Search

Many pivotal biological cell processes are affected by gravity. The aim of our study was to evaluate biological and functional effects, differentiation potential and exo-metabolome profile of simulated microgravity (SMG) on human hepatic cell line (HepG2) and human biliary tree stem/progenitor cells (hBTSCs). Both hBTSCs and HepG2 were cultured in a weightless and protected environment SGM produced by the Rotary Cell Culture System (Synthecon) and control condition in normal gravity (NG). Self-replication and differentiation toward mature cells were determined by culturing hBTSCs in Kubota’s Medium (KM) and in hormonally defined medium (HDM) tailored for hepatocyte differentiation. The effects on the expression and cell exo-metabolome profiles of SMG versus NG cultures were analyzed. SMG promotes tridimensional (3D) cultures of hBTSCs and HepG2. Significative increase of stemness gene expression (p in vitro toward mature hepatocytes when cultured in SMG condition. Furthermore, in HepG2 cells the SMG caused a lower (p

BackgroundCystic fibrosis (CF) is a disorder affecting the respiratory, digestive, reproductive systems and sweat glands. This lethal hereditary disease has known or suspected links to the dysbiosis gut microbiota. High-throughput meta-omics-based approaches may assist in unveiling this complex network of symbiosis modifications.ObjectivesThe aim of this study was to provide a predictive and functional model of the gut microbiota enterophenotype of pediatric patients affected by CF under clinical stability.MethodsThirty-one fecal samples were collected from CF patients and healthy children (HC) (age range, 1-6 years) and analysed using targeted-metagenomics and metabolomics to characterize the ecology and metabolism of CF-linked gut microbiota. The multidimensional data were low fused and processed by chemometric classification analysis.ResultsThe fused metagenomics and metabolomics based gut microbiota profile was characterized by a high abundance of Propionibacterium, Staphylococcus and Clostridiaceae, including Clostridium difficile, and a low abundance of Eggerthella, Eubacterium, Ruminococcus, Dorea, Faecalibacterium prausnitzii, and Lachnospiraceae, associated with overexpression of 4-aminobutyrate (GABA), choline, ethanol, propylbutyrate, and pyridine and low levels of sarcosine, 4-methylphenol, uracil, glucose, acetate, phenol, benzaldehyde, and methylacetate. The CF gut microbiota pattern revealed an enterophenotype intrinsically linked to disease, regardless of age, and with dysbiosis uninduced by reduced pancreatic function and only partially related to oral antibiotic administration or lung colonization/infection.ConclusionsAll together, the results obtained suggest that the gut microbiota enterophenotypes of CF, together with endogenous and bacterial CF biomarkers, are direct expression of functional alterations at the intestinal level. Hence, it's possible to infer that CFTR impairment causes the gut ecosystem imbalance.This new understanding of CF host-gut microbiota interactions may be helpful to rationalize novel clinical interventions to improve the affected children's nutritional status and intestinal function.

Metabolomics has the capability of providing predisposition, diagnostic, prognostic, and therapeutic biomarker profiles of individual patients, since a large number of metabolites can be measured in an unbiased manner from biological samples. In this setting, 1H-Nuclear Magnetic Resonance (NMR) spectroscopy of biofluids such as plasma, urine, and fecal water offers the opportunity to identify patterns of biomarker changes that reflects the physiological or pathological status of an individual patient.In this chapter, we show as a metabolomics study can be used to diagnose a disease, classifying patients as healthy or as pathological taking into account individual variability.

