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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.

This work presents a lab-on-chip system, which combines a glass-polydimethilsiloxane microfluidic network and an array of amorphous silicon photosensors for the diagnosis and follow-up of Celiac disease. The microfluidic chip implements an on-chip enzyme-linked immunosorbent assay (ELISA), relying on a sandwich immunoassay between antibodies against gliadin peptides (GPs) and a secondary antibody marked with horseradish peroxidase (Ig-HRP). This enzyme catalyzes a chemiluminescent reaction, whose light intensity is detected by the amorphous silicon photosensors and transduced into an electrical signal that can be processed to recognize the presence of antibodies against GPs in the serum of people affected by Celiac syndrome.The correct operation of the developed lab-on-chip has been demonstrated using rabbit serum in the microfluidic ELISA. In particular, optimizing the dilution factors of both sera and Ig-HRP samples in the flowing solutions, the specific and non-specific antibodies against GPs can be successfully distinguished, showing the suitability of the presented device to effectively screen celiac disease epitopes. Keywords: Lab-on-chip, Celiac disease, Microfluidics, On-chip detection, ELISA, Amorphous silicon photosensors

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.

In this paper, we present the preliminary results of an ELISA-on-chip device, intended as a technological demonstrator of a novel analytical system suitable for the diagnosis and follow-up of celiac disease. The idea of the work is to combine an array of amorphous silicon photosensors with a pattern of a poly(2-hydroxyethyl methacrylate) polymer brush film, which acts as anchor for the immobilization of gliadin peptides containing the celiac disease epitopes. Recognition relies on a sandwich immunoassay between antibodies against the peptides and secondary antibodies marked with horseradish peroxidase to obtain a chemiluminescent signal. Detection is based on the measurement of photocurrent induced in the array of amorphous silicon photosensors by the chemiluminescent signal. An ad-hoc procedure has been developed in order to enable the fabrication of the photodiode array and the polymer brush pattern on the two sides of the same glass substrate ensuring the compatibility of the different technological steps. The sensitivity and the selectivity of the chip for multiplex immunoassays were demonstrated using two gliadin peptides (VEA and DEC). In particular, we found that the average amount of the bound HRP revealed by our analytical protocol is 3.5(±0.3) × 10−6 pg μm−2 and 0.85(±0.3) × 10−6 pg μm−2 for specific and non-specific interactions, respectively.

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

To shed light on the specific contribution of HDA101 in modulating metabolic pathways in the maize seed, changes in the metabolic profiles of kernels obtained from hda101 mutant plants have been investigated by a metabonomic approach. Dynamic properties of chromatin folding can be mediated by enzymes that modify DNA and histones. The enzymes responsible for the steady-state of histone acetylation are histone acetyltransferase and histone deacetylase (HDA). Therefore, it is interesting to evaluate the effects of up- and down-regulation of a Rpd-3 type HDA on the development of maize seeds in terms of metabolic changes. This has been reached by analysing nuclear magnetic resonance spectra by different chemometrician approaches, such as Orthogonal Projection to Latent Structure-Discriminant Analysis, Parallel Factors Analysis, and Multi-way Partial Least Squares-Discriminant Analysis (N-PLS-DA). In particular, the latter approaches were chosen because they explicitly take time into account, organizing data into a set of slices that refer to different steps of the developing process. The results show the good discriminating capabilities of the N-PLS-DA approach, even if the number of samples ought be increased to obtain better predictive capabilities. However, using this approach, it was possible to show differences in the accumulation of metabolites during development and to highlight the changes occuring in the modified seeds. In particular, the results confirm the role of this gene in cell cycle control.

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: 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.

