A water-soluble tomato extract rich in secondary plant metabolites lowers trimethylamine-n-oxide and modulates gut microbiota: a randomized, double-blind, placebo-controlled cross-over study in overweight and obese adults.

BACKGROUND: Natural products rich in polyphenols have been shown to lower plasma trimethylamine-n-oxide (TMAO) known for its proatherogenic effects by modulating the intestinal microbiota. OBJECTIVES: We aimed to determine the impact of Fruitflow, a water-soluble tomato extract, on TMAO, fecal microbiota, and plasma and fecal metabolites. METHODS: Overweight and obese adults (n = 22, BMI 28-35 kg/m2) were included in a double-blind, placebo-controlled, cross-over study receiving 2×150 mg Fruitflow per day or placebo (maltodextrin) for 4 wk with a 6-week wash-out between interventions. Stool, blood, and urine samples were collected to assess changes in plasma TMAO (primary outcome) as well as fecal microbiota, fecal and plasma metabolites, and urine TMAO (secondary outcomes). In a subgroup (n = 9), postprandial TMAO was evaluated following a choline-rich breakfast (∼450 mg). Statistical methods included paired t-tests or Wilcoxon signed rank tests and permutational multivariate analysis of variance. RESULTS: Fruitflow, but not placebo, reduced fasting levels of plasma (-1.5 µM, P ≤ 0.05) and urine (-19.1 µM, P ≤ 0.01) TMAO as well as plasma lipopolysaccharides (-5.3 ng/mL, P ≤ 0.05) from baseline to the end of intervention. However, these changes were significant only for urine TMAO levels when comparing between the groups (P ≤ 0.05). Changes in microbial beta, but not alpha, diversity paralleled this with a significant difference in Jaccard distance-based Principal Component (P ≤ 0.05) as well as decreases in Bacteroides, Ruminococccus, and Hungatella and increases in Alistipes when comparing between and within groups (P ≤ 0.05, respectively). There were no between-group differences in SCFAs and bile acids (BAs) in both faces and plasma but several changes within groups such as an increase in fecal cholic acid or plasma pyruvate with Fruitflow (P ≤ 0.05, respectively). An untargeted metabolomic analysis revealed TMAO as the most discriminant plasma metabolite between groups (P ≤ 0.05). CONCLUSIONS: Our results support earlier findings that polyphenol-rich extracts can lower plasma TMAO in overweight and obese adults related to gut microbiota modulation. This trial was registered at clinicaltrials.gov as NCT04160481 (https://clinicaltrials.gov/ct2/show/NCT04160481?term= Fruitflow&draw= 2&rank= 2).

Structural characterization and anti-inflammatory activity of two novel polysaccharides from the sea squirt, Ascidiella aspersa.

It is now recognized that certain polysaccharides can exhibit anti-inflammatory activity, including the glycosaminoglycan (GAG) heparin that is widely used as an anti-coagulant drug. However, it would be desirable to identify molecules that retain the anti-inflammatory actions of heparin, but that are devoid of significant anti-coagulant activity. In the present study we have identified a number of novel GAG and GAG-like polysaccharides (VRP327) from marine organisms, most of which were resistant to digestion by heparinase II and chondroitinase ABC. Fourier transform infra-red spectrum (FTIR) revealed species with variable degrees of sulphation and monosaccharide analysis revealed a range of sugar compounds, which in some cases included sugars not present in mammalian GAGs. (1)H NMR spectra of these species are consistent with the structures of complex polysaccharides. From an initial screening cascade to remove compounds having significant anti-coagulant activity and no overt cytotoxicity, we identified a high molecular weight oversulphated dermatan sulphate (VRP327) isolated from the tunicate Ascidiella aspersa which was fully characterised by NMR spectroscopy. This material was depolymerised to produce well characterized low molecular weight fractions which were demonstrated to be non-toxic, with low levels of anti-coagulant activity, and to have demonstrable anti-inflammatory activity assessed in several in vitro and in vivo models. The identification of low molecular weight polysaccharides having significant anti-inflammatory activity without significant anti-coagulant activity may provide novel templates for the development of a novel class of anti-inflammatory drugs.