Background Solid tumours are less oxygenated than normal tissues. Consequently, cancer cells acquire to be adapted to a hypoxic environment. The poor oxygenation of solid tumours is also a major indicator of an adverse cancer prognosis and leads to resistance to conventional anticancer treatments. We previously showed the involvement of Che-1/AATF (Che-1) in cancer cell survival under stress conditions. Herein we hypothesized that Che-1 plays a role in the response of cancer cells to hypoxia. Methods The human colon adenocarcinoma HCT116 and HT29 cell lines undepleted or depleted for Che-1 expression by siRNA, were treated under normoxic and hypoxic conditions to perform studies regarding the role of this protein in metabolic adaptation and cell proliferation. Che-1 expression was detected using western blot assays; cell metabolism was assessed by NMR spectroscopy and functional assays. Additional molecular studies were performed by RNA seq, qRT-PCR and ChIP analyses. Results Here we report that Che-1 expression is required for the adaptation of cells to hypoxia, playing an important role in metabolic modulation. Indeed, Che-1 depletion impacted on HIF-1α stabilization, thus downregulating the expression of several genes involved in the response to hypoxia and affecting glucose metabolism. Conclusions We show that Che-1 a novel player in the regulation of HIF-1α in response to hypoxia. Notably, we found that Che-1 is required for SIAH-2 expression, a member of E3 ubiquitin ligase family that is involved in the degradation of the hydroxylase PHD3, the master regulator of HIF-1α stability. Electronic supplementary material The online version of this article (doi:10.1186/s13046-017-0497-1) contains supplementary material, which is available to authorized users.

// Mario Cioce 1* , MariaCristina Valerio 2* , Luca Casadei 2 , Claudio Pulito 3 , Andrea Sacconi 4 , Federica Mori 3 , Francesca Biagioni 4 , Cesare Manetti 2 , Paola Muti 5 , Sabrina Strano 3 , and Giovanni Blandino 4 . 1 Department of Cardiothoracic Surgery, NYU Langone Medical Center, New York, NY USA 2 Department of Chemistry, University of Rome ‘La Sapienza’, 00185 Rome, Italy 3 Molecular Chemoprevention Group, Italian National Cancer Institute “Regina Elena”, Rome, Italy 4 Translational Oncogenomic Unit, Italian National Cancer Institute “Regina Elena”, Rome, Italy 5 Department of Oncology, McMaster University, Hamilton, Ontario, L8V 5C2, Canada * These two authors contributed equally Correspondence: Giovanni Blandino , email: // Keywords : Metformin, metabolism, chemoresistance, ALDH, metabolic reprogramming, cancer Received : January 22, 2014 Accepted : March 24, 2014 Published : March 26, 2014 Abstract Metabolic remodeling is a hallmark of cancer progression and may affect tumor chemoresistance. Here we investigated by 1H-NMR/PCA analysis the metabolic profile of chemoresistant breast cancer cell subpopulations (ALDH bright cells) and their response to metformin, a promising anticancer metabolic modulator. The purified ALDH bright cells exhibited a different metabolic profile as compared to their chemosensitive ALDH low counterparts. Metformin treatment strongly affected the metabolism of the ALDHbright cells thereby affecting, among the others, the glutathione metabolism, whose upregulation is a feature of progenitor-like, chemoresistant cell subpopulations. Globally, metformin treatment reduced the differences between ALDH bright and ALDH low cells, making the former more similar to the latter. Metformin broadly modulated microRNAs in the ALDH bright cells, with a large fraction of them predicted to target the same metabolic pathways experimentally identified by 1H-NMR. Additionally, metformin modulated the levels of c-MYC and IRS-2, and this correlated with changes of the microRNA-33a levels. In summary, we observed, both by 1H-NMR and microRNA expression studies, that metformin treatment reduced the differences between the chemoresistant ALDH bright cells and the chemosensitive ALDH low cells. This works adds on the potential therapeutic relevance of metformin and shows the potential for metabolic reprogramming to modulate cancer chemoresistance.

