured detection limit was ca. 300 femtomols. Finally, the MIP-SE MALDI-TOF-MS was tested for its ability to enrich in the cTnI peptides from a complex sample, mimic of serum (i.e. 81 peptides of digested albumin). The MIP-SE MALDI-TOF-MS successfully enriched in cTnI peptides from the complex sample proving the technique could offer a flexible platform to prepare entailed materials suitable for diagnostic purposes.

The fatty acid composition of marine cold-water sponges of genus Latrunculia, namely Latrunculia bocagei Ridley and Dendy, 1886, and Latrunculia biformis Kirkpatrick, 1907, for which no fatty acid data had been previously reported, has been examined. High levels of long-chain fatty acids (C24–30) with high unsaturation levels (mainly polyunsaturation), and high incidence of branched- and odd-chain fatty acids in sponges are suggested to be partially connected with their specific cell membrane requirements to surmount the negative effects of low temperatures and to enable growth in extreme environments. Furthermore, variations in the fatty acid profile at the species level may reflect variations in compositions or quantities of symbiotic microorganisms that commonly represent up to 60 % of the sponge wet weight. The fatty acid compositions of lipid-rich sponge extracts from five Latrunculia specimens dredged from the Bransfield Strait near the Antarctic Peninsula, were obtained using supercritical carbon dioxide and analyzed using gas chromatography–mass spectrometry–flame ionization detection. Furthermore, the antioxidant activities of these extracts were determined using the photochemiluminescent method. Along with common fatty acids, the chemical analyses revealed very long-chain fatty acids (C22, C24). Differences were seen for the fatty acid levels between both species and different specimens of the same species. The observed differences do not seem connected to the habitat depth of the specimen, but rather indicate the variations within the associated microbiome. Furthermore, these sponge extracts showed antioxidant activities, confirming that they contain lipidic compounds that strongly scavenge free radicals.

In this paper, we report the development of a new method based on HILIC-ESI-MS for the separation of several different membrane lipid classes and their detection on a triple quadrupole mass spectrometer using Precursor Ion (PIS) and Neutral Loss (NL) scanning in positive ion mode. Four different columns were tested for their ability to separate, under different conditions, a mixture of 14 lipid standards containing 7 glycerophospholipids (GPL), 2 glycosphyngolipids (GSL), 3 glycolipids (GL) and 2 betaine lipids (BL). The best separation was obtained using a Lichrosphere DIOL column as stationary phase and water (10mm ammonium acetate)/acetonitrile gradient elution as mobile phase which allows the separation of the 14 lipid classes within 35min runtime. Our method was successfully tested for the separation and analysis of crude lipid extracts obtained from a green alga (Jaoa bullata), a dinoflagellate (Peridinium cinctum) and a plant (Vitis vinifera cv. Corvina).

Direct coupling of thin-layer chromatography (TLC) with matrix-assisted laser desorption ionization (MALDI) mass spectrometry allows fast and detailed characterization of a large variety of analytes. The use of this technique, however, presents great challenges in semiquantitative applications because of the complex phenomena occurring at the TLC surface. In our laboratory, we recently observed that the ion intensities of several alkali adduct ions were significantly different between the top and interior layer of the TLC plate. This indicates that the integrity of the TLC surface can have an important effect on the reproducibility of TLC- MALDI analyses. Graphical Abstract MALDI imaging reveals that surface integrity affects the detection of alkali adductions in TLC-MALDI.

The integration of molecularly imprinted polymers (MIPs) with MALDI-TOF mass spectrometry (MS) combines MIP selectivity with MS sensitivity. Whether the size of the MIP material-micro versus nano-has an effect on the MS analysis was the object of the study. MIPs, targeting respectively the epitope peptide NR11 of cardiac troponin I and the peptide CK13 of human serum transferrin, were synthesized and characterized. The size-related performance of the MIP materials hyphenated with MALDI-TOF-MS analysis was studied by the incubation of the target peptide with the respective micro- or nano-MIP, followed by rinsing to remove non-specific deposition of the MIP to the MALDI target plate, co-crystallization with the organic matrix, and mass analysis. The quality of the MS analysis was assessed comparing the S/N of the mass peaks of the MIP-bound peptide to that of the same quantity of free peptide. Sweet spots and lower S/N (~ 1 order of magnitude) were observed for micro-MIP materials, while in the case of nano-MIP-bound peptide, the S/N was comparable to that of the free peptide, indicating higher compatibility of the nano-MIPs to MALDI-TOF-MS. The nano-MIP/MALDI-TOF-MS permitted the selective determination of the target peptide in real serum samples. Graphical abstract ᅟ.