The aim of the research was to investigate metabolic variations associated with genetic modifications in the grains of Zea mays using metabonomic techniques. With this in mind, the non-targeted characteristic of the technique is useful to identify metabolites peculiar to the genetic modification and initially undefined. The results obtained showed that the genetic modification, introducing Cry1Ab gene expression, induces metabolic variations involving the primary nitrogen pathway. Concerning the methodological aspects, the experimental protocol used has been applied in this field for the first time. It consists of a combination of partial least square-discriminant analysis and principal component analysis. The most important metabolites for discrimination were selected and the metabolic correlations linking them are identified. Principal component analysis on selected signals confirms metabolic variations, highlighting important details about the changes induced on the metabolic network by the presence of a Bt transgene in the maize genome.

The aim of this research was to verify the possibility of identifying and classifying maize seeds obtained from transgenic plants, in different classes according to the modification, on the basis of the concerted variation in metabolite levels detected by NMR spectra. It was possible to recognise the discriminant metabolites of transgenic samples as well as to classify non-a priori defined samples of maize. It is important to underline that the obtained results are useful to point out the metabolic consequences of a specific genic modification on a plant, without using a targeted analysis of the different metabolites, in fact it was possible to classify the seeds also without the complete assignment of the spectra. The analysis was performed by applying multivariate techniques (principal component analysis and partial least squares-discriminant analysis) to NMR data.

This review considers the use of a nonlinear signal analysis tool, recurrence quantification analysis, as a method to study sequence/structure relationships of proteins. Four broad categories are discussed: (1) a point of view involving information contained in deterministic aspects of hydrophobicity; (2) the analysis of protein hydrophobicity "singularities"; (3) time-series analysis of protein dynamics simulations; and (4) prediction of protein secondary structure.

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.

Major changes in medical, intensive care and organ transplantation practices are drastically increasing the risk of fungal opportunistic infections. We designed and set-up a MALDI-TOF MS-based assay to identify the most isolated and emerging therapy-refractory/uncommon fungi from cystic fibrosis (CF) and immunocompromised patients. Two-hundred and thirty isolates from 10 different genera (Aspergillus, Emericella, Fusarium, Geosmithia, Neosartorya, Penicillium, Pseudallescheria, Scedosporium, Talaromyces, Fomitopsis), investigated during routine diagnostic efforts, were correlated to 22 laboratory-adapted reference MALDI-TOF MS "proteomic phenotypes". A growth time-course at 30°C on Sabouraud agar medium was performed for the 22 "phenotypes" at 48, 72, 96 and 120h points. The best peptide extraction conditions for full recovery of conidia- or asci-producing multihyphal morph structures and the highest intra- and inter-class profiling correlation were identified for the 120h point spectra dataset, from which an engineered library derived (pre-analytical phase). Fingerprinting classifiers, selected by Wilcoxon/Kruskal-Wallis algorithm, were computed by Genetic Algorithm, Support Vector Machine, Supervised Neuronal Network and Quick Classifier model construction. MS identification (ID) of clinical isolates was referred to genotyping (GT) and, retrospectively, compared to routine morphotyping (MT) IDs (analytical phase). Proteomic phenotyping is revolutionizing diagnostic mycology as fully reflecting species/morph varieties but often overcoming taxonomic hindrance.

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.

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 aim of this research was to show that the application of multiway partial least square-discriminant analysis to nuclear magnetic resonance spectra is a valuable tool to analyze metabonomic data of transgenic maize. We evaluated the effects, on the development of seeds, of the introduction of the antisense-mediated downregulation and overexpression of the Rpd3 gene (ZmRpd3) in the genome of a maize inbred line, we identified the metabolites involved in the differentiation between classes of samples, directly integrating the evolution of each metabolic perturbation over time in the model. Major differences were found at the beginning of development, confirming the results obtained by transcript analysis: ZmRpd3 transcripts and proteins accumulate during the initial stage of development, suggesting a role for this gene in cell cycle control.