Metabolomics belongs to the family of "-omics" sciences, also comprised of genomics, transcriptomics, and proteomics, all of which share the advantage of a non-targeted approach for identifying biomarkers and profiling the patient. This means that they do not require a preliminary knowledge of the substances to be studied. Moreover, even small quantities of biological fluids or tissues may be utilized for analysis. Metabolomic procedure has become feasible only recently with the advent and accessibility of new high-throughput technologies, including mass spectrometry and nuclear magnetic resonance. The methodology generally involves three defining steps: 1) the acquisition of experimental data, 2) the multivariate statistical analysis, and 3) the projection of the acquired information (profiles) to construct the patient map. Metabolomic analysis has been applied to several disorders: as far as rheumatic diseases are concerned, a few studies have focused on rheumatoid arthritis, spondyloarthritis, systemic lupus erythematosus, and osteoarthritis. Both murine models and clinical data have shown the potential of this novel tool to contribute to deciding a diagnosis, discriminate between patients based on disease activity, and even predict the response to a particular treatment. The present review fully reports these findings and offers a critical view of the challenges still to be met.

In this work we show the functionalization of the interior of microfluidic glass chips with poly(2-hydroxyethyl methacrylate) polymer brushes as anchors for co-immobilization of the enzymes glucose-oxidase and horseradish peroxidase. The formation of the brush layer and subsequent immobilization of these enzymes have been characterized on flat surfaces by atomic force microscopy and Fourier transform infrared spectroscopy, and studied inside glass chips by field emission scanning microscopy. Enzyme-functionalized glass chips have been applied for performing a multi-enzymatic cascade reaction for the fast (20 s) determination of glucose in human blood samples and the result is in excellent agreement with values obtained from the conventional hospital laboratory. The limit of detection of this bi-enzymatic method is 60 μM. With the advantages of high selectivity and reproducibility, this functionalization method can be used for improving the efficiency of glucose sensors.

Contents 8.1 Migraine and Genetic, Hematochemical, and Instrumental Parameters 172 8.2 Structure of the Database 173

Malignant pleural mesothelioma is a poorly treated neoplasia arising from the pleural mesothelial lining. Here we document that the leaf extract of Cynara scolymus exerts broad antitumoral effects both in vitro and in vivo on mesothelioma cell lines. We found that Cynara scolymus treatment affects strongly cell growth, migration and tumor engraftment of mesothelioma cell lines. Strikingly, dietary feeding with Cynara scolymus leaf extract reduces the growth of mesothelioma xenografted tumors similarly to pemetrexed, a commonly employed drug in the treatment of mesothelioma. In aggregate our findings suggest that leaf extract of Cynara scolymus holds therapeutic potential for the treatment of mesothelioma.

Objective A considerable proportion of patients with rheumatoid arthritis (RA) do not have a satisfactory response to biological therapies. We investigated the use of metabolomics approach to identify biomarkers able to anticipate the response to biologics in RA patients. Methods Due to gender differences in metabolomic profiling, the analysis was restricted to female patients starting etanercept as the first biological treatment and having a minimum of six months’ follow-up. Each patient was evaluated by the same rheumatologist before and after six months of treatment. At this time, the clinical response (good, moderate, none) was determined according to the EUropean League Against Rheumatism (EULAR) criteria, based on both erythrocyte sedimentation rate (EULAR-ESR) and C-reactive protein (EULAR-CRP). Sera collected prior and after six months of etanercept were analyzed by 1H-nuclear magnetic resonance (NMR) spectroscopy in combination with multivariate data analysis. Results Twenty-seven patients were enrolled: 18 had a good/moderate response and 9 were non responders according to both EULAR-ESR and EULAR-CRP after six months of etanercept. Metabolomic analysis at baseline was able to discriminate good, moderate, and non-responders with a very good predictivity (Q2 = 0.68) and an excellent sensitivity, specificity, and accuracy (100%). In good responders, we found an increase in isoleucine, leucine, valine, alanine, glutamine, tyrosine, and glucose levels and a decrease in 3-hydroxybutyrate levels after six months of treatment with etanercept with respect to baseline. Conclusion Our study confirms the potential of metabolomic analysis to predict the response to biological agents. Changes in metabolic profiles during treatment may help elucidate their mechanism of action.