Fucosylated chondroitin sulfates from the body wall of the sea cucumber Holothuria forskali: conformation, selectin binding, and biological activity.

Fucosylated chondroitin sulfate (fCS) extracted from the sea cucumber Holothuria forskali is composed of the following repeating trisaccharide unit: → 3)GalNAcß4,6S(1 → 4) [FucαX(1 → 3)]GlcAß(1 →, where X stands for different sulfation patterns of fucose (X = 3,4S (46%), 2,4S (39%), and 4S (15%)). As revealed by NMR and molecular dynamics simulations, the fCS repeating unit adopts a conformation similar to that of the Le(x) blood group determinant, bringing several sulfate groups into close proximity and creating large negative patches distributed along the helical skeleton of the CS backbone. This may explain the high affinity of fCS oligosaccharides for L- and P-selectins as determined by microarray binding of fCS oligosaccharides prepared by Cu(2+)-catalyzed Fenton-type and photochemical depolymerization. No binding to E-selectin was observed. fCS poly- and oligosaccharides display low cytotoxicity in vitro, inhibit human neutrophil elastase activity, and inhibit the migration of neutrophils through an endothelial cell layer in vitro. Although the polysaccharide showed some anti-coagulant activity, small oligosaccharide fCS fragments had much reduced anticoagulant properties, with activity mainly via heparin cofactor II. The fCS polysaccharides showed prekallikrein activation comparable with dextran sulfate, whereas the fCS oligosaccharides caused almost no effect. The H. forskali fCS oligosaccharides were also tested in a mouse peritoneal inflammation model, where they caused a reduction in neutrophil infiltration. Overall, the data presented support the action of fCS as an inhibitor of selectin interactions, which play vital roles in inflammation and metastasis progression. Future studies of fCS-selectin interaction using fCS fragments or their mimetics may open new avenues for therapeutic intervention.

Changes in primary metabolite content may affect thrips feeding preference in soybean crops.

Past research has characterized the induction of plant defenses in response to chewing insect damage. However, little is known about plant responses to piercing-sucking insects that feed on plant cell-contents like thrips (Caliothrips phaseoli). In this study, we used NMR spectroscopy to measure metabolite changes in response to six days of thrips damage from two field-grown soybean cultivars (cv.), known for their different susceptibility to Caliothrips phaseoli. We observed that thrips damage reduces sucrose concentration in both cultivars, while pinitol, the most abundant leaf soluble carbohydrate, is induced in cv. Charata but not in cv. Williams. Thrips did not show preference for leaves where sucrose or pinitol were externally added, at tested concentration. In addition, we also noted that cv. Charata was less naturally colonized and contained higher levels of trigonelline, tyrosine as well as several compounds that we have not yet identified. We have established that preference-feeding clues are not dependent on the plants major soluble carbohydrates but may depend on other types of compounds or leaf physical characteristics.

Digestive activity and organic compounds of Nezara viridula watery saliva induce defensive soybean seed responses.

The stink bug Nezara viridula is one of the most threatening pests for agriculture in North and South America, and its oral secretion may be responsible for the damage it causes in soybean (Glycine max) crop. The high level of injury to seeds caused by pentatomids is related to their feeding behavior, morphology of mouth parts, and saliva, though information on the specific composition of the oral secretion is scarce. Field studies were conducted to evaluate the biochemical damage produced by herbivory to developing soybean seeds. We measured metabolites and proteins to profile the insect saliva in order to understand the dynamics of soybean-herbivore interactions. We describe the mouth parts of N. viridula and the presence of metabolites, proteins and active enzymes in the watery saliva that could be involved in seed cell wall modification, thus triggering plant defenses against herbivory. We did not detect proteins from bacteria, yeasts, or soybean in the oral secretion after feeding. These results suggest that the digestive activity and organic compounds of watery saliva may elicit a plant self-protection response. This study adds to our understanding of stink bug saliva plasticity and its role in the struggle against soybean defenses.

A redox-active switch in fructosamine-3-kinases expands the regulatory repertoire of the protein kinase superfamily.

