Multi-Method Quantification of Acetyl-Coenzyme A and Further Acyl-Coenzyme A Species in Normal and Ischemic Rat Liver.

Thioesters of coenzyme A (CoA) carrying different acyl chains (acyl-CoAs) are central intermediates of many metabolic pathways and donor molecules for protein lysine acylation. Acyl-CoA species largely differ in terms of cellular concentrations and physico-chemical properties, rendering their analysis challenging. Here, we compare several approaches to quantify cellular acyl-CoA concentrations in normal and ischemic rat liver, using HPLC and LC-MS/MS for multi-acyl-CoA analysis, as well as NMR, fluorimetric and spectrophotometric techniques for the quantification of acetyl-CoAs. In particular, we describe a simple LC-MS/MS protocol that is suitable for the relative quantification of short and medium-chain acyl-CoA species. We show that ischemia induces specific changes in the short-chain acyl-CoA relative concentrations, while mild ischemia (1-2 min), although reducing succinyl-CoA, has little effects on acetyl-CoA, and even increases some acyl-CoA species upstream of the tricarboxylic acid cycle. In contrast, advanced ischemia (5-6 min) also reduces acetyl-CoA levels. Our approach provides the keys to accessing the acyl-CoA metabolome for a more in-depth analysis of metabolism, protein acylation and epigenetics.

NOX2 control over energy metabolism plays a role in acute myeloid leukaemia prognosis and survival.

Acute myeloid leukaemia (AML) is a highly heterogeneous disease, however the therapeutic approaches have hardly changed in the last decades. Metabolism rewiring and the enhanced production of reactive oxygen species (ROS) are hallmarks of cancer. A deeper understanding of these features could be instrumental for the development of specific AML-subtypes treatments. NADPH oxidases (NOX), the only cellular system specialised in ROS production, are also involved in leukemic metabolism control. NOX2 shows a variable expression in AML patients, so patients can be classified based on such difference. Here we have analysed whether NOX2 levels are important for AML metabolism control. The lack of NOX2 in AML cells slowdowns basal glycolysis and oxidative phosphorylation (OXPHOS), along with the accumulation of metabolites that feed such routes, and a sharp decrease of glutathione. In addition, we found changes in the expression of 725 genes. Among them, we have discovered a panel of 30 differentially expressed metabolic genes, whose relevance was validated in patients. This panel can segregate AML patients according to CYBB expression, and it can predict patient prognosis and survival. In summary, our data strongly support the relevance of NOX2 for AML metabolism, and highlights the potential of our discoveries in AML prognosis.

The fate of mitochondria during platelet activation.

Blood platelets undergo several successive motor-driven reorganizations of the cytoskeleton when they are recruited to an injured part of a vessel. These reorganizations take place during the platelet activation phase, the spreading process on the injured vessel or between fibrin fibers of the forming clot, and during clot retraction. All these steps require a lot of energy, especially the retraction of the clot when platelets develop strong forces similar to those of muscle cells. Platelets can produce energy through glycolysis and mitochondrial respiration. However, although resting platelets have only 5 to 8 individual mitochondria, they produce adenosine triphosphate predominantly via oxidative phosphorylation. Activated, spread platelets show an increase in size compared with resting platelets, and the question arises as to where the few mitochondria are located in these larger platelets. Using expansion microscopy, we show that the number of mitochondria per platelet is increased in spread platelets. Live imaging and focused ion beam-scanning electron microscopy suggest that a mitochondrial fission event takes place during platelet activation. Fission is Drp1 dependent because Drp1-deficient platelets have fused mitochondria. In nucleated cells, mitochondrial fission is associated with a shift to a glycolytic phenotype, and using clot retraction assays, we show that platelets have a more glycolytic energy production during clot retraction and that Drp1-deficient platelets show a defect in clot retraction.

Identification of Modulators of the C. elegans Aryl Hydrocarbon Receptor and Characterization of Transcriptomic and Metabolic AhR-1 Profiles.

