Metabolomics approach in allergic and rheumatic diseases.

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.

Glucose level determination with a multi-enzymatic cascade reaction in a functionalized glass chip.

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.

Biomolecular Monitoring Tool Based on Lab-on-Chip for Virus Detection.

Lab-on-Chip (LoC) devices for performing real-time PCR are advantageous compared to standard equipment since these systems allow to conduct in-field quick analysis. The development of LoCs, where the components for performing the nucleic acid amplification are all integrated, can be an issue. In this work, we present a LoC-PCR device where thermalization, temperature control and detection elements are all integrated on a single glass substrate named System-on-Glass (SoG) obtained using metal thin-film deposition. By using a microwell plate optically coupled with the SoG, real-time reverse transcriptase PCR of RNA extracted from both a plant and human virus has been carried out in the developed LoC-PCR device. The limit of detection and time of analysis for the detection of the two viruses by using the LoC-PCR were compared with those achieved by standard equipment. The results showed that the two systems can detect the same concentration of RNA; however, the LoC-PCR performs the analysis in half of the time compared to the standard thermocycler, with the advantage of the portability, leading to a point-of-care device for several diagnostic applications.

Split Aptamers Immobilized on Polymer Brushes Integrated in a Lab-on-Chip System Based on an Array of Amorphous Silicon Photosensors: A Novel Sensor Assay.

Innovative materials for the integration of aptamers in Lab-on-Chip systems are important for the development of miniaturized portable devices in the field of health-care and diagnostics. Herein we highlight a general method to tailor an aptamer sequence in two subunits that are randomly immobilized into a layer of polymer brushes grown on the internal surface of microfluidic channels, optically aligned with an array of amorphous silicon photosensors for the detection of fluorescence. Our approach relies on the use of split aptamer sequences maintaining their binding affinity to the target molecule. After binding the target molecule, the fragments, separately immobilized to the brush layer, form an assembled structure that in presence of a "light switching" complex [Ru(phen)2(dppz)]2+, emit a fluorescent signal detected by the photosensors positioned underneath. The fluorescent intensity is proportional to the concentration of the target molecule. As proof of principle, we selected fragments derived from an aptamer sequence with binding affinity towards ATP. Using this assay, a limit of detection down to 0.9 µM ATP has been achieved. The sensitivity is compared with an assay where the original aptamer sequence is used. The possibility to re-use both the aptamer assays for several times is demonstrated.

Fluorescent Label-Free Aptasensor Integrated in a Lab-on-Chip System for the Detection of Ochratoxin A in Beer and Wheat.

This paper reports on the development of a fluorescent label-free aptamer assay integrated in a lab-on-chip (LoC) system for the detection of Ochratoxin A (OTA). The detection system relies on the integration, on a single glass substrate, of an array of amorphous silicon photosensors and a long pass interferential filter. The aptamer assay, integrated into the microfluidic network, is an aptasensor having affinity versus OTA, selected as a case study. The fluorescent molecule is a "light switch" complex [Ru(phen)2(dppz)]2+. The aptamer is directly anchored into a layer of poly(2-hydroxyethyl methacrylate) polymer brushes grown inside the channels. The fluorophore is intercalated between the base pairs of the aptamer. Upon the interaction of OTA with the aptasensor, a change of the aptamer conformation causes the release of the fluorophore, yielding a decrease of the fluorescent signal detected by the array of the amorphous silicon photosensors positioned underneath the microfluidic network. The developed LoC is a portable system capable of performing the analysis with a small volume of sample (about 10 µL) in a short time (5 min) with a limit of detection for OTA equal to 1.3 ng/mL. The LoC has been applied for the detection of OTA (5-200 ng/mL) in beer and wheat samples.

Simulated microgravity promotes the formation of tridimensional cultures and stimulates pluripotency and a glycolytic metabolism in human hepatic and biliary tree stem/progenitor cells.

