Precision Medicine: Determination of Ribavirin Urinary Metabolites in Relation to Drug Adverse Effects in HCV Patients.

The most commonly used antiviral treatment against hepatitis C virus is a combination of direct-acting antivirals (DAAs) and ribavirin (RBV), which leads to a shortened duration of therapy and a sustained virologic response until 98%. Nonetheless, several dose-related side effects of RBV could limit its applications. This study aims to measure the urinary concentration of RBV and its main metabolites in order to evaluate the drug metabolism ability of HCV patients and to evaluate the adverse effects, such as anemia, with respect to RBV metabolite levels. RBV and its proactive and inactive metabolites were identified and quantified in the urine of 17 HCV males with severe liver fibrosis using proton nuclear magnetic resonance (1H-NMR) at the fourth week (TW4) and at the twelfth week of treatment (EOT). Four prodrug urinary metabolites, including RBV, were identified and three of them were quantified. At both the TW4 and EOT stages, six HCV patients were found to maintain high concentrations of RBV, while another six patients maintained a high level of RBV proactive metabolites, likely due to nucleosidase activity. Furthermore, a negative correlation between the reduction in hemoglobin (Hb) and proactive forms was observed, according to RBV-triphosphate accumulation causing the hemolysis. These findings represent a proof of concept regarding tailoring the drug dose in relation to the specific metabolic ability of the individual, as expected by the precision medicine approach.

Gut Microbiota Functional Traits, Blood pH, and Anti-GAD Antibodies Concur in the Clinical Characterization of T1D at Onset.

Alterations of gut microbiota have been identified before clinical manifestation of type 1 diabetes (T1D). To identify the associations amongst gut microbiome profile, metabolism and disease markers, the 16S rRNA-based microbiota profiling and 1H-NMR metabolomic analysis were performed on stool samples of 52 T1D patients at onset, 17 T1D siblings and 57 healthy subjects (CTRL). Univariate, multivariate analyses and classification models were applied to clinical and -omic integrated datasets. In T1D patients and their siblings, Clostridiales and Dorea were increased and Dialister and Akkermansia were decreased compared to CTRL, while in T1D, Lachnospiraceae were higher and Collinsella was lower, compared to siblings and CTRL. Higher levels of isobutyrate, malonate, Clostridium, Enterobacteriaceae, Clostridiales, Bacteroidales, were associated to T1D compared to CTRL. Patients with higher anti-GAD levels showed low abundances of Roseburia, Faecalibacterium and Alistipes and those with normal blood pH and low serum HbA1c levels showed high levels of purine and pyrimidine intermediates. We detected specific gut microbiota profiles linked to both T1D at the onset and to diabetes familiarity. The presence of specific microbial and metabolic profiles in gut linked to anti-GAD levels and to blood acidosis can be considered as predictive biomarker associated progression and severity of T1D.

Characterization of the gut-liver-muscle axis in cirrhotic patients with sarcopenia.

BACKGROUND & AIM: Sarcopenia is frequent in cirrhosis and is associated with unfavourable outcomes. The role of the gut-liver-muscle axis in this setting has been poorly investigated. The aim of this study was to identify gut microbiota, metabolic and inflammatory signatures associated with sarcopenia in cirrhotic patients. METHODS: Fifty cirrhotic patients assessed for the presence of sarcopenia by the quantification of muscle mass and strength were compared with age- and sex-matched controls. A multiomic analysis, including gut microbiota composition and metabolomics, serum myokines and systemic and intestinal inflammatory mediators, was performed. RESULTS: The gut microbiota of sarcopenic cirrhotic patients was poor in bacteria associated with physical function (Methanobrevibacter, Prevotella and Akkermansia), and was enriched in Eggerthella, a gut microbial marker of frailty. The abundance of potentially pathogenic bacteria, such as Klebsiella, was also increased, to the detriment of autochthonous ones. Sarcopenia was associated with elevated serum levels of pro-inflammatory mediators and of fibroblast growth factor 21 (FGF21) in cirrhotic patients. Gut microbiota metabolic pathways involved in amino acid, protein and branched-chain amino acid metabolism were up-regulated, in addition to ethanol, trimethylamine and dimethylamine production. Correlation networks and clusters of variables associated with sarcopenia were identified, including one centred on Klebsiella/ethanol/FGF21/Eggerthella/Prevotella. CONCLUSIONS: Alterations in the gut-liver-muscle axis are associated with sarcopenia in patients with cirrhosis. Detrimental but also compensatory functions are involved in this complex network.