The Aryl hydrocarbon Receptor (AhR) is a xenobiotic sensor in vertebrates, regulating the metabolism of its own ligands. However, no ligand has been identified to date for any AhR in invertebrates. In C. elegans, the AhR ortholog, AHR-1, displays physiological functions. Therefore, we compared the transcriptomic and metabolic profiles of worms expressing AHR-1 or not and investigated the putative panel of chemical AHR-1 modulators. The metabolomic profiling indicated a role for AHR-1 in amino acids, carbohydrates, and fatty acids metabolism. The transcriptional profiling in neurons expressing AHR-1, identified 95 down-regulated genes and 76 up-regulated genes associated with neuronal and metabolic functions in the nervous system. A gene reporter system allowed us to identify several AHR-1 modulators including bacterial, dietary, or environmental compounds. These results shed new light on the biological functions of AHR-1 in C. elegans and perspectives on the evolution of the AhR functions across species.

Effects of an Exercise and Nutritional Intervention on Circulating Biomarkers and Metabolomic Profiling During Adjuvant Treatment for Localized Breast Cancer: Results From the PASAPAS Feasibility Randomized Controlled Trial.

PURPOSE: Exercise has been shown to improve physical and psychological conditions during cancer therapy, but mechanisms remain poorly understood. The purpose of the present study was to report the results of cancer-related biomarkers and metabolomics outcomes from the PASAPAS feasibility study. METHODS: In the PASAPAS randomized controlled trial, 61 women beginning adjuvant chemotherapy for localized breast cancer were randomized in a 6-month program of weekly aerobic exercises associated with nutritional counseling versus usual care with nutritional counseling. In the present analysis of 58 women for whom blood samples were available, first, circulating levels of biomarkers (ie, insulin, insulin-like growth factor 1, estradiol, adiponectin, leptin, interleukin-6, and tumor necrosis factor α) were measured at baseline and 6-month follow-up. Changes in biomarkers were compared between exercisers (n = 40) and controls (n = 18) using mixed-effect models. Second, serum metabolites were studied using an untargeted 1H nuclear magnetic resonance spectroscopy, and orthogonal partial least squares analyses were performed to discriminate exercisers and controls at baseline and at 6 months. RESULTS: Over the 6-month intervention, no statistically significant differences were observed between exercisers and controls regarding changes in biomarkers and metabolomic profiles. CONCLUSION: The present analysis of the PASAPAS feasibility trial did not reveal any improvement in circulating biomarkers nor identified metabolic signatures in exercisers versus controls during adjuvant breast cancer treatment. Larger studies preferably in women with poor physical activity level to avoid ceiling effect, testing different doses and types of exercise on additional biological pathways, could allow to clarify the mechanisms mediating beneficial effects of physical exercise during cancer treatment. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01331772. Registered 8 April 2011, https://clinicaltrials.gov/ct2/show/NCT01331772?term=pasapas&rank=1.

Investigation of circulating metabolites associated with breast cancer risk by untargeted metabolomics: a case-control study nested within the French E3N cohort.

BACKGROUND: Perturbations in circulating metabolites prior to a breast cancer diagnosis are not well characterised. We aimed to gain more detailed knowledge to help understand and prevent the disease. METHODS: Baseline plasma samples from 791 breast cancer cases and 791 matched controls from the E3N (EPIC-France) cohort were profiled by nuclear magnetic resonance (NMR)-based untargeted metabolomics. Partial least-squares discriminant analysis (PLS-DA) models were built from NMR profiles to predict disease outcome, and odds ratios and false discovery rate (FDR)-adjusted CIs were calculated for 43 identified metabolites by conditional logistic regression. RESULTS: Breast cancer onset was predicted in the premenopausal subgroup with modest accuracy (AUC 0.61, 95% CI: 0.49-0.73), and 10 metabolites associated with risk, particularly histidine (OR = 1.70 per SD increase, FDR-adjusted CI 1.19-2.41), N-acetyl glycoproteins (OR = 1.53, FDR-adjusted CI 1.18-1.97), glycerol (OR = 1.55, FDR-adjusted CI 1.11-2.18) and ethanol (OR = 1.44, FDR-adjusted CI 1.05-1.97). No predictive capacity or significant metabolites were found overall or for postmenopausal women. CONCLUSIONS: Perturbed metabolism compared to controls was observed in premenopausal but not postmenopausal cases. Histidine and NAC have known involvement in inflammatory pathways, and the robust association of ethanol with risk suggests the involvement of alcohol intake.