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 < 0.05) has been observed in hBTSCs cultured in SMG when compared to NG condition. At the same time, the expression of hepatocyte lineage markers in hBTSCs differentiated by HDM was significantly lower (p < 0.05) in SMG compared to NG, demonstrating an impaired capability of hBTSCs to differentiate in vitro toward mature hepatocytes when cultured in SMG condition. Furthermore, in HepG2 cells the SMG caused a lower (p < 0.05 vs controls) transcription of CYP3A4, a marker of late-stage (i.e. Zone 3) hepatocytes. Exo-metabolome NMR-analysis showed that both cell cultures consumed a higher amount of glucose and lower glutamate in SMG respect to NG (p < 0.05). Moreover, hBTSCs media cultures resulted richer of released fermentation (lactate, acetate) and ketogenesis products (B-hydroxybutyrate) in SGM (p < 0.05) than NG. While, HepG2 cells showed higher consumption of amino acids and release of ketoacids (3-Methyl-2-oxovalerate, 2-oxo-4-methyl-valerate) and formiate with respect to normogravity condition (p < 0.05). Based on our results, SMG could be helpful for developing hBTSCs-derived liver devices. In conclusion, SMG favored the formation of hBTSCs and HepG2 3D cultures and the maintenance of stemness contrasting cell differentiation; these effects being associated with stimulation of glycolytic metabolism. Interestingly, the impact of SMG on stem cell biology should be taken into consideration for workers involved in space medicine programs.

Variation of metabolic profiles in developing maize kernels up- and down-regulated for the hda101 gene.

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.

Metabolomics: Challenges and Opportunities in Systems Biology Studies.

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.

Gut microbiota signatures in cystic fibrosis: Loss of host CFTR function drives the microbiota enterophenotype.

BACKGROUND: Cystic 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. OBJECTIVES: The 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. METHODS: Thirty-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. RESULTS: The 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. CONCLUSIONS: All 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.

An All-Glass Microfluidic Network with Integrated Amorphous Silicon Photosensors for on-Chip Monitoring of Enzymatic Biochemical Assay.

A lab-on-chip system, integrating an all-glass microfluidics and on-chip optical detection, was developed and tested. The microfluidic network is etched in a glass substrate, which is then sealed with a glass cover by direct bonding. Thin film amorphous silicon photosensors have been fabricated on the sealed microfluidic substrate preventing the contamination of the micro-channels. The microfluidic network is then made accessible by opening inlets and outlets just prior to the use, ensuring the sterility of the device. The entire fabrication process relies on conventional photolithographic microfabrication techniques and is suitable for low-cost mass production of the device. The lab-on-chip system has been tested by implementing a chemiluminescent biochemical reaction. The inner channel walls of the microfluidic network are chemically functionalized with a layer of polymer brushes and horseradish peroxidase is immobilized into the coated channel. The results demonstrate the successful on-chip detection of hydrogen peroxide down to 18 µM by using luminol and 4-iodophenol as enhancer agent.

Che-1 sustains hypoxic response of colorectal cancer cells by affecting Hif-1α stabilization.

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.

Early events in protein aggregation: molecular flexibility and hydrophobicity/charge interaction in amyloid peptides as studied by molecular dynamics simulations.

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.

¹H-NMR-Based Metabolomic Study for Identifying Serum Profiles Associated with the Response to Etanercept in Patients with Rheumatoid Arthritis.

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.

Cynara scolymus affects malignant pleural mesothelioma by promoting apoptosis and restraining invasion.

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.

Review of nonlinear analysis of proteins through recurrence quantification.

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.

Application of trilinear SLICING to analyse a single relaxation curve.

Determining the time constants and amplitudes of exponential decays from relaxation data is a common task in LF-NMR. In this communication, we present an application of the SLICING algorithm to evaluate its possibilities for solving this problem. The method, originally introduced to compare different samples, is applied here to analyse a single relaxation curve, using the embedding technique. To test this procedure, we acquired data sets from samples of liquids properly separated, and characterized by different relaxation times. The results show a good estimation of parameters, comparable with those obtained applying Marquardt's algorithm, when the components have sufficiently different relaxation times.

NMR-based metabonomic study of transgenic maize.

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.

Metformin-induced metabolic reprogramming of chemoresistant ALDHbright breast cancer cells.

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 (ALDHbright cells) and their response to metformin, a promising anticancer metabolic modulator. The purified ALDHbright cells exhibited a different metabolic profile as compared to their chemosensitive ALDHlow 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 ALDHbright and ALDHlow cells, making the former more similar to the latter. Metformin broadly modulated microRNAs in the ALDHbright 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 ALDHbright cells and the chemosensitive ALDHlow cells. This works adds on the potential therapeutic relevance of metformin and shows the potential for metabolic reprogramming to modulate cancer chemoresistance.

Metabolomics in rheumatic diseases: the potential of an emerging methodology for improved patient diagnosis, prognosis, and treatment efficacy.

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.

Metformin elicits anticancer effects through the sequential modulation of DICER and c-MYC.

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.