Blood biomarkers indicate that the preclinical stages of Alzheimer's disease present overlapping molecular features.

It is still debated whether non-specific preclinical symptoms of Alzheimer's disease (AD) can have diagnostic relevance. We followed the evolution from cognitively normal to AD by NMR-based metabolomics of blood sera. Multivariate statistical analysis of the NMR profiles yielded models that discriminated subjective memory decline (SMD), mild cognitive impairment (MCI) and AD. We validated a panel of six statistically significant metabolites that predicted SMD, MCI and AD in a blind cohort with sensitivity values ranging from 88 to 95% and receiver operating characteristic values from 0.88 to 0.99. However, lower values of specificity, accuracy and precision were observed for the models involving SMD and MCI, which is in line with the pathological heterogeneity indicated by clinical data. This excludes a "linear" molecular evolution of the pathology, pointing to the presence of overlapping "gray-zones" due to the reciprocal interference of the intermediate stages. Yet, the clear difference observed in the metabolic pathways of each model suggests that pathway dysregulations could be investigated for diagnostic purposes.

Modeling Acquired Resistance to the Second-Generation Androgen Receptor Antagonist Enzalutamide in the TRAMP Model of Prostate Cancer.

Enzalutamide (MDV3100) is a potent second-generation androgen receptor antagonist approved for the treatment of castration-resistant prostate cancer (CRPC) in chemotherapy-naïve as well as in patients previously exposed to chemotherapy. However, resistance to enzalutamide and enzalutamide withdrawal syndrome have been reported. Thus, reliable and integrated preclinical models are required to elucidate the mechanisms of resistance and to assess therapeutic settings that may delay or prevent the onset of resistance. In this study, the prostate cancer multistage murine model TRAMP and TRAMP-derived cells have been used to extensively characterize in vitro and in vivo the response and resistance to enzalutamide. The therapeutic profile as well as the resistance onset were characterized and a multiscale stochastic mathematical model was proposed to link the in vitro and in vivo evolution of prostate cancer. The model showed that all therapeutic strategies that use enzalutamide result in the onset of resistance. The model also showed that combination therapies can delay the onset of resistance to enzalutamide, and in the best scenario, can eliminate the disease. These results set the basis for the exploitation of this "TRAMP-based platform" to test novel therapeutic approaches and build further mathematical models of combination therapies to treat prostate cancer and CRPC.Significance: Merging mathematical modeling with experimental data, this study presents the "TRAMP-based platform" as a novel experimental tool to study the in vitro and in vivo evolution of prostate cancer resistance to enzalutamide.

Metabolomics of Exhaled Breath Condensate by Nuclear Magnetic Resonance Spectroscopy and Mass Spectrometry: A Methodological Approach.

Respiratory diseases present a very high prevalence in the general population, with an increase in morbidity, mortality and health-care expenses worldwide. They are complex and heterogeneous pathologies that may present different pathological facets in different subjects, often with personal evolution. Therefore, there is a need to identify patients with similar characteristics, prognosis or treatment, defining the so-called phenotype, but also to mark specific differences within each phenotype, defining the endotypes. Biomarkers are very useful to study respiratory phenotypes and endotypes. Metabolomics, one of the recently introduced "omics", is becoming a leading technique for biomarker discovery. For the airways, metabolomics appears to be well suited as the respiratory tract offers a natural matrix, the Exhaled Breath Condensate (EBC), in which several biomarkers can be measured. In this review, we will discuss the main methodological issues related to the application of Nuclear Magnetic Resonance (NMR) spectroscopy and Mass Spectrometry (MS) to EBC metabolomics for investigating respiratory diseases.

Metabolite variation in three edible Italian Allium cepa L. by NMR-based metabolomics: a comparative study in fresh and stored bulbs.