Metabolomic Approaches to Study Chemical Exposure-Related Metabolism Alterations in Mammalian Cell Cultures.

Biological organisms are constantly exposed to an immense repertoire of molecules that cover environmental or food-derived molecules and drugs, triggering a continuous flow of stimuli-dependent adaptations. The diversity of these chemicals as well as their concentrations contribute to the multiplicity of induced effects, including activation, stimulation, or inhibition of physiological processes and toxicity. Metabolism, as the foremost phenotype and manifestation of life, has proven to be immensely sensitive and highly adaptive to chemical stimuli. Therefore, studying the effect of endo- or xenobiotics over cellular metabolism delivers valuable knowledge to apprehend potential cellular activity of individual molecules and evaluate their acute or chronic benefits and toxicity. The development of modern metabolomics technologies such as mass spectrometry or nuclear magnetic resonance spectroscopy now offers unprecedented solutions for the rapid and efficient determination of metabolic profiles of cells and more complex biological systems. Combined with the availability of well-established cell culture techniques, these analytical methods appear perfectly suited to determine the biological activity and estimate the positive and negative effects of chemicals in a variety of cell types and models, even at hardly detectable concentrations. Metabolic phenotypes can be estimated from studying intracellular metabolites at homeostasis in vivo, while in vitro cell cultures provide additional access to metabolites exchanged with growth media. This article discusses analytical solutions available for metabolic phenotyping of cell culture metabolism as well as the general metabolomics workflow suitable for testing the biological activity of molecular compounds. We emphasize how metabolic profiling of cell supernatants and intracellular extracts can deliver valuable and complementary insights for evaluating the effects of xenobiotics on cellular metabolism. We note that the concepts and methods discussed primarily for xenobiotics exposure are widely applicable to drug testing in general, including endobiotics that cover active metabolites, nutrients, peptides and proteins, cytokines, hormones, vitamins, etc.

Fast and ergonomic extraction of adherent mammalian cells for NMR-based metabolomics studies.

Cellular metabolomics has become key to elucidate mechanistic aspects in various fields such as cancerology or pharmacology, and is rapidly becoming a standard phenotyping tool accessible to the broad biological community. Acquisition of reliable spectroscopic datasets, such as nuclear magnetic resonance (NMR) spectra, to characterize biological systems depends on the elaboration of robust methods for cellular metabolites extraction. Previous studies have addressed many issues raised by these protocols, however with little pondering on ergonomic and practical aspects of the methods that impact their scalability, reproducibility and hence their suitability to high-throughput studies or their use by non-metabolomics experts. Here, we optimize a fast and ergonomic protocol for extraction of metabolites from adherent mammalian cells for NMR metabolomics studies. The proposed extraction protocol, including cell washing, metabolism quenching and actual extraction of intracellular metabolites, was first optimized on HeLa cells. Efficiency of the protocol, in its globality and for the different individual steps, was assessed by NMR quantification of 27 metabolites from cellular extracts. We show that a single PBS wash provides a seemly compromise between contamination from growth medium and leakage of intracellular metabolites. In HeLa cells, extraction using pure methanol, without cell scraping, recovered a higher amount of intracellular metabolites than the reference methanol/water/chloroform method with cell scraping, with yields varying across metabolite classes. Optimized and reference protocols were further tested on eight cell lines of miscellaneous nature, and inter-operator reproducibility was demonstrated. Our results stress the need for tailored extraction protocols and show that fast protocols minimizing time-consuming steps, without compromising extraction yields, are suitable for high-throughput metabolomics studies. Graphical abstract.