Gut metabolomics profiling of non-small cell lung cancer (NSCLC) patients under immunotherapy treatment.

BACKGROUND: Despite the efficacy of immune checkpoint inhibitors (ICIs) only the 20-30% of treated patients present long term benefits. The metabolic changes occurring in the gut microbiota metabolome are herein proposed as a factor potentially influencing the response to immunotherapy. METHODS: The metabolomic profiling of gut microbiota was characterized in 11 patients affected by non-small cell lung cancer (NSCLC) treated with nivolumab in second-line treatment with anti-PD-1 nivolumab. The metabolomics analyses were performed by GC-MS/SPME and 1H-NMR in order to detect volatile and non-volatile metabolites. Metabolomic data were processed by statistical profiling and chemometric analyses. RESULTS: Four out of 11 patients (36%) presented early progression, while the remaining 7 out of 11 (64%) presented disease progression after 12 months. 2-Pentanone (ketone) and tridecane (alkane) were significantly associated with early progression, and on the contrary short chain fatty acids (SCFAs) (i.e., propionate, butyrate), lysine and nicotinic acid were significantly associated with long-term beneficial effects. CONCLUSIONS: Our preliminary data suggest a significant role of gut microbiota metabolic pathways in affecting response to immunotherapy. The metabolic approach could be a promising strategy to contribute to the personalized management of cancer patients by the identification of microbiota-linked "indicators" of early progressor and long responder patients.

Network Analysis of Gut Microbiome and Metabolome to Discover Microbiota-Linked Biomarkers in Patients Affected by Non-Small Cell Lung Cancer.

Several studies in recent times have linked gut microbiome (GM) diversity to the pathogenesis of cancer and its role in disease progression through immune response, inflammation and metabolism modulation. This study focused on the use of network analysis and weighted gene co-expression network analysis (WGCNA) to identify the biological interaction between the gut ecosystem and its metabolites that could impact the immunotherapy response in non-small cell lung cancer (NSCLC) patients undergoing second-line treatment with anti-PD1. Metabolomic data were merged with operational taxonomic units (OTUs) from 16S RNA-targeted metagenomics and classified by chemometric models. The traits considered for the analyses were: (i) condition: disease or control (CTRLs), and (ii) treatment: responder (R) or non-responder (NR). Network analysis indicated that indole and its derivatives, aldehydes and alcohols could play a signaling role in GM functionality. WGCNA generated, instead, strong correlations between short-chain fatty acids (SCFAs) and a healthy GM. Furthermore, commensal bacteria such as Akkermansia muciniphila, Rikenellaceae, Bacteroides, Peptostreptococcaceae, Mogibacteriaceae and Clostridiaceae were found to be more abundant in CTRLs than in NSCLC patients. Our preliminary study demonstrates that the discovery of microbiota-linked biomarkers could provide an indication on the road towards personalized management of NSCLC patients.

Gut microbiota profiling of pediatric nonalcoholic fatty liver disease and obese patients unveiled by an integrated meta-omics-based approach.