Objective: Microgravity was often shown to cause cell damage and impair cell cycle in a variety of biological systems. Since the effects on the neural system were poorly investigated, we aimed to gain insight into how biological processes such as cell cycle, cell damage, stemness features and metabolic status are involved in neural stem cells (NSC) when they experience simulated microgravity. We also wished to investigate whether these modulations were transient or permanent once cells were returned to normal gravity. Methods: NSC were isolated from mouse cerebella and cultured in the Rotary Cell Culture System (RCCS) to model microgravity. We analyzed cell cycle, stress and apoptotic response. We also performed a 1H NMR-based metabolomic analysis and evaluation of stemness features of NSC in simulated microgravity and once in the returned to normogravity cell culture. Results: Biological processes and metabolic status were modulated by simulated microgravity. Cells were arrested in S-phase together with enhanced apoptosis. Metabolic changes occurred in NSC after simulated microgravity. Interestingly, these modulations were transient. Indeed, stemness features and metabolic footprint returned to basal levels after few days of culture in normal conditions. Moreover NSC clonogenic ability was not impaired. Conclusions: Our data suggest that simulated microgravity impacts on NSC biological processes, including cell cycle and apoptosis. However, NSC does not suffer from permanent damage.

Metabolomics is the analysis of the concentration profiles of low molecular weight compounds present in biological fluids. Metabolites are nonpeptide molecules representing the end products of cellular activity. Therefore, changes in metabolite concentrations reveal the range of biochemical effects induced by a disease or its therapeutic intervention. Metabolomics has recently become feasible with the accessibility of new technologies, including mass spectrometry and high-resolution proton nuclear magnetic resonance, and has already been applied to several disorders. Indeed, it has the advantage of being a nontargeted approach for identifying potential biomarkers, which means that it does not require a preliminary knowledge of the substances to be studied. In this review, we summarize the main studies in which metabolomic approach was used in some allergic (asthma, atopic dermatitis) and rheumatic diseases (rheumatoid arthritis, systemic lupus erythematosus) to explore the feasibility of this technique as a novel diagnostic tool in these complex disorders.

Changes in brain lipid composition have been determined in 24 months-old Fischer rats with respect to 6 months-old ones. The cerebral levels of sphingomyelin and cholesterol were found to be significantly increased in aged rats, whereas the amount of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and phosphatidic acid appear to be unaffected by aging. Long-term feeding with acetyl-L-carnitine was able to reduce the age-dependent increase of both sphingomyelin and cholesterol cerebral levels with no effect on the other measured phospholipids. These findings shown that changes in membrane lipid metabolism and/or composition represent one of the alterations occurring in rat brain with aging, and that long-term feeding with acetyl-L-carnitine can be useful in normalizing these age-dependent disturbances.

The copper (II) complex of amikacin in water solution at pH 5.5 was investigated by 13 C-NMR. The temperature dependence of spin–lattice relaxation rates was measured and fast exchange conditions were shown to apply. The motional correlation time of the complex was approximated by the pseudo-isotropic rotational correlation time of free amikacin in water solution ( τ c =0.17 ns at 300 K). Formation of a pseudo-tetrahedral 1:1 complex was demonstrated by relaxation rates analysis and also by UV–Vis spectrophotometry. Two amino nitrogens of amikacin, together with the amide nitrogen and the hydroxyl in the hydroxyl–aminopropyl carbonyl side chain, were assigned as the copper-binding sites and a model of the complex was built by using copper–carbon distances obtained by NMR analysis as input parameters.

The astonishing development of high-throughput techniques in the last decades has fostered a renewed, dynamic comprehension of cell and tissue metabolism, giving unexpected insights into the ‘systemic aspects’ of cancer, namely pointing out that metabolism should be considered a truly “systems property”. Both internal and microenvironmental cues tightly cooperate in shaping the tissue metabolomic fingerprint. Tumour metabolome hardly could be mechanistically linked to the linear dynamics of few gene regulatory networks thus, it is more likely to be the complex end point of several interacting non-linear pathways, involving both cells and their microenvironment. As such, tumour metabolism might be considered an emerging, “systems property”, arising at the integrated scale of the whole system and behaving like an “attractor” in a specific space phase defined by thermodynamic constraints.