INTRODUCTION: In fruits and vegetables, comparative analysis of metabolic plant profiles has a high potential for quality control of active components. Onion (Allium cepa L.) is used fresh or stored as food, spice, and in traditional medicine. Its metabolic content, often with nutraceutical value, makes its level an important factor in agronomic production. OBJECTIVE: To describe for the first time the metabolome of "San Pietro" white onion (WP), and compare its chemical profile with the red onion var. Tropea (RT) and the yellow onion var. Montoro (CM). Furthermore, we also aim to obtain a multivariate model based on NMR fingerprints to discriminate the three Italian A. cepa L. cultivars. METHODS: For the chemical fingerprinting we used NMR-based metabolomics. We investigated the aqueous and chloroform extracts of fresh onion at harvesting time, and after 9-month storage. Principal component analysis (PCA), Partial least squares discriminant analysis (PLS-DA) and Orthogonal partial least squares (OPLS-DA) were used to build reliable models. RESULTS: We obtained a clear discrimination of A. cepa L. varieties for the fresh and stored batches. The statistical model highlighted higher levels of fructo-oligosaccharides (FOS) in the fresh WP; RT showed a high content of glucose, citrate and amino acids, while CM had many sulfur components. In the stored samples (CMS, RTS), carbohydrates and sulfur components decreased, while in WPS the free monosaccharides concentration increased. Linoleic acid was overexpressed in the apolar extracts of CMF and WPF cultivars. CONCLUSION: Metabolomics allows a reliable differentiation among onion varieties, and highlights the potential of fingerprinting for food authentication purposes.

Biomonitoring of workers using nuclear magnetic resonance-based metabolomics of exhaled breath condensate: A pilot study.

Exhaled breath condensate (EBC) is a non-invasive biological matrix that allows easy access to the lung epithelial lining fluid. It can provide, for instance, useful biochemical information in workers following inhalatory exposure. Recently, nuclear magnetic resonance (NMR)-based metabolomics has been applied to EBC profiling to identify metabolic phenotypes ("metabotypes") of relevance in respiratory medicine. We aimed at verifying if NMR-based metabolomics of EBC, combined with statistical analysis, could find differences in metabolomic profiles between groups of subjects occupationally exposed to levels of airborne inhalable dust, phenol, formaldehyde and volatile organic compounds (VOCs) below regulatory limits. Samples were collected from 20 blue-collar workers without wearing a mask (therefore "exposed" to ambient air) and after wearing a mask ("not exposed"). This procedure was only allowed after the standard air quality tests had shown levels of airborne xenobiotics below the occupational exposure limit or even below the LOD. Ten white-collar controls were also included. After partial least squares discriminant analysis, the exposed to the controlled plant environment and the not exposed groups were clearly separated, and discrimination was due to alteration of fatty acids and alcohols, whose statistical significance was also evaluated. Our results show that NMR-based metabolomics of EBC is a useful tool that has the potential to be successfully used in occupational health to distinguish between subjects exposed and non-exposed to very low airborne levels of chemicals.

Metabolomic Analysis by Nuclear Magnetic Resonance Spectroscopy as a New Approach to Understanding Inflammation and Monitoring of Pharmacological Therapy in Children and Young Adults With Cystic Fibrosis.

15-F2t-Isoprostane, a reliable biomarker of oxidative stress, has been found elevated in exhaled breath condensate (EBC), a non-invasive technique for sampling of airway secretions, in patients with cystic fibrosis (CF). Azithromycin has antioxidant properties in experimental models of CF, but its effects on oxidative stress in CF patients are largely unknown. Primary objective of this pilot, proof-of-concept, prospective, parallel group, pharmacological study, was investigating the potential antioxidant effects of azithromycin in CF patients as reflected by EBC 15-F2t-isoprostane. Secondary objectives included studying the effect of azithromycin on EBC and serum metabolic profiles, and on serum 15-F2t-isoprostane. In CF patients who were on maintenance treatment with oral vitamin E (200 UI once daily), treatment with oral azithromycin (250 or 500 mg depending on body weight) plus vitamin E (400 UI once daily) (group A) (n = 24) or oral vitamin E alone (400 UI once daily) (group B) (n = 21) was not associated with changes in EBC 15-F2t-isoprostane concentrations compared with baseline values after 8-weeks treatment or 2 weeks after treatment suspension. There was no between-group difference in post-treatment EBC 15-F2t-isoprostane. Likewise, no within- or between-group differences in serum 15-F2t-isoprostane concentrations were observed in either study group. NMR spectroscopy-based metabolomics of EBC shows that suspension of both azithromycin plus vitamin E and vitamin E alone has a striking effect on metabolic profiles in EBC. Between-group comparisons show that EBC metabolite distribution after treatment and 2 weeks after treatment suspension is different. Quantitative differences in ethanol, saturated fatty acids, acetate, acetoin/acetone, and methanol are responsible for these differences. Our study was unable to show antioxidant effect of azithromycin as add-on treatment with doubling the dose of oral vitamin E as reflected by 15-F2t-isoprostane concentrations in EBC. Add-on therapy with azithromycin itself does not induce EBC metabolite changes, but its suspension is associated with EBC metabolic profiles that are different from those observed after vitamin E suspension. The pathophysiological and therapeutic implications of these findings in patients with stable CF are unknown and require further research. Preliminary data suggest that EBC NMR-based metabolomics might be used for assessing the effects of pharmacological treatment suspension in stable CF patients.