There is evidence that nonalcoholic fatty liver disease (NAFLD) is affected by gut microbiota. Therefore, we investigated its modifications in pediatric NAFLD patients using targeted metagenomics and metabolomics. Stools were collected from 61 consecutive patients diagnosed with nonalcoholic fatty liver (NAFL), nonalcoholic steatohepatitis (NASH), or obesity and 54 healthy controls (CTRLs), matched in a case-control fashion. Operational taxonomic units were pyrosequenced targeting 16S ribosomal RNA and volatile organic compounds determined by solid-phase microextraction gas chromatography-mass spectrometry. The α-diversity was highest in CTRLs, followed by obese, NASH, and NAFL patients; and ß-diversity distinguished between patients and CTRLs but not NAFL and NASH. Compared to CTRLs, in NAFLD patients Actinobacteria were significantly increased and Bacteroidetes reduced. There were no significant differences among the NAFL, NASH, and obese groups. Overall NAFLD patients had increased levels of Bradyrhizobium, Anaerococcus, Peptoniphilus, Propionibacterium acnes, Dorea, and Ruminococcus and reduced proportions of Oscillospira and Rikenellaceae compared to CTRLs. After reducing metagenomics and metabolomics data dimensionality, multivariate analyses indicated a decrease of Oscillospira in NAFL and NASH groups and increases of Ruminococcus, Blautia, and Dorea in NASH patients compared to CTRLs. Of the 292 volatile organic compounds, 26 were up-regulated and 2 down-regulated in NAFLD patients. Multivariate analyses found that combination of Oscillospira, Rickenellaceae, Parabacteroides, Bacteroides fragilis, Sutterella, Lachnospiraceae, 4-methyl-2-pentanone, 1-butanol, and 2-butanone could discriminate NAFLD patients from CTRLs. Univariate analyses found significantly lower levels of Oscillospira and higher levels of 1-pentanol and 2-butanone in NAFL patients compared to CTRLs. In NASH, lower levels of Oscillospira were associated with higher abundance of Dorea and Ruminococcus and higher levels of 2-butanone and 4-methyl-2-pentanone compared to CTRLs. CONCLUSION: An Oscillospira decrease coupled to a 2-butanone up-regulation and increases in Ruminococcus and Dorea were identified as gut microbiota signatures of NAFL onset and NAFL-NASH progression, respectively. (Hepatology 2017;65:451-464).

Assessment of Fecal Microbiota and Fecal Metabolome in Symptomatic Uncomplicated Diverticular Disease of the Colon.

GOAL: The aim of this study was to assess fecal microbiota and metabolome in a population with symptomatic uncomplicated diverticular disease (SUDD). BACKGROUND: Whether intestinal microbiota and metabolic profiling may be altered in patients with SUDD is unknown. PATIENTS AND METHODS: Stool samples from 44 consecutive women [15 patients with SUDD, 13 with asymptomatic diverticulosis (AD), and 16 healthy controls (HCs)] were analyzed. Real-time polymerase chain reaction was used to quantify targeted microorganisms. High-resolution proton nuclear magnetic resonance spectroscopy associated with multivariate analysis with partial least-square discriminant analysis (PLS-DA) was applied on the metabolite data set. RESULTS: The overall bacterial quantity did not differ among the 3 groups (P=0.449), with no difference in Bacteroides/Prevotella, Clostridium coccoides, Bifidobacterium, Lactobacillus, and Escherichia coli subgroups. The amount of Akkermansia muciniphila species was significantly different between HC, AD, and SUDD subjects (P=0.017). PLS-DA analysis of nuclear magnetic resonance -based metabolomics associated with microbiological data showed significant discrimination between HCs and AD patients (R=0.733; Q=0.383; P<0.05, LV=2). PLS analysis showed lower N-acetyl compound and isovalerate levels in AD, associated with higher levels of A. municiphila, as compared with the HC group. PLS-DA applied on AD and SUDD samples showed a good discrimination between these 2 groups (R=0.69; Q=0.35; LV=2). SUDD patients were characterized by low levels of valerate, butyrate, and choline and by high levels of N-acetyl derivatives and U1. CONCLUSIONS: SUDD and AD do not show colonic bacterial overgrowth, but a significant difference in the levels of fecal A. muciniphila was observed. Moreover, increasing expression of some metabolites as expression of different AD and SUDD metabolic activity was found.