Multi-platform NMR Study of Pluripotent Stem Cells Unveils Complementary Metabolic Signatures towards Differentiation.

Stem cells, poised to revolutionize current medicine, stand as major workhorses for monitoring changes in cell fate. Characterizing metabolic phenotypes is key to monitor in differentiating cells transcriptional and epigenetic shifts at a functional level and provides a non-genetic means to control cell specification. Expanding the arsenal of analytical tools for metabolic profiling of cell differentiation is therefore of importance. Here, we describe the metabolome of whole pluripotent stem cells (PSCs) using high-resolution magic angle spinning (HR-MAS), a non-destructive approach for Nuclear Magnetic Resonance (NMR) analysis. The integrated 1H NMR analysis results in detection of metabolites of various groups, including energy metabolites, amino acids, choline derivatives and short chain fatty acids. It unveils new metabolites that discriminate PSCs from differentiated counterparts and directly measures substrates and co-factors of histone modifying enzymes, suggesting that NMR stands as a strategic technique for deciphering metabolic regulations of histone post-translational modifications. HR-MAS NMR analysis of whole PSCs complements the much used solution NMR of cell extracts. Altogether, our multi-platform NMR investigation provides a consolidated picture of PSC metabolic signatures and of metabolic pathways involved in differentiation.

General Guidelines for Sample Preparation Strategies in HR-µMAS NMR-based Metabolomics of Microscopic Specimens.

The study of the metabolome within tissues, organisms, cells or biofluids can be carried out by several bioanalytical techniques. Among them, nuclear magnetic resonance (NMR) is one of the principal spectroscopic methods. This is due to a sample rotation technique, high-resolution magic angle spinning (HR-MAS), which targets the analysis of heterogeneous specimens with a bulk sample mass from 5 to 10 mg. Recently, a new approach, high-resolution micro-magic angle spinning (HR-µMAS), has been introduced. It opens, for the first time, the possibility of investigating microscopic specimens (<500 µg) with NMR spectroscopy, strengthening the concept of homogeneous sampling in a heterogeneous specimen. As in all bioanalytical approaches, a clean and reliable sample preparation strategy is a significant component in designing metabolomics (or -omics, in general) studies. The sample preparation for HR-µMAS is consequentially complicated by the µg-scale specimen and has yet to be addressed. This report details the strategies for three specimen types: biofluids, fluid matrices and tissues. It also provides the basis for designing future µMAS NMR studies of microscopic specimens.

Discrimination of Escherichia coli and Shigella spp. by Nuclear Magnetic Resonance Based Metabolomic Characterization of Culture Media.

Dysentery is a major health threat that dramatically impacts childhood morbidity and mortality in developing countries. Various pathogenic agents cause dysentery, such as Shigella spp. and Escherichia coli, which are very closely related if not identical species. Sensitive and precise detection and identification of the infectious agent is important to target the best therapeutic strategy, but the differential diagnosis of these two groups remains a challenge using conventional methods. Here, we present a nuclear magnetic resonance (NMR) based multivariate classification model employing bacterial metabolic footprints in postculture growth media with remarkable segregation capability, including the discrimination of lactose negative E. coli and Shigella spp. Our results confirm the potential of metabolomic markers in the field of bacterial identification for the distinction of even very closely related species.

Metabolomic Profiling of Body Fluids in Mouse Models Demonstrates that Nuclear Magnetic Resonance Is a Putative Diagnostic Tool for the Presence of Thyroid Hormone Receptor α1 Mutations.