Enhancement of hepatic autophagy increases ureagenesis and protects against hyperammonemia.

Ammonia is a potent neurotoxin that is detoxified mainly by the urea cycle in the liver. Hyperammonemia is a common complication of a wide variety of both inherited and acquired liver diseases. If not treated early and thoroughly, it results in encephalopathy and death. Here, we found that hepatic autophagy is critically involved in systemic ammonia homeostasis by providing key urea-cycle intermediates and ATP. Hepatic autophagy is triggered in vivo by hyperammonemia through an α-ketoglutarate-dependent inhibition of the mammalian target of rapamycin complex 1, and deficiency of autophagy impairs ammonia detoxification. In contrast, autophagy enhancement by means of hepatic gene transfer of the master regulator of autophagy transcription factor EB or treatments with the autophagy enhancers rapamycin and Tat-Beclin-1 increased ureagenesis and protected against hyperammonemia in a variety of acute and chronic hyperammonemia animal models, including acute liver failure and ornithine transcarbamylase deficiency, the most frequent urea-cycle disorder. In conclusion, hepatic autophagy is an important mechanism for ammonia detoxification because of its support of urea synthesis, and its enhancement has potential for therapy of both primary and secondary causes of hyperammonemia.

Vitamin C and l-Proline Antagonistic Effects Capture Alternative States in the Pluripotency Continuum.

Metabolites and cofactors are emerging as key regulators of cell plasticity and reprogramming, and their role in the control of pluripotency is just being discovered. Here we provide unprecedented evidence that embryonic stem cell (ESC) pluripotency relies on the relative levels of two physiological metabolites, namely ascorbic acid (vitamin C, VitC) and l-proline (l-Pro), which affect global DNA methylation, transcriptional profile, and energy metabolism. Specifically, while a high VitC/l-Pro ratio drives ESCs toward a naive state, the opposite condition (l-Pro excess) captures a fully reversible early primed pluripotent state, which depends on autocrine fibroblast growth factor and transforming growth factor ß signaling pathways. Our findings highlight the pivotal role of metabolites availability in controlling the pluripotency continuum from naive to primed states.

Monoterpenoids from the traditional North Italian vegetable Aruncus dioicus (Walter) Fernald var. vulgaris (Maxim.) H.Hara (Rosaceae).

Investigations of young shoots of Aruncus dioicus (Walter) Fernald var. vulgaris (Maxim.) H.Hara (Rosaceae), collected from the wild and used as vegetables in alpine provinces of Italy, yielded eight monoterpenoids. Besides known compounds, aruncin A, aruncide A, and cimicifugolide, five previously undescribed substances, aruncins C, D, and E, and aruncides D and E, were identified. Based on results from the full synthesis of aruncin B, structures of aruncin A and aruncide A were revised. Structures were established by HR mass spectrometry and extensive 1D and 2D NMR spectroscopy and based on data from synthetic aruncin B. An HPLC-DAD-ESI-MS method was developed to investigate the distribution of the monoterpenoids in different organs of Aruncus dioicus var. vulgaris and in aerial parts of A. dioicus var. aethusifolius (H.Lév.) H.Hara [Syn.: Aruncus aethusifolius (H.Lév.) Nakai]. Preliminary bioactivity studies moreover indicated weak cytotoxicity for some of the compounds against human prostrate adenocarcinoma cells.

Coexistence of obesity and asthma determines a distinct respiratory metabolic phenotype.