Acetic acid acts as an elicitor exerting a chitosan-like effect on xanthone biosynthesis in Hypericum perforatum L. root cultures.

KEY MESSAGE: Acetic acid acts as a signal molecule, strongly enhancing xanthone biosynthesis in Hypericum perforatum root cultures. This activity is specific, as demonstrated by the comparison with other short-chain monocarboxylic acids. We have recently demonstrated that Hypericum perforatum root cultures constitutively produce xanthones at higher levels than the root of the plant and that they respond to chitosan (CHIT) elicitation with a noteworthy increase in xanthone production. In the present study, CHIT was administered to H. perforatum root cultures using three different elicitation protocols, and the increase in xanthone production was evaluated. The best results (550 % xanthone increase) were obtained by subjecting the roots to a single elicitation with 200 mg l(-1) CHIT and maintaining the elicitor in the culture medium for 7 days. To discriminate the effect of CHIT from that of the solvent, control experiments were performed by administering AcOH alone at the same concentration used for CHIT solubilization. Unexpectedly, AcOH caused an increase in xanthone production comparable to that observed in response to CHIT. Feeding experiments with (13)C-labeled AcOH demonstrated that this compound was not incorporated into the xanthone skeleton. Other short-chain monocarboxylic acids (i.e., propionic and butyric acid) have little or no effect on the production of xanthones. These results indicate that AcOH acts as a specific signal molecule, able to greatly enhance xanthone biosynthesis in H. perforatum root cultures.

Administration of a multistrain probiotic product (VSL#3) to women in the perinatal period differentially affects breast milk beneficial microbiota in relation to mode of delivery.

Probiotic supplementation to a mother during the perinatal period can have a positive impact on the breast milk composition. The aim of our study was to evaluate the effect of oral supplementation with the probiotic VSL#3, during late pregnancy and lactation, on breast milk levels of beneficial bacteria and some functional components (oligosaccharides and lactoferrin) potentially able to have a positive influence on the microbiota. Breast milk microbiota was analyzed by conventional and quantitative real-time PCR. In a double-blind, placebo-controlled, randomized trial, 66 women took daily either the probiotic (n=33) or a placebo (n=33). Intergroup analysis demonstrated that the amounts of both lactobacilli and bifidobacteria were significantly higher in the colostrum and mature milk of the mothers taking VSL#3 in comparison to those taking placebo. The analysis of bacterial strains and species present in breast milk of VSL#3 supplemented mothers indicated that the administered probiotic microorganisms did not pass from maternal gut to mammary gland. In women with vaginal delivery, significantly higher amounts of lactobacilli and bifidobacteria were detected in colostrum and mature milk of probiotic treated group in comparison to placebo group, whereas no significant difference was observed between groups in women who had caesarean section, neither in colostrum nor in mature milk. Milk levels of oligosaccharides and lactoferrin were similar in placebo and probiotic supplemented groups at all timepoints and regardless of the mode of delivery. Our results indicate a probiotic-dependent modulation of breast milk microbiota in vaginally delivering women, possibly exerted through a systemic effect.

1H NMR-based urinary metabolic profiling reveals changes in nicotinamide pathway intermediates due to postnatal stress model in rat.