Background: Resistance to thyroid hormone alpha (RTHα) is a rare genetic disease due to mutations in the THRA gene, which encodes thyroid hormone receptor alpha 1 (TRα1). Since its first description in 2012, 46 cases of RTHα have been reported worldwide, corresponding to 26 different mutations of TRα1. RTHα patients share some common symptoms with hypothyroid patients, without significant reduction in thyroid hormone level. The high variability of clinical features and the absence of reliable biochemical markers make the diagnosis of this disease difficult. Some of these mutations have been recently modeled in mice. Methods: In our study, we used four different mouse models heterozygous for frameshift mutations in the Thra gene. Two of them are very close to human mutations, while the two others have not yet been found in patients. We characterized the metabolic phenotypes of urine and plasma samples collected from these four animal models using an untargeted nuclear magnetic resonance (NMR)-based metabolomic approach. Results: Multivariate statistical analysis of the metabolomic profiles shows that biofluids of mice that carry human-like mutations can be discriminated from controls. Metabolic signatures associated with Thra mutations in urine and plasma are stable over time and clearly differ from the metabolic fingerprint of hypothyroidism in the mouse. Conclusion: Our results provide a proof-of-principle that easily accessible NMR-based metabolic fingerprints of biofluids could be used to diagnose RTHα in humans.

Metabolic Phenotyping of Adipose-Derived Stem Cells Reveals a Unique Signature and Intrinsic Differences between Fat Pads.

White adipose tissues are functionally heterogeneous and differently manage the excess of energy supply. While the expansion of subcutaneous adipose tissues (SAT) is protective in obesity, that of visceral adipose tissues (VAT) correlates with the emergence of metabolic diseases. Maintained in fat pads throughout life, adipose stem cells (ASC) are mesenchymal-like stem cells with adipogenesis and multipotent differentiation potential. ASC from distinct fat pads have long been reported to present distinct proliferation and differentiation potentials that are maintained in culture, yet the origins of these intrinsic differences are still unknown. Metabolism is central to stem cell fate decision in line with environmental changes. In this study, we performed high-resolution nuclear magnetic resonance (NMR) metabolomic analyses of ASC culture supernatants in order to characterize their metabolic phenotype in culture. We identified and quantified 29 ASC exometabolites and evaluated their consumption or secretion over 72 h of cell culture. Both ASC used glycolysis and mitochondrial metabolism, as evidenced by the high secretions of lactate and citrate, respectively, but V-ASC mostly used glycolysis. By varying the composition of the cell culture medium, we showed that glutaminolysis, rather than glycolysis, supported the secretion of pyruvate, alanine, and citrate, evidencing a peculiar metabolism in ASC cells. The comparison of the two types of ASC in glutamine-free culture conditions also revealed the role of glutaminolysis in the limitation of pyruvate routing towards the lactate synthesis, in S-ASC but not in V-ASC. Altogether, our results suggest a difference between depots in the capacity of ASC mitochondria to assimilate pyruvate, with probable consequences on their differentiation potential in pathways requiring an increased mitochondrial activity. These results highlight a pivotal role of metabolic mechanisms in the discrimination between ASC and provide new perspectives in the understanding of their functional differences.

Cell-Free Protein Synthesis Enhancement from Real-Time NMR Metabolite Kinetics: Redirecting Energy Fluxes in Hybrid RRL Systems.

A counterintuitive cell-free protein synthesis (CFPS) strategy, based on reducing the ribosomal fraction in rabbit reticulocyte lysate (RRL), triggers the development of hybrid systems composed of RRL ribosome-free supernatant complemented with ribosomes from different mammalian cell-types. Hybrid RRL systems maintain translational properties of the original ribosome cell types, and deliver protein expression levels similar to RRL. Here, we show that persistent ribosome-associated metabolic activity consuming ATP is a major obstacle for maximal protein yield. We provide a detailed picture of hybrid CFPS systems energetic metabolism based on real-time nuclear magnetic resonance (NMR) investigation of metabolites kinetics. We demonstrate that protein synthesis capacity has an upper limit at native ribosome concentration and that lower amounts of the ribosomal fraction optimize energy fluxes toward protein translation, consequently increasing CFPS yield. These results provide a rationalized strategy for further mammalian CFPS developments and reveal the potential of real-time NMR metabolism phenotyping for optimization of cell-free protein expression systems.

Longitudinal serum metabolomics evaluation of trastuzumab and everolimus combination as pre-operative treatment for HER-2 positive breast cancer patients.