Diabetic patients treated with metformin have a reduced incidence of cancer and cancer-related mortality. Here we show that metformin affects engraftment and growth of breast cancer tumours in mice. This correlates with the induction of metabolic changes compatible with clear anticancer effects. We demonstrate that microRNA modulation underlies the anticancer metabolic actions of metformin. In fact, metformin induces DICER expression and its effects are severely impaired in DICER knocked down cells. Conversely, ectopic expression of DICER recapitulates the effects of metformin in vivo and in vitro. The microRNAs upregulated by metformin belong mainly to energy metabolism pathways. Among the messenger RNAs downregulated by metformin, we found c-MYC, IRS-2 and HIF1alpha. Downregulation of c-MYC requires AMP-activated protein kinase-signalling and mir33a upregulation by metformin. Ectopic expression of c-MYC attenuates the anticancer metabolic effects of metformin. We suggest that DICER modulation, mir33a upregulation and c-MYC targeting have an important role in the anticancer metabolic effects of metformin.

The effects on metabolism by a synthetic corticosteroid drug, clobetasone-17-butyrate, were investigated in rabbit lenses by 31P, 1H, 13C nuclear magnetic resonance spectroscopy.

Spikes and seeds from diploid ‘einkorn’ wheat Triticum monococcum and two near-isogenic hard and soft common wheat (Triticum aestivum) lines were harvested at regular intervals from 7 days post-anthesis (dpa) and analysed by non-destructive magnetic resonance imaging (MRI) and time domain-nuclear magnetic resonance (TD-NMR). A large amount of free water occurred in rachises, glumes and awns of spikes collected at 7 dpa, and accumulated in the physiologically active cells of the endosperm at 21 dpa. In the final stages of kernel development, awns and seed embryos exhibited a high MR signal due to the presence of free water likely associated with biological activities. TD-NMR relaxation time distributions obtained by discrete exponential fitting, distributed exponential fitting and SLICING multivariate analysis offered detailed information on mobility behaviour of water molecules in developing seeds and were able to differentiate two soft and hard isolines from common wheat cv. Enesco at early stages of seed development.

Fractal analysis in cancer cell investigation provided meaningful insights into the relationship between morphology and phenotype. Some reports demonstrated that changes in cell shape precede and trigger dramatic modifications in both gene expression and enzymatic function. Nonetheless, metabolomic pattern in cells undergoing shape changes have been not still reported. Our study was aimed to investigate if modifications in cancer cell morphology are associated to relevant transition in tumour metabolome, analyzed by nuclear magnetic resonance spectroscopy and principal component analysis. MCF-7 and MDA-MB-231 breast cancer cells, exposed to an experimental morphogenetic field, undergo a dramatic change in their membrane profiles. Both cell lines recover a more rounded shape, loosing spindle and invasive protrusions, acquiring a quite “normal” morphology. This result, quantified by fractal analysis, shows that normalized bending energy (a global shape characterization expressing the amount of energy needed to transform a specific shape into its lowest energy state) decreases after 48 h. Later on, a significant shift from a high to a low glycolytic phenotype was observed on both cell lines: glucose flux begins to drop off at 48 h, leading to reduced lactate accumulation, and fatty acids and citrate synthesis slow-down after 72 h. Moreover, de novo lipidogenesis is inhibited and nucleotide synthesis is reduced, as indicated by the positive correlation between glucose and formate. In conclusion, these data indicate that the reorganization of cell membrane architecture, induced by environmental cues, is followed by a relevant transition of the tumour metabolome, suggesting cells undergo a dramatic phenotypic reversion.

The different swelling properties of ionic and non-ionic lenses, immersed in sodium chloride or artificial tears, were investigated by Low-Field Nuclear Magnetic Resonance (LF-NMR) through relaxation time and self-diffusion coefficient measurements. A new model was developed to estimate the water self-diffusion coefficient inside the lenses, the results of which revealed a different mechanism of interaction for ionic versus non-ionic materials with artificial tears. The consequence of this finding suggests clinical implications.