The maternal separation protocol in rodents is a widely recognized model of early life stress allowing acute and chronic physiological consequences to be studied. An (1)H NMR-based metabolomic approach was applied to urines to evaluate the systemic metabolic consequences of maternal separation stress in female rats after the beginning of weaning and 4 weeks later when the rats were reaching adulthood. Furthermore, because maternal separation is considered as a model mimicking the inflammatory bowel syndrome, the lactulose/mannitol test was used to evaluate the influence of postnatal maternal separation on gut permeability and mucosal barrier function by (1)H NMR spectroscopy analysis of urine. The results showed no statistical differences in gut permeability due to maternal separation. The application of ANOVA simultaneous component analysis allowed the contributions of physiological adaptations to the animal's development to be separated from the metabolic consequences due to postnatal stress. Systemic metabolic differences in the maternally separated pups were mainly due to the tryptophan/NAD pathway intermediate levels and to the methyladenosine level. Urinary NMR-based metabolic profiling allowed us to disentangle the metabolic adaptive response of the rats to postnatal stress during the animal's growth, highlighting the metabolic changes induced by weaning, gut closure, and maturity.

Application of NMR-based metabolomics to the study of gut microbiota in obesity.

Lifestyle habits, host gene repertoire, and alterations in the intestinal microbiota concur to the development of obesity. A great deal of research has recently been focused on investigating the role gut microbiota plays in the pathogenesis of metabolic dysfunctions and increased adiposity. Altered microbiota can affect host physiology through several pathways, including enhanced energy harvest, and perturbations in immunity, metabolic signaling, and inflammatory pathways. A broad range of "omics" technologies is now available to help decipher the interactions between the host and the gut microbiota at detailed genetic and functional levels. In particular, metabolomics--the comprehensive analysis of metabolite composition of biological fluids and tissues--could provide breakthrough insights into the links among the gut microbiota, host genetic repertoire, and diet during the development and progression of obesity. Here, we briefly review the most insightful findings on the involvement of gut microbiota in the pathogenesis of obesity. We also discuss how metabolomic approaches based on nuclear magnetic resonance spectroscopy could help understand the activity of gut microbiota in relation to obesity, and assess the effects of gut microbiota modulation in the treatment of this condition.

NMR Untargeted and HPLC-MS/MS Targeted Metabolomic Approaches for Evaluating Styrene Exposure in the Urine of Shipyard Workers.

Due to its chemical properties, styrene is largely employed in the manufacturing of several products including rubber, polymers and resins, and it is particularly suitable for shipbuilding industry purposes. In this context, the main exposure to styrene occurs in occupational settings. Despite its widespread use, its long-term effects on human health at the occupational level are still unclear. The aim of this pilot study was to evaluate changes in styrene exposure biomarkers related to the metabolic and oxidative stress profiles in the urine of seventeen shipyard workers and seventeen non-exposed subjects. Urinary metabolites were assessed by means of NMR spectroscopy, including mandelic and phenylglyoxylic acids; four oxidative stress biomarkers, namely 8-oxo-7,8-dihydroguanine, 8-oxo-7,8-dihydroguanosine, and 8-oxo-7,8-dihydro-2'-deoxyguanosine and 3-nitrotyrosine, were evaluated via HPLC-MS/MS. The metabolic profiles of exposed workers showed both long- and short-term metabolic responses to styrene exposure compared to non-exposed subjects. From the comparison between non-exposed and before-shift workers, only 8-oxo-7,8-dihydroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine levels were significantly different (long term exposure response). At the same time, comparing the non-exposed group with after-shift workers, we observed lower levels of pseudouridine and 1-methylnicotinamide and higher glutamine levels in after-shift workers. The comparison between before-shift and after-shift workers showed that 8-oxo-7,8-dihydroguanine significantly increased after the shift, suggesting its involvement in the exposure to styrene (short-term exposure response). The obtained results, although preliminary, allow us to lay the basis for further human studies aimed at establishing a global understanding of styrene metabolism.

Antibiotics treatment promotes vasculogenesis in the brain of glioma-bearing mice.