BACKGROUND: Epidemiologic and clinical evidence supports the existence of an obesity-related asthma phenotype. No distinct pathophysiologic elements or specific biomarkers have been identified thus far, but increased oxidative stress has been reported. OBJECTIVE: We aimed at verifying whether metabolomics of exhaled breath condensate from obese asthmatic (OA) patients, lean asthmatic (LA) patients, and obese nonasthmatic (ONA) subjects could recognize specific and statistically validated biomarkers for a separate "asthma-obesity" respiratory metabolic phenotype, here defined as "metabotype." METHODS: Twenty-five OA patients, 30 ONA subjects, and 30 mild-to-moderate LA age-matched patients participated in a cross-sectional study. Nuclear magnetic resonance (NMR) profiles were analyzed by using partial least-squares discriminant analysis, and the results were validated with an independent patient set. RESULTS: From NMR profiles, we obtained strong regression models that distinguished OA patients from ONA subjects (quality parameters: goodness-of-fit parameter [R2] = 0.81 and goodness-of-prediction parameter [Q2] = 0.79), as well as OA patients from LA patients (R2 = 0.91 and Q2 = 0.89). The all-classes comparison (R2 = 0.86 and Q2 = 0.83) indicated that OA patients possess a respiratory metabolic profile fully divergent from those obtained in the other patient groups. We also identified specific biomarkers for between-class separation, which are independent from clinical bias. They are involved in the methane, pyruvate, and glyoxylate and dicarboxylate metabolic pathways. CONCLUSIONS: NMR-based metabolomics indicates that OA patients are characterized by a respiratory metabolic fingerprint fully different from that of patients independently affected by asthma or obesity. Such a phenotypic difference strongly suggests unique pathophysiologic pathways involved in the pathogenesis of asthma in adult obese subjects. Furthermore, the OA metabotype could define a strategy for patient stratification based on unbiased biomarkers, with important diagnostic and therapeutic implications.

Neuroendocrine Transdifferentiation in Human Prostate Cancer Cells: An Integrated Approach.

Prostate cancer is highly sensitive to hormone therapy because androgens are essential for prostate cancer cell growth. However, with the nearly invariable progression of this disease to androgen independence, endocrine therapy ultimately fails to control prostate cancer in most patients. Androgen-independent acquisition may involve neuroendocrine transdifferentiation, but there is little knowledge about this process, which is presently controversial. In this study, we investigated this question in a novel model of human androgen-dependent LNCaP cells cultured for long periods in hormone-deprived conditions. Strikingly, characterization of the neuroendocrine phenotype by transcriptomic, metabolomic, and other statistically integrated analyses showed how hormone-deprived LNCaP cells could transdifferentiate to a nonmalignantneuroendocrine phenotype. Notably, conditioned media from neuroendocrine-like cells affected LNCaP cell proliferation. Predictive in silico models illustrated how after an initial period, when LNCaP cell survival was compromised by an arising population of neuroendocrine-like cells, a sudden trend reversal occurred in which the neuroendocrine-like cells functioned to sustain the remaining androgen-dependent LNCaP cells. Our findings provide direct biologic and molecular support for the concept that neuroendocrine transdifferentiation in prostate cancer cell populations influences the progression to androgen independence.

NADP(+)-dependent dehydrogenase activity of carbonyl reductase on glutathionylhydroxynonanal as a new pathway for hydroxynonenal detoxification.

An NADP(+)-dependent dehydrogenase activity on 3-glutathionyl-4-hydroxynonanal (GSHNE) was purified to electrophoretic homogeneity from a line of human astrocytoma cells (ADF). Proteomic analysis identified this enzymatic activity as associated with carbonyl reductase 1 (EC 1.1.1.184). The enzyme is highly efficient at catalyzing the oxidation of GSHNE (KM 33 µM, kcat 405 min(-1)), as it is practically inactive toward trans-4-hydroxy-2-nonenal (HNE) and other HNE-adducted thiol-containing amino acid derivatives. Combined mass spectrometry and nuclear magnetic resonance spectroscopy analysis of the reaction products revealed that carbonyl reductase oxidizes the hydroxyl group of GSHNE in its hemiacetal form, with the formation of the corresponding 3-glutathionylnonanoic-δ-lactone. The relevance of this new reaction catalyzed by carbonyl reductase 1 is discussed in terms of HNE detoxification and the recovery of reducing power.