Pseudokeronopsis erythrina produces three new secondary metabolites, erythrolactones A2, B2 and C2, and their respective sulfate esters (A1, B1, C1), the structures of which have been recently elucidated on the basis of NMR spectroscopic data coupled to high resolution mass measurements (HR-MALDI-TOF). An analysis of the discharge of the protozoan pigment granules revealed that the non-sulfonated erythrolactones are exclusively stored in these cortical organelles, which are commonly used by a number of ciliates as chemical weapons in offense/defense interactions with prey and predators. We evaluated the toxic activity of pigment granule discharge on a panel of free-living ciliates and micro-invertebrates, and the activity of each single purified erythrolactone on three ciliate species. We also observed predator-prey interactions of P. erythrina with unicellular and multicellular predators. Experimental results confirm that only P. erythrina cells with discharged pigment granules were preferentially or exclusively hunted and eaten by at least some of its predators, whereas almost all intact (fully pigmented) cells remained alive. Our results indicate that erythrolactones are very effective as a chemical defense in P. erythrina.

Induced hypoxia stress on cervical cancer derived cells (HeLa cells) leads to significant changes in their membrane lipid profiles. The lipidome of HeLa cells was characterized by a joint approach wherein liquid chromatography-mass spectrometry (LC-MS) analysis was followed by high resolution NMR measurements. Multivariate data analysis showed apparent separation between control and hypoxia-treated HeLa cells and thus demonstrated hypoxia effects on lipid metabolism. The most striking finding was that hypoxia stimulation significantly reduced the total amount of cellular phosphoinositols (PI) but caused a prominent increase in the amount of lyso phosphocholines (lyso-PC) and lyso phosphoethanolamines (lyso-PE). The observed decrease of PI amount under hypoxic conditions is probably due to the accumulation of cellular myo-inositol, which is known to play a critical role in de novo synthesis of PI. Moreover, our study suggests that polyunsaturated phospholipid species are stronger biomarkers for discriminating the effect of hypoxia treatment. The evaluation of changes in the average unsaturation index (UI) of the membrane lipids acyl chains reveals that UI slightly increases in several lipid classes, thus affecting membrane fluidity and further membrane-dependent functions. The plausible mechanisms by which HeLa cells adapt to hypoxia conditions are also briefly reported. This journal is © 2014 The Royal Society of Chemistry.

A chemotaxonomic study on the marine brown alga Cystoseira schiffneri collected from the Tunisian marine coast allowed us to identify kjellmanianone (1) and a new isololiolide derivative named schiffnerilolide (2). The structure elucidation and the assignment of relative configurations of the isolated natural products were based on advanced mass spectrometric and nuclear magnetic resonance techniques. This outcome suggested a close phylogenetic relationship of C. schiffneri with brown algae belonging to genus Sargassum C. Agardh. Molecular characterization using the nuclear small subunit rRNA (SSU rRNA) gene (18S) sequence as genetic marker was made. Pigment analysis showed a significant seasonal change of carotenoids, in particular of fucoxanthin and fucoxanthinol. Also galactolipids, the main constituents of the thylakoid membranes, showed remarkable seasonal changes.

The molecular events that characterize postripening grapevine berries have rarely been investigated and are poorly defined. In particular, a detailed definition of changes occurring during the postharvest dehydration, a process undertaken to make some particularly special wine styles, would be of great interest for both winemakers and plant biologists. We report an exhaustive survey of transcriptomic and metabolomic responses in berries representing six grapevine genotypes subjected to postharvest dehydration under identical controlled conditions. The modulation of phenylpropanoid metabolism clearly distinguished the behavior of genotypes, with stilbene accumulation as the major metabolic event, although the transient accumulation/depletion of anthocyanins and flavonols was the prevalent variation in genotypes that do not accumulate stilbenes. The modulation of genes related to phenylpropanoid/stilbene metabolism highlighted the distinct metabolomic plasticity of genotypes, allowing for the identification of candidate structural and regulatory genes. In addition to genotype-specific responses, a core set of genes was consistently modulated in all genotypes, representing the common features of berries undergoing dehydration and/or commencing senescence. This included genes controlling ethylene and auxin metabolism as well as genes involved in oxidative and osmotic stress, defense responses, anaerobic respiration, and cell wall and carbohydrate metabolism. Several transcription factors were identified that may control these shared processes in the postharvest berry. Changes representing both common and genotype-specific responses to postharvest conditions shed light on the cellular processes taking place in harvested berries stored under dehydrating conditions for several months.