In recent years, several studies described the close relationship between the composition of gut microbiota and brain functions, highlighting the importance of gut-derived metabolites in mediating neuronal and glial cells cross-talk in physiological and pathological condition. Gut dysbiosis may affects cerebral tumors growth and progression, but the specific metabolites involved in this modulation have not been identified yet. Using a syngeneic mouse model of glioma, we have investigated the role of dysbiosis induced by the administration of non-absorbable antibiotics on mouse metabolome and on tumor microenvironment. We report that antibiotics treatment induced: (1) alteration of the gut and brain metabolome profiles; (2) modeling of tumor microenvironment toward a pro-angiogenic phenotype in which microglia and glioma cells are actively involved; (3) increased glioma stemness; (4) trans-differentiation of glioma cells into endothelial precursor cells, thus increasing vasculogenesis. We propose glycine as a metabolite that, in ABX-induced dysbiosis, shapes brain microenvironment and contributes to glioma growth and progression.

Effects of resveratrol on HepG2 cells as revealed by (1)H-NMR based metabolic profiling.

BACKGROUND: Resveratrol, a polyphenol found in plant products, has been shown to regulate many cellular processes and to display multiple protective and therapeutic effects. Several in vitro and in vivo studies have demonstrated the influence of resveratrol on multiple intracellular targets that may regulate metabolic homeostasis. METHODS: We analysed the metabolic modifications induced by resveratrol treatment in a human hepatoblastoma line, HepG2 cells, using a (1)H-NMR spectroscopy-based metabolomics approach that allows the simultaneous screening of multiple metabolic pathways. RESULTS: Results demonstrated that cells cultured in the presence or absence of resveratrol displayed different metabolic profiles: the treatment induced a decreased utilisation of glucose and amino acids for purposes of energy production and synthesis associated to a decreased release of lactate in the culture medium and an increase in succinate utilisation. At the same time, resveratrol treatment slowed the cell cycle in the S phase without inducing apoptosis, and increased Sirt1 expression, also affecting its intracellular localisation. CONCLUSIONS: Our results show that the metabolomic analysis of the exometabolome of resveratrol-treated HepG2 cells indicates a metabolic switch from glucose and amino acid utilisation to fat utilisation for the production of energy, and seem in agreement with an effect mediated via AMPK- and Sirt1-activation. GENERAL SIGNIFICANCE: NMR-based metabolomics has been applied in a hepatocyte cell culture model in relation to resveratrol treatment; such an approach could be transferred to evaluate the effects of nutritional compounds with health impact.

Mitochondrial pathways in sarcopenia of aging and disuse muscle atrophy.

Muscle loss during aging and disuse is a highly prevalent and disabling condition, but knowledge about cellular pathways mediating muscle atrophy is still limited. Given the postmitotic nature of skeletal myocytes, the maintenance of cellular homeostasis relies on the efficiency of cellular quality control mechanisms. In this scenario, alterations in mitochondrial function are considered a major factor underlying sarcopenia and muscle atrophy. Damaged mitochondria are not only less bioenergetically efficient, but also generate increased amounts of reactive oxygen species, interfere with cellular quality control mechanisms, and display a greater propensity to trigger apoptosis. Thus, mitochondria stand at the crossroad of signaling pathways that regulate skeletal myocyte function and viability. Studies on these pathways have sometimes provided unexpected and counterintuitive results, which suggests that they are organized into a complex, heterarchical network that is currently insufficiently understood. Untangling the complexity of such a network will likely provide clinicians with novel and highly effective therapeutics to counter the muscle loss associated with aging and disuse. In this review, we summarize the current knowledge on the mechanisms whereby mitochondrial dysfunction intervenes in the pathogenesis of sarcopenia and disuse atrophy, and highlight the prospect of targeting specific processes to treat these conditions.

Lactobacillus acidophilus La5 and Bifidobacterium lactis Bb12 induce different age-related metabolic profiles revealed by 1H-NMR spectroscopy in urine and feces of mice.