Two non-psychoactive cannabinoids reduce intracellular lipid levels and inhibit hepatosteatosis.

BACKGROUND & AIMS: Obesity and associated metabolic syndrome have quickly become a pandemic and a major detriment to global human health. The presence of non-alcoholic fatty liver disease (NAFLD; hepatosteatosis) in obesity has been linked to the worsening of the metabolic syndrome, including the development of insulin resistance and cardiovascular disease. Currently, there are few options to treat NAFLD, including life style changes and insulin sensitizers. Recent evidence suggests that the cannabinoids Δ(9)-tetrahydrocannabivarin (THCV) and cannabidiol (CBD) improve insulin sensitivity; we aimed at studying their effects on lipid levels. METHODS: The effects of THCV and CBD on lipid levels were examined in a variety of in vitro and in vivo systems, with special emphasis on models of hepatosteatosis. Transcriptional, post-translational and metabolomic changes were assayed. RESULTS: THCV and CBD directly reduce accumulated lipid levels in vitro in a hepatosteatosis model and adipocytes. Nuclear magnetic resonance- (NMR) based metabolomics confirmed these results and further identified specific metabolic changes in THCV and CBD-treated hepatocytes. Treatment also induced post-translational changes in a variety of proteins such as CREB, PRAS40, AMPKa2 and several STATs indicating increased lipid metabolism and, possibly, mitochondrial activity. These results are supported by in vivo data from zebrafish and obese mice indicating that these cannabinoids are able to increase yolk lipid mobilization and inhibit the development of hepatosteatosis respectively. CONCLUSIONS: Our results suggest that THCV and CBD might be used as new therapeutic agents for the pharmacological treatment of obesity- and metabolic syndrome-related NAFLD/hepatosteatosis.

NMR metabolomic analysis of exhaled breath condensate of asthmatic patients at two different temperatures.

Exhaled breath condensate (EBC) collection is a noninvasive method to investigate lung diseases. EBC is usually collected with commercial/custom-made condensers, but the optimal condensing temperature is often unknown. As such, the physical and chemical properties of exhaled metabolites should be considered when setting the temperature, therefore requiring validation and standardization of the collecting procedure. EBC is frequently used in nuclear magnetic resonance (NMR)-based metabolomics, which unambiguously recognizes different pulmonary pathological states. Here we applied NMR-based metabolomics to asthmatic and healthy EBC samples collected with two commercial condensers operating at -27.3 and -4.8 °C. Thirty-five mild asthmatic patients and 35 healthy subjects were included in the study, while blind validation was obtained from 20 asthmatic and 20 healthy different subjects not included in the primary analysis. We initially analyzed the samples separately and assessed the within-day, between-day, and technical repeatabilities. Next, samples were interchanged, and, finally, all samples were analyzed together, disregarding the condensing temperature. Partial least-squares discriminant analysis of NMR spectra correctly classified samples, without any influence from the temperature. Input variables were either integral bucket areas (spectral bucketing) or metabolite concentrations (targeted profiling). We always obtained strong regression models (95%), with high average-quality parameters for spectral profiling (R(2) = 0.84 and Q(2) = 0.78) and targeted profiling (R(2) = 0.91 and Q(2) = 0.87). In particular, although targeted profiling clustering is better than spectral profiling, all models reproduced the relative metabolite variations responsible for class differentiation. This warrants that cross comparisons are reliable and that NMR-based metabolomics could attenuate some specific problems linked to standardization of EBC collection.

Brain synaptosomes harbor more than one cytoplasmic system of protein synthesis.

Synaptosomal protein synthesis from rat brain is selectively increased by learning and is massively enhanced during the recovery period from brain ischemia. To lay the groundwork for identification of the involved synaptic elements, we examined the effects induced by varying the concentrations of extracellular cations and endogenous calcium. Most of the recorded rate response curves exhibited biphasic profiles that suggested the presence of more than one translation system. Because comparable profiles were obtained by fully inhibiting mitochondrial translation, the data indicated the involvement of cytoplasmic translation systems present in different synaptosomal classes. Their properties may be individually investigated by exploiting the partially inhibited conditions we have described. The identification of the synaptic elements from which they originated and their newly synthesized proteins will significantly expand our understanding of the synaptic contribution to brain plastic events.