The mammalian target of rapamycin complex 1 (mTORC1) is an attractive target for HER-2 positive breast cancer therapy because of its key role in protein translation regulation, cell growth and metabolism. We present here a metabolomic investigation exploring the impact of mTOR inhibition on serum metabolic profiles from patients with non-metastatic breast cancer overexpressing HER-2. Baseline, treatment-related and post-treatment serum samples were analyzed for 79 patients participating in the French clinical trial RADHER, in which randomized patients with HER-2 positive breast cancer received either trastuzumab alone (arm T) or a trastuzumab and everolimus combination (arm T+E). Longitudinal series of NMR serum metabolic profiles were exploited to investigate treatment effects on the patients metabolism over time, in both group. Trastuzumab and everolimus combination induces faster changes in patients metabolism than trastuzumab alone, visible after only one week of treatment as well as a residual effect detectable up to three weeks after ending the treatment. These metabolic fingerprints highlight the involvement of several metabolic pathways reflecting a systemic effect, particularly on the liver and visceral fat. Comparison of serum metabolic profiles between the two arms shows that everolimus, an mTORC1 inhibitor, is responsible for host metabolism modifications observed in arm T+E. In HER-2 positive breast cancer, our metabolomic approach confirms a fast and persistent host metabolism modification caused by mTOR inhibition.

Evolution of Newborns' Urinary Metabolomic Profiles According to Age and Growth.

Improving the management of neonatal diseases and prevention of chronic diseases in adulthood requires a better comprehension of the complex maturational processes associated with newborns' development. Urine-based metabolomic studies play a promising role in the fields of pediatrics and neonatology, relying on simple and noninvasive collection procedures while integrating a variety of factors such as genotype, nutritional state, lifestyle, and diseases. Here, we investigate the influence of age, weight, height, and gender on the urine metabolome during the first 4 months of life. Untargeted analysis of urine was carried out by 1H-Nuclear Magnetic Resonance (NMR) spectroscopy for 90 newborns under 4 months of age, and free of metabolic, nephrologic, or urologic diseases. Supervised multivariate statistical analysis of the metabolic profiles revealed metabolites significantly associated with age, weight, and height, respectively. The tremendous growth occurring during the neonatal period is associated with specific modifications of newborns' metabolism. Conversely, gender appears to have no impact on the urine metabolome during early infancy. These results allow a deeper understanding of newborns' metabolic maturation and underline potential confounding factors in newborns' metabolomics studies. We emphasize the need to systematically and precisely report children age, height, and weight that impact urine metabolic profiles of infants.

A Systematic Evaluation of Blood Serum and Plasma Pre-Analytics for Metabolomics Cohort Studies.

The recent thriving development of biobanks and associated high-throughput phenotyping studies requires the elaboration of large-scale approaches for monitoring biological sample quality and compliance with standard protocols. We present a metabolomic investigation of human blood samples that delineates pitfalls and guidelines for the collection, storage and handling procedures for serum and plasma. A series of eight pre-processing technical parameters is systematically investigated along variable ranges commonly encountered across clinical studies. While metabolic fingerprints, as assessed by nuclear magnetic resonance, are not significantly affected by altered centrifugation parameters or delays between sample pre-processing (blood centrifugation) and storage, our metabolomic investigation highlights that both the delay and storage temperature between blood draw and centrifugation are the primary parameters impacting serum and plasma metabolic profiles. Storing the blood drawn at 4 °C is shown to be a reliable routine to confine variability associated with idle time prior to sample pre-processing. Based on their fine sensitivity to pre-analytical parameters and protocol variations, metabolic fingerprints could be exploited as valuable ways to determine compliance with standard procedures and quality assessment of blood samples within large multi-omic clinical and translational cohort studies.

Insights on the virulence of swine respiratory tract mycoplasmas through genome-scale metabolic modeling.