Age-related dysbioses of intestinal microbiota and decline in the overall metabolic homeostasis are frequently found in the elderly. Probiotic supplementation may represent a way to prevent or reduce the senescence-associated metabolic disorders. The present study evaluated the metabolic impact of Lactobacillus acidophilus La5 and Bifidobacterium lactis Bb12 supplementation in relation to age by analyzing urine and feces metabolic profiles using (1)H-nuclear magnetic resonance spectroscopy and multivariate analysis. Adult (3 mo old) and aged (16 mo old) mice received an oral supplementation of the 2 probiotics (1 × 10(9) colony-forming units/d each) or phosphate buffered saline (control) daily for 30 d. Urine and feces were collected for 48 h before the end of the study. Partial least squares-discriminant analysis showed that the urinary discriminant metabolites for the probiotic treatment included higher dimethylglycine in adult and aged mice, lower sarcosine and nicotinate in adult mice, higher N-methylnicotinamide in adult mice and lower N-methylnicotinamide in aged mice compared with their controls. These results indicate a probiotic-induced modulation of homocysteine and NAD metabolism pathways, which have important implications because these pathways are involved in essential cellular processes that can be altered in senescence. The probiotic supplementation also modified the fecal metabolic profiles, inducing in both adult and aged mice higher 4-hydroxyphenylacetate and lower xylose in treated mice compared with their control mice, whereas valerate was greater in treated adult mice and lower in treated aged mice compared with their controls. The ANOVA simultaneous component analysis on urinary and fecal metabolic profiling showed an age × treatment interaction (P < 0.05), confirming the age-related modulation of the metabolic response to probiotic supplementation. The results suggest that L. acidophilus and B. lactis may prevent or reduce age-related metabolic dysfunction.

Metabolic Biomarkers of Red Beetroot Juice Intake at Rest and after Physical Exercise.

BACKGROUND: Red beetroot is known to be a health-promoting food. However, little attention is placed on intestinal bioactive compound absorption. The aim of the study was to assess the urinary red beetroot juice (RBJ) intake biomarkers and possible differences in RBJ's micronutrient absorption at rest or after physical exercise. METHODS: This is a three-armed, single-blind study, involving seven healthy volunteers which were randomly divided into three groups and alternatively assigned to three experimental sessions: RBJ intake at rest, RBJ intake with physical activity, and placebo intake with physical activity. For each session, urine samples were collected before and 120, 180, and 240 min after the intake of RBJ or placebo. The same sampling times were employed for the experimental session at rest. The RBJ metabolic composition was also characterized to identify the urinary biomarkers derived from the intake. RESULTS: 4-methylpyridine-2-carboxylic acid, dopamine-3-O-sulfate, glutamine, and 3-hydroxyisobutyrate were identified as RBJ intake biomarkers. Physical activity significantly increased only the dopamine-3-O-sulfate excretion 120 min after RBJ intake. CONCLUSIONS: Urinary dopamine-3-O-sulfate is related to RBJ dopamine content, while 4-methylpyridine-2-carboxylic acid is a betanin or betalamic acid catabolite. The different excretions of these metabolites following physical activity suggest a possible effect on the RBJ uptake depending on different transport processes through the mucosa, namely diffusion-mediated transport for dopamine and saturable transcellular transport for betalamic acid derivatives. These results open new perspectives in improving the absorption of natural bioactive molecules through physical activity.

In Vitro Probiotic Properties and In Vivo Anti-Ageing Effects of Lactoplantibacillus plantarum PFA2018AU Strain Isolated from Carrots on Caenorhabditis elegans.