Lentisone, a new phytotoxic anthraquinone produced by Ascochyta lentis, the causal agent of Ascochyta blight in Lens culinaris.

An aggressive isolate of Ascochyta lentis obtained from lentil (Lens culinaris L.) produced various metabolites in vitro. The metabolites were isolated from the culture filtrates and characterized by spectroscopic, chemical, and optical methods. A new phytotoxic anthraquinone, named lentisone, was isolated and characterized as (1S*,2S*,3S*)-1,2,3,8-tetrahydroxy-1,2,3,4-tetrahydro-6-methylanthraquinone together with the well-known pachybasin (1-hydroxy-3-methylanthraquinone), tyrosol, and pseurotin A. Lentisone, tyrosol, and pseurotin A were phytotoxic to lentil, with lentisone the most toxic of all. The toxicity of these compounds is light-dependent. Finally, lentisone was also found to be phytotoxic to chickpea, pea, and faba bean, with toxicity in the latter higher than in any other tested legume, including lentil.

Separating smoking-related diseases using NMR-based metabolomics of exhaled breath condensate.

Nuclear magnetic resonance (NMR)-based metabolomics separates exhaled breath condensate (EBC) profiles of patients affected by pulmonary disease from those of healthy subjects. Here we show the discriminatory ability of NMR-based metabolomics in separating patients exposed to the same risk factor, namely, smoking habit in smoking-related diseases. Fifty duplicated EBC samples from a cohort of current smokers without chronic obstructive pulmonary disease (COPD, henceforth HS), COPD smokers, and subjects with established pulmonary Langerhans cell histiocytosis (PLCH) were analyzed by means of NMR spectroscopy followed by principal component analysis (PCA) and projection to latent structures discriminant analysis (PLS-DA). Clusterization of EBC spectra was disease-specific. COPD and PLCH samples present a profile different from that of HS, showing acetate increase and 1-methylimidazole reduction. An inverse behavior of 2-propanol and isobutyrate characterized COPD with respect to PLCH (high/low in COPD, low/high in PLCH). Both the 2-component and the 3-component PLS-DA models showed a 96% cross-validated accuracy, presenting R(2) and Q(2) values in the ranges of 0.97-0.87 and 0.91-0.78, respectively, and R(2) = 0.87 and Q(2) = 0.78, indicating that data variation is well explained by each model (R(2)), with a good predictivity (Q(2)). NMR spectra of EBC discriminate COPD and PLCH patients from HS and between them, with well-defined metabolic profiles for each class. The specificity of EBC profiles suggests that disease itself drives metabolic separation overwhelming the "common background" due to smoking habit. EBC-NMR investigation offers a powerful tool for assessing the evolution of airway diseases even in the presence of a strong common factor.

Cyclobotryoxide, a phytotoxic metabolite produced by the plurivorous pathogen Neofusicoccum australe.

Two isolates of Neofusicoccum australe belonging to ITS haplotypes H4 and H1 and associated with grapevine cordon dieback and branch dieback of Phoenicean juniper, respectively, have been shown to produce in vitro structurally different secondary metabolites. From the strain BOT48 of N. australe (haplotype H4) a new cyclohexenone oxide, namely, cyclobotryoxide, was isolated together with 3-methylcatechol and tyrosol. Cyclobotryoxide was characterized as (1S,5R,6S)-5-hydroxy-3-methoxy-4-methyl-7-oxabicyclo[4.1.0]hept-3-en-2-one by spectroscopic, optical, and chemical methods. The strain BL24 (haplotype H1) produced tyrosol along with botryosphaerone D and (3S,4S)-3,4,8-trihydroxy-6-methoxy-3,4-dihydro-1(2H)-naphthalenone. The metabolites obtained from both strains were tested at four concentrations on leaves of grapevine cv. Cannonau, holm oak, and cork oak by the leaf puncture assay. Cyclobotryoxide proved to be the most phytotoxic compound. Tyrosol and cyclobotryoxide were also tested on detached grapevine leaves at concentrations of 0.25 and 0.5 mg/mL. Only cyclobotryoxide was found to be active in this bioassay.