The goal of this group is to define the reporting requirements associated with the statistical analysis (including univariate, multivariate, informatics, machine learning etc.) of metabolite data with respect to other measured/collected experimental data (often called meta-data). These definitions will embrace as many aspects of a complete metabolomics study as possible at this time. In chronological order this will include: Experimental Design, both in terms of sample collection/ matching, and data acquisition scheduling of samples through whichever spectroscopic technology used; Deconvolution (if required); Pre-processing, for example, data cleaning, outlier detection, row/column scaling, or other transformations; Definition and parameterization of subsequent visualizations and Statistical/Machine learning Methods applied to the dataset; If required, a clear definition of the Model Validation Scheme used (including how data are split into training/validation/test sets); Formal indication on whether the data analysis has been Independently Tested (either by experimental reproduction, or blind hold out test set). Finally, data interpretation and the visual representations and hypotheses obtained from the data analyses. © Springer Science+Business Media, LLC 2007.

In a previous article (Zbilut et al., Biophys J 2003;85:3544-3557), we demonstrated how an aggregation versus folding choice could be approached considering hydrophobicity distribution and charge. In this work, our aim is highlighting the mutual interaction of charge and hydrophobicity distribution in the aggregation process. Use was made of two different peptides, both derived from a transmembrane protein (amyloid precursor protein; APP), namely, Abeta(1-28) and Abeta(1-40). Abeta(1-28) has a much lower aggregation propensity than Abeta(1-40). The results obtained by means of molecular dynamics simulations show that, when submitted to the most "aggregation-prone" environment, corresponding to the isoelectric point and consequently to zero net charge, both peptides acquire their maximum flexibility, but Abeta(1-40) has a definitely higher conformational mobility than Abeta(1-28). The absence of a hydrophobic "tail," which is the most mobile part of the molecule in Abeta(1-40), is the element lacking in Abeta(1-28) for obtaining a "fully aggregating" phenotype. Our results suggest that conformational flexibility, determined by both hydrophobicity and charge effect, is the main mechanistic determinant of aggregation propensity.

Two naphthyridines interacting with Torpedo californica acetylcholinesterase (AChE) were investigated. 1 H NMR spectra were recorded and nonselective, selective, and double-selective spin–lattice relaxation rates were measured. The enhancement of selective relaxation rates could be titrated by different ligand concentrations at constant AChE (yielding 0.22 and 1.53 mM for the dissociation constants) and was providing evidence of a diverse mode of interaction. The double-selective relaxation rates were used to evaluate the motional correlation times of bound ligands at 34.9 and 36.5 ns at 300 K. Selective relaxation rates of bound inhibitors could be interpreted also in terms of dipole–dipole interactions with protons in the enzyme active site.

The purification and structural characterization of phytotoxic lipodepsipeptides from some Pseudomonas syringae pv. syringae strains have been conducted in italy, USA and Japan during the past decade. The main results of the chemical investigations on these metabolites are reported in this paper together with some data of their biological activities.

Syringopeptin 25-A is a phytotoxic amphiphilic lipodepsipeptide containing 25 amino acid residues, produced by some isolates of the plant pathogenic bacterium Pseudomonas syringae pv. syringae. Previous papers have reported its covalent structure and some of its biological properties. Attention has now been directed to define its conformation in solution, a structural feature regarded as important for understanding its possible role in the bacterial colonization of host plants, and its toxic action on the plant cell. Here we report the stereochemistry of its amino acid components, the complete interpretation of the two-dimensional NMR spectra and NOE data, and finally the structure obtained by computer simulations applying distance geometry and molecular dynamics procedures. The conformation of syringopeptin 25-A in aqueous solution includes three different structural regions interrupted by rigid 2,3-dehydro-2-aminobutyric acid residues: a loop from residue 2 to 6, a helicoidal zone from 8 to 15, and the lactone ring from 18 to 25. The three-dimensional structure of the lactone moiety is very similar to that of two previously studied bioactive lipodepsinonapeptides. Preliminary circular dichroism evidence of conformational variations in solution of trifluoroethanol, which stimulates a membrane-like environment, are also reported.

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.