BACKGROUND: The respiratory tract of swine is colonized by several bacteria among which are three Mycoplasma species: Mycoplasma flocculare, Mycoplasma hyopneumoniae and Mycoplasma hyorhinis. While colonization by M. flocculare is virtually asymptomatic, M. hyopneumoniae is the causative agent of enzootic pneumonia and M. hyorhinis is present in cases of pneumonia, polyserositis and arthritis. The genomic resemblance among these three Mycoplasma species combined with their different levels of pathogenicity is an indication that they have unknown mechanisms of virulence and differential expression, as for most mycoplasmas. METHODS: In this work, we performed whole-genome metabolic network reconstructions for these three mycoplasmas. Cultivation tests and metabolomic experiments through nuclear magnetic resonance spectroscopy (NMR) were also performed to acquire experimental data and further refine the models reconstructed in silico. RESULTS: Even though the refined models have similar metabolic capabilities, interesting differences include a wider range of carbohydrate uptake in M. hyorhinis, which in turn may also explain why this species is a widely contaminant in cell cultures. In addition, the myo-inositol catabolism is exclusive to M. hyopneumoniae and may be an important trait for virulence. However, the most important difference seems to be related to glycerol conversion to dihydroxyacetone-phosphate, which produces toxic hydrogen peroxide. This activity, missing only in M. flocculare, may be directly involved in cytotoxicity, as already described for two lung pathogenic mycoplasmas, namely Mycoplasma pneumoniae in human and Mycoplasma mycoides subsp. mycoides in ruminants. Metabolomic data suggest that even though these mycoplasmas are extremely similar in terms of genome and metabolism, distinct products and reaction rates may be the result of differential expression throughout the species. CONCLUSIONS: We were able to infer from the reconstructed networks that the lack of pathogenicity of M. flocculare if compared to the highly pathogenic M. hyopneumoniae may be related to its incapacity to produce cytotoxic hydrogen peroxide. Moreover, the ability of M. hyorhinis to grow in diverse sites and even in different hosts may be a reflection of its enhanced and wider carbohydrate uptake. Altogether, the metabolic differences highlighted in silico and in vitro provide important insights to the different levels of pathogenicity observed in each of the studied species.

Structure elucidation of a complex CO2-based organic framework material by NMR crystallography.

A three-dimensional structural model of a complex CO2-based organic framework made from high molecular weight, self-assembled, flexible and multi-functional oligomeric constituents has been determined de novo by solid-state NMR including DNP-enhanced experiments. The complete assignment of the 15N, 13C and 1H resonances was obtained from a series of two-dimensional through space and through bond correlation experiments. MM-QM calculations were used to generate different model structures for the material which were then evaluated by comparing multiple experimental and calculated NMR parameters. Both NMR and powder X-ray diffraction were evaluated as tools to determine the packing by crystal modelling, and at the level of structural modelling used here PXRD was found not to be a useful complement. The structure determined reveals a highly optimised H-bonding network that explains the unusual selectivity of the self-assembly process which generates the material. The NMR crystallography approach used here should be applicable for the structure determination of other complex solid materials.

Metabolomic profiles of hepatocellular carcinoma in a European prospective cohort.

BACKGROUND: Hepatocellular carcinoma (HCC), the most prevalent form of liver cancer, is difficult to diagnose and has limited treatment options with a low survival rate. Aside from a few key risk factors, such as hepatitis, high alcohol consumption, smoking, obesity, and diabetes, there is incomplete etiologic understanding of the disease and little progress in identification of early risk biomarkers. METHODS: To address these aspects, an untargeted nuclear magnetic resonance metabolomic approach was applied to pre-diagnostic serum samples obtained from first incident, primary HCC cases (n = 114) and matched controls (n = 222) identified from amongst the participants of a large European prospective cohort. RESULTS: A metabolic pattern associated with HCC risk comprised of perturbations in fatty acid oxidation and amino acid, lipid, and carbohydrate metabolism was observed. Sixteen metabolites of either endogenous or exogenous origin were found to be significantly associated with HCC risk. The influence of hepatitis infection and potential liver damage was assessed, and further analyses were made to distinguish patterns of early or later diagnosis. CONCLUSION: Our results show clear metabolic alterations from early stages of HCC development with application for better etiologic understanding, prevention, and early detection of this increasingly common cancer.