Aberrant regulation of metabolic kinases by altered redox homeostasis substantially contributes to aging and various diseases, such as diabetes. We found that the catalytic activity of a conserved family of fructosamine-3-kinases (FN3Ks), which are evolutionarily related to eukaryotic protein kinases, is regulated by redox-sensitive cysteine residues in the kinase domain. The crystal structure of the FN3K homolog from Arabidopsis thaliana revealed that it forms an unexpected strand-exchange dimer in which the ATP-binding P-loop and adjoining ß strands are swapped between two chains in the dimer. This dimeric configuration is characterized by strained interchain disulfide bonds that stabilize the P-loop in an extended conformation. Mutational analysis and solution studies confirmed that the strained disulfides function as redox "switches" to reversibly regulate the activity and dimerization of FN3K. Human FN3K, which contains an equivalent P-loop Cys, was also redox sensitive, whereas ancestral bacterial FN3K homologs, which lack a P-loop Cys, were not. Furthermore, CRISPR-mediated knockout of FN3K in human liver cancer cells altered the abundance of redox metabolites, including an increase in glutathione. We propose that redox regulation evolved in FN3K homologs in response to changing cellular redox conditions. Our findings provide insights into the origin and evolution of redox regulation in the protein kinase superfamily and may open new avenues for targeting human FN3K in diabetic complications.

NMR metabolomics reveals effects of Cryptosporidium infections on host cell metabolome.

BACKGROUND: Cryptosporidium is an important gut microbe whose contributions towards infant and immunocompromise patient mortality rates are steadily increasing. Over the last decade, we have seen the development of various tools and methods for studying Cryptosporidium infection and its interactions with their hosts. One area that is sorely overlooked is the effect infection has on host metabolic processes. RESULTS: Using a 1H nuclear magnetic resonance approach to metabolomics, we have explored the nature of the mouse gut metabolome as well as providing the first insight into the metabolome of an infected cell line. Statistical analysis and predictive modelling demonstrated new understandings of the effects of a Cryptosporidium infection, while verifying the presence of known metabolic changes. Of note is the potential contribution of host derived taurine to the diarrhoeal aspects of the disease previously attributed to a solely parasite-based alteration of the gut environment, in addition to other metabolites involved with host cell catabolism. CONCLUSION: This approach will spearhead our understanding of the Cryptosporidium-host metabolic exchange and provide novel targets for tackling this deadly parasite.

Photochemical depolymerisation of dermatan sulfate and analysis of the generated oligosaccharides.

Radical depolymerisation is the method of choice for the depolymerisation of glycosaminoglycans (GAGs), especially when enzymatic depolymerisation cannot be performed due to the lack of suitable enzymes. The established Fenton type free radical depolymerisation generates radicals from a solution of H2O2 in the presence of Cu(2+) or Fe(2+). When applied to dermatan sulfate (DS), the Fenton type depolymerisation of DS (Panagos, Thomson, Bavington, & Uhrin, 2012) produced exclusively oligosaccharides with reducing end GalNAc, which was partially oxidised to acetylgalactosaminic acid. We report here the results of the TiO2 catalysed photochemical depolymerisation of DS. NMR analysis of these DS oligosaccharides revealed the presence of reducing end IdoA, observed for the first time. The reducing end acetylgalactosaminic acid was also detected. The photochemical depolymerisation method thus enables preparation of new types of GAG oligosaccharides suitable for further biochemical and biological investigation.

Characterisation of hyaluronic acid and chondroitin/dermatan sulfate from the lumpsucker fish, C. lumpus.

The lumpsucker, Cyclopterus lumpus, a cottoid teleost fish found in the cold waters of the North Atlantic, and North Pacific, was identified as a possible source of GAGs. The GAGs present in the C. lumpus dorsal hump and body wall tissue were isolated and purified. Two fractions were analysed by NMR and their GAG structures determined as hyaluronic acid and CS/DS chains. The latter fraction contained GlcA (65% of the total uronic acids) and IdoA (the remaining 35%). All uronic acid residues were unsulfated, whilst 86% of the GalNAc was 4-sulfated and 14% was 6-sulfated. The presence of GlcA-GalNAc4S, IdoA-GalNAc4S and GlcA-GalNAc6S disaccharide fragments was confirmed. The isolated GAGs obtained from each tissue were biochemically characterised. The lumpsucker offers a high yield source of GAGs, which compares favourably with other sources such as shark cartilage.