Background Metabolomics belongs to the family of “-omics” sciences, all of which share the advantage of a non-targeted approach for identifying biomarkers and profiling the patient. Metabolomic procedure has become feasible recently with the advent and accessibility of new high-throughput technologies, including mass spectrometry and 1H Nuclear Magnetic Resonance (1H NMR) and a few studies have been published in rheumatic disorders. Objectives To evaluate whether a 1H NMR-based metabolomic analysis in serum from patients with rheumatoid arthritis (RA) could predict the response to etanercept evaluated at 6 months. Methods Adult patients fulfilling the 1987 ACR revised criteria for the classification of RA and designated to start anti-TNF therapy were prospectively enrolled. The analysis was restricted to female patients with active disease starting etanercept as the first biological treatment and having a minimum of 6 months9 follow-up. Each patient was evaluated by the same rheumatologist at baseline before starting etanercept and after 6 months following the onset of biological treatment. DAS28 was calculated and the clinical response (good, moderate, none) was evaluated according to the EULAR criteria, based on both erythrocyte sedimentation rate (EULAR-ESR) and C-reactive protein (EULAR-CRP). We merged good and moderate categories as response in comparison with no response. Sera collected prior to the onset of etanercept were analyzed via 1H NMR-based metabolomics. Discriminating metabolites were identified, and the relationship between metabolic profiles and clinical outcomes was assessed. Results Twenty-seven patients were included (mean age 57.8 years, ± SD 12.5; mean disease duration 102.5 months, ± SD 78.05). Eighteen patients had a good/moderate response and 9 were non responders according to both EULAR-ESR and EULAR-CRP after 6 months of etanercept. Baseline serum metabolic profiles discriminated between RA patients who did or did not have a response to etanercept. Unsupervised Principal Component Analysis (PCA) results according to EULAR-ESR or EULAR-CRP response criteria highlighted significant differences between the metabolic profiles of responders and non responders (p Conclusions Metabolomics approach is a potentially useful technique for predicting the response of RA patients to etanercept, as demonstrated by differences in serum metabolic profiles at baseline. This observation deserves further investigation in larger cohorts of patients to confirm the capability of predicting clinical response without the need for empirical treatment. Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.5080

Metabolic changes in the rabbit lens have been studied by means of nuclear magnetic resonance spectroscopy. These changes have been induced by prolonged topical treatment with dexamethasone. Our results demonstrate an increase in sorbitol, sorbitol-3-phosphate, fructose-3-phosphate, glycerol-3-phosphate and glucose-6-phosphate levels and a decrease in glutathione sulphate (GSH) and myo-inositol levels, in agreement with what was observed in lenses from streptozocin-diabetic rats before lens opacity. The hyperglycaemia can only partially explain all these observed biochemical variations. The lack of increase in the intermediates of pentose cycle, such as sedoheptulose-7-phosphate, seems to support the hypothesis of an inhibition of glucose-6-phosphate dehydrogenase by dexamethasone treatment. Finally dexamethasone treatment induces a decrease in GSH. The decreasing or the loss of GSH has been suggested as a possible pathogenic mechanism in the cataract formation.

The effect of dexamethasone on lipid composition of Epstein-Barr virus transformed human B lymphocytes have been investigated by 31P- and 1H-NMR spectroscopy and compared to the effects due to exogenous sphingosine treatment. Furthermore, the effects of dexamethasone and sphingosine on membrane structure was evaluated by fluorimetry. No significant changes were evidenced in phospholipid composition and in the ratio of unsaturated to total fatty-acid chains. A significant increase in total cholesterol levels was evident at 30 min incubation with dexamethasone or sphingosine; a parallel increase in DPH polarization at 30 min was also demonstrated. TMA-DPH intensity measurements suggest a slowing of vesicular intracellular traffic due to the treatment. The results suggest a dexamethasone- and sphingosine-dependent inhibition of intracellular cholesterol transport.