Lactic acid bacteria (LAB) share and provide several beneficial effects on human health, such as the release of bioactive metabolites, pathogen competition, and immune stimulation. The two major reservoirs of probiotic microorganisms are the human gastro-intestinal tract and fermented dairy products. However, other sources, such as plant-based foods, represent important alternatives thanks to their large distribution and nutritive value. Here, the probiotic potential of autochthonous Lactiplantibacillus plantarum PFA2018AU, isolated from carrots harvested in Fucino highland, Abruzzo (Italy), was investigated through in vitro and in vivo approaches. The strain was sent to the biobank of Istituto Zooprofilattico Sperimentale della Lombardia ed Emilia Romagna in Italy for the purpose of patent procedures under the Budapest Treaty. The isolate showed high survival capability under in vitro simulated gastro-intestinal conditions, antibiotic susceptibility, hydrophobicity, aggregation, and the ability to inhibit the in vitro growth of Salmonella enterica serovar Typhimurium, Listeria monocytogenes, Pseudomonas aeruginosa, and Staphylococcus aureus pathogens. Caenorhabditis elegans was used as the in vivo model in order to analyse prolongevity and anti-ageing effects. L. plantarum PFA2018AU significantly colonised the gut of the worms, extended their lifespan, and stimulated their innate immunity. Overall, these results showed that autochthonous LAB from vegetables, such as carrots, have functional features that can be considered novel probiotic candidates.

Biostimulant Effects of Chaetomium globosum and Minimedusa polyspora Culture Filtrates on Cichorium intybus Plant: Growth Performance and Metabolomic Traits.

In this study, we investigated the biostimulant effect of fungal culture filtrates obtained from Chaetomium globosum and Minimedusa polyspora on growth performance and metabolomic traits of chicory (Cichorium intybus) plants. For the first time, we showed that M. polyspora culture filtrate exerts a direct plant growth-promoting effect through an increase of biomass, both in shoots and roots, and of the leaf area. Conversely, no significant effect on morphological traits and biomass yield was observed in C. intybus plants treated with C. globosum culture filtrate. Based on 1H-NMR metabolomics data, differential metabolites and their related metabolic pathways were highlighted. The treatment with C. globosum and M. polyspora culture filtrates stimulated a common response in C. intybus roots involving the synthesis of 3-OH-butyrate through the decrease in the synthesis of fatty acids and sterols, as a mechanism balancing the NADPH/NADP+ ratio. The fungal culture filtrates differently triggered the phenylpropanoid pathway in C. intybus plants: C. globosum culture filtrate increased phenylalanine and chicoric acid in the roots, whereas M. polyspora culture filtrate stimulated an increase of 4-OH-benzoate. Chicoric acid, whose biosynthetic pathway in the chicory plant is putative and still not well known, is a very promising natural compound playing an important role in plant defense. On the contrary, benzoic acids serve as precursors for a wide variety of essential compounds playing crucial roles in plant fitness and defense response activation. To the best of our knowledge, this is the first study that shows the biostimulant effect of C. globosum and M. polyspora culture filtrates on C. intybus growth and metabolome, increasing the knowledge on fungal bioresources for the development of biostimulants.

Hyperbaric Exposure of Scuba Divers Affects the Urinary Excretion of Nucleic Acid Oxidation Products and Hypoxanthine.

In recent studies, oxidative stress after scuba diving has been explored by measuring urinary biomarkers in volunteers under controlled conditions. Dive depth and duration, water temperature, and workload are all variables that can elicit metabolic responses. A controlled diving experiment was performed in an indoor pool at 20, 30, and 40 m depths at a water temperature of 32 °C, on three different days. Samples of urine from five male scuba divers were taken before diving and at four time points after diving, and then tested for their concentration of five different oxidative stress biomarkers by means of liquid chromatography tandem mass spectrometry and by 1H nuclear magnetic resonance metabolomics analysis. The results showed no variation in the five biomarkers after diving, but a decreasing trend was observed over the three days, with no differences among the three depths. The lack of effect on oxidative stress biomarkers has been attributed to the comfortable water temperature and to the absence of exercise in the divers during the experiment. Instead, an increase in hypoxanthine excretion, which can be considered a biomarker sensitive to hyperbaric exposure, was found after diving. Finally, the results suggest a physiological mechanism of metabolic adaptation to a new condition.