The shape recognition model of olfaction maintains that odorant reception probes physicochemical properties such as size, shape, electric charge and hydrophobicity of the ligand. Recently, insects were shown to distinguish common from deuterated isotopomers of the same odorant, suggesting the involvement of other molecular properties to odorant reception. Via two-photon functional microscopy we investigated how common and deuterated isoforms of natural odorants are coded within the honeybee brain. Our results provide evidence that (i) different isotopomers generate different neuronal activation maps, (ii) isotopomer sensitivity is a general mechanism common to multiple odorant receptors and (iii) isotopomer specificity is highly consistent across individuals. This indicates that honeybee’s olfactory system discriminates between isotopomers of the same odorant, suggesting that other features, such as molecular vibrations, may contribute to odour signal transduction.

Terroir refers to the combination of environmental factors that affect the characteristics of crops such as grapevine (Vitis vinifera) according to particular habitats and management practices. This article shows how certain terroir signatures can be detected in the berry metabolome and transcriptome of the grapevine cultivar Corvina using multivariate statistical analysis. The method first requires an appropriate sampling plan. In this case study, a specific clone of the Corvina cultivar was selected to minimize genetic differences, and samples were collected from seven vineyards representing three different macro-zones during three different growing seasons. An untargeted LC-MS metabolomics approach is recommended due to its high sensitivity, accompanied by efficient data processing using MZmine software and a metabolite identification strategy based on fragmentation tree analysis. Comprehensive transcriptome analysis can be achieved using microarrays containing probes covering ~99% of all predicted grapevine genes, allowing the simultaneous analysis of all differentially expressed genes in the context of different terroirs. Finally, multivariate data analysis based on projection methods can be used to overcome the strong vintage-specific effect, allowing the metabolomics and transcriptomics data to be integrated and analyzed in detail to identify informative correlations.

We established a step-by-step, experiment-guided metabolomics procedure, based on LC-ESI-MS analysis, to generate a detailed picture of the changing metabolic profiles during late berry development in the important Italian grapevine cultivar Corvina. We sampled berries from four developmental time points and three post-harvest time points during the withering process, and used chromatograms of methanolic extracts to test the performance of the MetAlign and MZmine data mining programs. MZmine achieved a better resolution and therefore generated a more useful data matrix. Then both the quantitative performance of the analytical platform and the matrix effect were assessed, and the final dataset was investigated by multivariate data analysis. Our analysis confirmed the results of previous studies but also revealed some novel findings, including the prevalence of two specific flavonoids in unripe berries and important differences between the developmental profiles of flavones and flavanones, suggesting that specific individual metabolites could have different functions, and that flavones and flavanones probably play quite distinct biological roles. Moreover, the hypothesis-free multivariate analysis of subsets of the wide data matrix evidentiated the relationships between the various classes of metabolites, such as those between anthocyanins and hydroxycinnamic acids and between flavan-3-ols and anthocyanins.

Background The definition of the terroir concept is one of the most debated issues in oenology and viticulture. The dynamic interaction among diverse factors including the environment, the grapevine plant and the imposed viticultural techniques means that the wine produced in a given terroir is unique. However, there is an increasing interest to define and quantify the contribution of individual factors to a specific terroir objectively. Here, we characterized the metabolome and transcriptome of berries from a single clone of the Corvina variety cultivated in seven different vineyards, located in three macrozones, over a 3-year trial period. Results To overcome the anticipated strong vintage effect, we developed statistical tools that allowed us to identify distinct terroir signatures in the metabolic composition of berries from each macrozone, and from different vineyards within each macrozone. We also identified non-volatile and volatile components of the metabolome which are more plastic and therefore respond differently to terroir diversity. We observed some relationships between the plasticity of the metabolome and transcriptome, allowing a multifaceted scientific interpretation of the terroir concept. Conclusions Our experiments with a single Corvina clone in different vineyards have revealed the existence of a clear terroir-specific effect on the transcriptome and metabolome which persists over several vintages and allows each vineyard to be characterized by the unique profile of specific metabolites. Electronic supplementary material The online version of this article (doi:10.1186/s12870-015-0584-4) contains supplementary material, which is available to authorized users.

Life at low temperature imposes many constraints linked to sustaining cellular functions. The cold-adapted freshwater dinoflagellate Peridinium aciculiferum has overcome these barriers, often causing blooms in winter but forming resting cysts in spring. Little is known of the biochemical changes that accompany this temperature-induced transformation from vegetative cells to resting cysts. We investigated how the profiles of lipids and mycosporine-like amino acids (MAAs) vary with temperature in vegetative cells and resting cysts of P. aciculiferum. The freshwater dinoflagellate was grown at four temperatures (2.7-7.7 °C), simulating the seasonal changes from winter to spring that also induce the transition from cells to cysts. Biochemical profiles were established by liquid chromatography/mass spectrometry with the simultaneous detection of polar and non-polar compounds. Data were analysed by non-metric multidimensional scaling and ANOVA. Over 100 species of galactolipids, betaine lipids, phospholipids and triacylglycerols (TAGs) were found, and many were strong biomarkers for specific temperatures and life stage. Variations in galactolipids, betaine lipids and phospholipids were unidirectional, as shown by an overall decrease in the unsaturation index with temperature. In contrast, changes in TAGs were specific to life stages: short-chain TAGs (cumulative acyl length of 44-52 carbon atoms) decreased in cysts with respect to vegetative cells, while long-chain TAGs (54-62) showed the opposite pattern. The concentration of MAAs decreased with increasing temperature. Final cell yield, a measure of population fitness, also decreased with increasing temperature, confirming the psychrophilic status of P. aciculiferum. We report the first detailed biochemical profiles of vegetative cells and resting cysts for a dinoflagellate and show how small-scale temperature variations alter the biochemical make-up within and between life stages, thus contributing to our understanding of seasonal succession of species. © 2014 John Wiley & Sons Ltd.

Supercritical carbon dioxide (SC-CO2) treatment is one of the most promising alternative techniques for pasteurization of both liquid and solid food products. The inhibitory effect of SC-CO2 on bacterial growth has been investigated in different species, but the precise mechanism of action remains unknown. Membrane permeabilization has been proposed to be the first event in SC-CO2-mediated inactivation. Flow cytometry, high performance liquid chromatography–electrospray ionization–mass spectrometry and NMR analyses were performed to investigate the effect of SC-CO2 treatment on membrane lipid profile and membrane permeability in Escherichia coli K12. After 15 min of SC-CO2 treatment at 120 bar and 35 °C, the majority of bacterial cells dissipated their membrane potential (95 %) and lost membrane integrity, as 81 % become partially permeabilized and 18 % fully permeabilized. Membrane permeabilization was associated with a 20 % decrease in bacterial biovolume and to a strong (>50 %) reduction in phosphatidylglycerol (PG) membrane lipids, without altering the fatty acid composition and the degree of unsaturation of acyl chains. PGs are thought to play an important role in membrane stability, by reducing motion of phosphatidylethanolamine (PE) along the membrane bilayer, therefore promoting the formation of inter-lipid hydrogen bonds. In addition, the decrease in intracellular pH induced by SC-CO2 likely alters the chemical properties of phospholipids and the PE/PG ratio. Biophysical effects of SC-CO2 thus cause a strong perturbation of membrane architecture in E. coli, and such alterations are likely associated with its strong inactivation effect.

Phenotypic plasticity refers to the range of phenotypes a single genotype can express as a function of its environment. These phenotypic variations are attributable to the effect of the environment on the expression and function of genes influencing plastic traits. We investigated phenotypic plasticity in grapevine by comparing the berry transcriptome in a single clone of the vegetatively-propagated common grapevine species Vitis vinifera cultivar Corvina through 3 consecutive growth years cultivated in 11 different vineyards in the Verona area of Italy. Most of the berry transcriptome clustered by year of growth rather than common environmental conditions or viticulture practices, and transcripts related to secondary metabolism showed high sensitivity towards different climates, as confirmed also by metabolomic data obtained from the same samples. When analyzed in 11 vineyards during 1 growth year, the environmentally-sensitive berry transcriptome comprised 5% of protein-coding genes and 18% of the transcripts modulated during berry development. Plastic genes were particularly enriched in ontology categories such as transcription factors, translation, transport, and secondary metabolism. Specific plastic transcripts were associated with groups of vineyards sharing common viticulture practices or environmental conditions, and plastic transcriptome reprogramming was more intense in the year characterized by extreme weather conditions. We also identified a set of genes that lacked plasticity, showing either constitutive expression or similar modulation in all berries. Our data reveal candidate genes potentially responsible for the phenotypic plasticity of grapevine and provide the first step towards the characterization of grapevine transcriptome plasticity under different agricultural systems.